Early Placenta Insulin-like Growth Factor (pro-EPIL) is Overexpressed and Secreted by C-erbB-2-positive Cells with High Invasion Potential

Cancer Research. Feb, 2002  |  Pubmed ID: 11861376

By differential-display PCR a subclone of the SKBR3 cell line with high in vitro transendothelial invasiveness was identified to express increased levels of the INSL-4 gene. This new member of the insulin-like growth factor family encodes for a peptide, designated early placenta insulin-like (EPIL), being expressed in the so-called "invasive" phase of the placental development. Immunohistochemistry on tissue microarrays revealed a heterogeneous expression of EPIL in breast cancer tissue and no expression in the surrounding stroma cells. A coexpression of pro-EPIL and c-erbB-2 could be observed predominantly in cell clusters at the infiltrating edge of the tumor. Our results give new suggestions for the presence of a signaling network of receptor tyrosine kinases underlying breast cancer invasion and metastasis.

Combined Anti-fetal Liver Kinase 1 Monoclonal Antibody and Continuous Low-dose Doxorubicin Inhibits Angiogenesis and Growth of Human Soft Tissue Sarcoma Xenografts by Induction of Endothelial Cell Apoptosis

Cancer Research. Apr, 2002  |  Pubmed ID: 11929822

Vascular endothelial growth factor (VEGF) and VEGF receptor 2 [fetal liver kinase 1 (Flk-1)/kinase insert domain-containing receptor] have been shown to play a major role in tumor angiogenesis. In this study, we investigated whether anti-Flk-1 monoclonal antibody DC101 could therapeutically inhibit growth and angiogenesis of human soft tissue sarcoma, and we explored its capacity to enhance the tumoricidal effects of doxorubicin. Treatment of well-established leiomyosarcoma SKLMS-1 and rhabdomyosarcoma RD xenografts in severe combined immunodeficient mice with DC101 resulted in significant antitumor activity. In a parallel study, we compared tumor inhibition with continuous low-dose "antiangiogenic" schedule versus once-every-2-weeks high-dose standard schedule of doxorubicin. We found that continuous low-dose treatment inhibited the tumor growth of RD xenografts about 46.5% of that with standard-schedule treatment, but that continuous low-dose treatment did not inhibit the tumor growth of SKLMS-1 xenografts. Notably, combined DC101 and continuous low-dose doxorubicin resulted in more effective growth inhibition of SKLMS-1 and RD xenografts than has been observed with any agent alone in a long-term s.c. tumor xenograft model. The combination therapy was associated with no additional toxicity to the host animal compared with low-dose doxorubicin alone. Histological examination of xenografts showed significantly reduced microvessel counts in the tumors given combined therapy compared with the tumors given either agent alone. These results are consistent with an enhanced inhibition of angiogenesis in vivo by combined DC101 and doxorubicin using Matrigel plug assay. Additionally, DC101 plus doxorubicin directly exerted enhanced inhibitory effects on endothelial cell migration, proliferation, and tube-like formation in vitro. Furthermore, the combination induced an enhanced apoptosis of endothelial cells that was associated with an increase of capase-3 activity. Thus, the inhibition of angiogenesis and induction of endothelial cell apoptosis are likely important mechanisms for the antitumor activity of combined DC101 and doxorubicin. Collectively, our data suggested that anti-VEGF receptor 2 in combination with continuous low-dose doxorubicin may provide a new therapeutic approach for human soft tissue sarcoma in the clinic.

Phosphorylation on Tyrosine-15 of P34(Cdc2) by ErbB2 Inhibits P34(Cdc2) Activation and is Involved in Resistance to Taxol-induced Apoptosis

Molecular Cell. May, 2002  |  Pubmed ID: 12049736

ErbB2 overexpression confers resistance to taxol-induced apoptosis by inhibiting p34(Cdc2) activation. One mechanism is via ErbB2-mediated upregulation of p21(Cip1), which inhibits Cdc2. Here, we report that the inhibitory phosphorylation on Cdc2 tyrosine (Y)15 (Cdc2-Y15-p) is elevated in ErbB2-overexpressing breast cancer cells and primary tumors. ErbB2 binds to and colocalizes with cyclin B-Cdc2 complexes and phosphorylates Cdc2-Y15. The ErbB2 kinase domain is sufficient to directly phosphorylate Cdc2-Y15. Increased Cdc2-Y15-p in ErbB2-overexpressing cells corresponds with delayed M phase entry. Expressing a nonphosphorylatable mutant of Cdc2 renders cells more sensitive to taxol-induced apoptosis. Thus, ErbB2 membrane RTK can confer resistance to taxol-induced apoptosis by directly phosphorylating Cdc2.

ErbB2 Overexpression Correlates with Increased Expression of Vascular Endothelial Growth Factors A, C, and D in Human Breast Carcinoma

Cancer. Jun, 2002  |  Pubmed ID: 12115372

The angiogenic factor vascular endothelial growth factor (VEGF)-A plays an important role in breast cancer progression. However, the involvement of VEGF-C and VEGF-D, two newer members of the VEGF family, in breast carcinoma and their relationship with clinicopathologic parameters have not been clearly demonstrated.

Characterization of 11 Human Sarcoma Cell Strains: Evaluation of Cytogenetics, Tumorigenicity, Metastasis, and Production of Angiogenic Factors

Cancer. Oct, 2002  |  Pubmed ID: 12237927

Human sarcomas have a propensity for aggressive local invasion and early pulmonary metastasis. Frequently, deaths are due to uncontrolled pulmonary metastases. The purpose of the current study was to evaluate cytogenetics, tumorigenicity, metastatic potential, and production of angiogenic factors in human sarcoma cell strains. A secondary purpose was to establish low passage cell strains for studying new therapeutic approaches.

Enhanced Sensitization to Taxol-induced Apoptosis by Herceptin Pretreatment in ErbB2-overexpressing Breast Cancer Cells

Cancer Research. Oct, 2002  |  Pubmed ID: 12384528

The recombinant humanized anti-ErbB2/HER2 monoclonal antibody Herceptin (Trastuzumab) has been shown to significantly enhance the tumoricidaleffects of antitumor drugs such as paclitaxel (Taxol) in patients with ErbB2-overexpressing breast cancers. Here, we investigated the molecular mechanisms by which Herceptin enhances the antitumor effects of Taxol. Because activation of p34(Cdc2) is required for Taxol-induced apoptosis and because overexpression of ErbB2 blocks Taxol-induced apoptosis by inhibiting p34(Cdc2) activation, we studied the effect of Herceptin treatment on p34(Cdc2) kinase activation and apoptosis in Taxol-treated human breast carcinoma cell lines MDA-MB-435, SKBr3, MDA-MB-453, and 435.eB, which is an ErbB2 transfectant of MDA-MB-435. Herceptin treatment down-regulated ErbB2, reduced the inhibitory phosphorylation of Cdc2 on Tyr-15, and down-regulated the expression of p21(Cip1), a Cdc2 inhibitor. Herceptin plus Taxol treatment led to higher levels of p34(Cdc2) kinase activity and apoptosis in ErbB2-overexpressing breast cancer cells, which is likely attributable to inhibition of Cdc2-Tyr-15 phosphorylation and p21(Cip1) expression. Because significant dephosphorylation of Cdc2-Tyr-15 and down-regulation of p21(Cip1) occur at least 24 h after Herceptin treatment, we investigated whether 24 h Herceptin pretreatment will render ErbB2-overexpressing breast cancer cells more sensitive to Taxol-induced apoptosis compared with the simultaneous treatment of Herceptin plus Taxol. Indeed, Herceptin pretreatment increased Taxol-induced apoptosis and cytotoxicity in vitro and more effectively inhibited the growth of tumor xenografts with enhanced in vivo apoptosis. Thus, Herceptin treatment of ErbB2-overexpressing cells can inhibit ErbB2-mediated Cdc2-Tyr-15 phosphorylation and p21(Cip1) up-regulation, which allows effective p34(Cdc2) activation and induction of apoptosis upon Taxol treatment. Herceptin pretreatment renders ErbB2-overexpressing breast cancers more susceptible to Taxol-induced cell death, which may have important clinical therapeutic implications.

ErbB2 Overexpression in Human Breast Carcinoma is Correlated with P21Cip1 Up-regulation and Tyrosine-15 Hyperphosphorylation of P34Cdc2: Poor Responsiveness to Chemotherapy with Cyclophoshamide Methotrexate, and 5-fluorouracil is Associated with Erb2 Overexpression and with P21Cip1 Overexpression

Cancer. Sep, 2003  |  Pubmed ID: 12973835

Clinical investigations have shown that in patients with breast carcinoma, tumors that overexpress the erb-B2 gene are less responsive to certain chemotherapy regimens compared with tumors that express low levels of ErbB2, suggesting that ErbB2 overexpression may be used as a marker for poor response to chemotherapy in patients with breast carcinoma. The combination of cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) is one of the most widely used chemotherapy regimens in patients with breast carcinoma. Patients who have ErbB2-overexpressing breast carcinomas have poorer responses to CMF compared with patients who have breast carcinomas with low ErbB2 expression. ErbB2-overexpressing breast tumor cells are resistant to taxol-induced apoptotic cell death. The underlying molecular mechanism is that ErbB2 inhibits p34(Cdc2) activation, which is required for taxol-induced apoptosis, by up-regulating p21(Cip1) and by hyperphosphorylating p34(Cdc2) on tyrosine-15. However, the relation between ErbB2, p21(Cip1), and p34(Cdc2) in patients with breast carcinoma remains elusive. The contribution of these molecular alterations to ErbB2-mediated CMF resistance has not been examined.

Combined Trastuzumab and Paclitaxel Treatment Better Inhibits ErbB-2-mediated Angiogenesis in Breast Carcinoma Through a More Effective Inhibition of Akt Than Either Treatment Alone

Cancer. Oct, 2003  |  Pubmed ID: 14508823

Trastuzumab (Herceptin; Genentech, South San Francisco, CA) is a humanized anti-ErbB-2 monoclonal antibody that has demonstrated antitumor function, especially in combination with other chemotherapies such as paclitaxel (Taxol; Bristol Myers-Squibb, Princeton, NJ), in patients with tumors that overexpress ErbB-2. Because the repeated administration of low-dose chemotherapy, such as paclitaxel, endorsed an antiangiogenic effect in vitro, and because trastuzumab was shown to inhibit angiogenesis in tumor xenografts, the authors investigated whether ErbB-2-mediated angiogenic responses would be inhibited more effectively by the combined treatment of paclitaxel plus trastuzumab.

