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Find video protocols related to scientific articles indexed in Pubmed.
The induction of the p53 tumor suppressor protein bridges the apoptotic and autophagic signaling pathways to regulate cell death in prostate cancer cells.
Oncotarget
PUBLISHED: 10-10-2014
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The p53 tumor suppressor protein plays a crucial role in influencing cell fate decisions in response to cellular stress. As p53 elicits cell cycle arrest, senescence or apoptosis, the integrity of the p53 pathway is considered a key determinant of anti-tumor responses. p53 can also promote autophagy, however the role of p53-dependent autophagy in chemosensitivity is poorly understood. VMY-1-103 (VMY), a dansylated analog of purvalanol B, displays rapid and potent anti-tumor activities, however the pathways by which VMY works are not fully defined. Using established prostate cancer cell lines and novel conditionally reprogrammed cells (CRCs) derived from prostate cancer patients; we have defined the mechanisms of VMY-induced prostate cancer cell death. Herein, we show that the cytotoxic effects of VMY required a p53-dependent induction of autophagy, and that inhibition of autophagy abrogated VMY-induced cell death. Cancer cell lines harboring p53 missense mutations evaded VMY toxicity and treatment with a small molecule compound that restores p53 activity re-established VMY-induced cell death. The elucidation of the molecular mechanisms governing VMY-dependent cell death in cell lines, and importantly in CRCs, provides the rationale for clinical studies of VMY, alone or in combination with p53 reactivating compounds, in human prostate cancer.
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The potential to target CCL5/CCR5 in breast cancer.
Expert Opin. Ther. Targets
PUBLISHED: 09-26-2014
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Chemokines play a crucial role in breast cancer tumorigenesis and progression. Recently, the chemokine (C-C motif) ligand 5 (CCL5), which can be secreted either by tumor cells or by mesenchymal stromal cells recruited to the tumor, has been identified as a key node in the bidirectional communication between breast cancer and normal cells.
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Acetylation-defective mutant of Ppar? is associated with decreased lipid synthesis in breast cancer cells.
Oncotarget
PUBLISHED: 09-18-2014
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In our prior publications we characterized a conserved acetylation motif (K(R)xxKK) of evolutionarily related nuclear receptors. Recent reports showed that peroxisome proliferator activated receptor gamma (PPAR?) deacetylation by SIRT1 is involved in delaying cellular senescence and maintaining the brown remodeling of white adipose tissue. However, it still remains unknown whether lysyl residues 154 and 155 (K154/155) of the conserved acetylation motif (RIHKK) in Ppar?1 are acetylated. Herein, we demonstrate that Ppar?1 is acetylated and regulated by both endogenous TSA-sensitive and NAD-dependent deacetylases. Acetylation of lysine 154 was identified by mass spectrometry (MS) while deacetylation of lysine 155 by SIRT1 was confirmed by in vitro deacetylation assay. An in vivo labeling assay revealed K154/K155 as bona fide acetylation sites. The conserved acetylation sites of Ppar?1 and the catalytic domain of SIRT1 are both required for the interaction between Ppar?1 and SIRT1. Sirt1 and Ppar?1 converge to govern lipid metabolism in vivo. Acetylation-defective mutants of Ppar?1 were associated with reduced lipid synthesis in ErbB2 overexpressing breast cancer cells. Together, these results suggest that the conserved lysyl residues K154/K155 of Ppar?1 are acetylated and play an important role in lipid synthesis in ErbB2-positive breast cancer cells.
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Cyclin D1 integrates estrogen-mediated DNA damage repair signaling.
Cancer Res.
PUBLISHED: 05-15-2014
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The cyclin D1 gene encodes the regulatory subunit of a holoenyzme that phosphorylates the retinoblastoma protein (pRb) and nuclear respiratory factor (NRF1) proteins. The abundance of cyclin D1 determines estrogen-dependent gene expression in the mammary gland of mice. Using estradiol (E2) and an E2-dendrimer conjugate that is excluded from the nucleus, we demonstrate that E2 delays the DNA damage response (DDR) via an extranuclear mechanism. The E2-induced DDR required extranuclear cyclin D1, which bound ER? at the cytoplasmic membrane and augmented AKT phosphorylation (Ser473) and ?H2AX foci formation. In the nucleus, E2 inhibited, whereas cyclin D1 enhanced homology-directed DNA repair. Cyclin D1 was recruited to ?H2AX foci by E2 and induced Rad51 expression. Cyclin D1 governs an essential role in the E2-dependent DNA damage response via a novel extranuclear function. The dissociable cytoplasmic function to delay the E2-mediated DDR together with the nuclear enhancement of DNA repair uncovers a novel extranuclear function of cyclin D1 that may contribute to the role of E2 in breast tumorigenesis.
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miR-221/222 promotes S-phase entry and cellular migration in control of basal-like breast cancer.
Molecules
PUBLISHED: 04-02-2014
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The miR-221/222 cluster has been demonstrated to function as oncomiR in human cancers. miR-221/222 promotes epithelial-to-mesenchymal transition (EMT) and confers tamoxifen resistance in breast cancer. However, the effects and mechanisms by which miR-221/222 regulates breast cancer aggressiveness remain unclear. Here we detected a much higher expression of miR-221/222 in highly invasive basal-like breast cancer (BLBC) cells than that in non-invasive luminal cells. A microRNA dataset from breast cancer patients indicated an elevated expression of miR-221/222 in BLBC subtype. S-phase entry of the cell cycle was associated with the induction of miR-221/222 expression. miRNA inhibitors specially targeting miR-221 or miR-222 both significantly suppressed cellular migration, invasion and G1/S transition of the cell cycle in BLBC cell types. Proteomic analysis demonstrated the down-regulation of two tumor suppressor genes, suppressor of cytokine signaling 1 (SOCS1) and cyclin-dependent kinase inhibit 1B (CDKN1B), by miR-221/222. This is the first report to reveal miR-221/222 regulation of G1/S transition of the cell cycle. These findings demonstrate that miR-221/222 contribute to the aggressiveness in control of BLBC.
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miR-17/20 sensitization of breast cancer cells to chemotherapy-induced apoptosis requires Akt1.
Oncotarget
PUBLISHED: 03-25-2014
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The serine threonine kinase Akt1 has been implicated in the control of cellular metabolism, survival and growth. Herein, disruption of the ubiquitously expressed member of the Akt family of genes, Akt1, in the mouse, demonstrates a requirement for Akt1 in miRNA-mediated cellular apoptosis. The miR-17/20 cluster is known to inhibit breast cancer cellular proliferation through G1/S cell cycle arrest via binding to the cyclin D1 3'UTR. Here we show that miR-17/20 overexpression sensitizes cells to apoptosis induced by either Doxorubicin or UV irradiation in MCF-7 cells via Akt1. miR-17/20 mediates apoptosis via increased p53 expression which promotes Akt degradation. Akt1?/? mammary epithelial cells which express Akt2 and Akt3 demonstrated increased apoptosis to DNA damaging agents. Akt1 deficiency abolished the miR-17/20-mediated apoptosis. These results demonstrated a novel pathway through which miR17/20 regulate p53 and Akt controlling breast cancer cell apoptosis.
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Caveolin-1 regulates the anti-atherogenic properties of macrophages.
Cell Tissue Res.
PUBLISHED: 03-11-2014
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Atherosclerosis is a complex disease initiated by the vascular accumulation of lipoproteins in the sub-endothelial space, followed by the infiltration of monocytes into the arterial intima. Caveolin-1 (Cav-1) plays an essential role in the regulation of cellular cholesterol metabolism and of various signaling pathways. In order to study specifically the role of macrophage Cav-1 in atherosclerosis, we used Cav-1 (-/-) Apoe (-/-) mice and transplanted them with bone marrow (BM) cells obtained from Cav-1 (+/+) Apoe (-/-) or Cav-1 (-/-) Apoe (-/-) mice and vice versa. We found that Cav-1 (+/+) mice harboring Cav-1 (-/-) BM-derived macrophages developed significantly larger lesions than Cav-1 (+/+) mice harboring Cav-1 (+/+) BM-derived macrophages. Cav-1 (-/-) macrophages were more susceptible to apoptosis and more prone to induce inflammation. The present study provides clear evidence that the absence of Cav-1 in macrophage is pro-atherogenic, whereas its absence in endothelial cells protects against atherosclerotic lesion formation. These findings demonstrate the cell-specific role of Cav-1 during the development of this disease.
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CAPER, a novel regulator of human breast cancer progression.
Cell Cycle
PUBLISHED: 02-17-2014
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CAPER is an estrogen receptor (ER) co-activator that was recently shown to be involved in human breast cancer pathogenesis. Indeed, we reported increased expression of CAPER in human breast cancer specimens. We demonstrated that CAPER was undetectable or expressed at relatively low levels in normal breast tissue and assumed a cytoplasmic distribution. In contrast, CAPER was expressed at higher levels in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) specimens, where it assumed a predominantly nuclear distribution. However, the functional role of CAPER in human breast cancer initiation and progression remained unknown. Here, we used a lentiviral-mediated gene silencing approach to reduce the expression of CAPER in the ER-positive human breast cancer cell line MCF-7. The proliferation and tumorigenicity of MCF-7 cells stably expressing control or human CAPER shRNAs was then determined via both in vitro and in vivo experiments. Knockdown of CAPER expression significantly reduced the proliferation of MCF-7 cells in vitro. Importantly, nude mice injected with MCF-7 cells harboring CAPER shRNAs developed smaller tumors than mice injected with MCF-7 cells harboring control shRNAs. Mechanistically, tumors derived from mice injected with MCF-7 cells harboring CAPER shRNAs displayed reduced expression of the cell cycle regulators PCNA, MCM7, and cyclin D1, and the protein synthesis marker 4EBP1. In conclusion, knockdown of CAPER expression markedly reduced human breast cancer cell proliferation in both in vitro and in vivo settings. Mechanistically, knockdown of CAPER abrogated the activity of proliferative and protein synthesis pathways.
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The metastatic potential of triple-negative breast cancer is decreased via caloric restriction-mediated reduction of the miR-17~92 cluster.
Breast Cancer Res. Treat.
PUBLISHED: 02-01-2014
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Caloric restriction (CR) has been shown to cause tumor regression in models of triple-negative breast cancer (TNBC), and the regression is augmented when coupled with ionizing radiation (IR). In this study, we sought to determine if the molecular interaction between CR and IR could be mediated by microRNA (miR). miR arrays revealed 3 miRs in the miR-17~92 cluster as most significantly down regulated when CR is combined with IR. In vivo, CR and IR down regulated miR-17/20 in 2 TNBC models. To elucidate the mechanism by which this cluster regulates the response to CR, cDNA arrays were performed and the top 5 statistically significant gene ontology terms with high fold changes were all associated with extracellular matrix (ECM) and metastases. In silico analysis revealed 4 potential targets of the miR-17~92 cluster related to ECM: collagen 4 alpha 3, laminin alpha 3, and metallopeptidase inhibitors 2 and 3, which were confirmed by luciferase assays. The overexpression or silencing of miR-17/20a demonstrated that those miRs directly affected the ECM proteins. Furthermore, we found that CR-mediated inhibition of miR-17/20a can regulate the expression of ECM proteins. Functionally, we demonstrate that CR decreases the metastatic potential of cells which further demonstrates the importance of the ECM. In conclusion, CR can be used as a potential treatment for cancer because it may alter many molecular targets concurrently and decrease metastatic potential for TNBC.
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Long and noncoding RNAs (lnc-RNAs) determine androgen receptor dependent gene expression in prostate cancer growth in vivo.
Asian J. Androl.
PUBLISHED: 01-18-2014
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Hyperactive androgen receptor (AR) activity remains a key determinant of the onset and progression of prostate cancer and resistance to current therapies. The mechanisms governing castrate resistant prostate cancer are poorly understood, but defining these molecular events is essential in order to impact deaths from prostate cancer. Yang et al. demonstrate that two lnc-RNAs known to be overexpressed in therapy resistant prostate cancer, PRNCR1 (also known as PCAT8) and PCGEM1, bound to the AR to enhance ligand-dependent and ligand-independent AR gene expression and proliferation of prostate cancer cells.1 The sequence of these interactions involved the binding of PRNCR1 to the acetylated AR and a subsequent association of DOT1L, which was required for the sequential recruitment of the lncRNA PCGEM1 to the AR amino terminus, which in turn was methylated by DOT1L.
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MicroRNA-mediated cancer metastasis regulation via heterotypic signals in the microenvironment.
