utilizing-functional-genomics-screening-to-identify-potentially-novel

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

The Institute of Cancer Research

Gefitinib (Iressa) is a specific and effective epidermal growth factor receptor inhibitor. An understanding of the downstream cellular targets of gefitinib will allow the discovery of biomarkers for predicting outcomes and monitoring anti-epidermal growth factor receptor therapies and provide information for overcoming gefitinib resistance. In this study, we investigated the role and regulation of FOXM1 in response to gefitinib treatment in breast cancer. Using the gefitinib-sensitive breast carcinoma cell lines BT474 and SKBR3 as well as the resistant lines MCF-7, MDA-MB-231, and MDA-MB-453, we showed that gefitinib represses the expression of the transcription factor FOXM1 in sensitive, but not resistant, cells. FOXM1 repression by gefitinib is associated with FOXO3a activation and is mediated at the transcriptional level and gene promoter level. These results were verified by immunohistochemical staining of biopsy samples from primary breast cancer patients obtained from a gefitinib neoadjuvant study. We also showed that ectopic expression of an active FOXO3a represses FOXM1 expression, whereas knockdown of FOXO3a expression using small interfering RNA can up-regulate FOXM1 and its downstream targets polo-like kinase, cyclin B1, and CDC25B and rescue sensitive BT474 cells from gefitinib-induced cell proliferative arrest. These results suggest that gefitinib represses FOXM1 expression via FOXO3a in breast cancer. We further showed that overexpression of a wild-type FOXM1 or a constitutively active FOXM1, DeltaN-FOXM1, abrogates the cell death induced by gefitinib, indicating that FOXM1 has a functional role in mediating the gefitinib-induced proliferative arrest and in determining sensitivity to gefitinib. In summary, our study defined FOXM1 as a cellular target and marker of gefitinib activity in breast cancer.

Gefitinib (Iressa) represses FOXM1 expression via FOXO3a in breast cancer.

The tyrosine kinase receptor, HER2 is a crucial prognostic marker and therapeutic target for breast cancer; however, the downstream targets and biological effectors of HER2 remain unclear. We investigated the relationship between HER2 and the transcription factor FoxM1 in breast cancer. HER2 and FoxM1 expression levels were compared in breast carcinoma cell lines, paraffin-embedded breast cancer patient samples and at the mRNA level in purified breast epithelial cells. To further examine the relationship between HER2 and FoxM1 expression, we either overexpressed or siRNA-mediated depleted endogenous HER2 in breast cancer cell lines. Additionally, a mammary epithelium-targeted HER2 (neu) transgenic mouse model was also used to assess the effect of HER2 on FoxM1 levels. Furthermore, the effect of the HER2-tyrosine kinase inhibitor lapatinib on FoxM1 in HER2 positive breast cancer cells was investigated. HER2 protein levels directly correlated with FoxM1 expression in both breast carcinoma cell lines and paraffin-embedded breast cancer patient samples. Moreover, in purified breast epithelial cells, overexpression of HER2 was associated with high levels of FoxM1 mRNA, suggesting that the upregulation of FoxM1 expression is at least partially mediated transcriptionally. Furthermore, overexpression or ablation of endogenous HER2 resulted in parallel changes in FoxM1 expression. Critically, mammary epithelium-targeted HER2 mouse tumours also resulted in increased FoxM1 expression, suggesting that HER2 directed FoxM1 expression occurs in vivo and may be a critical downstream effector of HER2-targeting therapies. Indeed, treatment of breast cancer cells with lapatinib reduced FoxM1 expression at protein, mRNA and gene promoter levels. Moreover, analysis of normal and breast cancer patient samples revealed that elevated FoxM1 expression at protein and mRNA levels correlated with breast cancer development, but not significantly with cancer progression and survival. Our results indicate that the HER2 receptor regulates the expression of the FoxM1 transcription factor, which has a role in breast cancer development.

FoxM1 is a downstream target and marker of HER2 overexpression in breast cancer.

