Selvakumar Elangovan

SIRT1 is essential for oncogenic signaling by estrogen/estrogen receptor α in breast cancer

The NAD-dependent histone deacetylase silent information regulator 1 (SIRT1) is overexpressed and catalytically activated in a number of human cancers, but recent studies have actually suggested that it may function as a tumor suppressor and metastasis inhibitor in vivo. In breast cancer, SIRT1 stabilization has been suggested to contribute to the oncogenic potential of the estrogen receptor α (ERα), but SIRT1 activity has also been associated with ERα deacetylation and inactivation. In this study, we show that SIRT1 is critical for estrogen to promote breast cancer. ERα physically interacted and functionally cooperated with SIRT1 in breast cancer cells. ERα also bound to the promoter for SIRT1 and increased its transcription. SIRT1 expression induced by ERα was sufficient to activate antioxidant and prosurvival genes in breast cancer cells, such as catalase and glutathione peroxidase, and to inactivate tumor suppressor genes such as cyclin G2 (CCNG2) and p53. Moreover, SIRT1 inactivation eliminated estrogen/ERα-induced cell growth and tumor development, triggering apoptosis. Taken together, these results indicated that SIRT1 is required for estrogen-induced breast cancer growth. Our findings imply that the combination of SIRT1 inhibitors and antiestrogen compounds may offer more effective treatment strategies for breast cancer.

DNMT1 is essential for mammary and cancer stem cell maintenance and tumorigenesis

Mammary stem/progenitor cells (MaSCs) maintain self-renewal of the mammary epithelium during puberty and pregnancy. DNA methylation provides a potential epigenetic mechanism for maintaining cellular memory during self-renewal. Although DNA methyltransferases (DNMTs) are dispensable for embryonic stem cell maintenance, their role in maintaining MaSCs and cancer stem cells (CSCs) in constantly replenishing mammary epithelium is unclear. Here we show that DNMT1 is indispensable for MaSC maintenance. Furthermore, we find that DNMT1 expression is elevated in mammary tumors, and mammary gland-specific DNMT1 deletion protects mice from mammary tumorigenesis by limiting the CSC pool. Through genome-scale methylation studies, we identify ISL1 as a direct DNMT1 target, hypermethylated and downregulated in mammary tumors and CSCs. DNMT inhibition or ISL1 expression in breast cancer cells limits CSC population. Altogether, our studies uncover an essential role for DNMT1 in MaSC and CSC maintenance and identify DNMT1-ISL1 axis as a potential therapeutic target for breast cancer treatment.

SLC6A14 (ATB0,+) Protein, a Highly Concentrative and Broad Specific Amino Acid Transporter, Is a Novel and Effective Drug Target for Treatment of Estrogen Receptor-positive Breast Cancer

SLC6A14, also known as ATB0,+, is an amino acid transporter with unique characteristics. It transports 18 of the 20 proteinogenic amino acids. However, this transporter is expressed only at low levels in normal tissues. Here, we show that the transporter is up-regulated specifically in estrogen receptor (ER)-positive breast cancer, demonstrable with primary human breast cancer tissues and human breast cancer cell lines. SLC6A14 is an estrogen/ER target. The transport features of SLC6A14 include concentrative transport of leucine (an activator of mTOR), glutamine (an essential amino acid for nucleotide biosynthesis and substrate for glutaminolysis), and arginine (an essential amino acid for tumor cells), suggesting that ER-positive breast cancer cells up-regulate SLC6A14 to meet their increased demand for these amino acids. Consequently, treatment of ER-positive breast cancer cells in vitro with α-methyl-dl-tryptophan (α-MT), a selective blocker of SLC6A14, induces amino acid deprivation, inhibits mTOR, and activates autophagy. Prolongation of the treatment with α-MT causes apoptosis. Addition of an autophagy inhibitor (3-methyladenine) during α-MT treatment also induces apoptosis. These effects of α-MT are specific to ER-positive breast cancer cells, which express the transporter. The ability of α-MT to cause amino acid deprivation is significantly attenuated in MCF-7 cells, an ER-positive breast cancer cell line, when SLC6A14 is silenced with shRNA. In mouse xenograft studies, α-MT by itself is able to reduce the growth of the ER-positive ZR-75-1 breast cancer cells. These studies identify SLC6A14 as a novel and effective drug target for the treatment of ER-positive breast cancer.