Curr Pharm Biotechnol
PUBLISHED: 01-14-2014
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MicroRNAs (miRNAs) are thought to regulate tumor progression and metastasis via direct interaction with target genes within cells. Emerging evidence has demonstrated the secretion of miRNAs into environment via cancer cell exosomes, called "exosomal shuttle small RNA". Microenvironmental miRNAs are important mediators of cell-to-cell communication, and they play important roles in regulating cancer metastasis. RNA analysis indicates enrichment of the miRNA population in cell-culturing medium. miRNA-conditioned medium is able to mediate the function of miRNAs in regulating cancer cell migration and invasion. Here we combine our recent work with literature discussing multiple mechanisms through which exosomal miRNAs regulate cancer cell migration, invasion and metastasis. We summarize a heterotypic signaling pathway by which miRNA regulates the cellular secretion and tumor microenvironment in control of breast cancer cell migration and invasion. In conclusion, exosomal miRNAs are able to regulate cancer metastasis via heterotypic signals in the microenvironment.
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Overview of cyclins D1 function in cancer and the CDK inhibitor landscape: past and present.
Expert Opin Investig Drugs
PUBLISHED: 01-03-2014
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Intensive efforts, over the last decade, have been made to inhibit the kinase activity of cyclins that act as mediators during cell-cycle progression. Activation of the cyclin D1 oncogene, often by amplification or rearrangement, is a major driver of multiple types of human tumors including breast and squamous cell cancers, B-cell lymphoma, myeloma and parathyroid adenoma.
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Cell Fate Factor DACH1 Represses YB-1-mediated Oncogenic Transcription and Translation.
Cancer Res.
PUBLISHED: 12-12-2013
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The epithelial-mesenchymal transition (EMT) enhances cellular invasiveness and confers tumor cells with cancer stem cell like characteristics, through transcriptional and translational mechanisms. The mechanisms maintaining transcriptional and translational repression of EMT and cellular invasion are poorly understood. Herein, the cell fate-determination factor Dachshund (DACH1), suppressed EMT via repression of cytoplasmic translational induction of Snail by inactivating the Y box-binding protein (YB-1). In the nucleus, DACH1 antagonized YB-1-mediated oncogenic transcriptional modules governing cell invasion. DACH1 blocked YB-1-induced mammary tumor growth and EMT in mice. In basal-like breast cancer (BLBC) the reduced expression of DACH1 and increased YB-1, correlated with poor metastasis free survival. The loss of DACH1 suppression of both cytoplasmic translational and nuclear transcriptional events governing EMT and tumor invasion may contribute to poor prognosis in basal-like forms of breast cancer, a relatively aggressive disease subtype.
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Identification of a cyclin D1 network in prostate cancer that antagonizes epithelial-mesenchymal restraint.
Cancer Res.
PUBLISHED: 11-26-2013
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Improved clinical management of prostate cancer (PCa) has been impeded by an inadequate understanding of molecular genetic elements governing tumor progression. Gene signatures have provided improved prognostic indicators of human PCa. The TGF?/BMP-SMAD4 signaling pathway, which induces epithelial mesenchymal transition (EMT), is known to constrain prostate cancer progression induced by Pten deletion. Herein, cyclin D1 inactivation reduced cellular proliferation in the murine prostate in vivo and in isogenic oncogene-transformed prostate cancer cell lines. The in vivo cyclin D1-mediated molecular signature predicted poor outcome of recurrence free survival for prostate cancer patients (K-means hazard ratio 3.75, P-value=0.02) and demonstrated that endogenous cyclin D1 restrains TGF?, Snail, Twist and Goosecoid signaling. Endogenous cyclin D1 enhanced Wnt and ES cell gene expression and expanded a prostate stem cell population. In ChIP-Seq, cyclin D1 occupied genes governing stem cell expansion and induced their transcription. The coordination of EMT restraining and stem cell expanding gene expression by cyclin D1 in the prostate may contribute to its strong prognostic value for poor outcome in biochemical free recurrence in human prostate cancer.
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Cyclin D1 determines estrogen signaling in the mammary gland in vivo.
Mol. Endocrinol.
PUBLISHED: 07-17-2013
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The CCND1 gene, which is frequently overexpressed in cancers, encodes the regulatory subunit of a holoenzyme that phosphorylates the retinoblastoma protein. Although it is known that cyclin D1 regulates estrogen receptor (ER)? transactivation using heterologous reporter systems, the in vivo biological significance of cyclin D1 to estrogen-dependent signaling, and the molecular mechanisms by which cyclin D1 is involved, are yet to be elucidated. Herein, genome-wide expression profiling conducted of 17?-estradiol-treated castrated virgin mice deleted of the Ccnd1 gene demonstrated that cyclin D1 determines estrogen-dependent gene expression for 88% of estrogen-responsive genes in vivo. In addition, expression profiling of 17?-estradiol-stimulated cyclin D1 small interfering RNA treated MCF7 cells shows cyclin D1 is required for estrogen-mediated gene expression in vitro. Genome-wide chromatin immunoprecipitation-Seq analysis revealed a cyclin D1-DNA bound form associated with genes that were regulated by estrogen in a cyclin D1-dependent manner. The cyclin D1-dependent estrogen signaling pathways identified in vivo were highly enriched for extracellular membrane-associated growth factor receptors (epidermal growth factor receptor, ErbB3, and EphB3) and their ligands (amphiregulin, encoded by AREG gene), and matrix metalloproteinase. The AREG protein, a pivotal ligand for epidermal growth factor receptors to promote cellular proliferation, was induced by cyclin D1 via the AREG promoter. Chromatin immunoprecipitation analysis demonstrated the recruitment of cyclin D1 to the breast cancer 1 (Brca1)/ER? binding site of the Areg gene. Cyclin D1 genetic deletion demonstrated the in vivo requirement for cyclin D1 in assembling the estrogen-dependent amplified in breast cancer 1-associated multiprotein complex. The current studies define a requirement for cyclin D1 in estrogen-dependent signaling modules governing growth factor receptor and ligand expression in vivo and reveal a noncanonical function of cyclin D1 at ER? target gene promoters. Cyclin D1 mediates the convergence of ER? and growth factor signaling at a common cis-element of growth factor genes.
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Oncogenes and inflammation rewire host energy metabolism in the tumor microenvironment: RAS and NF?B target stromal MCT4.
Cell Cycle
PUBLISHED: 07-08-2013
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Here, we developed a model system to evaluate the metabolic effects of oncogene(s) on the host microenvironment. A matched set of "normal" and oncogenically transformed epithelial cell lines were co-cultured with human fibroblasts, to determine the "bystander" effects of oncogenes on stromal cells. ROS production and glucose uptake were measured by FACS analysis. In addition, expression of a panel of metabolic protein biomarkers (Caveolin-1, MCT1, and MCT4) was analyzed in parallel. Interestingly, oncogene activation in cancer cells was sufficient to induce the metabolic reprogramming of cancer-associated fibroblasts toward glycolysis, via oxidative stress. Evidence for "metabolic symbiosis" between oxidative cancer cells and glycolytic fibroblasts was provided by MCT1/4 immunostaining. As such, oncogenes drive the establishment of a stromal-epithelial "lactate-shuttle", to fuel the anabolic growth of cancer cells. Similar results were obtained with two divergent oncogenes (RAS and NF?B), indicating that ROS production and inflammation metabolically converge on the tumor stroma, driving glycolysis and upregulation of MCT4. These findings make stromal MCT4 an attractive target for new drug discovery, as MCT4 is a shared endpoint for the metabolic effects of many oncogenic stimuli. Thus, diverse oncogenes stimulate a common metabolic response in the tumor stroma. Conversely, we also show that fibroblasts protect cancer cells against oncogenic stress and senescence by reducing ROS production in tumor cells. Ras-transformed cells were also able to metabolically reprogram normal adjacent epithelia, indicating that cancer cells can use either fibroblasts or epithelial cells as "partners" for metabolic symbiosis. The antioxidant N-acetyl-cysteine (NAC) selectively halted mitochondrial biogenesis in Ras-transformed cells, but not in normal epithelia. NAC also blocked stromal induction of MCT4, indicating that NAC effectively functions as an "MCT4 inhibitor". Taken together, our data provide new strategies for achieving more effective anticancer therapy. We conclude that oncogenes enable cancer cells to behave as selfish "metabolic parasites", like foreign organisms (bacteria, fungi, viruses). Thus, we should consider treating cancer like an infectious disease, with new classes of metabolically targeted "antibiotics" to selectively starve cancer cells. Our results provide new support for the "seed and soil" hypothesis, which was first proposed in 1889 by the English surgeon, Stephen Paget.
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Acetylation of the cell-fate factor dachshund determines p53 binding and signaling modules in breast cancer.
Oncotarget
PUBLISHED: 06-27-2013
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Breast cancer is a leading form of cancer in the world. The Drosophila Dac gene was cloned as an inhibitor of the hyperactive epidermal growth factor (EGFR), ellipse. Herein, endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar compartment and bound to p53 in human breast cancer cell lines, p53 and DACH1 bound common genes in Chip-Seq. Full inhibition of breast cancer contact-independent growth by DACH1 required p53. The p53 breast cancer mutants R248Q and R273H, evaded DACH1 binding. DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding. DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628. DACH1 repressed p21CIP1 and induced RAD51, an association found in basal breast cancer. DACH1 inhibits breast cancer cellular growth in an NAD and p53-dependent manner through direct protein-protein association.
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Cyclin D1 induction of Dicer governs microRNA processing and expression in breast cancer.
Nat Commun
PUBLISHED: 06-14-2013
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Cyclin D1 encodes the regulatory subunit of a holoenzyme that phosphorylates the pRB protein and promotes G1/S cell-cycle progression and oncogenesis. Dicer is a central regulator of miRNA maturation, encoding an enzyme that cleaves double-stranded RNA or stem-loop-stem RNA into 20-25 nucleotide long small RNA, governing sequence-specific gene silencing and heterochromatin methylation. The mechanism by which the cell cycle directly controls the non-coding genome is poorly understood. Here we show that cyclin D1(-/-) cells are defective in pre-miRNA processing which is restored by cyclin D1a rescue. Cyclin D1 induces Dicer expression in vitro and in vivo. Dicer is transcriptionally targeted by cyclin D1, via a cdk-independent mechanism. Cyclin D1 and Dicer expression significantly correlates in luminal A and basal-like subtypes of human breast cancer. Cyclin D1 and Dicer maintain heterochromatic histone modification (Tri-m-H3K9). Cyclin D1-mediated cellular proliferation and migration is Dicer-dependent. We conclude that cyclin D1 induction of Dicer coordinates microRNA biogenesis.
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Caloric restriction augments radiation efficacy in breast cancer.
Cell Cycle
PUBLISHED: 05-21-2013
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Dietary modification such as caloric restriction (CR) has been shown to decrease tumor initiation and progression. We sought to determine if nutrient restriction could be used as a novel therapeutic intervention to enhance cytotoxic therapies such as radiation (IR) and alter the molecular profile of triple-negative breast cancer (TNBC), which displays a poor prognosis. In two murine models of TNBC, significant tumor regression is noted with IR or diet modification, and a greater regression is observed combining diet modification with IR. Two methods of diet modification were compared, and it was found that a daily 30% reduction in total calories provided more significant tumor regression than alternate day feeding. At the molecular level, tumors treated with CR and IR showed less proliferation and more apoptosis. cDNA array analysis demonstrated the IGF-1R pathway plays a key role in achieving this physiologic response, and multiple members of the IGF-1R pathway including IGF-1R, IRS, PIK3ca and mTOR were found to be downregulated. The innovative use of CR as a novel therapeutic option has the potential to change the biology of tumors and enhance the opportunity for clinical benefit in the treatment of patients with TNBC.
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EYA1 phosphatase function is essential to drive breast cancer cell proliferation through cyclin D1.
Cancer Res.
PUBLISHED: 05-01-2013
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The Drosophila Eyes Absent Homologue 1 (EYA1) is a component of the retinal determination gene network and serves as an H2AX phosphatase. The cyclin D1 gene encodes the regulatory subunits of a holoenzyme that phosphorylates and inactivates the pRb protein. Herein, comparison with normal breast showed that EYA1 is overexpressed with cyclin D1 in luminal B breast cancer subtype. EYA1 enhanced breast tumor growth in mice in vivo, requiring the phosphatase domain. EYA1 enhanced cellular proliferation, inhibited apoptosis, and induced contact-independent growth and cyclin D1 abundance. The induction of cellular proliferation and cyclin D1 abundance, but not apoptosis, was dependent upon the EYA1 phosphatase domain. The EYA1-mediated transcriptional induction of cyclin D1 occurred via the AP-1-binding site at -953 and required the EYA1 phosphatase function. The AP-1 mutation did not affect SIX1-dependent activation of cyclin D1. EYA1 was recruited in the context of local chromatin to the cyclin D1 AP-1 site. The EYA1 phosphatase function determined the recruitment of CBP, RNA polymerase II, and acetylation of H3K9 at the cyclin D1 gene AP-1 site regulatory region in the context of local chromatin. The EYA1 phosphatase regulates cell-cycle control via transcriptional complex formation at the cyclin D1 promoter.
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Caveolin-1 is a negative regulator of tumor growth in glioblastoma and modulates chemosensitivity to temozolomide.