Silibinin: a Thorny Therapeutic for EGF-R Expressing Tumors?

Cancer Biology & Therapy. Sep-Oct, 2003  |  Pubmed ID: 14614321

Liposomal Mediated Transfer of ErbB2 Antisense DNA: Coming of Age in the War Against Cancer

Cancer Biology & Therapy. Feb, 2004  |  Pubmed ID: 14764991

Lineage Infidelity of MDA-MB-435 Cells: Expression of Melanocyte Proteins in a Breast Cancer Cell Line

Cancer Research. May, 2004  |  Pubmed ID: 15150101

The origin of cell lines is critical in defining cell type-specific biological functions. Several reports suggested that the MDA-MB-435 cell line, a cell line extensively used for studying breast cancer biology, has a gene expression pattern most compatible with melanocyte origin. However, we demonstrate that MDA-MB-435 cells express breast-specific or epithelial-specific markers. Also, MDA-MB-435 cells were induced to express breast differentiation-specific proteins and secrete milk lipids as observed in other well-established breast cancer cell lines. Notably, MDA-MB-435 cells also expressed melanocyte-specific proteins as did another highly aggressive breast cancer cell line. MDA-MB-435 xenograft tissue sections stained entirely positive for epithelium-specific markers but only partially positive for melanocyte-specific markers. Thus, MDA-MB-435 is most likely a breast epithelial cell line that has undergone lineage infidelity.

PI3K: Missense Mutation Motivates Malignancy

Cancer Biology & Therapy. Aug, 2004  |  Pubmed ID: 15280668

PTEN Activation Contributes to Tumor Inhibition by Trastuzumab, and Loss of PTEN Predicts Trastuzumab Resistance in Patients

Cancer Cell. Aug, 2004  |  Pubmed ID: 15324695

The ErbB2-targeting antibody, trastuzumab (Herceptin), has remarkable therapeutic efficacy in certain patients with ErbB2-overexpressing tumors. The overall trastuzumab response rate, however, is limited and what determines trastuzumab response is poorly understood. Here we report that PTEN activation contributes to trastuzumab's antitumor activity. Trastuzumab treatment quickly increased PTEN membrane localization and phosphatase activity by reducing PTEN tyrosine phosphorylation via Src inhibition. Reducing PTEN in breast cancer cells by antisense oligonucleotides conferred trastuzumab resistance in vitro and in vivo. Patients with PTEN-deficient breast cancers had significantly poorer responses to trastuzumab-based therapy than those with normal PTEN. Thus, PTEN deficiency is a powerful predictor for trastuzumab resistance. Additionally, PI3K inhibitors rescued PTEN loss-induced trastuzumab resistance, suggesting that PI3K-targeting therapies could overcome this resistance.

Activation of the Akt/mammalian Target of Rapamycin/4E-BP1 Pathway by ErbB2 Overexpression Predicts Tumor Progression in Breast Cancers

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Oct, 2004  |  Pubmed ID: 15501954

The Akt/mammalian target of rapamycin (mTOR)/4E-BP1 pathway is considered to be a central regulator of protein synthesis, involving the regulation of cell proliferation, differentiation, and survival. The inhibitors of mTOR as anticancer reagents are undergoing active evaluation in various malignancies including breast cancer. However, the activation status of the Akt/mTOR/4E-BP1 pathway and its potential roles in breast cancers remain unknown. Thus, we examined 165 invasive breast cancers with specific antibodies for the phosphorylation of Akt, mTOR, and 4E-BP1 by immunohistochemistry and compared them with normal breast epithelium, fibroadenoma, intraductal hyperplasia, and ductal carcinoma in situ. We discovered that the phosphorylation of Akt, mTOR, and 4E-BP1 increased progressively from normal breast epithelium to hyperplasia and abnormal hyperplasia to tumor invasion. Phosphorylated Akt, mTOR, and 4E-BP1 were positively associated with ErbB2 overexpression. Survival analysis showed that phosphorylation of each of these three markers was associated with poor disease-free survival independently. In vitro, we further confirmed the causal relationship between ErbB2 overexpression and mTOR activation, which was associated with enhanced invasive ability and sensitivity to a mTOR inhibitor, rapamycin. Our results, for the first time, demonstrate the following: (a) high levels of phosphorylation of Akt, mTOR, and 4E-BP1 in breast cancers, indicating activation of the Akt/mTOR/4E-BP1 pathway in breast cancer development and progression; (b) a link between ErbB2 and the Akt/mTOR/4E-BP1 pathway in breast cancers in vitro and in vivo, indicating the possible role of Akt/mTOR activation in ErbB2-mediated breast cancer progression; and (c) a potential role for this pathway in predicting the prognosis of patients with breast cancer, especially those treated with mTOR inhibitors.

Upregulation of CXCR4 is Essential for HER2-mediated Tumor Metastasis

Cancer Cell. Nov, 2004  |  Pubmed ID: 15542430

The receptor tyrosine kinase HER2 enhances tumor metastasis; however, its role in homing to metastatic organs is poorly understood. The chemokine receptor CXCR4 has recently been shown to mediate the movement of malignant cancer cells to specific organs. Here, we show that HER2 enhances the expression of CXCR4, which is required for HER2-mediated invasion in vitro and lung metastasis in vivo. HER2 also inhibits ligand-induced CXCR4 degradation. Finally, a significant correlation between HER2 and CXCR4 expression was observed in human breast tumor tissues, and CXCR4 expression correlated with a poor overall survival rate in patients with breast cancer. These results provide a plausible mechanism for HER2-mediated breast tumor metastasis and establish a functional link between HER2 and CXCR4 signaling pathways.

Transcriptional Repression of Protein Kinase Calpha Via Sp1 by Wild Type P53 is Involved in Inhibition of Multidrug Resistance 1 P-glycoprotein Phosphorylation

The Journal of Biological Chemistry. Feb, 2005  |  Pubmed ID: 15563462

The protein kinase C (PKC) family consists of serine/threonine protein kinases that play important roles in signal transduction, cell proliferation, and tumor formation. Recent studies found that PKCs are commonly overexpressed in human tumors, including soft tissue sarcoma (STS). Overexpression of PKCs contributes to invasion and migration of tumor cells and induction of angiogenesis. PKC can also phosphorylate the multidrug resistance (MDR) gene-encoded P-glycoprotein and induce MDR phenotype. Our previous studies showed that mutation of p53 enhanced STS metastasis and mediated the MDR phenotype. Restoring wild type (WT) p53 in STS cells containing mutant p53 sensitized the cells to chemotherapy. In the present study, we found that PKCalpha protein expression is inhibited by WT p53 partly due to reduced PKCalpha mRNA expression in STS cells, but p53 does not affect PKCalpha mRNA stability. Deletion and mutation analysis of the PKCalpha promoter fused to the luciferase reporter gene identified a Sp1 binding site (-244/-234) in the PKCalpha promoter that is required for p53-mediated inhibition of PKCalpha promoter activity. More importantly, PKCalpha phosphorylates and activates MDR1 P-glycoprotein, whereas inhibition of PKCalpha by p53 leads to decreased MDR1 phosphorylation in STS cells, which sensitizes STS cells to chemotherapeutic agents. These data indicate that WT p53 may resensitize STS to chemotherapeutic agents by reducing MDR1 phosphorylation via transcriptional repression of PKCalpha expression. Thus, molecular-based therapies targeting mutant p53 and PKCalpha may be an effective new strategy to improve chemotherapeutic efficacy in STS.

Expression of Receptor Tyrosine Kinases Epidermal Growth Factor Receptor and HER-2/neu in Synovial Sarcoma

Cancer. Feb, 2005  |  Pubmed ID: 15641030

Synovial sarcomas are high-grade soft tissue neoplasms often characterized by a biphasic spindle and epithelioid cell morphology. The majority of synovial sarcomas harbor a specific chromosomal translocation in which the proximal portion of the SYT gene at chromosome 18q11 is fused to the distal portion of one of several duplicated SSX genes (most notably SSX1 and SSX2) at chromosome Xp11. SYT/SSX1 translocations are seen in nearly three times as many synovial sarcomas as SYT/SSX2 translocations. Although the SYT/SSX2 fusion is usually associated with the monophasic disease pattern, the SYT/SSX1 fusion is present in tumors with biphasic or monophasic patterns. The SYT/SSX1 fusion gene is associated with more aggressive tumor growth and poor outcome. Despite advances in the therapy of local disease, distant metastasis remains the predominant cause of death. Accordingly, there is a need for alternate therapies, such as those recently developed against the receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and HER-2/neu.

A Robust Assay for Alternative Lengthening of Telomeres in Tumors Shows the Significance of Alternative Lengthening of Telomeres in Sarcomas and Astrocytomas

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Jan, 2005  |  Pubmed ID: 15671549

Telomeres of tumor cells may be maintained by telomerase or by alternative lengthening of telomeres (ALT). The standard ALT assay requires Southern analysis of high molecular weight genomic DNA. We aimed to establish and validate an ALT assay suitable for archived paraffin-embedded tumors and to use it to examine the prevalence and clinical significance of ALT in various types of tumors that are often telomerase negative.