The Niacin/Butyrate Receptor GPR109A Suppresses Mammary Tumorigenesis by Inhibiting Cell Survival

GPR109A, a G-protein-coupled receptor, is activated by niacin and butyrate. Upon activation in colonocytes, GPR109A potentiates anti-inflammatory pathways, induces apoptosis, and protects against inflammation-induced colon cancer. In contrast, GPR109A activation in keratinocytes induces flushing by activation of Cox-2-dependent inflammatory signaling, and the receptor expression is upregulated in human epidermoid carcinoma. Thus, depending on the cellular context and tissue, GPR109A functions either as a tumor suppressor or a tumor promoter. However, the expression status and the functional implications of this receptor in the mammary epithelium are not known. Here, we show that GPR109A is expressed in normal mammary tissue and, irrespective of the hormone receptor status, its expression is silenced in human primary breast tumor tissues, breast cancer cell lines, and in tumor tissues of three different murine mammary tumor models. Functional expression of this receptor in human breast cancer cell lines decreases cyclic AMP production, induces apoptosis, and blocks colony formation and mammary tumor growth. Transcriptome analysis revealed that GPR109A activation inhibits genes, which are involved in cell survival and antiapoptotic signaling, in human breast cancer cells. In addition, deletion of Gpr109a in mice increased tumor incidence and triggered early onset of mammary tumorigenesis with increased lung metastasis in MMTV-Neu mouse model of spontaneous breast cancer. These findings suggest that GPR109A is a tumor suppressor in mammary gland and that pharmacologic induction of this gene in tumor tissues followed by its activation with agonists could be an effective therapeutic strategy to treat breast cancer.

Role of SLC5A8, a plasma membrane transporter and a tumor suppressor, in the antitumor activity of dichloroacetate

There has been growing interest among the public and scientists in dichloroacetate (DCA) as a potential anticancer drug. Credible evidence exists for the antitumor activity of this compound, but high concentrations are needed for significant therapeutic effect. Unfortunately, these high concentrations produce detrimental side effects involving the nervous system, thereby precluding its use for cancer treatment. The mechanistic basis of the compounds antitumor activity is its ability to activate the pyruvate dehydrogenase complex through inhibition of pyruvate dehydrogenase kinase. As the compound inhibits the kinase at micromolar concentrations, it is not known why therapeutically prohibitive high doses are needed for suppression of tumor growth. We hypothesized that lack of effective mechanisms for the entry of DCA into tumor cells may underlie this phenomenon. Here we show that SLC5A8 transports DCA very effectively with high affinity. This transporter is expressed in normal cells, but expression is silenced in tumor cells by epigenetic mechanisms. The lack of the transporter makes tumor cells resistant to the antitumor activity of DCA. However, if the transporter is expressed in tumor cells ectopically, the cells become sensitive to the drug at low concentrations. This is evident in breast cancer cells, colon cancer cells and prostate cancer cells. Normal cells, which constitutively express the transporter, are however not affected by the compound, indicating tumor cell-selective therapeutic activity. The mechanism of the compounds antitumor activity still remains its ability to inhibit pyruvate dehydrogenase kinase and force mitochondrial oxidation of pyruvate. As silencing of SLC5A8 in tumors involves DNA methylation and its expression can be induced by treatment with DNA methylation inhibitors, our findings suggest that combining DCA with a DNA methylation inhibitor would offer a means to reduce the doses of DCA to avoid detrimental effects associated with high doses but without compromising antitumor activity.

Transport by SLC5A8 with subsequent inhibition of histone deacetylase 1 (HDAC1) and HDAC3 underlies the antitumor activity of 3-bromopyruvate

3‐Bromopyruvate is an alkylating agent with antitumor activity. It is currently believed that blockade of adenosine triphosphate production from glycolysis and mitochondria is the primary mechanism responsible for this antitumor effect. The current studies uncovered a new and novel mechanism for the antitumor activity of 3‐bromopyruvate.