Cell Cycle
PUBLISHED: 04-17-2013
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Caveolin-1 (Cav-1) is a critical regulator of tumor progression in a variety of cancers where it has been shown to act as either a tumor suppressor or tumor promoter. In glioblastoma multiforme, it has been previously demonstrated to function as a putative tumor suppressor. Our studies here, using the human glioblastoma-derived cell line U-87MG, further support the role of Cav-1 as a negative regulator of tumor growth. Using a lentiviral transduction approach, we were able to stably overexpress Cav-1 in U-87MG cells. Gene expression microarray analyses demonstrated significant enrichment in gene signatures corresponding to downregulation of MAPK, PI3K/AKT and mTOR signaling, as well as activation of apoptotic pathways in Cav-1-overexpressing U-87MG cells. These same gene signatures were later confirmed at the protein level in vitro. To explore the ability of Cav-1 to regulate tumor growth in vivo, we further show that Cav-1-overexpressing U-87MG cells display reduced tumorigenicity in an ectopic xenograft mouse model, with marked hypoactivation of MAPK and PI3K/mTOR pathways. Finally, we demonstrate that Cav-1 overexpression confers sensitivity to the most commonly used chemotherapy for glioblastoma, temozolomide. In conclusion, Cav-1 negatively regulates key cell growth and survival pathways and may be an effective biomarker for predicting response to chemotherapy in glioblastoma.
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Compartment-specific activation of PPAR? governs breast cancer tumor growth, via metabolic reprogramming and symbiosis.
Cell Cycle
PUBLISHED: 04-10-2013
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The role of PPAR? in cancer therapy is controversial, with studies showing either pro-tumorigenic or antineoplastic effects. This debate is very clinically relevant, because PPAR? agonists are used as antidiabetic drugs. Here, we evaluated if the effects of PPAR? on tumorigenesis are determined by the cell type in which PPAR? is activated. Second, we examined if the metabolic changes induced by PPAR?, such as glycolysis and autophagy, play any role in the tumorigenic process. To this end, PPAR? was overexpressed in breast cancer cells or in stromal cells. PPAR?-overexpressing cells were examined with respect to (1) their tumorigenic potential, using xenograft models, and (2) regarding their metabolic features. In xenograft models, we show that when PPAR? is activated in cancer cells, tumor growth is inhibited by 40%. However, when PPAR? is activated in stromal cells, the growth of co-injected breast cancer cells is enhanced by 60%. Thus, the effect(s) of PPAR? on tumorigenesis are dependent on the cell compartment in which PPAR? is activated. Mechanistically, stromal cells with activated PPAR? display metabolic features of cancer-associated fibroblasts, with increased autophagy, glycolysis and senescence. Indeed, fibroblasts overexpressing PPAR? show increased expression of autophagic markers, increased numbers of acidic autophagic vacuoles, increased production of L-lactate, cell hypertrophy and mitochondrial dysfunction. In addition, PPAR? fibroblasts show increased expression of CDKs (p16/p21) and ?-galactosidase, which are markers of cell cycle arrest and senescence. Finally, PPAR? induces the activation of the two major transcription factors that promote autophagy and glycolysis, i.e., HIF-1? and NF?B, in stromal cells. Thus, PPAR? activation in stromal cells results in the formation of a catabolic pro-inflammatory microenvironment that metabolically supports cancer growth. Interestingly, the tumor inhibition observed when PPAR? is expressed in epithelial cancer cells is also associated with increased autophagy, suggesting that activation of an autophagic program has both pro- or antitumorigenic effects depending on the cell compartment in which it occurs. Finally, when PPAR? is expressed in epithelial cancer cells, the suppression of tumor growth is associated with a modest inhibition of angiogenesis. In conclusion, these data support the "two-compartment tumor metabolism" model, which proposes that metabolic coupling exists between catabolic stromal cells and oxidative cancer cells. Cancer cells induce autophagy, glycolysis and senescence in stromal cells. In return, stromal cells generate onco-metabolites and mitochondrial fuels (L-lactate, ketones, glutamine/aminoacids and fatty acids) that are used by cancer cells to enhance their tumorigenic potential. Thus, as researchers design new therapies, they must be conscious that cancer is not a cell-autonomous disease, but rather a tumor is an ecosystem of many different cell types, which engage in metabolic symbiosis.
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Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity.
Sci Rep
PUBLISHED: 04-05-2013
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Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzymes activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans.
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Dachshund binds p53 to block the growth of lung adenocarcinoma cells.
Cancer Res.
PUBLISHED: 03-14-2013
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Hyperactive EGF receptor (EGFR) and mutant p53 are common genetic abnormalities driving the progression of non-small cell lung cancer (NSCLC), the leading cause of cancer deaths in the world. The Drosophila gene Dachshund (Dac) was originally cloned as an inhibitor of hyperactive EGFR alleles. Given the importance of EGFR signaling in lung cancer etiology, we examined the role of DACH1 expression in lung cancer development. DACH1 protein and mRNA expression was reduced in human NSCLC. Reexpression of DACH1 reduced NSCLC colony formation and tumor growth in vivo via p53. Endogenous DACH1 colocalized with p53 in a nuclear, extranucleolar location, and shared occupancy of -15% of p53-bound genes in ChIP sequencing. The C-terminus of DACH1 was necessary for direct p53 binding, contributing to the inhibition of colony formation and cell-cycle arrest. Expression of the stem cell factor SOX2 was repressed by DACH1, and SOX2 expression was inversely correlated with DACH1 in NSCLC. We conclude that DACH1 binds p53 to inhibit NSCLC cellular growth.
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New roles of cyclin D1.
Am. J. Pathol.
PUBLISHED: 02-26-2013
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Cyclins encode regulatory subunits of holoenzymes that phosphorylate a variety of cellular substrates. Although the classic role of cyclins in cell cycle progression and tumorigenesis has been well characterized, new functions have been identified, including the induction of cellular migration and invasion, enhancement of angiogenesis, inhibition of mitochondrial metabolism, regulation of transcription factor signaling via a DNA-bound form, the induction of chromosomal instability, enhancement of DNA damage sensing and DNA damage repair, and feedback governing expression of the noncoding genome. This review describes the mechanisms of these new functions of cyclin D1.
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The CCL5/CCR5 axis promotes metastasis in basal breast cancer.
Oncoimmunology
PUBLISHED: 01-17-2013
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Recently, we have shown that the CCL5/CCR5 axis is active in patients affected by an aggressive basal subtype of breast cancer. Using preclinical models, we have demonstrated that CCR5 promotes breast cancer invasiveness and metastatic potential, while CCR5 inhibition abrogates them. Thus, CCR5 antagonists may constitute an alternative therapeutic approach for patients affected by metastatic basal breast cancer.
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Nutrient restriction and radiation therapy for cancer treatment: when less is more.
Oncologist
PUBLISHED: 01-08-2013
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Calorie restriction (CR), or a diet modification aiming to reduce the total intake of calories by 20%-40%, has been shown to increase longevity across multiple species. Recently, there has been growing interest in investigating the potential role of CR as a treatment intervention for age-related diseases, such as cancer, because an increasing body of literature has demonstrated a metabolic component to both carcinogenesis and tumor progression. In fact, many of the molecular pathways that are altered with CR are also known to be altered in cancer. Therefore, manipulation of these pathways using CR can render cancer cells, and most notably breast cancer cells, more susceptible to standard cytotoxic treatment with radiation and chemotherapy. In this review article we demonstrate the laboratory and clinical evidence that exists for CR and show compelling evidence through the molecular pathways CR induces about how it may be used as a treatment in tandem with radiation therapy to improve our rates of disease control.
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Glutamine fuels a vicious cycle of autophagy in the tumor stroma and oxidative mitochondrial metabolism in epithelial cancer cells: implications for preventing chemotherapy resistance.
Cancer Biol. Ther.
PUBLISHED: 12-15-2011
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Glutamine metabolism is crucial for cancer cell growth via the generation of intermediate molecules in the tricarboxylic acid (TCA) cycle, antioxidants and ammonia. The goal of the current study was to evaluate the effects of glutamine on metabolism in the breast cancer tumor microenvironment, with a focus on autophagy and cell death in both epithelial and stromal compartments. For this purpose, MCF7 breast cancer cells were cultured alone or co-cultured with non-transformed fibroblasts in media containing high glutamine and low glucose (glutamine +) or under control conditions, with no glutamine and high glucose (glutamine -). Here, we show that MCF7 cells maintained in co-culture with glutamine display increased mitochondrial mass, as compared with control conditions. Importantly, treatment with the autophagy inhibitor chloroquine abolishes the glutamine-induced augmentation of mitochondrial mass. It is known that loss of caveolin-1 (Cav-1) expression in fibroblasts is associated with increased autophagy and an aggressive tumor microenvironment. Here, we show that Cav-1 downregulation which occurs in fibroblasts maintained in co-culture specifically requires glutamine. Interestingly, glutamine increases the expression of autophagy markers in fibroblasts, but decreases expression of autophagy markers in MCF7 cells, indicating that glutamine regulates the autophagy program in a compartment-specific manner. Functionally, glutamine protects MCF7 cells against apoptosis, via the upregulation of the anti-apoptotic and anti-autophagic protein TIGAR. Also, we show that glutamine cooperates with stromal fibroblasts to confer tamoxifen-resistance in MCF7 cancer cells. Finally, we provide evidence that co-culture with fibroblasts (1) promotes glutamine catabolism, and (2) decreases glutamine synthesis in MCF7 cancer cells. Taken together, our findings suggest that autophagic fibroblasts may serve as a key source of energy-rich glutamine to fuel cancer cell mitochondrial activity, driving a vicious cycle of catabolism in the tumor stroma and anabolic tumor cell expansion.
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Pyruvate kinase expression (PKM1 and PKM2) in cancer-associated fibroblasts drives stromal nutrient production and tumor growth.
Cancer Biol. Ther.
PUBLISHED: 12-15-2011
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We have previously demonstrated that enhanced aerobic glycolysis and/or autophagy in the tumor stroma supports epithelial cancer cell growth and aggressive behavior, via the secretion of high-energy metabolites. These nutrients include lactate and ketones, as well as chemical building blocks, such as amino acids (glutamine) and nucleotides. Lactate and ketones serve as fuel for cancer cell oxidative metabolism, and building blocks sustain the anabolic needs of rapidly proliferating cancer cells. We have termed these novel concepts the "Reverse Warburg Effect," and the "Autophagic Tumor Stroma Model of Cancer Metabolism." We have also identified a loss of stromal caveolin-1 (Cav-1) as a marker of stromal glycolysis and autophagy. The aim of the current study was to provide genetic evidence that enhanced glycolysis in stromal cells favors tumorigenesis. To this end, normal human fibroblasts were genetically-engineered to express the two isoforms of pyruvate kinase M (PKM1 and PKM2), a key enzyme in the glycolytic pathway. In a xenograft model, fibroblasts expressing PKM1 or PKM2 greatly promoted the growth of co-injected MDA-MB-231 breast cancer cells, without an increase in tumor angiogenesis. Interestingly, PKM1 and PKM2 promoted tumorigenesis by different mechanism(s). Expression of PKM1 enhanced the glycolytic power of stromal cells, with increased output of lactate. Analysis of tumor xenografts demonstrated that PKM1 fibroblasts greatly induced tumor inflammation, as judged by CD45 staining. In contrast, PKM2 did not lead to lactate accumulation, but triggered a "pseudo-starvation" response in stromal cells, with induction of an NF?B-dependent autophagic program, and increased output of the ketone body 3-hydroxy-buryrate. Strikingly, in situ evaluation of Complex IV activity in the tumor xenografts demonstrated that stromal PKM2 expression drives mitochondrial respiration specifically in tumor cells. Finally, immuno-histochemistry analysis of human breast cancer samples lacking stromal Cav-1 revealed PKM1 and PKM2 expression in the tumor stroma. Thus, our data indicate that a subset of human breast cancer patients with a loss of stromal Cav-1 show profound metabolic changes in the tumor microenvironment. As such, this subgroup of patients may benefit therapeutically from potent inhibitors targeting glycolysis, autophagy and/or mitochondrial activity (such as metformin).
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Mammary gland selective excision of c-jun identifies its role in mRNA splicing.
Cancer Res.
PUBLISHED: 12-15-2011
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The c-jun gene regulates cellular proliferation and apoptosis via direct regulation of cellular gene expression. Alternative splicing of pre-mRNA increases the diversity of protein functions, and alternate splicing events occur in tumors. Here, by targeting the excision of the endogenous c-jun gene within the mouse mammary epithelium, we have identified its selective role as an inhibitor of RNA splicing. Microarray-based assessment of gene expression, on laser capture microdissected c-jun(-/-) mammary epithelium, showed that endogenous c-jun regulates the expression of approximately 50 genes governing RNA splicing. In addition, genome-wide splicing arrays showed that endogenous c-jun regulated the alternate exon of approximately 147 genes, and 18% of these were either alternatively spliced in human tumors or involved in apoptosis. Endogenous c-jun also was shown to reduce splicing activity, which required the c-jun dimerization domain. Together, our findings suggest that c-jun directly attenuates RNA splicing efficiency, which may be of broad biologic importance as alternative splicing plays an important role in both cancer development and therapy resistance.