ErbB2 Promotes Src Synthesis and Stability: Novel Mechanisms of Src Activation That Confer Breast Cancer Metastasis

Cancer Research. Mar, 2005  |  Pubmed ID: 15753384

Activation of Src kinase plays important roles in the development of many neoplasias. Most of the previous Src studies focused on the deregulation of Src kinase activity. The deregulated Src protein synthesis and stability in mediating malignant phenotypes of cancer cells, however, have been neglected. While investigating the signal transduction pathways contributing to ErbB2-mediated metastasis, we found that ErbB2-activated breast cancer cells that had higher metastatic potentials also had increased Src activity compared with ErbB2 low-expressing cells. The increased Src activity in ErbB2-activated cells paralleled higher Src protein levels, whereas Src RNA levels were not significantly altered. Our studies revealed two novel mechanisms that are involved in Src protein up-regulation and activation by ErbB2: (a) ErbB2 increased Src translation through activation of the Akt/mammalian target of rapamycin/4E-BP1 pathway and (b) ErbB2 increased Src stability most likely through the inhibition of the calpain protease. Furthermore, inhibition of Src activity by a Src-specific inhibitor, PP2, or a Src dominant-negative mutant dramatically reduced ErbB2-mediated cancer cell invasion in vitro and metastasis in an experimental metastasis animal model. Together, activation of ErbB2 and downstream signaling pathways can lead to increased Src protein synthesis and decreased Src protein degradation resulting in Src up-regulation and activation, which play critical roles in ErbB2-mediated breast cancer invasion and metastasis.

A Knotty Turnabout?: Akt1 As a Metastasis Suppressor

Cancer Cell. Dec, 2005  |  Pubmed ID: 16338656

Akt is well known to enhance malignancy and is recognized as a key target for antineoplastic therapies. However, intriguing findings reported by Yoeli-Lerner et al. in the November 23, 2005 issue of Molecular Cell, suggest a novel, antimetastasis function of Akt: activation of Akt1 inhibited invasion in some cancer cells. One possible mechanism for this surprising phenotype was that Akt activated the E3 ubiquitin ligase HDM2, causing ubiquitination and degradation of NFAT, an invasion-promoting factor. These findings clearly justify further investigations and, if validated in vivo, call for reevaluation of some Akt-targeting therapeutic strategies currently under development.

Mechanisms of Trastuzumab Resistance and Their Clinical Implications

Annals of the New York Academy of Sciences. Nov, 2005  |  Pubmed ID: 16382045

Trastuzumab (Herceptin) is an excellent model of rationally designed targeted cancer treatment. However, less than 35% of patients with ErbB2-positive breast tumors respond to trastuzumab as a single agent, and 2-5% of trastuzumab-treated patients suffer from severe side effects, including cardiac dysfunction. Recent progress in understanding the mechanisms of trastuzumab antitumor function and cellular defects leading to trastuzumab resistance is summarized. Also explored is the potential of combination therapies for reversing trastuzumab resistance.

ErbB2 Increases Vascular Endothelial Growth Factor Protein Synthesis Via Activation of Mammalian Target of Rapamycin/p70S6K Leading to Increased Angiogenesis and Spontaneous Metastasis of Human Breast Cancer Cells

Cancer Research. Feb, 2006  |  Pubmed ID: 16489002

ErbB2 overexpression in breast tumors results in increased metastasis and angiogenesis and reduced survival. To study ErbB2 signaling mechanisms in metastasis and angiogenesis, we did a spontaneous metastasis assay using MDA-MB-435 human breast cancer cells stably transfected with constitutively active ErbB2 kinase (V659E), a kinase-dead mutant of ErbB2 (K753M), or vector control (neo). Mice injected with V659E had increased metastasis incidence and tumor microvessel density than mice injected with K753M or control. Increased angiogenesis in vivo from the V659E transfectants paralleled increased angiogenic potential in vitro. V659E produced increased vascular endothelial growth factor (VEGF) through increased VEGF protein synthesis. This was mediated through signaling events involving extracellular signal-regulated kinase, phosphatidylinositol 3-kinase/Akt, mammalian target of rapamycin (mTOR), and p70S6K. The V659E xenografts also had significantly increased phosphorylated Akt, phosphorylated p70S6K, and VEGF compared with controls. To validate the clinical relevance of these findings, we examined 155 human breast tumor samples. Human tumors that overexpressed ErbB2, which have been previously shown to have higher VEGF expression, showed significantly higher p70S6K phosphorylation as well. Increased VEGF expression also significantly correlated with higher levels of Akt and mTOR phosphorylation. Additionally, patients with tumors having increased p70S6K phosphorylation showed a trend for worse disease-free survival and increased metastasis. Our findings show that ErbB2 increases VEGF protein production by activating p70S6K in cell lines, xenografts, and in human cancers and suggest that these signaling molecules may serve as targets for antiangiogenic and antimetastatic therapies.

Synthetic Triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic Acid Induces Growth Arrest in HER2-overexpressing Breast Cancer Cells

Molecular Cancer Therapeutics. Feb, 2006  |  Pubmed ID: 16505105

HER2 overexpression is one of the most recognizable molecular alterations in breast tumors known to be associated with a poor prognosis. In the study described here, we explored the effect of HER2 overexpression on the sensitivity of breast cancer cells to the growth-inhibitory effects of 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), a synthetic triterpenoid, both in vitro and in vivo in a xenograft model of breast cancer. Both cell growth and colony formation in the soft agar assay, a hallmark of the transformation phenotype, were preferentially suppressed in HER2-overexpressing cell lines at low concentrations of CDDO, whereas growth-inhibitory effects at high concentrations did not correlate with the expression level of HER2. CDDO dose-dependently inhibited phosphorylation of HER2 in HER2-overexpressing cells and diminished HER2 kinase activity in vitro. CDDO induced the transactivation of the nuclear receptor peroxisome proliferator-activated receptor-gamma in both vector control and HER2-transfected MCF7 cells. Dose-response studies showed that the growth inhibition seen at lower concentrations of CDDO correlated with induction of the tumor suppressor gene caveolin-1, which is known to inhibit breast cancer cell growth. CDDO also reduced cyclin D1 mRNA and protein expression. In vivo studies with liposomally encapsulated CDDO showed complete abrogation of the growth of the highly tumorigenic MCF7/HER2 cells in a xenograft model of breast cancer. These findings provide the first in vitro and in vivo evidence that CDDO effectively inhibits HER2 tyrosine kinase activity and potently suppresses the growth of HER2-overexpressing breast cancer cells and suggest that CDDO has a therapeutic potential in advanced breast cancer.

Selective Inhibition of ErbB2-overexpressing Breast Cancer in Vivo by a Novel TAT-based ErbB2-targeting Signal Transducers and Activators of Transcription 3-blocking Peptide

Cancer Research. Apr, 2006  |  Pubmed ID: 16585203

ErbB2 is an excellent target for cancer therapies. Unfortunately, the outcome of current therapies for ErbB2-positive breast cancers remains unsatisfying due to resistance and side effects. New therapies for ErbB2-overexpressing breast cancers continue to be in great need. Peptide therapy using cell-penetrating peptides (CPP) as peptide carriers is promising because the internalization is highly efficient, and the cargoes delivered can be bioactive. However, the major obstacle in using these powerful CPPs for therapy is their lack of specificity. Here, we sought to develop a peptide carrier that could introduce therapeutics specifically to ErbB2-overexpressing breast cancer cells. By modifying the HIV TAT-derived CPP and conjugating anti-HER-2/neu peptide mimetic (AHNP), we developed the peptide carrier (P3-AHNP) that specifically targeted ErbB2-overexpressing breast cancer cells in vitro and in vivo. A signal transducers and activators of transcription 3 (STAT3)-inhibiting peptide conjugated to this peptide carrier (P3-AHNP-STAT3BP) was delivered more efficiently into ErbB2-overexpressing than ErbB2 low-expressing cancer cells in vitro and successfully decreased STAT3 binding to STAT3-interacting DNA sequence. P3-AHNP-STAT3BP inhibited cell growth in vitro, with ErbB2-overexpressing 435.eB breast cancer cells being more sensitive to the treatment than the ErbB2 low-expressing MDA-MB-435 cells. Compared with ErbB2 low-expressing MDA-MB-435 xenografts, i.p. injected P3-AHNP-STAT3BP preferentially accumulated in 435.eB xenografts, which led to more reduction of proliferation and increased apoptosis and targeted inhibition of tumor growth. This novel peptide delivery system provided a sound basis for the future development of safe and effective new-generation therapeutics to cancer-specific molecular targets.

Mechanisms of Disease: Understanding Resistance to HER2-targeted Therapy in Human Breast Cancer

Nature Clinical Practice. Oncology. May, 2006  |  Pubmed ID: 16683005

Trastuzumab is a monoclonal antibody targeted against the human epidermal growth factor receptor (HER) 2 tyrosine kinase receptor, which is overexpressed in approximately 25% of invasive breast cancers. The majority of patients with metastatic breast cancer who initially respond to trastuzumab, however, demonstrate disease progression within 1 year of treatment initiation. Preclinical studies have indicated several molecular mechanisms that could contribute to the development of trastuzumab resistance. Increased signaling via the phosphatidylinositol 3-kinase/Akt pathway could contribute to trastuzumab resistance because of activation of multiple receptor pathways that include HER2-related receptors or non-HER receptors such as the insulin-like growth factor 1 receptor, which appears to be involved in a cross-talk with HER2 in resistant cells. Additionally, loss of function of the tumor suppressor PTEN gene, the negative regulator of Akt, results in heightened Akt signaling that leads to decreased sensitivity to trastuzumab. Decreased interaction between trastuzumab and its target receptor HER2, which is due to steric hindrance of HER2 by cell surface proteins such as mucin-4 (MUC4), may block the inhibitory actions of trastuzumab. Novel therapies targeted against these aberrant molecular pathways offer hope that the effectiveness and duration of response to trastuzumab can be greatly improved.

