Tumor cells have an increased demand for amino acids because of their rapid proliferation rate. In addition to their need in protein synthesis, several amino acids have other roles in supporting cancer growth. There are approximately two-dozen amino acid transporters in humans, and tumor cells must upregulate one or more of these transporters to satisfy their demand for amino acids. If the transporters that specifically serve this purpose in tumor cells are identified, they can be targeted for the development of a brand new class of anticancer drugs; the logical basis of such a strategy would be to starve the tumor cells of an important class of nutrients. To date, four amino acid transporters have been found to be expressed at high levels in cancer: SLC1A5, SLC7A5, SLC7A11, and SLC6A14. Their induction occurs in a cancer typespecific manner with a direct or indirect involvement of the oncogene c-Myc. Further, these transporters are functionally coupled, thus maximizing their ability to promote cancer growth and chemoresistance. Progress has been made in preclinical studies, exploiting these transporters as drug targets in cancer therapy. These transporters also show promise in development of new tumor-imaging probes and in tumor-specific delivery of appropriately designed chemotherapeutic agents. Cancer Res; 75(9);

ATB 0,ϩ (amino acid transporter responsible for the activity of system B 0,ϩ ) was named "B 0,ϩ " to indicate its broad substrate selectivity (denoted by "B"), accepting neutral (denoted by "0") and cationic (denoted by "ϩ") amino acids as substrates (1, 2). Its transport function is coupled to a Na ϩ gradient, a Cl Ϫ gradient, and membrane potential. ATB 0,ϩ is identified as SLC6A14 according to the Human Genome Organization nomenclature. This transporter has potential for delivery of a wide variety of drugs and prodrugs into cells (3-7). The substrate selectivity of SLC6A14 is interesting (1, 2). It transports all essential amino acids. The only excluded amino acids are glutamate and aspartate, which are nonessential. It also transports glutamine (an important precursor for nucleotide synthesis) and arginine (an amino acid essential for tumor growth). However, the transporter is expressed only at low levels in normal tissues. Tumor cells have an increased requirement for essential amino acids as well as glutamine and arginine to support their rapid growth. Essential amino acids are obligatory for protein synthesis. Leucine, an essential amino acid, is also a potent activator of mTOR (mammalian target of rapamycin) (8). Certain tumor cells metabolize glutamine at a rate far exceeding the requirement for protein and nucleotide synthesis, a phenomenon known as "glutamine addiction" (9 -11). Glutamine metabolism through a set of biochemical reactions called "glutaminolysis" provides a carbon source for tumor cells, thereby sparing glucose-derived carbon for the synthesis of lipids and other essential biomolecules. Arginine is essential for several types of cancer due to lack of the arginine-synthesizing enzyme argininosuccinate synthetase (12, 13). On the basis of these findings, we hypothesized that the expression of SLC6A14 may be up-regulated in cancer to meet the increasing demand for all essential amino acids as well as glutamine and arginine. In support of this hypothesis, past studies from our laboratory have shown that SLC6A14 is up-regulated in colon cancer (14) and cervical cancer (15). The transporter is also up-regulated in breast cancer cell lines, but interestingly only in estrogen receptor (ER) 2 -positive cell lines (16). Furthermore, we showed that blockade of SLC6A14 in ER-positive breast cancer cells by treatment with a selective blocker (␣-methyl-DLtryptophan (␣-MT)) starved the cells of glutamine, arginine, and essential amino acids, decreased cell proliferation, and caused apoptotic cell death (16). In the present study, we investigated the expression of SLC6A14 in primary breast cancer * This work was supported, in whole or in part, by National Institutes of Health Grant CA152396. 1 To whom correspondence should be addressed. E-mail: vganapat@ georgiahealth.edu.2 The abbreviations used are: ER, estrogen receptor; ␣-MT, ␣-methyl-DL-tryptophan; CHOP, CCAAT/enhancer-binding protein homologous protein; 3-MA, 3-methyladenine.

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.

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The NAD-dependent histone deacetylase SIRT1 is overexpressed and catalytically activated in a number of human cancers, but recent studies argue 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 anti-oxidant and pro-survival 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 anti-estrogen compounds may offer more effective treatment strategies for breast cancer.

Recently, impressive technical advancements have been made in the isolation and validation of mammary stem cells and cancer stem cells (CSCs), but the signaling pathways that regulate stem cell self-renewal are largely unknown. Further, CSCs are believed to contribute to chemo- and radioresistance. In this study, we used the MMTV-Neu-Tg mouse mammary tumor model to identify potential new strategies for eliminating CSCs. We found that both luminal progenitor and basal stem cells are susceptible to genetic and epigenetic modifications, which facilitate oncogenic transformation and tumorigenic potential. A combination of the DNMT inhibitor 5-azacytidine and the HDAC inhibitor butyrate markedly reduced CSC abundance and increased the overall survival in this mouse model. RNA-seq analysis of CSCs treated with 5-azacytidine plus butyrate provided evidence that inhibition of chromatin modifiers blocks growth-promoting signaling molecules such as RAD51AP1 and SPC25, which play key roles in DNA damage repair and kinetochore assembly. Moreover, RAD51AP1 and SPC25 were significantly overexpressed in human breast tumor tissues and were associated with reduced overall patient survival. In conclusion, our studies suggest that breast CSCs are intrinsically sensitive to genetic and epigenetic modifications and can therefore be significantly affected by epigenetic-based therapies, warranting further investigation of combined DNMT and HDAC inhibition in refractory or drug-resistant breast cancer.

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 cAMP 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 anti-apoptotic 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 pharmacological induction of this gene in tumor tissues followed by its activation with agonists could be an effective therapeutic strategy to treat breast cancer.

SLC6A14 mediates Na(+)/Cl(-)-coupled concentrative uptake of a broad-spectrum of amino acids. It is expressed at low levels in many tissues but up-regulated in certain cancers. Pharmacological blockade of SLC6A14 causes amino acid starvation in estrogen receptor positive (ER+) breast cancer cells and suppresses their proliferation in vitro and in vivo. In the present study, we interrogated the role of this transporter in breast cancer by deleting Slc6a14 in mice and monitoring the consequences of this deletion in models of spontaneous breast cancer (Polyoma middle T oncogene-transgenic mouse and mouse mammary tumour virus promoter-Neu-transgenic mouse). Slc6a14-knockout mice are viable, fertile and phenotypically normal. The plasma amino acids were similar in wild-type and knockout mice and there were no major compensatory changes in the expression of other amino acid transporter mRNAs. There was also no change in mammary gland development in the knockout mouse. However, when crossed with PyMT-Tg mice or MMTV/Neu (mouse mammary tumour virus promoter-Neu)-Tg mice, the development and progression of breast cancer were markedly decreased on Slc6a14(-/-) background. Analysis of transcriptomes in tumour tissues from wild-type mice and Slc6a14-null mice indicated no compensatory changes in the expression of any other amino acid transporter mRNA. However, the tumours from the null mice showed evidence of amino acid starvation, decreased mTOR signalling and decreased cell proliferation. These studies demonstrate that SLC6A14 is critical for the maintenance of amino acid nutrition and optimal mammalian target of rapamycin (mTOR) signalling in ER+ breast cancer and that the transporter is a potential target for development of a novel class of anti-cancer drugs targeting amino acid nutrition in tumour cells.