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Energy transfer in "parasitic" cancer metabolism: mitochondria are the powerhouse and Achilles heel of tumor cells.
Cell Cycle
PUBLISHED: 12-15-2011
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It is now widely recognized that the tumor microenvironment promotes cancer cell growth and metastasis via changes in cytokine secretion and extracellular matrix remodeling. However, the role of tumor stromal cells in providing energy for epithelial cancer cell growth is a newly emerging paradigm. For example, we and others have recently proposed that tumor growth and metastasis is related to an energy imbalance. Host cells produce energy-rich nutrients via catabolism (through autophagy, mitophagy, and aerobic glycolysis), which are then transferred to cancer cells to fuel anabolic tumor growth. Stromal cell-derived L-lactate is taken up by cancer cells and is used for mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP efficiently. However, "parasitic" energy transfer may be a more generalized mechanism in cancer biology than previously appreciated. Two recent papers in Science and Nature Medicine now show that lipolysis in host tissues also fuels tumor growth. These studies demonstrate that free fatty acids produced by host cell lipolysis are re-used via beta-oxidation (beta-OX) in cancer cell mitochondria. Thus, stromal catabolites (such as lactate, ketones, glutamine and free fatty acids) promote tumor growth by acting as high-energy onco-metabolites. As such, host catabolism, via autophagy, mitophagy and lipolysis, may explain the pathogenesis of cancer-associated cachexia and provides exciting new druggable targets for novel therapeutic interventions. Taken together, these findings also suggest that tumor cells promote their own growth and survival by behaving as a "parasitic organism." Hence, we propose the term "Parasitic Cancer Metabolism" to describe this type of metabolic coupling in tumors. Targeting tumor cell mitochondria (OXPHOS and beta-OX) would effectively uncouple tumor cells from their hosts, leading to their acute starvation. In this context, we discuss new evidence that high-energy onco-metabolites (produced by the stroma) can confer drug resistance. Importantly, this metabolic chemo-resistance is reversed by blocking OXPHOS in cancer cell mitochondria with drugs like Metformin, a mitochondrial "poison." In summary, parasitic cancer metabolism is achieved architecturally by dividing tumor tissue into at least two well-defined opposing "metabolic compartments:" catabolic and anabolic.
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SIRT1 modulates aggregation and toxicity through deacetylation of the androgen receptor in cell models of SBMA.
J. Neurosci.
PUBLISHED: 12-02-2011
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Posttranslational protein modifications can play a major role in disease pathogenesis; phosphorylation, sumoylation, and acetylation modulate the toxicity of a variety of proteotoxic proteins. The androgen receptor (AR) is substantially modified, in response to hormone binding, by phosphorylation, sumoylation, and acetylation; these modifications might thus contribute to DHT-dependent polyglutamine (polyQ)-expanded AR proteotoxicity in spinal and bulbar muscular atrophy (SBMA). SIRT1, a nuclear protein and deacetylase of the AR, is neuroprotective in many neurodegenerative disease models. Our studies reveal that SIRT1 also offers protection against polyQ-expanded AR by deacetylating the AR at lysines 630/632/633. This finding suggested that nuclear AR acetylation plays a role in the aberrant metabolism and toxicity of polyQ-expanded AR. Subsequent studies revealed that the polyQ-expanded AR is hyperacetylated and that pharmacologic reduction of acetylation reduces mutant AR aggregation. Moreover, genetic mutation to inhibit polyQ-expanded AR acetylation of lysines 630/632/633 substantially decreased its aggregation and completely abrogated its toxicity in cell lines and motor neurons. Our studies also reveal one means by which the AR acetylation state likely modifies polyQ-expanded AR metabolism and toxicity, through its effect on DHT-dependent AR stabilization. Overall, our findings reveal a neuroprotective function of SIRT1 that operates through its deacetylation of polyQ-expanded AR and highlight the potential of both SIRT1 and AR acetylation as powerful therapeutic targets in SBMA.
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Hyperactivation of oxidative mitochondrial metabolism in epithelial cancer cells in situ: visualizing the therapeutic effects of metformin in tumor tissue.
Cell Cycle
PUBLISHED: 12-01-2011
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We have recently proposed a new mechanism for explaining energy transfer in cancer metabolism. In this scenario, cancer cells behave as metabolic parasites, by extracting nutrients from normal host cells, such as fibroblasts, via the secretion of hydrogen peroxide as the initial trigger. Oxidative stress in the tumor microenvironment then leads to autophagy-driven catabolism, mitochondrial dys-function, and aerobic glycolysis. This, in turn, produces high-energy nutrients (such as L-lactate, ketones, and glutamine) that drive the anabolic growth of tumor cells, via oxidative mitochondrial metabolism. A logical prediction of this new "parasitic" cancer model is that tumor-associated fibroblasts should show evidence of mitochondrial dys-function (mitophagy and aerobic glycolysis). In contrast, epithelial cancer cells should increase their oxidative mitochondrial capacity. To further test this hypothesis, here we subjected frozen sections from human breast tumors to a staining procedure that only detects functional mitochondria. This method detects the in situ enzymatic activity of cytochrome C oxidase (COX), also known as Complex IV. Remarkably, cancer cells show an over-abundance of COX activity, while adjacent stromal cells remain essentially negative. Adjacent normal ductal epithelial cells also show little or no COX activity, relative to epithelial cancer cells. Thus, oxidative mitochondrial activity is selectively amplified in cancer cells. Although COX activity staining has never been applied to cancer tissues, it could now be used routinely to distinguish cancer cells from normal cells, and to establish negative margins during cancer surgery. Similar results were obtained with NADH activity staining, which measures Complex I activity, and succinate dehydrogenase (SDH) activity staining, which measures Complex II activity. COX and NADH activities were blocked by electron transport inhibitors, such as Metformin. This has mechanistic and clinical implications for using Metformin as an anti-cancer drug, both for cancer therapy and chemo-prevention. We also immuno-stained human breast cancers for a series of well-established protein biomarkers of metabolism. More specifically, we now show that cancer-associated fibroblasts over-express markers of autophagy (cathepsin B), mitophagy (BNIP3L), and aerobic glycolysis (MCT4). Conversely, epithelial cancer cells show the over-expression of a mitochondrial membrane marker (TOMM20), as well as key components of Complex IV (MT-CO1) and Complex II (SDH-B). We also validated our observations using a bioinformatics approach with data from > 2,000 breast cancer patients, which showed the transcriptional upregulation of mitochondrial oxidative phosphorylation (OXPHOS) in human breast tumors (p < 10(-20)), and a specific association with metastasis. Therefore, upregulation of OXPHOS in epithelial tumor cells is a common feature of human breast cancers. In summary, our data provide the first functional in vivo evidence that epithelial cancer cells perform enhanced mitochondrial oxidative phosphorylation, allowing them to produce high amounts of ATP. Thus, we believe that mitochondria are both the "powerhouse" and "Achilles heel" of cancer cells.
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Mitochondrial oxidative stress in cancer-associated fibroblasts drives lactate production, promoting breast cancer tumor growth: understanding the aging and cancer connection.
Cell Cycle
PUBLISHED: 12-01-2011
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Increasing chronological age is the most significant risk factor for cancer. Recently, we proposed a new paradigm for understanding the role of the aging and the tumor microenvironment in cancer onset. In this model, cancer cells induce oxidative stress in adjacent stromal fibroblasts. This, in turn, causes several changes in the phenotype of the fibroblast including mitochondrial dysfunction, hydrogen peroxide production, and aerobic glycolysis, resulting in high levels of L-lactate production. L-lactate is then transferred from these glycolytic fibroblasts to adjacent epithelial cancer cells and used as "fuel" for oxidative mitochondrial metabolism.  Here, we created a new pre-clinical model system to directly test this hypothesis experimentally. To synthetically generate glycolytic fibroblasts, we genetically-induced mitochondrial dysfunction by knocking down TFAM using an sh-RNA approach.  TFAM is mitochondrial transcription factor A, which is important in functionally maintaining the mitochondrial respiratory chain. Interestingly, TFAM-deficient fibroblasts showed evidence of mitochondrial dysfunction and oxidative stress, with the loss of certain mitochondrial respiratory chain components, and the over-production of hydrogen peroxide and L-lactate. Thus, TFAM-deficient fibroblasts underwent metabolic reprogramming towards aerobic glycolysis.  Most importantly, TFAM-deficient fibroblasts significantly promoted tumor growth, as assayed using a human breast cancer (MDA-MB-231) xenograft model. These increases in glycolytic fibroblast driven tumor growth were independent of tumor angiogenesis. Mechanistically, TFAM-deficient fibroblasts increased the mitochondrial activity of adjacent epithelial cancer cells in a co-culture system, as seen using MitoTracker. Finally, TFAM-deficient fibroblasts also showed a loss of caveolin-1 (Cav-1), a known breast cancer stromal biomarker. Loss of stromal fibroblast Cav-1 is associated with early tumor recurrence, metastasis, and treatment failure, resulting in poor clinical outcome in breast cancer patients. Thus, this new experimental model system, employing glycolytic fibroblasts, may be highly clinically relevant. These studies also have implications for understanding the role of hydrogen peroxide production in oxidative damage and "host cell aging," in providing a permissive metabolic microenvironment for promoting and sustaining tumor growth.
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Warburg meets autophagy: cancer-associated fibroblasts accelerate tumor growth and metastasis via oxidative stress, mitophagy, and aerobic glycolysis.
Antioxid. Redox Signal.
PUBLISHED: 11-17-2011
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Here, we review certain recent advances in oxidative stress and tumor metabolism, which are related to understanding the contributions of the microenvironment in promoting tumor growth and metastasis. In the early 1920s, Otto Warburg, a Nobel Laureate, formulated a hypothesis to explain the "fundamental basis" of cancer, based on his observations that tumors displayed a metabolic shift toward glycolysis. In 1963, Christian de Duve, another Nobel Laureate, first coined the phrase auto-phagy, derived from the Greek words "auto" and "phagy," meaning "self" and "eating."
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Anti-estrogen resistance in breast cancer is induced by the tumor microenvironment and can be overcome by inhibiting mitochondrial function in epithelial cancer cells.
Cancer Biol. Ther.
PUBLISHED: 11-15-2011
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Here, we show that tamoxifen resistance is induced by cancer-associated fibroblasts (CAFs). Coculture of estrogen receptor positive (ER+) MCF7 cells with fibroblasts induces tamoxifen and fulvestrant resistance with 4.4 and 2.5-fold reductions, respectively, in apoptosis compared with homotypic MCF7 cell cultures. Treatment of MCF7 cells cultured alone with high-energy mitochondrial "fuels" (L-lactate or ketone bodies) is sufficient to confer tamoxifen resistance, mimicking the effects of coculture with fibroblasts. To further demonstrate that epithelial cancer cell mitochondrial activity is the origin of tamoxifen resistance, we employed complementary pharmacological and genetic approaches. First, we studied the effects of two mitochondrial "poisons," namely metformin and arsenic trioxide (ATO), on fibroblast-induced tamoxifen resistance. We show here that treatment with metformin or ATO overcomes fibroblast-induced tamoxifen resistance in MCF7 cells. Treatment with the combination of tamoxifen plus metformin or ATO leads to increases in glucose uptake in MCF7 cells, reflecting metabolic uncoupling between epithelial cancer cells and fibroblasts. In coculture, tamoxifen induces the upregulation of TIGAR (TP53-induced glycolysis and apoptosis regulator), a p53 regulated gene that simultaneously inhibits glycolysis, autophagy and apoptosis and reduces ROS generation, thereby promoting oxidative mitochondrial metabolism. To genetically mimic the effects of coculture, we next recombinantly overexpressed TIGAR in MCF7 cells. Remarkably, TIGAR overexpression protects epithelial cancer cells from tamoxifen-induced apoptosis, providing genetic evidence that increased mitochondrial function confers tamoxifen resistance. Finally, CAFs also protect MCF7 cells against apoptosis induced by other anticancer agents, such as the topoisomerase inhibitor doxorubicin (adriamycin) and the PARP-1 inhibitor ABT-888. These results suggest that the tumor microenvironment may be a general mechanism for conferring drug resistance. In summary, we have discovered that mitochondrial activity in epithelial cancer cells drives tamoxifen resistance in breast cancer and that mitochondrial "poisons" are able to re-sensitize these cancer cells to tamoxifen. In this context, TIGAR may be a key "druggable" target for preventing drug resistance in cancer cells, as it protects cancer cells against the onset of stress-induced mitochondrial dys-function and aerobic glycolysis.