Vascular Endothelial Growth Factor Overexpression by Soft Tissue Sarcoma Cells: Implications for Tumor Growth, Metastasis, and Chemoresistance

Cancer Research. Sep, 2006  |  Pubmed ID: 16951193

To better elucidate the role of vascular endothelial growth factor (VEGF)(165) in soft tissue sarcoma (STS) growth, metastasis, and chemoresistance, we generated stably transfected human STS cell lines with VEGF(165) to study the effect of VEGF(165) on STS cells in vitro and the effect of culture medium from these cells on human umbilical vascular endothelial cells. Severe combined immunodeficient mice bearing xenografts of transfected cell lines were used to assess the effect of VEGF overexpression and the effect of VEGF receptor (VEGFR) 2 inhibition on STS growth, metastasis, and response to doxorubicin. VEGF(165)-transfected xenografts formed highly vascular tumors with shorter latency, accelerated growth, enhanced chemoresistance, and increased incidence of pulmonary metastases. Blockade of VEGFR2 signaling using DC101 anti-VEGFR2 monoclonal antibody enhanced doxorubicin chemoresponse; this combined biochemotherapy inhibited tumor growth and decreased pulmonary metastases without overt toxicity. Combined therapy reduced microvessel counts while increasing vessel maturation index. VEGF overexpression did not affect on the sarcoma cells per se; however, conditioned medium from VEGF transfectants caused increased endothelial cell proliferation, migration, and chemoresistance. Addition of DC101 induced endothelial cell sensitivity to doxorubicin and suppressed the activity of matrix metalloproteinases secreted by endothelial cells. We therefore conclude that VEGF is a critical determinant of STS growth and metastasis and that STS chemoresistance, in our model, is a process induced by the interplay between STS cells and tumor-associated endothelial cells. STS growth and metastasis can be interrupted by combined low-dose doxorubicin and anti-VEGFR2, a strategy that attacks STS-associated endothelial cells. In the future, such therapeutic approaches may be useful in treating STS before the development of clinically apparent metastases.

Cytoplasmic Expression of P21CIP1/WAF1 is Correlated with IKKbeta Overexpression in Human Breast Cancers

International Journal of Oncology. Nov, 2006  |  Pubmed ID: 17016640

Regulation of cytoplasmic p21CIP/WAF1 (p21) is of great clinical significance in molecular oncology due to its identification as an antiapoptotic factor, a poor survival predictor as well as a drug-resistance inducer. A retrospective study of the immunohistochemical (IHC) profiles of 128 human primary breast cancers showed that increased total and cytoplasmic p21 expression were highly associated with the expression of IkappaB kinase beta (IKKbeta), the major catalytic subunit of the IKK complex and another crucial player in tumorigenesis and drug resistance. The causal relationship study based on cultured cell lines, MDA-MB-453 and MCF-7, confirmed that IKKbeta overexpression did upregulate protein levels of total and cytoplasmic p21. Mechanistic investigation demonstrated that IKKbeta increased p21 expression through upregulation of p21 mRNA level. Moreover, by Western blotting, IKKbeta was found to be able to upregulate Akt phosphorylation on Ser 473. This novel finding indicated that IKKbeta could mediate cytoplasmic p21 accumulation via activation of its downstream target Akt, which was known to phosphorylate p21 and lead to cytoplasmic localization of p21.

Wild-type P53 Inhibits Nuclear Factor-kappaB-induced Matrix Metalloproteinase-9 Promoter Activation: Implications for Soft Tissue Sarcoma Growth and Metastasis

Molecular Cancer Research : MCR. Nov, 2006  |  Pubmed ID: 17077165

Human soft tissue sarcoma (STS) is a highly lethal malignancy in which control of metastasis determines survival. Little is known about the molecular determinants of STS dissemination. Here, we show that human STS express high levels of matrix metalloproteinase-9 (MMP-9) and that MMP-9 expression levels correlate with sequence analysis-defined p53 mutational status. Reintroduction of wild-type p53 (wtp53) into mutant p53 STS cell lines decreased MMP-9 mRNA and protein levels, decreased zymography-assessed MMP-9 proteolytic activity, and decreased tumor cell invasiveness. Reintroduction of wtp53 into STS xenografts decreased tumor growth and MMP-9 protein expression. Luciferase reporter studies showed that reintroduction of wtp53 into mutant p53 STS cells decreased MMP-9 promoter activity. Deletion constructs of the MMP-9 promoter identified a region containing a p53-responsive element that lacked a p53 consensus binding site but did contain a nuclear factor-kappaB (NF-kappaB) site. Mutating this NF-kappaB binding site eliminated the wtp53-repressive effect. Electrophoretic mobility shift assays confirmed decreased NF-kappaB binding in STS cells in the presence of wtp53. Our findings suggest a role for MMP-9 in STS progression and expand the role of p53 in molecular control of STS growth and metastasis. Therapeutic interventions in human STS targeting MMP-9 activity directly or via reintroduction of wtp53 merit further investigation.

Adenoviral-Vector Based SiRNA for Mutant K-ras As a Promising Tool for Lung Cancer Gene Therapy: A License to Kill

Cancer Biology & Therapy. Dec, 2006  |  Pubmed ID: 17224631

Tumor Progression and Metastasis from Genetic to Microenvironmental Determinants: a Workshop of the Tumor Progression and Metastasis NIH Study Section in Honor of Dr. Martin L. Padarathsingh, May 31, 2006, Georgetown, Washington, DC

Cancer Biology & Therapy. Dec, 2006  |  Pubmed ID: 17224636

Prostate Tumor Cells Infected with a Recombinant Influenza Virus Expressing a Truncated NS1 Protein Activate Cytolytic CD8+ Cells to Recognize Noninfected Tumor Cells

Journal of Virology. Jan, 2006  |  Pubmed ID: 16352563

Many viral oncolytic approaches against cancer are based on the ability of specific viruses to replicate in tumors expressing components of the constitutively activated Ras/mitogen-activated protein kinase (MAPK) pathways and/or inhibited or dysregulated alpha/beta interferon (IFN-alpha/beta) response pathways. A major issue when considering these approaches is their applicability to tumors that lack activated Ras. To identify the effector mechanisms activated by oncolytic viruses, we investigated inhibition of proliferation of the prostate cancer line LNCap by the recombinant TR-NS1 influenza A virus, a genetically attenuated influenza A/PR8/34 virus expressing a truncated nonstructural protein (NS1) of 126 amino acids. LNCap cells lack constitutively activated MAPK, extracellular signal-regulated kinase (ERK), and p38 and are resistant to death by IFN-alpha. Truncation of the NS1 protein of influenza viruses is known to result in viral attenuation due to a reduced ability of the NS1 to inhibit the IFN-alpha/beta response. Infection with TR-NS1 virus rapidly activated ERK-1 more than ERK-2 in LNCap cells. Importantly, TR-NS1 virus infection transiently inhibited cell proliferation and induced apoptosis in LNCap cells. Addition of peripheral blood mononuclear cells (PBMC) and interleukin 12 (IL-12) to TR-NS1 virus-infected LNCap cells (TR-NS1-LNCap) resulted in faster elimination of TR-NS1-LNCap cells compared with LNCap cells. Moreover, TR-NS1-LNCap cells induced IFN-gamma in PBMC. The levels of IFN-gamma were amplified by IL-12. TR-NS1-LNCap cells also induced tumor-lytic cytotoxic T lymphocytes (CTL). These CTL lysed noninfected LNCap cells in a CD8-dependent manner. Activation of cellular immunity to tumor cells by viruses is an intriguing effector pathway, which should be especially significant for elimination of human tumors that lack activated Ras.

High Prevalence of P53 Exon 4 Mutations in Soft Tissue Sarcoma

Cancer. Jun, 2007  |  Pubmed ID: 17429838

p53 is the most commonly mutated gene in cancer, including soft tissue sarcoma (STS). The authors characterized p53 alterations (protein accumulation and gene mutation) in STS to evaluate possible associations with patient outcomes.

Rad51 Overexpression Contributes to Chemoresistance in Human Soft Tissue Sarcoma Cells: a Role for P53/activator Protein 2 Transcriptional Regulation

Molecular Cancer Therapeutics. May, 2007  |  Pubmed ID: 17513613

We investigated whether Rad51 overexpression plays a role in soft tissue sarcoma (STS) chemoresistance as well as the regulatory mechanisms underlying its expression. The studies reported here show that Rad51 protein is overexpressed in a large panel of human STS specimens. Human STS cell lines showed increased Rad51 protein expression, as was also observed in nude rat STS xenografts. STS cells treated with doxorubicin exhibited up-regulation of Rad51 protein while arrested in the S-G(2) phase of the cell cycle. Treatment with anti-Rad51 small interfering RNA decreased Rad51 protein expression and increased chemosensitivity to doxorubicin. Because we previously showed that reintroduction of wild-type p53 (wtp53) into STS cells harboring a p53 mutation led to increased doxorubicin chemosensitivity, we hypothesized that p53 participates in regulating Rad51 expression in STS. Reintroduction of wtp53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression. Using luciferase reporter assays, we showed that reconstitution of wtp53 function decreased Rad51 promoter activity. Deletion constructs identified a specific Rad51 promoter region containing a p53-responsive element but no p53 consensus binding site. Electrophoretic mobility shift assays verified activator protein 2 (AP2) binding to this region and increased AP2 binding to the promoter in the presence of wtp53. Mutating this AP2 binding site eliminated the wtp53 repressive effect. Furthermore, AP2 knockdown resulted in increased Rad51 expression. In light of the importance of Rad51 in modulating STS chemoresistance, these findings point to a potential novel strategy for molecular-based treatments that may be of relevance to patients burdened by STS.

Preclinical Testing of Clinically Applicable Strategies for Overcoming Trastuzumab Resistance Caused by PTEN Deficiency

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Oct, 2007  |  Pubmed ID: 17908983

We have previously shown that PTEN loss confers trastuzumab resistance in ErbB2-overexpressing breast cancer using cell culture, xenograft models, and patient samples. This is a critical clinical problem because trastuzumab is used in a variety of therapeutic regimens, and at the current time, there are no established clinical strategies to overcome trastuzumab resistance. Here, we did preclinical studies on the efficacy of clinically applicable inhibitors of the Akt/mammalian target of rapamycin (mTOR) pathway to restore trastuzumab sensitivity to PTEN-deficient cells.