The plasma membrane transporter SLC5A8 suppresses tumour progression through depletion of survivin without involving its transport function

SLC5A8 (solute carrier gene family 5A, member 8) is a sodium-coupled transporter for monocarboxylates. Among its substrates are the HDAC (histone deacetylase) inhibitors butyrate, propionate and pyruvate. Expression of SLC5A8 is silenced in cancers via DNA methylation, and ectopic expression of SLC5A8 in cancer cells induces apoptosis in the presence of its substrates that are HDAC inhibitors. In the present study we show that ectopic expression of SLC5A8 in cancer cells translocates the anti-apoptotic protein survivin to the plasma membrane through protein-protein interaction resulting in depletion of nuclear survivin and also decreases cellular levels of survivin through inhibition of transcription. These SLC5A8-induced changes in the location and levels of survivin result in cell-cycle arrest, disruption of the chromosome passenger complex involved in mitosis, induction of apoptosis and enhancement in chemosensitivity. These effects are seen independently of the transport function of SLC5A8 and histone acetylation status of the cell; in the presence of pyruvate, a SLC5A8 substrate and also an HDAC inhibitor, these effects are amplified. Ectopic expression of SLC5A8 in the breast cancer cell line MB231 inhibits the ability of cells to form colonies in vitro and to form tumours in mouse xenografts in vivo. The suppression of survivin transcription occurs independently of HDAC inhibition, and the underlying mechanism is associated with decreased phosphorylation of STAT3 (signal transducer and activator of transcription 3). The observed effects are specific for survivin with no apparent changes in expression of other inhibitor-of-apoptosis proteins. The present study unravels a novel, hitherto unrecognized, mechanism for the tumour-suppressive role of a plasma membrane transporter independent of its transport function.

Molecular mechanism of SLC5A8 inactivation in breast cancer.

ABSTRACT SLC5A8 is a putative tumor suppressor that is inactivated in more than 10 different types of cancer, but neither the oncogenic signaling responsible for SLC5A8 inactivation nor the functional relevance of SLC5A8 loss to tumor growth has been elucidated. Here, we identify oncogenic HRAS (HRASG12V) as a potent mediator of SLC5A8 silencing in human nontransformed normal mammary epithelial cell lines and in mouse mammary tumors through DNMT1. Further, we demonstrate that loss of Slc5a8 increases cancer-initiating stem cell formation and promotes mammary tumorigenesis and lung metastasis in an HRAS-driven murine model of mammary tumors. Mammary-gland-specific overexpression of Slc5a8 (mouse mammary tumor virus-Slc5a8 transgenic mice), as well as induction of endogenous Slc5a8 in mice with inhibitors of DNA methylation, protects against HRAS-driven mammary tumors. Collectively, our results provide the tumor-suppressive role of SLC5A8 and identify the oncogenic HRAS as a mediator of tumor-associated silencing of this tumor suppressor in mammary glands. These findings suggest that pharmacological approaches to reactivate SLC5A8 expression in tumor cells have potential as a novel therapeutic strategy for breast cancer treatment.

Abstract 33: Mammary gland-specific deletion of Sirt1 delays mammary tumor growth and progression