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Caveolin-1 and cancer metabolism in the tumor microenvironment: markers, models, and mechanisms.
Annu Rev Pathol
PUBLISHED: 11-07-2011
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Caveolins are a family of membrane-bound scaffolding proteins that compartmentalize and negatively regulate signal transduction. Recent studies have implicated a loss of caveolin-1 (Cav-1) expression in the pathogenesis of human cancers. Loss of Cav-1 expression in cancer-associated fibroblasts results in an activated tumor microenvironment, thereby driving early tumor recurrence, metastasis, and poor clinical outcome in breast and prostate cancers. We describe various paracrine signaling mechanism(s) by which the loss of stromal Cav-1 promotes tumor progression, including fibrosis, extracellular matrix remodeling, and the metabolic/catabolic reprogramming of cancer-associated fibroblast, to fuel the growth of adjacent tumor cells. It appears that oxidative stress is the root cause of initiation of the loss of stromal Cav-1 via autophagy, which provides further impetus for the use of antioxidants in anticancer therapy. Finally, we discuss the functional role of Cav-1 in epithelial cancer cells.
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Caveolin-1 promotes pancreatic cancer cell differentiation and restores membranous E-cadherin via suppression of the epithelial-mesenchymal transition.
Cell Cycle
PUBLISHED: 11-01-2011
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Pancreatic cancer is one of the deadliest cancers due to early rapid metastasis and chemoresistance. Recently, epithelial to mesenchymal transition (EMT) was shown to play a key role in the pathogenesis of pancreatic cancer. To understand the role of caveolin-1 (Cav-1) in EMT, we over-expressed Cav-1 in a pancreatic cancer cell line, Panc 10.05, that does not normally express Cav-1. Here, we show that Cav-1 expression in pancreatic cancer cells induces an epithelial phenotype and promotes cell-cell contact, with increased expression of plasma membrane bound E-cadherin and beta-catenin. Mechanistically, Cav-1 induces Snail downregulation and decreased activation of AKT, MAPK and TGF-beta-Smad signaling pathways. In vitro, Cav-1 expression reduces cell migration and invasion, and attenuates doxorubicin-chemoresistance of pancreatic cancer cells. Importantly, in vivo studies revealed that Cav-1 expression greatly suppresses tumor formation in a xenograft model. Most interestingly, Panc/Cav-1 tumors displayed organized nests of differentiated cells that were totally absent in control tumors. Confirming our in vitro results, these nests of differentiated cells showed reexpression of E-cadherin and beta-catenin at the cell membrane. Thus, we provide evidence that Cav-1 functions as a crucial modulator of EMT and cell differentiation in pancreatic cancer.
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Hormonal therapy promotes hormone-resistant phenotype by increasing DNMT activity and expression in prostate cancer models.
Endocrinology
PUBLISHED: 10-11-2011
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We hypothesized that hormonal therapy favors the development of the hormone-resistant phenotype through epigenetic mechanisms. Human prostate cancer tissues and in vitro and in vivo models were used to verify this hypothesis. We demonstrated that tumor cells continuously treated with bicalutamide (BCLT) or cultured in androgen-depleted medium progressively acquire higher DNA methyltransferase (DNMT) activity and expression than cells cultured in standard condition. Increased DNMT expression and activity also paralleled the up-regulation of truncated AR isoforms, which favors the development of the hormone-resistant phenotype. After androgen stimulation with 10(-12) m dihydrotestosterone, DNMT activity was significantly reduced in comparison with hormonal therapy. Consistent with these observations, the silencing of DNMT3a and DNMT3b significantly decreased the DNMT activity levels. These findings were also directly correlated with phosphatase and tensin homolog down-regulation and activation of ERK and phosphatidylinositol 3-kinases/AKT8 virus oncogene cellular homolog pathways. The use of a pan-DNMT inhibitor (5-Azacitidine) greatly reduced the development of the hormone-resistant phenotype induced by long-term BCLT treatment, and this finding correlated with low DNMT activity. The regulation of DNMT activity was, in some measure, dependent on the androgen receptor, as small interfering RNA treatment targeting the androgen receptor greatly decreased the modulation of DNMT activity under androgenic and antiandrogenic stimulation. These observations were correlated in vivo in patients, as demonstrated by immunohistochemistry. Patients treated by BCLT before surgery had higher DNMT3a and DNMT3b expression than patients who had not undergone this treatment. Our findings provide evidence of a relationship between the castration-resistant phenotype and DNMT expression and activity in human prostate cancer.
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Cancer cells metabolically "fertilize" the tumor microenvironment with hydrogen peroxide, driving the Warburg effect: implications for PET imaging of human tumors.
Cell Cycle
PUBLISHED: 08-01-2011
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Previously, we proposed that cancer cells behave as metabolic parasites, as they use targeted oxidative stress as a "weapon" to extract recycled nutrients from adjacent stromal cells. Oxidative stress in cancer-associated fibroblasts triggers autophagy and  mitophagy, resulting in compartmentalized cellular catabolism, loss of mitochondrial function, and the onset of aerobic glycolysis, in the tumor stroma. As such, cancer-associated fibroblasts produce high-energy nutrients (such as lactate and ketones) that fuel mitochondrial biogenesis, and oxidative metabolism in cancer cells. We have termed this new energy-transfer mechanism the "reverse Warburg effect." To further test the validity of this hypothesis, here we used an in vitro MCF7-fibroblast co-culture system, and quantitatively measured a variety of metabolic parameters by FACS analysis (analogous to laser-capture micro-dissection).  Mitochondrial activity, glucose uptake, and ROS production were measured with highly-sensitive fluorescent probes (MitoTracker, NBD-2-deoxy-glucose, and DCF-DA). Interestingly, using this approach, we directly show that cancer cells initially secrete hydrogen peroxide that then triggers oxidative stress in neighboring fibroblasts. Thus, oxidative stress is contagious (spreads like a virus) and is propagated laterally and vectorially from cancer cells to adjacent fibroblasts. Experimentally, we show that oxidative stress in cancer-associated fibroblasts quantitatively reduces mitochondrial activity, and increases glucose uptake, as the fibroblasts become more dependent on aerobic glycolysis.  Conversely, co-cultured cancer cells show significant increases in mitochondrial activity, and corresponding reductions in both glucose uptake and GLUT1 expression. Pre-treatment of co-cultures with extracellular catalase (an anti-oxidant enzyme that detoxifies hydrogen peroxide) blocks the onset of oxidative stress, and potently induces the death of cancer cells, likely via starvation.  Given that cancer-associated fibroblasts show the largest increases in glucose uptake, we suggest that PET imaging of human tumors, with Fluoro-2-deoxy-D-glucose (F-2-DG), may be specifically detecting the tumor stroma, rather than epithelial cancer cells.
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Understanding the metabolic basis of drug resistance: therapeutic induction of the Warburg effect kills cancer cells.
Cell Cycle
PUBLISHED: 08-01-2011
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Previously, we identified a form of epithelial-stromal metabolic coupling, in which cancer cells induce aerobic glycolysis in adjacent stromal fibroblasts, via oxidative stress, driving autophagy and mitophagy. In turn, these cancer-associated fibroblasts provide recycled nutrients to epithelial cancer cells, "fueling" oxidative mitochondrial metabolism and anabolic growth. An additional consequence is that these glycolytic fibroblasts protect cancer cells against apoptosis, by providing a steady nutrient stream of to mitochondria in cancer cells. Here, we investigated whether these interactions might be the basis of tamoxifen-resistance in ER(+) breast cancer cells. We show that MCF7 cells alone are Tamoxifen-sensitive, but become resistant when co-cultured with hTERT-immortalized human fibroblasts. Next, we searched for a drug combination (Tamoxifen + Dasatinib) that could over-come fibroblast-induced Tamoxifen-resistance. Importantly, we show that this drug combination acutely induces the Warburg effect (aerobic glycolysis) in MCF7 cancer cells, abruptly cutting off their ability to use their fuel supply, effectively killing these cancer cells. Thus, we believe that the Warburg effect in tumor cells is not the "root cause" of cancer, but rather it may provide the necessary clues to preventing chemo-resistance in cancer cells. Finally, we observed that this drug combination (Tamoxifen + Dasatinib) also had a generalized anti-oxidant effect, on both co-cultured fibroblasts and cancer cells alike, potentially reducing tumor-stroma co-evolution. Our results are consistent with the idea that chemo-resistance may be both a metabolic and stromal phenomenon that can be overcome by targeting mitochondrial function in epithelial cancer cells. Thus, simultaneously targeting both (1) the tumor stroma and (2) the epithelial cancer cells, with combination therapies, may be the most successful approach to anti-cancer therapy. This general strategy of combination therapy for overcoming drug resistance could be applicable to many different types of cancer.
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Hydrogen peroxide fuels aging, inflammation, cancer metabolism and metastasis: the seed and soil also needs "fertilizer".
Cell Cycle
PUBLISHED: 08-01-2011
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In 1889, Dr. Stephen Paget proposed the "seed and soil" hypothesis, which states that cancer cells (the seeds) need the proper microenvironment (the soil) for them to grow, spread and metastasize systemically. In this hypothesis, Dr. Paget rightfully recognized that the tumor microenvironment has an important role to play in cancer progression and metastasis. In this regard, a series of recent studies have elegantly shown that the production of hydrogen peroxide, by both cancer cells and cancer-associated fibroblasts, may provide the necessary "fertilizer," by driving accelerated aging, DNA damage, inflammation and cancer metabolism, in the tumor microenvironment. By secreting hydrogen peroxide, cancer cells and fibroblasts are mimicking the behavior of immune cells (macrophages/neutrophils), driving local and systemic inflammation, via the innate immune response (NF?B). Thus, we should consider using various therapeutic strategies (such as catalase and/or other anti-oxidants) to neutralize the production of cancer-associated hydrogen peroxide, thereby preventing tumor-stroma co-evolution and metastasis. The implications of these findings for overcoming chemo-resistance in cancer cells are also discussed in the context of hydrogen peroxide production and cancer metabolism.
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Analysis of nuclear receptor acetylation.
Methods Mol. Biol.
PUBLISHED: 07-29-2011
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Acetylation is an essential post-translational modification in which an acetyl group is covalently conjugated to a protein substrate. Histone acetylation was first proposed nearly half a century ago by Dr. Vincent Allfrey. Subsequent studies have shown that acetylated core histones are often associated with transcriptionally active chromatin. Acetylation at lysine residues of histone tails neutralizes the positive charge, which decreases the binding ability to DNA and increases the accessibility of transcription factors and co-activators to the chromatin template. In addition to histones, a number of non-histone substrates are acetylated. Acetylation of non-histone proteins governs biological processes, including cellular proliferation and survival, transcriptional activity, and intracellular trafficking. We demonstrated that acetylation of transcription factors can regulate cellular growth. Further, we have shown that nuclear receptors are acetylated at a phylogenetically conserved motif. Since our initial observations with the estrogen and androgen receptors, more than a dozen nuclear receptors have been shown to function as substrates for acetyltransferases with a variety of new methods (Fig. 11.1). This chapter focuses on the protocol used in the studies of NR acetylation and de-acetylation. We will discuss the potential pitfalls of each method.
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Understanding the Warburg effect and the prognostic value of stromal caveolin-1 as a marker of a lethal tumor microenvironment.
Breast Cancer Res.
PUBLISHED: 07-08-2011
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Cancer cells show a broad spectrum of bioenergetic states, with some cells using aerobic glycolysis while others rely on oxidative phosphorylation as their main source of energy. In addition, there is mounting evidence that metabolic coupling occurs in aggressive tumors, between epithelial cancer cells and the stromal compartment, and between well-oxygenated and hypoxic compartments. We recently showed that oxidative stress in the tumor stroma, due to aerobic glycolysis and mitochondrial dysfunction, is important for cancer cell mutagenesis and tumor progression. More specifically , increased autophagy/mitophagy in the tumor stroma drives a form of parasitic epithelial-stromal metabolic coupling. These findings explain why it is effective to treat tumors with either inducers or inhibitors of autophagy, as both would disrupt this energetic coupling. We also discuss evidence that glutamine addiction in cancer cells produces ammonia via oxidative mitochondrial metabolism. Ammonia production in cancer cells, in turn, could then help maintain autophagy in the tumor stromal compartment. In this vicious cycle, the initial glutamine provided to cancer cells would be produced by autophagy in the tumor stroma. Thus, we believe that parasitic epithelial-stromal metabolic coupling has important implications for cancer diagnosis and therapy, for example, in designing novel metabolic imaging techniques and establishing new targeted therapies. In direct support of this notion, we identified a loss of stromal caveolin-1 as a marker of oxidative stress, hypoxia, and autophagy in the tumor microenvironment, explaining its powerful predictive value. Loss of stromal caveolin-1 in breast cancers is associated with early tumor recurrence, metastasis, and drug resistance, leading to poor clinical outcome.