The transcription factor Forkhead box M1 (FOXM1) is a key regulator of cell proliferation and is overexpressed in many forms of primary cancers, leading to uncontrolled cell division and genomic instability. To address the role of FOXM1 in chemoresistance, we generated a cisplatin-resistant breast cancer cell line (MCF-7-CIS(R)), which had an elevated level of FOXM1 protein and mRNA expression relative to the parental MCF-7 cells. A close correlation was observed between FOXM1 and the expression of its proposed downstream targets that are involved in DNA repair; breast cancer-associated gene 2 (BRCA2) and X-ray cross-complementing group 1 (XRCC1) were expressed at higher levels in the resistant cell lines compared with the sensitive MCF-7 cells. Moreover, cisplatin treatment induced DNA damage repair in MCF-7-CIS(R) and not in MCF-7 cells. Furthermore, the expression of a constitutively active FOXM1 (DeltaN-FOXM1) in MCF-7 cells alone was sufficient to confer cisplatin resistance. Crucially, the impairment of DNA damage repair pathways through the small interfering RNA knockdown inhibition of either FOXM1 or BRCA2/XRCC1 showed that only the silencing of FOXM1 could significantly reduce the rate of proliferation in response to cisplatin treatment in the resistant cells. This suggests that the targeting of FOXM1 is a viable strategy in circumventing acquired cisplatin resistance. Consistently, the FOXM1 inhibitor thiostrepton also showed efficacy in causing cell death and proliferative arrest in the cisplatin-resistant cells through the downregulation of FOXM1 expression. Taken together, we have identified a novel mechanism of acquired cisplatin resistance in breast cancer cells through the induction of FOXM1.

FOXM1 confers acquired cisplatin resistance in breast cancer cells.

SIRT proteins play an important role in the survival and drug resistance of tumor cells, especially during chemotherapy. In this study, we investigated the potency, specificity, and cellular targets of three SIRT inhibitors, Sirtinol, Salermide, and EX527. Cell proliferative and cell cycle analyses showed that Sirtinol and Salermide, but not EX527, were effective in inducing cell death at concentrations of 50 micromol/L or over in MCF-7 cells. Instead, EX527 caused cell cycle arrest at G(1) at comparable concentrations. In vitro SIRT assays using a p53 peptide substrate showed that all three compounds are potent SIRT1/2 inhibitors, with EX527 having the highest inhibitory activity for SIRT1. Computational docking analysis showed that Sirtinol and Salermide have high degrees of selectivity for SIRT1/2, whereas EX527 has high specificity for SIRT1 but not SIRT2. Consistently, Sirtinol and Salermide, but not EX527, treatment resulted in the in vivo acetylation of the SIRT1/2 target p53 and SIRT2 target tubulin in MCF-7 cells, suggesting that EX527 is ineffective in inhibiting SIRT2 and that p53 mediates the cytotoxic function of Sirtinol and Salermide. Studies using breast carcinoma cell lines and p53-deficient mouse fibroblasts confirmed that p53 is essential for the Sirtinol and Salermide-induced apoptosis. Further, we showed using small interfering RNA that silencing both SIRTs, but not SIRT1 and SIRT2 individually, can induce cell death in MCF-7 cells. Together, our results identify the specificity and cellular targets of these novel inhibitors and suggest that SIRT inhibitors require combined targeting of both SIRT1 and SIRT2 to induce p53 acetylation and cell death. Mol Cancer Ther; 9(4); 844-55. (c)2010 AACR.

SIRT inhibitors induce cell death and p53 acetylation through targeting both SIRT1 and SIRT2.

A role for the cancer-associated miR-106b~25 cluster in neuronal stem cells.

Alterations in cellular metabolism are a key feature of the transformed phenotype. Enhanced macromolecule synthesis is a prerequisite for rapid proliferation but may also contribute to induction of angiogenesis, metastasis formation, and tumor progression, thereby leading to a poorer clinical outcome. Metabolic adaptations enable cancer cells to survive in suboptimal growth conditions, such as the limited supply of nutrient and oxygen often found in the tumor microenvironment. Metabolic changes, including activation of glycolysis and inhibition of mitochondrial ATP production, are induced under hypoxia to promote survival in low oxygen. FOXO3a, a transcription factor that is inhibited by the phosphatidylinositol 3-kinase/Akt pathway and is upregulated in hypoxia, has emerged as an important negative regulator of MYC function. Recent studies have revealed that FOXO3a acts as a negative regulator of mitochondrial function through inhibition of MYC. Ablation of FOXO3a prevents the inhibition of mitochondrial function induced by hypoxia and results in enhanced oxidative stress. This review will focus on the antagonism between FOXO3a and MYC and discuss their role in cellular bioenergetics, reactive oxygen metabolism, and adaptation to hypoxia, raising questions about the role of FOXO proteins in cancer.