SIRT1, a type III HDAC, is involved in regulation of several cellular processes including longevity, inflammatory response, energy metabolism and stem cell maintenance. SIRT1 first emerged as a potential anti-aging factor but now it is implicated in a number of age-related diseases including carcinogenesis and also in cancer stem cell maintenance. SIRT1 is over-expressed and/or catalytically activated in a variety of human cancers, including breast cancer. Several lines of evidence suggest that SIRT1 plays an important role in the activation of oncogenic signalling in mammary epithelial cells and that inhibition of SIRT1 has a direct effect on tumor cell growth and apoptosis. Despite the fact that SIRT1 is mainly involved in the promotion of oncogenic signaling, it can also function as a tumor suppressor via deacetylation and subsequent destabilization of various oncoproteins such as c-Myc, β-catenin, NF-κB, and HIF1α. Further, SIRT1 activation by resveratrol protects BRCA1 mutation-associated breast cancer. Thus, the precise role of SIRT1 in cancer is still unclear and remains debatable; it is likely that SIRT1 function in cancer is tissue/context-dependent. Therefore, understanding the precise role of SIRT1 in mammary tumorigenesis is essential for the rational design of SIRT1-based novel therapeutic drugs to combat breast cancer. To understand the functional implications of SIRT1 in breast cancer, we generated mammary gland-specific Sirt1-conditional knockout mice. Sirt1 deletion is associated with reduced mammary gland outgrowth and terminal end bud development, leading to significant reduction in mammary stem and progenitor cell formation. This perturbed mammary gland outgrowth and reduced mammary stem and progenitor cell formation recovers either by puberty or by exogenous administration of estrogen, suggesting that Sirt1 plays a key role in mediating estrogen signalling in mammary gland. To assess the functional role of SIRT1 in mammary tumor growth and progression, we crossed Sirt1-knockout mice with MMTV-PyMT-Tg mice and C3(1)-SV40-Tg mice, two spontaneous murine mammary tumor models. Sirt1 deletion significantly reduces the development and growth of spontaneous mammary tumors in both mouse models. This phenomenon is associated with significant reduction in cancer stem cell (CSC) formation with dramatic reduction in CSC markers Slug and Sox2 expression. We also found that Sirt1 deletion reactivates tumor suppressor proteins p53 and Foxo3a. In conclusion, our study provides direct evidence that SIRT1 plays a key role in breast cancer growth and progression by maintaining cancer stem cells via stabilization of CSC drivers and inactivation of tumor suppressors in mammary gland. Thus, pharmacological agents targeted to disrupt SIRT1 function and signalling might have potential in breast cancer treatment. Citation Format: Sabarish Ramachandran, Rajneesh Pathania, Selvakumar Elangovan, Ganapathy Vadivel, Muthusamy Thangaraju. Mammary gland-specific deletion of Sirt1 delays mammary tumor growth and progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 33. doi:10.1158/1538-7445.AM2015-33

Abstract 2461: SLC5A8: A strategic target for advanced metastatic breast cancer

Despite intense efforts and great advances in cancer research, breast cancer remains the leading cause of death among women worldwide. Most breast cancer-related deaths are not due to cancer at the primary site, but rather due to metastasis, a process in which cancer cells spread from the primary site to distant secondary sites like lung, bones and brain. However, the molecular mechanism by which tumor cells invade from primary tumor site to distant metastasis has not been identified. Recently, we identified a tumor suppressor SLC5A8, which is not only prevent the mammary tumor incidence but also blocks tumor-metastasis by inactivating several metastasis-deriving molecules. SLC5A8, a transporter for small-chain fatty acids (SCFA) and monocarboxylates, is silenced in more than 10 different types of cancers including breast cancer. In breast cancer, irrespective of estrogen-receptor status SLC5A8 is inactivated in more than 90% of breast tumor tissues and in breast cancer cell lines. Ectopic expression of SLC5A8 in human breast cancer cells leads to translocation of the anti-apoptotic protein survivin to the plasma membrane through protein-protein interaction, thereby depleting nuclear survivin level. Further, tetracycline-inducible SLC5A8 expression in human breast cancer cells significantly reduced mammary tumor growth. In addition, functional inactivation of SLC5A8 in human immortalized normal mammary epithelial cells by lentivirus expressing shRNA showed differential regulation of genes that are involved in cellular transformation, oncogenesis, epithelial-mesenchymal-transition (EMT) and tumor metastasis. This is a totally unexpected finding and represents first of its kind for a plasma membrane transporter where mere expression itself, independent of its substrates, leads to tumor suppression. Reinforcing our findings further, deletion of Slc5a8 in mice is associated with increased stem/progenitor cells and mammary gland hyperplasia. By crossing the Slc5a8-null mice with spontaneous mouse mammary tumor mice, we observed increased cancer-initiating stem cells, early onset of mammary tumor formation and increased incidence of lung metastasis. More fascinatingly, mammary gland-specific overexpression of Slc5a8 or induction of endogenous Slc5a8 expression efficiently protects mice from breast cancer and associated lung metastasis resulting in extended life-span. Overall, our study provide a strong mechanism based evidence that SLC5A8 is a novel tumor suppressor in the mammary epithelium and it could be used as a potential new therapeutic target for treatment of breast cancer. Citation Format: Sabarish Ramachandran, Rajneesh Pathania, Ravi N. Padia, Selvakumar Elangovan, Veena Coothankandaswamy, Puttur D. Prasad, Vadivel Ganapathy, Muthusamy Thangaraju. SLC5A8: A strategic target for advanced metastatic breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2461. doi:10.1158/1538-7445.AM2014-2461