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Accelerated aging in the tumor microenvironment: connecting aging, inflammation and cancer metabolism with personalized medicine.
Cell Cycle
PUBLISHED: 07-01-2011
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Cancer is thought to be a disease associated with aging. Interestingly, normal aging is driven by the production of ROS and mitochondrial oxidative stress, resulting in the cumulative accumulation of DNA damage. Here, we discuss how ROS signaling, NF?B- and HIF1-activation in the tumor microenvironment induces a form of "accelerated aging," which leads to stromal inflammation and changes in cancer cell metabolism. Thus, we present a unified model where aging (ROS), inflammation (NF?B) and cancer metabolism (HIF1), act as co-conspirators to drive autophagy ("self-eating") in the tumor stroma. Then, autophagy in the tumor stroma provides high-energy "fuel" and the necessary chemical building blocks, for accelerated tumor growth and metastasis. Stromal ROS production acts as a "mutagenic motor" and allows cancer cells to buffer-at a distance-exactly how much of a mutagenic stimulus they receive, further driving tumor cell selection and evolution. Surviving cancer cells would be selected for the ability to induce ROS more effectively in stromal fibroblasts, so they could extract more nutrients from the stroma via autophagy. If lethal cancer is a disease of "accelerated host aging" in the tumor stroma, then cancer patients may benefit from therapy with powerful antioxidants. Antioxidant therapy should block the resulting DNA damage, and halt autophagy in the tumor stroma, effectively "cutting off the fuel supply" for cancer cells. These findings have important new implications for personalized cancer medicine, as they link aging, inflammation and cancer metabolism with novel strategies for more effective cancer diagnostics and therapeutics.
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Examining the role of cyclin D1 in breast cancer.
Future Oncol
PUBLISHED: 06-17-2011
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Cyclin D1 overexpression is found in more than 50% of human breast cancers and causes mammary cancer in transgenic mice. Dysregulation of cyclin D1 gene expression or function contributes to the loss of normal cell cycle control during tumorigenesis. Recent studies have demonstrated that cyclin D1 conducts additional specific functions to regulate gene expression in the context of local chromatin, promote cellular migration and inhibit mitochondrial metabolism. It is anticipated that these additional functions contribute to the pathology associated with dysregulated cyclin D1 abundance. This article discusses evidence that examines the significance of cyclin D1 in breast cancer with emphasis on its role in breast cancer stem cell expansion.
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Matrix remodeling stimulates stromal autophagy, "fueling" cancer cell mitochondrial metabolism and metastasis.
Cell Cycle
PUBLISHED: 06-15-2011
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We have previously demonstrated that loss of stromal caveolin-1 (Cav-1) in cancer-associated fibroblasts is a strong and independent predictor of poor clinical outcome in human breast cancer patients. However, the signaling mechanism(s) by which Cav-1 downregulation leads to this tumor-promoting microenvironment are not well understood. To address this issue, we performed an unbiased comparative proteomic analysis of wild-type (WT) and Cav-1(-/-) null mammary stromal fibroblasts (MSFs). Our results show that plasminogen activator inhibitor type 1 and type 2 (PAI-1 and PAI-2) expression is significantly increased in Cav-1(-/-) MSFs. To establish a direct cause-effect relationship, we next generated immortalized human fibroblast lines stably overexpressing either PAI-1 or PAI-2. Importantly, PAI-1/2(+) fibroblasts promote the growth of MDA-MB-231 tumors (a human breast cancer cell line) in a murine xenograft model, without any increases in angiogenesis. Similarly, PAI-1/2(+) fibroblasts stimulate experimental metastasis of MDA-MB-231 cells using an in vivo lung colonization assay. Further mechanistic studies revealed that fibroblasts overexpressing PAI-1 or PAI-2 display increased autophagy ("self-eating") and are sufficient to induce mitochondrial biogenesis/activity in adjacent cancer cells, in co-culture experiments. In xenografts, PAI-1/2(+) fibroblasts significantly reduce the apoptosis of MDA-MB-231 tumor cells. The current study provides further support for the "Autophagic Tumor Stroma Model of Cancer" and identifies a novel "extracellular matrix"-based signaling mechanism, by which a loss of stromal Cav-1 generates a metastatic phenotype. Thus, the secretion and remodeling of extracellular matrix components (such as PAI-1/2) can directly regulate both (1) autophagy in stromal fibroblasts and (2) epithelial tumor cell mitochondrial metabolism.
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The type 1 insulin-like growth factor receptor and resistance to DACH1.
Cell Cycle
PUBLISHED: 06-15-2011
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The mammalian homolog of the Drosophila dachshund gene (DACH1) has been reported as a tumor suppressor in human breast and prostate cancers. It downregulates the epidermal growth factor receptor (EGFR) and cyclin D1. The signaling pathway of the type 1 insulin-like growth factor receptor (IGF-IR) is known to be responsible for the development of resistance to treatment of human cancer with antibodies to the EGFR. We have asked whether DACH1 still exerts its tumor suppressor activity in cells dependent on the IGF-IR for growth. We find that in cells growing in IGF-1 (and unresponsive to EGF), DACH1 is devoid of tumor suppressor activity.
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Cytokine production and inflammation drive autophagy in the tumor microenvironment: role of stromal caveolin-1 as a key regulator.
Cell Cycle
PUBLISHED: 06-01-2011
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Recently, we proposed a new paradigm for understanding the role of the tumor microenvironment in breast cancer onset and progression. In this model, cancer cells induce oxidative stress in adjacent fibroblasts. This, in turn, results in the onset of stromal autophagy, which produces recycled nutrients to "feed" anabolic cancer cells. However, it remains unknown how autophagy in the tumor microenvironment relates to inflammation, another key driver of tumorigenesis. To address this issue, here we employed a well-characterized co-culture system in which cancer cells induce autophagy in adjacent fibroblasts via oxidative stress and NF?B-activation. We show, using this co-culture system, that the same experimental conditions that result in an autophagic microenvironment, also drive in the production of numerous inflammatory mediators (including IL-6, IL-8, IL-10, MIP1a, IFNg, RANTES (CCL5) and GMCSF). Furthermore, we demonstrate that most of these inflammatory mediators are individually sufficient to directly induce the onset of autophagy in fibroblasts. To further validate the in vivo relevance of these findings, we assessed the inflammatory status of Cav-1 (-/-) null mammary fat pads, which are a model of a bonafide autophagic microenvironment. Notably, we show that Cav-1 (-/-) mammary fat pads undergo infiltration with numerous inflammatory cell types, including lymphocytes, T-cells, macrophages and mast cells. Taken together, our results suggest that cytokine production and inflammation are key drivers of autophagy in the tumor microenvironment. These results may explain why a loss of stromal Cav-1 is a powerful predictor of poor clinical outcome in breast cancer patients, as it is a marker of both (1) autophagy and (2) inflammation in the tumor microenvironment. Lastly, hypoxia in fibroblasts was not sufficient to induce the full-blown inflammatory response that we observed during the co-culture of fibroblasts with cancer cells, indicating that key reciprocal interactions between cancer cells and fibroblasts may be required.
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Evidence for a stromal-epithelial "lactate shuttle" in human tumors: MCT4 is a marker of oxidative stress in cancer-associated fibroblasts.
Cell Cycle
PUBLISHED: 06-01-2011
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Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to "feed" adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal physiological function of muscle tissue and brain. These data are consistent with the "reverse Warburg effect," which states that cancer-associated fibroblasts undergo aerobic glycolysis, thereby producing lactate, which is utilized as a metabolic substrate by adjacent cancer cells. In this model, "energy transfer" or "metabolic-coupling" between the tumor stroma and epithelial cancer cells "fuels" tumor growth and metastasis, via oxidative mitochondrial metabolism in anabolic cancer cells. Most importantly, our current findings provide a new rationale and novel strategy for anti-cancer therapies, by employing MCT inhibitors.
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Ketones and lactate increase cancer cell "stemness," driving recurrence, metastasis and poor clinical outcome in breast cancer: achieving personalized medicine via Metabolo-Genomics.
Cell Cycle
PUBLISHED: 04-23-2011
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Previously, we showed that high-energy metabolites (lactate and ketones) "fuel" tumor growth and experimental metastasis in an in vivo xenograft model, most likely by driving oxidative mitochondrial metabolism in breast cancer cells. To mechanistically understand how these metabolites affect tumor cell behavior, here we used genome-wide transcriptional profiling. Briefly, human breast cancer cells (MCF7) were cultured with lactate or ketones, and then subjected to transcriptional analysis (exon-array). Interestingly, our results show that treatment with these high-energy metabolites increases the transcriptional expression of gene profiles normally associated with "stemness," including genes upregulated in embryonic stem (ES) cells. Similarly, we observe that lactate and ketones promote the growth of bonafide ES cells, providing functional validation. The lactate- and ketone-induced "gene signatures" were able to predict poor clinical outcome (including recurrence and metastasis) in a cohort of human breast cancer patients. Taken together, our results are consistent with the idea that lactate and ketone utilization in cancer cells promotes the "cancer stem cell" phenotype, resulting in significant decreases in patient survival. One possible mechanism by which these high-energy metabolites might induce stemness is by increasing the pool of Acetyl-CoA, leading to increased histone acetylation, and elevated gene expression. Thus, our results mechanistically imply that clinical outcome in breast cancer could simply be determined by epigenetics and energy metabolism, rather than by the accumulation of specific "classical" gene mutations. We also suggest that high-risk cancer patients (identified by the lactate/ketone gene signatures) could be treated with new therapeutics that target oxidative mitochondrial metabolism, such as the anti-oxidant and "mitochondrial poison" metformin. Finally, we propose that this new approach to personalized cancer medicine be termed "Metabolo-Genomics," which incorporates features of both 1) cell metabolism and 2) gene transcriptional profiling. Importantly, this powerful new approach directly links cancer cell metabolism with clinical outcome, and new therapeutic strategies for inhibiting the TCA cycle and mitochondrial oxidative phosphorylation in cancer cells.
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c-Jun is required for TGF-?-mediated cellular migration via nuclear Ca²? signaling.
Int. J. Biochem. Cell Biol.
PUBLISHED: 03-18-2011
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Tumor progression involves the acquisition of invasiveness through a basement membrane. The c-jun proto-oncogene is overexpressed in human tumors and has been identified at the leading edge of human breast tumors. TGF-? plays a bifunctional role in tumorigenesis and cellular migration. Although c-Jun and the activator protein 1 (AP-1) complex have been implicated in human cancer, the molecular mechanisms governing cellular migration via c-Jun and the role of c-Jun in TGF-? signaling remains poorly understood. Here, we analyze TGF-? mediated cellular migration in mouse embryo fibroblasts using floxed c-jun transgenic mice. We compared the c-jun wild type with the c-jun knockout cells through the use of Cre recombinase. Herein, TGF-? stimulated cellular migration and intracellular calcium release requiring endogenous c-Jun. TGF-? mediated Ca(2+) release was independent of extracellular calcium and was suppressed by both U73122 and neomycin, pharmacological inhibitors of the breakdown of PIP(2) into IP(3). Unlike TGF-?-mediated Ca(2+) release, which was c-Jun dependent, ATP mediated Ca(2+) release was c-Jun independent. These studies identify a novel pathway by which TGF-? regulates cellular migration and Ca(2+) release via endogenous c-Jun.
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Caveolin-1 and mitochondrial SOD2 (MnSOD) function as tumor suppressors in the stromal microenvironment: a new genetically tractable model for human cancer associated fibroblasts.
Cancer Biol. Ther.
PUBLISHED: 02-15-2011
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We have recently proposed a new model for understanding tumor metabolism, termed: "The Autophagic Tumor Stroma Model of Cancer Metabolism". In this new paradigm, catabolism (autophagy) in the tumor stroma fuels the anabolic growth of aggressive cancer cells. Mechanistically, tumor cells induce autophagy in adjacent cancer-associated fibroblasts via the loss of caveolin-1 (Cav-1), which is sufficient to promote oxidative stress in stromal fibroblasts. To further test this hypothesis, here we created human Cav-1 deficient immortalized fibroblasts using a targeted sh-RNA knock-down approach. Relative to control fibroblasts, Cav-1 deficient fibroblasts dramatically promoted tumor growth in xenograft assays employing an aggressive human breast cancer cell line, namely MDA-MB-231 cells. Co-injection of Cav-1 deficient fibroblasts, with MDA-MB-231 cells, increased both tumor mass and tumor volume by ~4-fold. Immuno-staining with CD31 indicated that this paracrine tumor promoting effect was clearly independent of angiogenesis. Mechanistically, proteomic analysis of these human Cav-1 deficient fibroblasts identified > 40 protein biomarkers that were upregulated, most of which were associated with i) myofibroblast differentiation, or ii) oxidative stress/hypoxia. In direct support of these findings, the tumor promoting effects of Cav-1 deficient fibroblasts could be functionally suppressed (nearly 2-fold) by the recombinant over-expression of SOD2 (superoxide dismutase 2), a known mitochondrial enzyme that de-activates superoxide, thereby reducing mitochondrial oxidative stress. In contrast, cytoplasmic soluble SOD1 had no effect, further highlighting a specific role for mitochondrial oxidative stress in this process. In summary, here we provide new evidence directly supporting a key role for a loss of stromal Cav-1 expression and oxidative stress in cancer-associated fibroblasts, in promoting tumor growth, which is consistent with "The Autophagic Tumor Stroma Model of Cancer". The human Cav-1 deficient fibroblasts that we have generated are a new genetically tractable model system for identifying other suppressors of the cancer-associated fibroblast phenotype, via a genetic "complementation" approach. This has important implications for understanding the pathogenesis of triple negative and basal breasts cancers, as well as tamoxifen-resistance in ER+ breast cancers, which are all associated with a Cav-1 deficient "lethal" tumor micro-environment, driving poor clinical outcome.