Molecular Mechanisms of ErbB2-mediated Breast Cancer Chemoresistance

Advances in Experimental Medicine and Biology. 2007  |  Pubmed ID: 17993237

Novel Approaches for Chemosensitization of Breast Cancer Cells: the E1A Story

Advances in Experimental Medicine and Biology. 2007  |  Pubmed ID: 17993239

The adenoviral E1A-mediated sensitization to a variety of anti-cancer drug-induced apoptosis is a well-established phenomenon on different types of cell systems. However, the mechanisms underlying E1A-mediated chemosensitization are still not fully understood. Recent studies demonstrate that E1A-mediated sensitization to drug-induced apoptosis can occur via multiple pathways; some of which depend on the expression of functional p53 and/or p19ARF proteins, while some are not. In human breast cancer cells with Her-2/neu overexpression, which usually are more resistance to anti-cancer drugs than cells without Her-2/ neu overexpression, may be sensitized through E1A-mediated downregulation of Her-2/neu. Alternatively, E1A can induce sensitization to anticancer drugs in cancer cells or normal diploid fibroblast cells through upregulating the expression of caspase proenzymes, or downregulating the activity of a critical survival factor Akt and/or upregulating the activities of a pro-apoptotic kinase p38 and a protein phosphatase PP2A, etc. This review summarizes these progresses and proposes a plausible feed-forward model for E1A-mediated chemosensitization in human breast cancer cells.

Loss of Trimethylation at Lysine 27 of Histone H3 is a Predictor of Poor Outcome in Breast, Ovarian, and Pancreatic Cancers

Molecular Carcinogenesis. Sep, 2008  |  Pubmed ID: 18176935

Methylation of lysine 27 on histone H3 (H3K27) by the EZH2 complex is an epigenetic mark that mediates gene silencing. EZH2 is overexpressed in many cancers and correlates with poor prognosis in both breast and prostate cancers. However, the status of H3K27 methylation and its clinical implication in cancer patients have not been reported. We thus examined trimethylation of H3K27 (H3K27me3) by immunohistochemistry and its association with clinical variables and prognosis in breast, ovarian, and pancreatic cancers. We found that H3K27me3 expression was significantly lower in breast, ovarian and pancreatic cancers than in normal tissues (62% in breast cancer vs. 88% in normal breast tissue, P = 0.001; 38.4% in ovarian cancer vs. 83.3% in normal ovarian tissue, P < 0.05; and 26% in pancreatic cancer vs. 89% in normal pancreatic tissue, P < 0.001). H3K27me3 expression showed significant prognostic impact in breast, ovarian and pancreatic cancers in univariate survival analyses. In all three cancer types, patients with low expression of H3K27me3 had significantly shorter overall survival time when compared with those with high H3K27me3 expression. In a multivariate model, H3K27me3 expression was an independent prognostic value for overall survival in all three cancer types. These results suggest that H3K27me3 expression is a prognostic indicator for clinical outcome in patients with breast, ovarian, and pancreatic cancers.

ERK Promotes Tumorigenesis by Inhibiting FOXO3a Via MDM2-mediated Degradation

Nature Cell Biology. Feb, 2008  |  Pubmed ID: 18204439

The RAS-ERK pathway is known to play a pivotal role in differentiation, proliferation and tumour progression. Here, we show that Erk downregulates Forkhead box O 3a (FOXO3a) by directly interacting with and phosphorylating FOXO3a at Ser 294, Ser 344 and Ser 425, which consequently promotes cell proliferation and tumorigenesis. The ERK-phosphorylated FOXO3a degrades via an MDM2-mediated ubiquitin-proteasome pathway. However, the non-phosphorylated FOXO3a mutant is resistant to the interaction and degradation by murine double minute 2 (MDM2), thereby resulting in a strong inhibition of cell proliferation and tumorigenicity. Taken together, our study elucidates a novel pathway in cell growth and tumorigenesis through negative regulation of FOXO3a by RAS-ERK and MDM2.

14-3-3 Zeta Down-regulates P53 in Mammary Epithelial Cells and Confers Luminal Filling

Cancer Research. Mar, 2008  |  Pubmed ID: 18339856

Recent progress in diagnostic tools allows many breast cancers to be detected at an early preinvasive stage. Thus, a better understanding of the molecular basis of early breast cancer progression is essential. Previously, we discovered that 14-3-3 zeta is overexpressed in >40% of advanced breast cancers, and this overexpression predicts poor patient survival. Here, we examined at what stage of breast disease 14-3-3 zeta overexpression occurs, and we found that increased expression of 14-3-3 zeta begins at atypical ductal hyperplasia, an early stage of breast disease. To determine whether 14-3-3 zeta overexpression is a decisive early event in breast cancer, we overexpressed 14-3-3 zeta in MCF10A cells and examined its effect in a three-dimensional culture model. We discovered that 14-3-3 zeta overexpression severely disrupted the acini architecture resulting in luminal filling. Proper lumen formation is a result of anoikis, apoptosis due to detachment from the basement membrane. We found that 14-3-3 zeta overexpression conferred resistance to anoikis. Additionally, 14-3-3 zeta overexpression in MCF10A cells and in mammary epithelial cells (MEC) from 14-3-3 zeta transgenic mice reduced expression of p53, which is known to mediate anoikis. Mechanistically, 14-3-3 zeta induced hyperactivation of the phosphoinositide 3-kinase/Akt pathway which led to phosphorylation and translocation of the MDM2 E3 ligase resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14-3-3 zeta-overexpressing MCF10A acini in three-dimensional cultures. These data suggest that 14-3-3 zeta overexpression is a critical event in early breast disease, and down-regulation of p53 is one of the mechanisms by which 14-3-3 zeta alters MEC acini structure and increases the risk of breast cancer.

Ph.D. Training in Cancer Biology

Cancer Research. Nov, 2008  |  Pubmed ID: 19010881

Mitotic Deregulation by Survivin in ErbB2-overexpressing Breast Cancer Cells Contributes to Taxol Resistance

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Feb, 2009  |  Pubmed ID: 19228734

Taxol resistance remains a major obstacle to improve the benefit of breast cancer patients. Here, we studied whether overexpression of ErbB2 may lead to mitotic deregulation in breast cancer cells via up-regulation of survivin that confers Taxol resistance.

14-3-3zeta Overexpression Defines High Risk for Breast Cancer Recurrence and Promotes Cancer Cell Survival

Cancer Research. Apr, 2009  |  Pubmed ID: 19318578

The ubiquitously expressed 14-3-3 proteins are involved in numerous important cellular functions. The loss of 14-3-3sigma is a common event in breast cancer; however, the role of other 14-3-3s in breast cancer is unclear. Recently, we found that 14-3-3zeta overexpression occurs in early stage breast diseases and contributes to transformation of human mammary epithelial cells. Here, we show that 14-3-3zeta overexpression also persisted in invasive ductal carcinoma and contributed to the further progression of breast cancer. To examine the clinical effect of 14-3-3zeta overexpression in advanced stage breast cancer, we performed immunohistochemical analysis of 14-3-3zeta expression in primary breast carcinomas. 14-3-3zeta overexpression occurred in 42% of breast tumors and was determined to be an independent prognostic factor for reduced disease-free survival. 14-3-3zeta overexpression combined with ErbB2 overexpression and positive lymph node status identified a subgroup of patients at high risk for developing distant metastasis. To investigate whether 14-3-3zeta overexpression causally promotes breast cancer progression, we overexpressed 14-3-3zeta by stable transfection or reduced 14-3-3zeta expression by siRNA in cancer cell lines. Increased 14-3-3zeta expression enhanced anchorage-independent growth and inhibited stress-induced apoptosis, whereas down-regulation of 14-3-3zeta reduced anchorage-independent growth and sensitized cells to stress-induced apoptosis via the mitochondrial apoptotic pathway. Transient blockade of 14-3-3zeta expression by siRNA in cancer cells effectively reduced the onset and growth of tumor xenografts in vivo. Therefore, 14-3-3zeta overexpression is a novel molecular marker for disease recurrence in breast cancer patients and may serve as an effective therapeutic target in patients whose tumors overexpress 14-3-3zeta.

14-3-3zeta/tau Heterodimers Regulate Slingshot Activity in Migrating Keratinocytes

Biochemical and Biophysical Research Communications. Jun, 2009  |  Pubmed ID: 19371722

Defining the pathways required for keratinocyte cell migration is important for understanding mechanisms of wound healing and tumor cell metastasis. We have recently identified an alpha6beta4 integrin-Rac1 signaling pathway via which the phosphatase Slingshot (SSH) activates/dephosphorylates cofilin, thereby determining keratinocyte migration behavior. Here, we assayed the role of 14-3-3 isoforms in regulating the activity of SSH1. Using amino or carboxy terminal domains of 14-3-3zeta, we demonstrate that in keratinocytes 14-3-3zeta/tau heterodimers bind SSH1, in the absence of Rac1 signaling. This interaction leads to an inhibition of SSH1 activity, as measured by an increase in phosphorylated cofilin levels. Overexpression of the carboxy terminal domain of 14-3-3zeta acts as a dominant negative and inhibits the interaction between 14-3-3tau and SSH1. These results implicate 14-3-3zeta/tau heterodimers as key regulators of SSH1 activity in keratinocytes and suggest they play a role in cytoskeleton remodeling during cell migration.

ErbB2-mediated Src and Signal Transducer and Activator of Transcription 3 Activation Leads to Transcriptional Up-regulation of P21Cip1 and Chemoresistance in Breast Cancer Cells

Molecular Cancer Research : MCR. Apr, 2009  |  Pubmed ID: 19372587

Overexpression of the ErbB2 receptor tyrosine kinase is prevalent in approximately 30% of human breast cancers and confers Taxol resistance. Our previous work has shown that ErbB2 inhibits Taxol-induced apoptosis in breast cancer cells by transcriptionally up-regulating p21(Cip1). However, the mechanism of ErbB2-mediated p21(Cip1) up-regulation is unclear. Here, we show that ErbB2 up-regulates p21(Cip1) transcription through increased Src activity in ErbB2-overexpressing cells. Src activation further activated signal transducer and activator of transcription 3 (STAT3) that recognizes a SIE binding site on the p21(Cip1) promoter required for ErbB2-mediated p21(Cip1) transcriptional up-regulation. Both Src and STAT3 inhibitors restored Taxol sensitivity in resistant ErbB2-overexpressing breast cancer cells. Our data suggest that ErbB2 overexpression can activate STAT3 through Src leading to transcriptional up-regulation of p21(Cip1) that confers Taxol resistance of breast cancer cells. Our study suggests a potential clinical application of Src and STAT3 inhibitors in Taxol sensitization of ErbB2-overexpressing breast cancers.