Antagonism between FOXO and MYC Regulates Cellular Powerhouse.

An increased rate of lipid synthesis in cancerous tissues has long been recognised as an important aspect of the rewired metabolism of transformed cells. However, the contribution of lipids to cellular transformation, tumour development and tumour progression, as well as their potential role in facilitating the spread of cancerous cells to secondary sites, are not yet fully understood. In this article, we review the recent findings that support the importance of lipid synthesis and metabolism in tumorigenesis. Specifically, we explore the role of aberrant lipid biosynthesis in cancer cell migration and invasion, and in the induction of tumour angiogenesis. These processes are crucial for the dissemination of tumour cells and formation of metastases, which constitute the main cause of cancer mortality.

Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development.

Regulation of lipid metabolism via activation of sterol regulatory element binding proteins (SREBPs) has emerged as an important function of the Akt/mTORC1 signaling axis. Although the contribution of dysregulated Akt/mTORC1 signaling to cancer has been investigated extensively and altered lipid metabolism is observed in many tumors, the exact role of SREBPs in the control of biosynthetic processes required for Akt-dependent cell growth and their contribution to tumorigenesis remains unclear.

Sterol regulatory element binding protein-dependent regulation of lipid synthesis supports cell survival and tumor growth.

Tumors display distinct metabolic programs, and altered lipid metabolism is emerging as an important process in cancer. In this issue, Yue et al. (2014) report that aberrant cholesteryl ester accumulation is found in advanced prostate cancers with PTEN loss and PI3K/AKT activation. Importantly, inhibition of cholesterol esterification impaired cancer aggressiveness.

Cholesteryl esters: fueling the fury of prostate cancer.

An in vivo model of antiangiogenic therapy allowed us to identify genes upregulated by bevacizumab treatment, including Fatty Acid Binding Protein 3 (FABP3) and FABP7, both of which are involved in fatty acid uptake. In vitro, both were induced by hypoxia in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. There was a significant lipid droplet (LD) accumulation in hypoxia that was time and O2 concentration dependent. Knockdown of endogenous expression of FABP3, FABP7, or Adipophilin (an essential LD structural component) significantly impaired LD formation under hypoxia. We showed that LD accumulation is due to FABP3/7-dependent fatty acid uptake while de novo fatty acid synthesis is repressed in hypoxia. We also showed that ATP production occurs via β-oxidation or glycogen degradation in a cell-type-dependent manner in hypoxia-reoxygenation. Finally, inhibition of lipid storage reduced protection against reactive oxygen species toxicity, decreased the survival of cells subjected to hypoxia-reoxygenation in vitro, and strongly impaired tumorigenesis in vivo.

Fatty acid uptake and lipid storage induced by HIF-1α contribute to cell growth and survival after hypoxia-reoxygenation.

A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment.

Acetyl-CoA synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress.

Metabolic reprogramming is a central feature of transformed cells. Cancer metabolism is now fully back in the focus of cancer research, as the interactions between oncogenic signalling and cellular metabolic processes are uncovered. One aspect of metabolic reprogramming in cancer is alterations in lipid metabolism. In contrast to most untransformed tissues, which satisfy their demand from dietary lipids, cancer cells frequently re-activate de novo lipogenesis. However, compounds targeting fatty acid synthase (FASN), a multiprotein complex integral to lipogenesis, have so far shown limited efficacy in pre-clinical cancer models and to date only one FASN inhibitor has entered clinical trials. Recently, a number of studies have suggested that enhanced production of fatty acids in cancer cells could also increases their dependence on the activity of desaturases, a class of enzymes that insert double bonds into acyl-CoA chains. Targeting desaturase activity could provide a window of opportunity to selectively interfere with the metabolic activity of cancer cells. This review will summarise some key findings that implicate altered lipid metabolism in cancer and investigate the molecular interactions between lipid desaturation and cancer cell survival.