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The role of breast cancer stem cells in metastasis and therapeutic implications.
Am. J. Pathol.
PUBLISHED: 02-07-2011
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Cancer stem cells (CSCs) possess the capacity to self-renew and to generate heterogeneous lineages of cancer cells that comprise tumors. A substantial body of evidence supports a model in which CSCs play a major role in the initiation, maintenance, and clinical outcome of cancers. In contrast, analysis of the role of CSCs in metastasis has been mainly conceptual and speculative. This review summarizes recent data that support the theory of CSCs as the source of metastatic lesions in breast cancer, with a focus on the key role of the microenvironment in the stemness-metastasis link.
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Stromal-epithelial metabolic coupling in cancer: integrating autophagy and metabolism in the tumor microenvironment.
Int. J. Biochem. Cell Biol.
PUBLISHED: 01-19-2011
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Cancer cells do not exist as pure homogeneous populations in vivo. Instead they are embedded in "cancer cell nests" that are surrounded by stromal cells, especially cancer associated fibroblasts. Thus, it is not unreasonable to suspect that stromal fibroblasts could influence the metabolism of adjacent cancer cells, and visa versa. In accordance with this idea, we have recently proposed that the Warburg effect in cancer cells may be due to culturing cancer cells by themselves, out of their normal stromal context or tumor microenvironment. In fact, when cancer cells are co-cultured with fibroblasts, then cancer cells increase their mitochondrial mass, while fibroblasts lose their mitochondria. An in depth analysis of this phenomenon reveals that aggressive cancer cells are "parasites" that use oxidative stress as a "weapon" to extract nutrients from surrounding stromal cells. Oxidative stress in fibroblasts induces the autophagic destruction of mitochondria, by mitophagy. Then, stromal cells are forced to undergo aerobic glycolysis, and produce energy-rich nutrients (such as lactate and ketones) to "feed" cancer cells. This mechanism would allow cancer cells to seed anywhere, without blood vessels as a food source, as they could simply induce oxidative stress wherever they go, explaining how cancer cells survive during metastasis. We suggest that stromal catabolism, via autophagy and mitophagy, fuels the anabolic growth of tumor cells, promoting tumor progression and metastasis. We have previously termed this new paradigm "The Autophagic Tumor Stroma Model of Cancer Metabolism", or the "Reverse Warburg Effect". We also discuss how glutamine addiction (glutaminolysis) in cancer cells fits well with this new model, by promoting oxidative mitochondrial metabolism in aggressive cancer cells.
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MEK/ERK inhibitor U0126 increases the radiosensitivity of rhabdomyosarcoma cells in vitro and in vivo by downregulating growth and DNA repair signals.
Mol. Cancer Ther.
PUBLISHED: 01-12-2011
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Multimodal treatment has improved the outcome of many solid tumors, and in some cases the use of radiosensitizers has significantly contributed to this gain. Activation of the extracellular signaling kinase pathway (MEK/ERK) generally results in stimulation of cell growth and confers a survival advantage playing the major role in human cancer. The potential involvement of this pathway in cellular radiosensitivity remains unclear. We previously reported that the disruption of c-Myc through MEK/ERK inhibition blocks the expression of the transformed phenotype; affects in vitro and in vivo growth and angiogenic signaling; and induces myogenic differentiation in the embryonal rhabdomyosarcoma (ERMS) cell lines (RD). This study was designed to examine whether the ERK pathway affects intrinsic radiosensitivity of rhabdomyosarcoma cancer cells. Exponentially growing human ERMS, RD, xenograft-derived RD-M1, and TE671 cell lines were used. The specific MEK/ERK inhibitor, U0126, reduced the clonogenic potential of the three cell lines, and was affected by radiation. U0126 inhibited phospho-active ERK1/2 and reduced DNA protein kinase catalytic subunit (DNA-PKcs) suggesting that ERKs and DNA-PKcs cooperate in radioprotection of rhabdomyosarcoma cells. The TE671 cell line xenotransplanted in mice showed a reduction in tumor mass and increase in the time of tumor progression with U0126 treatment associated with reduced DNA-PKcs, an effect enhanced by radiotherapy. Thus, our results show that MEK/ERK inhibition enhances radiosensitivity of rhabdomyosarcoma cells suggesting a rational approach in combination with radiotherapy.
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PACSIN 2 represses cellular migration through direct association with cyclin D1 but not its alternate splice form cyclin D1b.
Cell Cycle
PUBLISHED: 01-01-2011
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Cyclin D1 overexpression is a common feature of many human malignancies. Genomic deletion analysis has demonstrated a key role for cyclin D1 in cellular proliferation, angiogenesis, and cellular migration. To investigate the mechanisms contributing to cyclin D1 functions, we purified cyclin D1a-associated complexes by affinity chromatography and identified the PACSIN 2 (protein kinase C and casein kinase substrate in neurons 2) protein by mass spectrometry. The PACSIN 2, but not the related PACSIN 1 and 3, directly bound wild-type cyclin D1 (cyclin D1a) at the carboxyl terminus, and failed to bind cyclin D1b, the alternative splicing variant of cyclin D1. PACSIN 2 knockdown induced cellular migration and reduced cell spreading in LNCaP cells expressing cyclin D1a. In cyclin D1(-/-) mouse embryonic fibroblasts (MEFs), cyclin D1a, but not cyclin D1b, reduced the cell spreading to a polarized morphology. siPACSIN 2 had no effect on cellular migration of cyclin D1(-/-) MEFs. Cyclin D1a restored the migratory ability of cyclin D1(-/-) MEFs, which was further enhanced by knocking down PACSIN 2 with siRNA. The cyclin D1-associated protein, PACSIN 2, regulates cell spreading and migration, which are dependent on cyclin D1 expression.
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Disruption of a Sirt1-dependent autophagy checkpoint in the prostate results in prostatic intraepithelial neoplasia lesion formation.
Cancer Res.
PUBLISHED: 12-28-2010
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The Sirtuin family of proteins (SIRT) encode a group of evolutionarily conserved, NAD-dependent histone deacetylases, involved in many biological pathways. SIRT1, the human homologue of the yeast Silent Information Regulator 2 (Sir2) gene, deacetylates histones, p300, p53, and the androgen receptor. Autophagy is required for the degradation of damaged organelles and long-lived proteins, as well as for the development of glands such as the breast and prostate. Herein, homozygous deletion of the Sirt1 gene in mice resulted in prostatic intraepithelial neoplasia (PIN) associated with reduced autophagy. Genome-wide gene expression analysis of Sirt1(-/-) prostates demonstrated that endogenous Sirt1 repressed androgen responsive gene expression and induced autophagy in the prostate. Sirt1 induction of autophagy occurred at the level of autophagosome maturation and completion in cultured prostate cancer cells. These studies provide novel evidence for a checkpoint function of Sirt1 in the development of PIN and further highlight a role for SIRT1 as a tumor suppressor in the prostate.
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The canonical NF-kappaB pathway governs mammary tumorigenesis in transgenic mice and tumor stem cell expansion.
Cancer Res.
PUBLISHED: 12-17-2010
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The role of mammary epithelial cell (MEC) NF-?B in tumor progression in vivo is unknown, as murine NF-?B components and kinases either are required for murine survival or interfere with normal mammary gland development. As NF-?B inhibitors block both tumor-associated macrophages (TAM) and MEC NF-?B, the importance of MEC NF-?B to tumor progression in vivo remained to be determined. Herein, an MEC-targeted inducible transgenic inhibitor of NF-?B (I?B?SR) was developed in ErbB2 mammary oncomice. Inducible suppression of NF-?B in the adult mammary epithelium delayed the onset and number of new tumors. Within similar sized breast tumors, TAM and tumor neoangiogenesis was reduced. Coculture experiments demonstrated MEC NF-?B enhanced TAM recruitment. Genome-wide expression and proteomic analysis showed that I?B?SR inhibited tumor stem cell pathways. I?B?SR inhibited breast tumor stem cell markers in transgenic tumors, reduced stem cell expansion in vitro, and repressed expression of Nanog and Sox2 in vivo and in vitro. MEC NF-?B contributes to mammary tumorigenesis. As we show that NF-?B contributes to expansion of breast tumor stem cells and heterotypic signals that enhance TAM and vasculogenesis, these processes may contribute to NF-?B-dependent mammary tumorigenesis.
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The autophagic tumor stroma model of cancer or "battery-operated tumor growth": A simple solution to the autophagy paradox.
Cell Cycle
PUBLISHED: 11-30-2010
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The role of autophagy in tumorigenesis is controversial. Both autophagy inhibitors (chloroquine) and autophagy promoters (rapamycin) block tumorigenesis by unknown mechanism(s). This is called the "Autophagy Paradox". We have recently reported a simple solution to this paradox. We demonstrated that epithelial cancer cells use oxidative stress to induce autophagy in the tumor microenvironment. As a consequence, the autophagic tumor stroma generates recycled nutrients that can then be used as chemical building blocks by anabolic epithelial cancer cells. This model results in a net energy transfer from the tumor stroma to epithelial cancer cells (an energy imbalance), thereby promoting tumor growth. This net energy transfer is both unilateral and vectorial, from the tumor stroma to the epithelial cancer cells, representing a true host-parasite relationship. We have termed this new paradigm "The Autophagic Tumor Stroma Model of Cancer Cell Metabolism" or "Battery-Operated Tumor Growth". In this sense, autophagy in the tumor stroma serves as a "battery" to fuel tumor growth, progression and metastasis, independently of angiogenesis. Using this model, the systemic induction of autophagy will prevent epithelial cancer cells from using recycled nutrients, while the systemic inhibiton of autophagy will prevent stromal cells from producing recycled nutrients-both effectively "starving" cancer cells. We discuss the idea that tumor cells could become resistant to the systemic induction of autophagy, by the upregulation of natural endogenous autophagy inhibitors in cancer cells. Alternatively, tumor cells could also become resistant to the systemic induction of autophagy, by the genetic silencing/deletion of pro-autophagic molecules, such as Beclin1. If autophagy resistance develops in cancer cells, then the systemic inhibition of autophagy would provide a therapeutic solution to this type of drug resistance, as it would still target autophagy in the tumor stroma. As such, an anti-cancer therapy that combines the alternating use of both autophagy promoters and autophagy inhibitors would be expected to prevent the onset of drug resistance. We also discuss why anti-angiogenic therapy has been found to promote tumor recurrence, progression and metastasis. More specifically, anti-angiogenic therapy would induce autophagy in the tumor stroma via the induction of stromal hypoxia, thereby converting a non-aggressive tumor type to a "lethal" aggressive tumor phenotype. Thus, uncoupling the metabolic parasitic relationship between cancer cells and an autophagic tumor stroma may hold great promise for anti-cancer therapy. Finally, we believe that autophagy in the tumor stroma is the local microscopic counterpart of systemic wasting (cancer-associated cachexia), which is associated with advanced and metastatic cancers. Cachexia in cancer patients is not due to decreased energy intake, but instead involves an increased basal metabolic rate and increased energy expenditures, resulting in a negative energy balance. Importantly, when tumors were surgically excised, this increased metabolic rate returned to normal levels. This view of cachexia, resulting in energy transfer to the tumor, is consistent with our hypothesis. So, cancer-associated cachexia may start locally as stromal autophagy, and then spread systemically. As such, stromal autophagy may be the requisite precursor of systemic cancer-associated cachexia.
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Regulation of the androgen receptor by SET9-mediated methylation.
Nucleic Acids Res.
PUBLISHED: 10-19-2010
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The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors that plays a critical role in regulating expression of genes involved in prostate development and transformation. Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor. Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9. Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes. We also show that SET9 is pro-proliferative and anti-apoptotic in prostate cancer cells and demonstrates up-regulated nuclear expression in prostate cancer tissue. In all, our date indicate a new mechanism of AR regulation that may be therapeutically exploitable for prostate cancer treatment.