14-3-3zeta Cooperates with ErbB2 to Promote Ductal Carcinoma in Situ Progression to Invasive Breast Cancer by Inducing Epithelial-mesenchymal Transition

Cancer Cell. Sep, 2009  |  Pubmed ID: 19732720

ErbB2, a metastasis-promoting oncoprotein, is overexpressed in approximately 25% of invasive/metastatic breast cancers, but in 50%-60% of noninvasive ductal carcinomas in situ (DCIS). It has been puzzling how a subset of ErbB2-overexpressing DCIS develops into invasive breast cancer (IBC). We found that co-overexpression of 14-3-3zeta in ErbB2-overexpressing DCIS conferred a higher risk of progression to IBC. ErbB2 and 14-3-3zeta overexpression, respectively, increased cell migration and decreased cell adhesion, two prerequisites of tumor cell invasion. 14-3-3zeta overexpression reduced cell adhesion by activating the TGF-beta/Smads pathway that led to ZFHX1B/SIP-1 upregulation, E-cadherin loss, and epithelial-mesenchymal transition. Importantly, patients whose breast tumors overexpressed both ErbB2 and 14-3-3zeta had higher rates of metastatic recurrence and death than those whose tumors overexpressed only one.

Upregulation of Neutrophil Gelatinase-associated Lipocalin by ErbB2 Through Nuclear Factor-kappaB Activation

Cancer Research. Dec, 2009  |  Pubmed ID: 19951994

ErbB2 (HER2, neu) is a receptor tyrosine kinase overexpressed in about 25% of invasive breast carcinomas. Neutrophil gelatinase-associated lipocalin (NGAL) is a secreted glycoprotein expressed in a variety of cancers, including breast carcinomas. NGAL can inhibit erythroid cell production, leading to anemia. Anemia usually occurs in cancer patients and negatively affects quality of life. However, current treatment for cancer-related anemia has potential complications. ErbB2, NGAL, and anemia have all been associated with increased metastasis and poor prognosis in breast cancer patients, although the relationship between ErbB2 and NGAL expression is not clear. Here, using breast cancer cell lines in vitro and transgenic mice carrying the activated c-neu oncogene driven by a mouse mammary tumor virus (MMTV-neu) in vivo, we show that ErbB2 overexpression leads to NGAL upregulation, which is dependent on nuclear factor-kappaB (NF-kappaB) activity. MMTV-neu transgenic mice developed anemia after tumor onset, and anemia progression could be partially arrested by a NF-kappaB inhibitor and ErbB2-targeted therapy. Taken together, upregulation of NGAL by ErbB2 through NF-kappaB activation is involved in cancer-related anemia, and the ErbB2, NF-kappaB, and NGAL pathways may serve as potential therapeutic targets for cancer-related anemia.

Oxygen Sensor Boosts Growth Factor Signaling

Nature Medicine. Mar, 2009  |  Pubmed ID: 19265822

Breast Cancer Metastasis: Challenges and Opportunities

Cancer Research. Jun, 2009  |  Pubmed ID: 19470768

Molecular Predictors of Response to Trastuzumab and Lapatinib in Breast Cancer

Nature Reviews. Clinical Oncology. Feb, 2010  |  Pubmed ID: 20027191

Trastuzumab is a monoclonal antibody directed against the human EGFR2 (HER2) protein that has been shown to improve survival in patients with HER2-positive breast cancer. Lapatinib is an oral small-molecule tyrosine kinase inhibitor directed against EGFR and HER2. Lapatinib therapy was shown to prolong the time to progression and increase the rate of response to capecitabine in patients who had received anthracycline-based and taxane-based chemotherapy, and whose tumors had progressed on trastuzumab. HER2 status, either gene copy number or the protein expression level, is the best predictive marker available for assessing response to trastuzumab and lapatinib. Whether the power of this predictive marker is the same in advanced and early-stage cancers is unknown. There is great interest in developing diagnostic tests that predict which patients are more likely to benefit from specific HER2-directed therapies. Novel therapeutics that will overcome resistance to trastuzumab and lapatinib are under intense clinical development. In the future, it will be important to characterize mechanisms of resistance in metastatic tumors to determine which novel targeted therapy will be most appropriate for individual patients.

Invasive Breast Cancer Development: "fatal Accident" from Malfunctions in Both "motor" and "brake"

Cell Cycle (Georgetown, Tex.). Feb, 2010  |  Pubmed ID: 20090417

PI(3)king Apart PTEN's Role in Cancer

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Sep, 2010  |  Pubmed ID: 20622047

The tumor suppressor phosphatase and tensin homolog (PTEN) is a nonredundant phosphatase, counteracting one of the most critical cancer-promoting pathways: the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. In addition to the canonical function of dephosphorylation of phosphatidylinositol-3,4,5-trisphosphate (PIP3), recent studies showed the intriguing roles of PTEN in regulating genomic instability, DNA repair, stem cell self-renewal, cellular senescence, and cell migration and/or metastasis. Clinically, PTEN mutations and deficiencies are prevalent in many types of human cancers. Severe PTEN deficiency is also associated with advanced tumor stage and therapeutic resistance, such as the resistance to trastuzumab, an anti-HER2 therapy. Currently, targeting the deregulated PI3K/PTEN-Akt signaling axis has emerged as one of the major tenets in anticancer drug development. In this review, we highlight our current knowledge of PTEN function and the recent discoveries in dissecting the PTEN signaling pathway. The deregulations of PTEN in cancers, clinical lessons, and new prospects of rationally designed PI3K/Akt-targeted therapy for effective cancer treatment are also discussed.

Activation of Murine Double Minute 2 by Akt in Mammary Epithelium Delays Mammary Involution and Accelerates Mammary Tumorigenesis

Cancer Research. Oct, 2010  |  Pubmed ID: 20841468

Amplification or overexpression of murine double minute 2 (MDM2) promotes a variety of human tumors by degrading tumor suppressor proteins such as p53. Phosphorylation of MDM2 on Ser(166) and Ser(186) by the survival kinase Akt inhibits p53-mediated apoptosis. However, it is unclear whether this pathway contributes to normal or malignant pathophysiology in vivo. To address these questions, we generated transgenic mice expressing the Akt-phosphorylated form of MDM2 (MDM2DDS166D/S186D) in the mammary epithelium. Activation of MDM2 delayed mammary gland involution and accelerated tumor progression in mouse mammary tumor virus/neu transgenic mice by inhibiting apoptosis in a manner associated with decreased p53 expression. Our findings offer in vivo evidence that activation of MDM2 by Akt contributes to mammary development and tumorigenesis.

Cancer Cell Stiffness: Integrated Roles of Three-dimensional Matrix Stiffness and Transforming Potential

Biophysical Journal. Oct, 2010  |  Pubmed ID: 20923638

While significant advances have been made toward revealing the molecular mechanisms that influence breast cancer progression, much less is known about the associated cellular mechanical properties. To this end, we use particle-tracking microrheology to investigate the interplay among intracellular mechanics, three-dimensional matrix stiffness, and transforming potential in a mammary epithelial cell (MEC) cancer progression series. We use a well-characterized model system where human-derived MCF10A MECs overexpress either ErbB2, 14-3-3ζ, or both ErbB2 and 14-3-3ζ, with empty vector as a control. Our results show that MECs possessing ErbB2 transforming potential stiffen in response to elevated matrix stiffness, whereas non-transformed MECs or those overexpressing only 14-3-3ζ do no exhibit this response. We further observe that overexpression of ErbB2 alone is associated with the highest degree of intracellular sensitivity to matrix stiffness, and that the effect of transforming potential on intracellular stiffness is matrix-stiffness-dependent. Moreover, our intracellular stiffness measurements parallel cell migration behavior that has been previously reported for these MEC sublines. Given the current knowledge base of breast cancer mechanobiology, these findings suggest that there may be a positive relationship among intracellular stiffness sensitivity, cell motility, and perturbed mechanotransduction in breast cancer.

Tissue Transglutaminase Promotes Drug Resistance and Invasion by Inducing Mesenchymal Transition in Mammary Epithelial Cells

PloS One. 2010  |  Pubmed ID: 20967228

Recent observations that aberrant expression of tissue transglutaminase (TG2) promotes growth, survival, and metastasis of multiple tumor types is of great significance and could yield novel therapeutic targets for improved patient outcomes. To accomplish this, a clear understanding of how TG2 contributes to these phenotypes is essential. Using mammary epithelial cell lines (MCF10A, MCF12A, MCF7 and MCF7/RT) as a model system, we determined the impact of TG2 expression on cell growth, cell survival, invasion, and differentiation. Our results show that TG2 expression promotes drug resistance and invasive functions by inducing epithelial-mesenchymal transition (EMT). Thus, TG2 expression supported anchorage-independent growth of mammary epithelial cells in soft-agar, disrupted the apical-basal polarity, and resulted in disorganized acini structures when grown in 3D-culture. At molecular level, TG2 expression resulted in loss of E-cadherin and increased the expression of various transcriptional repressors (Snail1, Zeb1, Zeb2 and Twist1). Tumor growth factor-beta (TGF-β) failed to induce EMT in cells lacking TG2 expression, suggesting that TG2 is a downstream effector of TGF-β-induced EMT. Moreover, TG2 expression induced stem cell-like phenotype in mammary epithelial cells as revealed by enrichment of CD44(+)/CD24(-/low) cell populations. Overall, our studies show that aberrant expression of TG2 is sufficient for inducing EMT in epithelial cells and establish a strong link between TG2 expression and progression of metastatic breast disease.