Lipid desaturation - the next step in targeting lipogenesis in cancer?

Enhanced macromolecule biosynthesis is integral to growth and proliferation of cancer cells. Lipid biosynthesis has been predicted to be an essential process in cancer cells. However, it is unclear which enzymes within this pathway offer the best selectivity for cancer cells and could be suitable therapeutic targets.

Inhibition of fatty acid desaturation is detrimental to cancer cell survival in metabolically compromised environments.

The initiation and progression of breast cancer from the transformation of the normal epithelium to ductal carcinoma in situ (DCIS) and invasive disease is a complex process involving the acquisition of genetic alterations and changes in gene expression, alongside microenvironmental and recognized histological alterations. Here, we sought to comprehensively characterise the genomic and transcriptomic features of the MCF10 isogenic model of breast cancer progression, and to functionally validate potential driver alterations in three-dimensional (3D) spheroids that may provide insights into breast cancer progression, and identify targetable alterations in conditions more similar to those encountered in vivo. We performed whole genome, exome and RNA sequencing of the MCF10 progression series to catalogue the copy number and mutational and transcriptomic landscapes associated with progression. We identified a number of predicted driver mutations (including PIK3CA and TP53) that were acquired during transformation of non-malignant MCF10A cells to their malignant counterparts that are also present in analysed primary breast cancers from The Cancer Genome Atlas (TCGA). Acquisition of genomic alterations identified MYC amplification and previously undescribed RAB3GAP1-HRAS and UBA2-PDCD2L expressed in-frame fusion genes in malignant cells. Comparison of pathway aberrations associated with progression showed that, when cells are grown as 3D spheroids, they show perturbations of cancer-relevant pathways. Functional interrogation of the dependency on predicted driver events identified alterations in HRAS, PIK3CA and TP53 that selectively decreased cell growth and were associated with progression from preinvasive to invasive disease only when cells were grown as spheroids. Our results have identified changes in the genomic repertoire in cell lines representative of the stages of breast cancer progression, and demonstrate that genetic dependencies can be uncovered when cells are grown in conditions more like those in vivo. The MCF10 progression series therefore represents a good model with which to dissect potential biomarkers and to evaluate therapeutic targets involved in the progression of breast cancer. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Article	Year
Gefitinib (Iressa) represses FOXM1 expression via FOXO3a in breast cancer. Molecular cancer therapeutics\| PubMed ID: 19276163	2009
FoxM1 is a downstream target and marker of HER2 overexpression in breast cancer. International journal of oncology\| PubMed ID: 19513552	2009
FOXM1 confers acquired cisplatin resistance in breast cancer cells. Molecular cancer research : MCR\| PubMed ID: 20068070	2010
SIRT inhibitors induce cell death and p53 acetylation through targeting both SIRT1 and SIRT2. Molecular cancer therapeutics\| PubMed ID: 20371709	2010
A role for the cancer-associated miR-106b~25 cluster in neuronal stem cells. Aging\| PubMed ID: 21483041	2011
Antagonism between FOXO and MYC Regulates Cellular Powerhouse. Frontiers in oncology\| PubMed ID: 23630664	2013
Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development. Disease models & mechanisms\| PubMed ID: 24203995	2013
Sterol regulatory element binding protein-dependent regulation of lipid synthesis supports cell survival and tumor growth. Cancer & metabolism\| PubMed ID: 24280005	2013
Cholesteryl esters: fueling the fury of prostate cancer. Cell metabolism\| PubMed ID: 24606894	2014
Fatty acid uptake and lipid storage induced by HIF-1α contribute to cell growth and survival after hypoxia-reoxygenation. Cell reports\| PubMed ID: 25263561	2014
Acetyl-CoA synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress. Cancer cell\| PubMed ID: 25584894	2015
Lipid desaturation - the next step in targeting lipogenesis in cancer? The FEBS journal\| PubMed ID: 26881388	2016
Inhibition of fatty acid desaturation is detrimental to cancer cell survival in metabolically compromised environments. Cancer & metabolism\| PubMed ID: 27042297	2016
Three-dimensional modelling identifies novel genetic dependencies associated with breast cancer progression in the isogenic MCF10 model. The Journal of pathology\| PubMed ID: 27512948	2016

Barrie Peck

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