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Transcription elongation regulator 1 is a co-integrator of the cell fate determination factor Dachshund homolog 1.
J. Biol. Chem.
PUBLISHED: 10-18-2010
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DACH1 (Dachshund homolog 1) is a key component of the retinal determination gene network and regulates gene expression either indirectly as a co-integrator or through direct DNA binding. The current studies were conducted to understand, at a higher level of resolution, the mechanisms governing DACH1-mediated transcriptional repression via DNA sequence-specific binding. DACH1 repressed gene transcription driven by the DACH1-responsive element (DRE). Recent genome-wide ChIP-Seq analysis demonstrated DACH1 binding sites co-localized with Forkhead protein (FOX) binding sites. Herein, DACH1 repressed, whereas FOX proteins enhanced, both DRE and FOXA-responsive element-driven gene expression. Reduced DACH1 expression using a shRNA approach enhanced FOX protein activity. As DACH1 antagonized FOX target gene expression and attenuated FOX signaling, we sought to identify limiting co-integrator proteins governing DACH1 signaling. Proteomic analysis identified transcription elongation regulator 1 (TCERG1) as the transcriptional co-regulator of DACH1 activity. The FF2 domain of TCERG1 was required for DACH1 binding, and the deletion of FF2 abolished DACH1 trans-repression function. The carboxyl terminus of DACH1 was necessary and sufficient for TCERG1 binding. Thus, DACH1 represses gene transcription through direct DNA binding to the promoter region of target genes by recruiting the transcriptional co-regulator, TCERG1.
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Alternative cyclin D1 splice forms differentially regulate the DNA damage response.
Cancer Res.
PUBLISHED: 10-12-2010
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The DNA damage response (DDR) activates downstream pathways including cell cycle checkpoints. The cyclin D1 gene is overexpressed or amplified in many human cancers and is required for gastrointestinal, breast, and skin tumors in murine models. A common polymorphism in the human cyclin D1 gene is alternatively spliced, resulting in cyclin D1a and D1b proteins that differ in their carboxyl terminus. Cyclin D1 overexpression enhances DNA damage-induced apoptosis. The role of cyclin D1 and the alternative splice form in regulating the DDR is not well understood. Herein cyclin D1a overexpression enhanced the DDR as characterized by induction of ?H2AX phosphorylation, the assembly of DNA repair foci, specific recruitment of DNA repair factors to chromatin, and G(2)-M arrest. Cyclin D1 deletion in fibroblasts or small interfering RNA-mediated reduction of endogenous cyclin D1 in colon cancer cells reduced the 5-fluorouracil-mediated DDR. Mechanistic studies showed that cyclin D1a, like DNA repair factors, elicited the DDR when stably associated with chromatin.
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Cell fate determination factor Dachshund reprograms breast cancer stem cell function.
J. Biol. Chem.
PUBLISHED: 10-11-2010
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The cell fate determination factor Dachshund was cloned as a dominant inhibitor of the hyperactive epidermal growth factor receptor ellipse. The expression of Dachshund is lost in human breast cancer associated with poor prognosis. Breast tumor-initiating cells (TIC) may contribute to tumor progression and therapy resistance. Here, endogenous DACH1 was reduced in breast cancer cell lines with high expression of TIC markers and in patient samples of the basal breast cancer phenotype. Re-expression of DACH1 reduced new tumor formation in serial transplantations in vivo, reduced mammosphere formation, and reduced the proportion of CD44(high)/CD24(low) breast tumor cells. Conversely, lentiviral shRNA to DACH1 increased the breast (B)TIC population. Genome-wide expression studies of mammary tumors demonstrated DACH1 repressed a molecular signature associated with stem cells (SOX2, Nanog, and KLF4) and genome-wide ChIP-seq analysis identified DACH1 binding to the promoter of the Nanog, KLF4, and Lin28 genes. KLF4/c-Myc and Oct4/Sox2 antagonized DACH1 repression of BTIC. Mechanistic studies demonstrated DACH1 directly repressed the Nanog and Sox2 promoters via a conserved domain. Endogenous DACH1 regulates BTIC in vitro and in vivo.
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C-jun inhibits mammary apoptosis in vivo.
Mol. Biol. Cell
PUBLISHED: 10-06-2010
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c-jun, which is overexpressed in a number of human cancers encodes a critical component of the AP-1 complex. c-jun has been shown to either induce or inhibit cellular apoptosis. Germ line deletion of both c-jun alleles is embryonically lethal. To determine the role of the endogenous c-jun gene in apoptosis, we performed mammary epithelial cell-targeted somatic deletion using floxed c-jun (c-jun(f/f)) conditional knockout mice. Laser capture microdissection demonstrated endogenous c-jun inhibits expression of apoptosis inducing genes and reactive oxygen species (ROS)-reducing genes (MnSOD, catalase). ROS have been implicated in apoptosis and undergo enzymatic elimination via MnSOD and CuZnSOD with further detoxification via catalase. c-jun-mediated survival was in part dependent on ROS production. c-jun-mediated repression of MnSOD and catalase occurred via mitochondrial complex I and NOX I. Collectively, these studies define a pivotal role of endogenous c-jun in promoting cell survival via maintaining mitochondrial integrity and expression of the key regulators of ROS production.
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The autophagic tumor stroma model of cancer: Role of oxidative stress and ketone production in fueling tumor cell metabolism.
Cell Cycle
PUBLISHED: 09-24-2010
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A loss of stromal Cav-1 in the tumor fibroblast compartment is associated with early tumor recurrence, lymph-node metastasis, and tamoxifen-resistance, resulting in poor clinical outcome in breast cancer patients. Here, we have used Cav-1 (-/-) null mice as a pre-clinical model for this "lethal tumor micro-environment." Metabolic profiling of Cav-1 (-/-) mammary fat pads revealed the upregulation of numerous metabolites (nearly 100), indicative of a major catabolic phenotype. Our results are consistent with the induction of oxidative stress, mitochondrial dysfunction, and autophagy/mitophagy. The two most prominent metabolites that emerged from this analysis were ADMA (asymmetric dimethyl arginine) and BHB (beta-hydroxybutyrate; a ketone body), which are markers of oxidative stress and mitochondrial dysfunction, respectively. Transcriptional profiling of Cav-1 (-/-) stromal cells and human tumor stroma from breast cancer patients directly supported an association with oxidative stress, mitochondrial dysfunction, and autophagy/mitophagy, as well as ADMA and ketone production. MircoRNA profiling of Cav-1 (-/-) stromal cells revealed the upregulation of two key cancer-related miRs, namely miR-31 and miR-34c. Consistent with our metabolic findings, these miRs are associated with oxidative stress (miR-34c) or activation of the hypoxic response/HIF1a (miR-31), which is sufficient to drive authophagy/mitophagy. Thus, via an unbiased comprehensive analysis of a lethal tumor micro-environment, we have identified a number of candidate biomarkers (ADMA, ketones, and miR-31/34c) that could be used to identify high-risk cancer patients at diagnosis, for treatment stratification and/or for evaluating therapeutic efficacy during anti-cancer therapy. We propose that the levels of these key biomarkers (ADMA, ketones/BHB, miR-31, and miR-34c) could be (1) assayed using serum or plasma from cancer patients, or (2) performed directly on excised tumor tissue. Importantly, induction of oxidative stress and autophagy/mitophagy in the tumor stromal compartment provides a means by which epithelial cancer cells can directly "feed off" of stromal-derived essential nutrients, chemical building blocks (amino acids, nucleotides), and energy-rich metabolites (glutamine, pyruvate, ketones/BHB), driving tumor progression and metastasis. Essentially, aggressive cancer cells are "eating" the cancer-associated fibroblasts via autophagy/mitophagy in the tumor micro-environment. Lastly, we discuss that this "Autophagic Tumor Stroma Model of Cancer Metabolism" provides a viable solution to the "Autophagy Paradox" in cancer etiology and chemo-therapy.
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Ketones and lactate "fuel" tumor growth and metastasis: Evidence that epithelial cancer cells use oxidative mitochondrial metabolism.
Cell Cycle
PUBLISHED: 09-21-2010
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Previously, we proposed a new model for understanding the "Warburg effect" in tumor metabolism. In this scheme, cancer-associated fibroblasts undergo aerobic glycolysis and the resulting energy-rich metabolites are then transferred to epithelial cancer cells, where they enter the TCA cycle, resulting in high ATP production via oxidative phosphorylation. We have termed this new paradigm "The Reverse Warburg Effect." Here, we directly evaluate whether the end-products of aerobic glycolysis (3-hydroxy-butyrate and L-lactate) can stimulate tumor growth and metastasis, using MDA-MB-231 breast cancer xenografts as a model system. More specifically, we show that administration of 3-hydroxy-butyrate (a ketone body) increases tumor growth by ?2.5-fold, without any measurable increases in tumor vascularization/angiogenesis. Both 3-hydroxy-butyrate and L-lactate functioned as chemo-attractants, stimulating the migration of epithelial cancer cells. Although L-lactate did not increase primary tumor growth, it stimulated the formation of lung metastases by ?10-fold. Thus, we conclude that ketones and lactate fuel tumor growth and metastasis, providing functional evidence to support the "Reverse Warburg Effect". Moreover, we discuss the possibility that it may be unwise to use lactate-containing i.v. solutions (such as Lactated Ringers or Hartmanns solution) in cancer patients, given the dramatic metastasis-promoting properties of L-lactate. Also, we provide evidence for the up-regulation of oxidative mitochondrial metabolism and the TCA cycle in human breast cancer cells in vivo, via an informatics analysis of the existing raw transcriptional profiles of epithelial breast cancer cells and adjacent stromal cells. Lastly, our findings may explain why diabetic patients have an increased incidence of cancer, due to increased ketone production, and a tendency towards autophagy/mitophagy in their adipose tissue.
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Autophagy in cancer associated fibroblasts promotes tumor cell survival: Role of hypoxia, HIF1 induction and NF?B activation in the tumor stromal microenvironment.
Cell Cycle
PUBLISHED: 09-09-2010
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Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1? and NF?B. In addition, pharmacological activation of HIF-1? drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NF?B--another inducer of autophagy-prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NF?B-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1?- and NF?B-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knock-down of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (-/-) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the "Autophagic Tumor Stroma Model of Cancer Metabolism", and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NF?B-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a "lethal" tumor microenvironment.
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HIF1-alpha functions as a tumor promoter in cancer associated fibroblasts, and as a tumor suppressor in breast cancer cells: Autophagy drives compartment-specific oncogenesis.
Cell Cycle
PUBLISHED: 09-04-2010
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Our recent studies have mechanistically implicated a loss of stromal Cav-1 expression and HIF1-alpha-activation in driving the cancer-associated fibroblast phenotype, through the paracrine production of nutrients via autophagy and aerobic glycolysis. However, it remains unknown if HIF1a-activation is sufficient to confer the cancer-associated fibroblast phenotype. To test this hypothesis directly, we stably-expressed activated HIF1a in fibroblasts and then examined their ability to promote tumor growth using a xenograft model employing human breast cancer cells (MDA-MB-231). Fibroblasts harboring activated HIF1a showed a dramatic reduction in Cav-1 levels and a shift towards aerobic glycolysis, as evidenced by a loss of mitochondrial activity, and an increase in lactate production. Activated HIF1a also induced BNIP3 and BNIP3L expression, markers for the autophagic destruction of mitochondria. Most importantly, fibroblasts expressing activated HIF1a increased tumor mass by ?2-fold and tumor volume by ?3-fold, without a significant increase in tumor angiogenesis. In this context, HIF1a also induced an increase in the lymph node metastasis of cancer cells. Similar results were obtained by driving NF?B activation in fibroblasts, another inducer of autophagy. Thus, activated HIF1a is sufficient to functionally confer the cancer-associated fibroblast phenotype. It is also known that HIF1a expression is required for the induction of autophagy in cancer cells. As such, we next directly expressed activated HIF1a in MDA-MB-231 cells and assessed its effect on tumor growth via xenograft analysis. Surprisingly, activated HIF1a in cancer cells dramatically suppressed tumor growth, resulting in a 2-fold reduction in tumor mass and a 3-fold reduction in tumor volume. We conclude that HIF1a activation in different cell types can either promote or repress tumorigenesis. Based on these studies, we suggest that autophagy in cancer-associated fibroblasts promotes tumor growth via the paracrine production of recycled nutrients, which can directly "feed" cancer cells. Conversely, autophagy in cancer cells represses tumor growth via their "self-digestion". Thus, we should consider that the activities of various known oncogenes and tumor-suppressors may be compartment and cell-type specific, and are not necessarily an intrinsic property of the molecule itself. As such, other "classic" oncogenes and tumor suppressors will have to be re-evaluated to determine their compartment specific effects on tumor growth and metastasis. Lastly, our results provide direct experimental support for the recently proposed "Autophagic Tumor Stroma Model of Cancer".
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