PTEN, PIK3CA, P-AKT, and P-p70S6K Status: Association with Trastuzumab Response and Survival in Patients with HER2-positive Metastatic Breast Cancer

The American Journal of Pathology. Oct, 2010  |  Pubmed ID: 20813970

Phosphatase and tensin homolog (PTEN) is a key modulator of trastuzumab sensitivity in HER2-overexpressing breast cancer. Because PTEN opposes the downstream signaling of phosphoinositide 3-kinase (PI3K), we investigated the role of PTEN and other components of the PI3K pathway in trastuzumab resistance. We analyzed the status of PTEN, p-AKT-Ser473, and p-p70S6K-Thr389 using immunohistochemistry. PIK3CA mutation status was analyzed by direct sequencing. Primary tumor tissue was available from 137 patients with HER2-overexpressing metastatic breast cancer who had received trastuzumab-based chemotherapy. We observed that each of the four biomarkers alone did not significantly correlate with trastuzumab response, whereas PTEN loss alone significantly correlated with shorter survival times (P = 0.023). PI3K pathway activation, defined as PTEN loss and/or PIK3CA mutation, was associated with a poor response to trastuzumab (P = 0.047) and a shorter survival time (P = 0.015). PTEN loss was significantly associated with a poor response to trastuzumab (P = 0.028) and shorter survival time (P = 0.008) in patients who had received first-line trastuzumab and in patients with estrogen receptor- (P = 0.029) and progesterone receptor-negative tumors (P = 0.033). p-AKT-Ser473 and p-p70S6K-Thr389 each had a limited correlation with trastuzumab response. When these markers were combined with PTEN loss, an increased correlation with patient outcome was observed. In conclusion, PI3K pathway activation plays a pivotal role in trastuzumab resistance. Our findings may facilitate the evaluation of tumor response to trastuzumab-based and targeted therapies.

Activation of P21(CIP1/WAF1) in Mammary Epithelium Accelerates Mammary Tumorigenesis and Promotes Lung Metastasis

Biochemical and Biophysical Research Communications. Dec, 2010  |  Pubmed ID: 21040707

While p21 is well known to inhibit cyclin-CDK activity in the nucleus and it has also been demonstrated to have oncogenic properties in different types of human cancers. In vitro studies showed that the oncogenic function of p21is closely related to its cytoplasmic localization. However, it is unclear whether cytoplasmic p21 contributes to tumorigenesis in vivo. To address this question, we generated transgenic mice expressing the Akt-phosphorylated form of p21 (p21T145D) in the mammary epithelium. The results showed that Akt-activated p21 was expressed in the cytoplasm of mammary epithelium. Overexpression of Akt-activated p21 accelerated tumor onset and promoted lung metastasis in MMTV/neu mice, providing evidence that p21, especially cytoplasmic phosphorylated p21, has an oncogenic role in promoting mammary tumorigenesis and metastasis.

14-3-3ζ As a Prognostic Marker and Therapeutic Target for Cancer

Expert Opinion on Therapeutic Targets. Dec, 2010  |  Pubmed ID: 21058923

The ubiquitously expressed 14-3-3ζ protein is involved in numerous important cellular pathways involved in cancer. Recent research suggests 14-3-3ζ may play a central role regulating multiple pathways responsible for cancer initiation and progression. This review will provide an overview of 14-3-3 proteins and address the role of 14-3-3ζ overexpression in cancer.

Cytokine Receptor CXCR4 Mediates Estrogen-independent Tumorigenesis, Metastasis, and Resistance to Endocrine Therapy in Human Breast Cancer

Cancer Research. Jan, 2011  |  Pubmed ID: 21123450

Estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer patients. Metastasis has been associated with chemokine signaling through the SDF-1-CXCR4 axis. Thus, the development of estrogen independence and endocrine therapy resistance in breast cancer patients may be driven by SDF-1-CXCR4 signaling. Here we report that CXCR4 overexpression is indeed correlated with worse prognosis and decreased patient survival irrespective of the status of the estrogen receptor (ER). Constitutive activation of CXCR4 in poorly metastatic MCF-7 cells led to enhanced tumor growth and metastases that could be reversed by CXCR4 inhibition. CXCR4 overexpression in MCF-7 cells promoted estrogen independence in vivo, whereas exogenous SDF-1 treatment negated the inhibitory effects of treatment with the anti-estrogen ICI 182,780 on CXCR4-mediated tumor growth. The effects of CXCR4 overexpression were correlated with SDF-1-mediated activation of downstream signaling via ERK1/2 and p38 MAPK (mitogen activated protein kinase) and with an enhancement of ER-mediated gene expression. Together, these results show that enhanced CXCR4 signaling is sufficient to drive ER-positive breast cancers to a metastatic and endocrine therapy-resistant phenotype via increased MAPK signaling. Our findings highlight CXCR4 signaling as a rational therapeutic target for the treatment of ER-positive, estrogen-independent breast carcinomas needing improved clinical management.

Protein Microarray Analysis of Mammary Epithelial Cells from Obese and Nonobese Women at High Risk for Breast Cancer: Feasibility Data

Cancer Epidemiology, Biomarkers & Prevention : a Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology. Mar, 2011  |  Pubmed ID: 21242333

Obesity is a well-established risk factor for cancer, accounting for up to 20% of cancer deaths in women. Studies of women with breast cancer have shown obesity to be associated with an increased risk of dying from breast cancer and increased risk of developing distant metastasis. While previous studies have focused on differences in circulating hormone levels as a cause for increased breast cancer incidence in postmenopausal women, few studies have focused on potential differences in the protein expression patterns of mammary epithelial cells obtained from obese versus nonobese women.

P53 Regulates Epithelial-mesenchymal Transition and Stem Cell Properties Through Modulating MiRNAs

Nature Cell Biology. Mar, 2011  |  Pubmed ID: 21336307

The epithelial-mesenchymal transition (EMT) has recently been linked to stem cell phenotype. However, the molecular mechanism underlying EMT and regulation of stemness remains elusive. Here, using genomic approaches, we show that tumour suppressor p53 has a role in regulating both EMT and EMT-associated stem cell properties through transcriptional activation of the microRNA miR-200c. p53 transactivates miR-200c through direct binding to the miR-200c promoter. Loss of p53 in mammary epithelial cells leads to decreased expression of miR-200c and activates the EMT programme, accompanied by an increased mammary stem cell population. Re-expressing miR-200c suppresses genes that mediate EMT and stemness properties and thereby reverts the mesenchymal and stem-cell-like phenotype caused by loss of p53 to a differentiated epithelial cell phenotype. Furthermore, loss of p53 correlates with a decrease in the level of miR-200c, but an increase in the expression of EMT and stemness markers, and development of a high tumour grade in a cohort of breast tumours. This study elucidates a role for p53 in regulating EMT-MET (mesenchymal-epithelial transition) and stemness or differentiation plasticity, and reveals a potential therapeutic implication to suppress EMT-associated cancer stem cells through activation of the p53-miR-200c pathway.

Combating Trastuzumab Resistance by Targeting SRC, a Common Node Downstream of Multiple Resistance Pathways

Nature Medicine. Apr, 2011  |  Pubmed ID: 21399647

Trastuzumab is a successful rationally designed ERBB2-targeted therapy. However, about half of individuals with ERBB2-overexpressing breast cancer do not respond to trastuzumab-based therapies, owing to various resistance mechanisms. Clinically applicable regimens for overcoming trastuzumab resistance of different mechanisms are not yet available. We show that the nonreceptor tyrosine kinase c-SRC (SRC) is a key modulator of trastuzumab response and a common node downstream of multiple trastuzumab resistance pathways. We find that SRC is activated in both acquired and de novo trastuzumab-resistant cells and uncover a novel mechanism of SRC regulation involving dephosphorylation by PTEN. Increased SRC activation conferred considerable trastuzumab resistance in breast cancer cells and correlated with trastuzumab resistance in patients. Targeting SRC in combination with trastuzumab sensitized multiple lines of trastuzumab-resistant cells to trastuzumab and eliminated trastuzumab-resistant tumors in vivo, suggesting the potential clinical application of this strategy to overcome trastuzumab resistance.

Cancer Cell Migration: Integrated Roles of Matrix Mechanics and Transforming Potential

PloS One. 2011  |  Pubmed ID: 21647371

Significant progress has been achieved toward elucidating the molecular mechanisms that underlie breast cancer progression; yet, much less is known about the associated cellular biophysical traits. To this end, we use time-lapsed confocal microscopy to investigate the interplay among cell motility, three-dimensional (3D) matrix stiffness, matrix architecture, and transforming potential in a mammary epithelial cell (MEC) cancer progression series. We use a well characterized breast cancer progression model where human-derived MCF10A MECs overexpress either ErbB2, 14-3-3ζ, or both ErbB2 and 14-3-3ζ, with empty vector as a control. Cell motility assays showed that MECs overexpressing ErbB2 alone exhibited notably high migration speeds when cultured atop two-dimensional (2D) matrices, while overexpression of 14-3-3ζ alone most suppressed migration atop 2D matrices (as compared to non-transformed MECs). Our results also suggest that co-overexpression of the 14-3-3ζ and ErbB2 proteins facilitates cell migratory capacity in 3D matrices, as reflected in cell migration speed. Additionally, 3D matrices of sufficient stiffness can significantly hinder the migratory ability of partially transformed cells, but increased 3D matrix stiffness has a lesser effect on the aggressive migratory behavior exhibited by fully transformed cells that co-overexpress both ErbB2 and 14-3-3ζ. Finally, this study shows that for MECs possessing partial or full transforming potential, those overexpressing ErbB2 alone show the greatest sensitivity of cell migration speed to matrix architecture, while those overexpressing 14-3-3ζ alone exhibit the least sensitivity to matrix architecture. Given the current knowledge of breast cancer mechanobiology, these findings overall suggest that cell motility is governed by a complex interplay between matrix mechanics and transforming potential.

Pilot and Feasibility Study: Prospective Proteomic Profiling of Mammary Epithelial Cells from High-risk Women Provides Evidence of Activation of Pro-survival Pathways

Breast Cancer Research and Treatment. Jun, 2011  |  Pubmed ID: 21647677

Normal mammary gland homeostasis requires the coordinated regulation of protein signaling networks. However, we have little prospective information on whether activation of protein signaling occurs in premalignant mammary epithelial cells, as represented by cells with cytological atypia from women who are at high risk for breast cancer. This information is critical for understanding the role of deregulated signaling pathways in the initiation of breast cancer and for developing targeted prevention and/or treatment strategies for breast cancer in the future. In this pilot and feasibility study, we examined the expression of 52 phosphorylated, total, and cleaved proteins in 31 microdissected Random Periareolar Fine Needle Aspiration (RPFNA) samples by high-throughput Reverse Phase Protein Microarray. Unsupervised hierarchical clustering analysis indicated the presence of four clusters of proteins that represent the following signaling pathways: (1) receptor tyrosine kinase/Akt/mammalian target of rapamycin (RTK/Akt/mTOR), (2) RTK/Akt/extracellular signal-regulated kinase (RTK/Akt/ERK), (3) mitochondrial apoptosis, and (4) indeterminate. Clusters 1 through 3 comprised moderately to highly expressed proteins, while Cluster 4 comprised proteins that are lowly expressed in a majority of RPFNA samples. Our exploratory study showed that the interlinked components of mitochondrial apoptosis pathway are highly expressed in all mammary epithelial cells obtained from high-risk women. In particular, the expression levels of anti-apoptotic Bcl-xL and pro-apoptotic Bad are positively correlated in both non-atypical and atypical samples (unadjusted P < 0.0001), suggesting a delicate balance between the pro-apoptotic and anti-apoptotic regulation of cell proliferation during the early steps of mammary carcinogenesis. Our feasibility study suggests that the activation of key proteins along the RTK/Akt pathway may tip this balance to cell survival. Taken together, our results demonstrate the feasibility of mapping proteomic signaling networks in limited RPFNA samples obtained from high-risk women and the promise of developing rational drug targets or preventative strategies for breast cancer in future proteomic studies with a larger cohort of high-risk women.

Evidence That Aberrant Expression of Tissue Transglutaminase Promotes Stem Cell Characteristics in Mammary Epithelial Cells

PloS One. 2011  |  Pubmed ID: 21687668

Cancer stem cells (CSCs) or tumor initiating cells (TICs) make up only a small fraction of total tumor cell population, but recent evidence suggests that they are responsible for tumor initiation and the maintenance of tumor growth. Whether CSCs/TICs originate from normal stem cells or result from the dedifferentiation of terminally differentiated cells remains unknown. Here we provide evidence that sustained expression of the proinflammatory protein tissue transglutaminase (TG2) confers stem cell like properties in non-transformed and transformed mammary epithelial cells. Sustained expression of TG2 was associated with increase in CD44(high)/CD24(low/-) subpopulation, increased ability of cells to form mammospheres, and acquisition of self-renewal ability. Mammospheres derived from TG2-transfected mammary epithelial cells (MCF10A) differentiated into complex secondary structures when grown in Matrigel cultures. Cells in these secondary structures differentiated into Muc1-positive (luminal marker) and integrin α6-positive (basal marker) cells in response to prolactin treatment. Highly aggressive MDA-231 and drug-resistant MCF-7/RT breast cancer cells, which express high basal levels of TG2, shared many traits with TG2-transfected MCF10A stem cells but unlike MCF10A-derived stem cells they failed to form the secondary structures and to differentiate into Muc1-positive luminal cells when grown in Matrigel culture. Downregulation of TG2 attenuated stem cell properties in both non-transformed and transformed mammary epithelial cells. Taken together, these results suggested a new function for TG2 and revealed a novel mechanism responsible for promoting the stem cell characteristics in adult mammary epithelial cells.

Microenvironment Determinants of Brain Metastasis

Cell & Bioscience. 2011  |  Pubmed ID: 21711688

Metastasis accounts for 90% of cancer-related mortality. Brain metastases generally present during the late stages in the natural history of cancer progression. Recent advances in cancer treatment and management have resulted in better control of systemic disease metastatic to organs other than the brain and improved patient survival. However, patients who experience recurrent disease manifest an increasing number of brain metastases, which are usually refractory to therapies. To meet the new challenges of controlling brain metastasis, the research community has been tackling the problem with novel experimental models and research tools, which have led to an improved understanding of brain metastasis. The time-tested "seed-and-soil" hypothesis of metastasis indicates that successful outgrowth of deadly metastatic tumors depends on permissible interactions between the metastatic cancer cells and the site-specific microenvironment in the host organs. Consistently, recent studies indicate that the brain, the major component of the central nervous system, has unique physiological features that can determine the outcome of metastatic tumor growth. The current review summarizes recent discoveries on these tumor-brain interactions, and the potential clinical implications these novel findings could have for the better treatment of patients with brain metastasis.

Phase I/II Study of Trastuzumab in Combination with Everolimus (RAD001) in Patients with HER2-overexpressing Metastatic Breast Cancer Who Progressed on Trastuzumab-based Therapy

Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology. Aug, 2011  |  Pubmed ID: 21730275

Trastuzumab resistance has been linked to activation of the phosphoinositol 3-kinase (PI3K) pathway. Phosphatase and tensin homolog (PTEN) is a dual phosphatase that counteracts the PI3K function; PTEN loss leads to activation of the Akt cascade and the downstream mammalian target of rapamycin (mTOR). Preclinical studies demonstrated that mTOR inhibition sensitized the response to trastuzumab in mice with HER2 overexpressing and PTEN-deficient breast xenografts. Our trial evaluated the safety and efficacy of the combination of everolimus and trastuzumab in women with HER2-overexpressing metastatic breast cancer (MBC) that progressed on trastuzumab-based therapy.

BikDD Eliminates Breast Cancer Initiating Cells and Synergizes with Lapatinib for Breast Cancer Treatment

Cancer Cell. Sep, 2011  |  Pubmed ID: 21907925

Breast cancer initiating cells (BCICs), which can fully recapitulate the tumor origin and are often resistant to chemo- and radiotherapy, are currently considered as a major obstacle for breast cancer treatment. Here, we show that BIKDD, a constitutively active mutant form of proapoptotic gene, BIK, effectively induces apoptosis of breast cancer cells and synergizes with lapatinib. Most importantly, BikDD significantly reduces BCICs through co-antagonism of its binding partners Bcl-2, Bcl-xL, and Mcl-1, suggesting a potential therapeutic strategy targeting BCICs. Furthermore, we developed a cancer-specific targeting approach for breast cancer that selectively expresses BikDD in breast cancer cells including BCICs, and demonstrated its potent antitumor activity and synergism with lapatinib in vitro and in vivo.

Nucleolin Protein Interacts with Microprocessor Complex to Affect Biogenesis of MicroRNAs 15a and 16

The Journal of Biological Chemistry. Dec, 2011  |  Pubmed ID: 22049078

MicroRNAs (miRNA) are endogenous, short, non-coding RNA that undergo a multistep biogenesis before generating the functional, mature sequence. The core components of the microprocessor complex, consisting of Drosha and DGCR8, are both necessary and sufficient for this process, although accessory proteins have been found that modulate the biogenesis of a subset of miRNA. Curiously, many of the proteins involved in miRNA biogenesis are also needed for ribosomal RNA processing. Here we show that nucleolin, another protein critical for rRNA processing, is involved in the biogenesis of microRNA 15a/16 (miR-15a/16), specifically at the primary to precursor stage of processing. Through overexpression and knockdown studies, we show that miR-15a/16 levels are directly correlated to nucleolin expression. Furthermore, we found that cellular localization is critical for the proper functioning of nucleolin in this pathway and that nucleolin directly interacts with DGCR8 and Drosha in the nucleus. Nucleolin can bind to the primary miRNA both directly and specifically. Finally, we show that in the absence of nucleolin, cell extracts are unable to process miR-15a/16 in vitro and that this can be rescued by the addition of nucleolin. Our findings offer a new protein component in the microRNA biogenesis pathway and lend insight into miRNA dysregulation in certain cancers.

Targeting Src Family Kinases in Anti-cancer Therapies: Turning Promise into Triumph

Trends in Pharmacological Sciences. Dec, 2011  |  Pubmed ID: 22153719

Src is a non-receptor tyrosine kinase that is deregulated in many types of cancer. Decades of research have revealed the crucial role of Src in many aspects of tumor development, including proliferation, survival, adhesion, migration, invasion and, most importantly, metastasis, in multiple tumor types. Despite extensive preclinical evidence that warrants targeting Src as a promising therapeutic approach for cancer, Src inhibitor(s) showed only minimal therapeutic activity in various types of solid tumors when used as a single agent in recent early-phase clinical trials. In this review, we highlight the most recent advances from preclinical studies and clinical trials that shed light on potential clinical use of Src inhibitor-containing combinatorial regimens in overcoming resistance to current anticancer therapies and in preventing metastatic recurrence.

Evidence That GTP-binding Domain but Not Catalytic Domain of Transglutaminase 2 is Essential for Epithelial-to-mesenchymal Transition in Mammary Epithelial Cells

Breast Cancer Research : BCR. Jan, 2012  |  Pubmed ID: 22225906

ABSTRACT: INTRODUCTION: The expression of proinflammatory protein tissue transglutaminase (TG2) is frequently upregulated in multiple cancer cell types. However, the exact role of TG2 in cancer cells is not well understood. We recently initiated studies to determine the significance of TG2 in cancer cells and observed that sustained expression of TG2 resulted in epithelial-to-mesenchymal transition (EMT) and promoted stem cell (CSC) traits in mammary epithelial cells. These results suggested that TG2 could serve as a promising therapeutic target for overcoming chemoresistance and inhibiting metastatic spread of cancer cells. METHODS: Using various mutant constructs, we analyzed the activity of TG2 that is essential for promoting the EMT/CSC phenotype. RESULTS: Our results suggest that catalytically inactive TG2 (TG2-C277S) is as effective as wild-type TG2 (TG2-WT) in inducing the EMT/CSC in mammary epithelial cells. In contrast, overexpression of a GTP-binding-deficient mutant (TG2-R580A) was completely incompetent in this regard. Moreover, TG2-dependent activation of the proinflammatory transcription factor, NF-kappaB, is deemed essential for promoting the EMT/CSC phenotype in mammary epithelial cells. CONCLUSIONS: Our results suggest that transamidation activity of TG2 is not essential for promoting its oncogenic functions and provide strong rationale for developing small molecule inhibitors to block GTP-binding pockets of TG2. Such inhibitors may have great potential for inhibiting the TG2-regulated pathways and for reversing drug resistance and inhibiting metastasis of cancer cells.