Short-chain fatty acids, generated in colon by bacterial fermentation of dietary fiber, protect against colorectal cancer and inflammatory bowel disease. Among these bacterial metabolites, butyrate is biologically most relevant. GPR109A is a G-protein-coupled receptor for nicotinate, but recognizes butyrate with low affinity. Millimolar concentrations of butyrate are needed to activate the receptor. Although concentrations of butyrate in colonic lumen are sufficient to activate the receptor maximally, there have been no reports on the expression/function of GPR109A in this tissue. Here we show that GPR109A is expressed in the lumen-facing apical membrane of colonic and intestinal epithelial cells and that the receptor recognizes butyrate as a ligand. The expression of GPR109A is silenced in colon cancer in humans, in a mouse model of intestinal/colon cancer, and in colon cancer cell lines. The tumor-associated silencing of GPR109A involves DNA methylation directly or indirectly. Re-expression of GPR109A in colon cancer cells induces apoptosis, but only in the presence of its ligands butyrate and nicotinate. Butyrate is an inhibitor of histone deacetylases, but apoptosis induced by activation of GPR109A with its ligands in colon cancer cells does not involve inhibition of histone deacetylation. The primary changes in this apoptotic process include downregulation of Bcl-2, Bcl-xL, and cyclin D1, and upregulation of death receptor pathway. In addition, GPR109A/butyrate suppresses NF-κB activation in normal and cancer colon cell lines as well as in normal mouse colon. These studies show that GPR109A mediates the tumor-suppressive effects of the bacterial fermentation product butyrate in colon.

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.

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.

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.

SummaryHepcidin is a hormone central to the regulation of iron homeostasis in the body. It is believed to be produced exclusively by the liver. Ferroportin, an iron exporter, is the receptor for hepcidin. This transporter/receptor is expressed in Müller cells, photoreceptor cells, and retinal pigment epithelium (RPE) within the retina. Since the retina is protected by the retinal-blood barriers, we asked whether ferroportin in the retina is regulated by hepcidin in the circulation or whether the retina produces hepcidin for regulation of its own iron homeostasis. Here we show that hepcidin is expressed robustly in Müller cells, photoreceptor cells, and RPE, closely resembling the expression pattern of ferroportin. We also show that bacterial lipopolysaccharide (LPS) is a regulator of hepcidin expression in Müller cells and RPE, both in vitro and in vivo, and that the regulation occurs at the transcriptional level. The action of LPS on hepcidin expression is mediated by the Toll-like receptor-4. The upregulation of hepcidin by LPS occurs independent of Hfe (Human leukocyte antigen-like protein involved in Fe homeostasis). The increase in hepcidin levels in retinal cells in response to LPS treatment is associated with a decrease in ferroportin levels. The LPS-induced upregulation of hepcidin and consequent downregulation of ferroportin is associated with increased oxidative stress and apoptosis within the retina in vivo. We conclude that retinal iron homeostasis may be regulated in an autonomous manner by hepcidin generated within the retina and that chronic bacterial infection/inflammation of the retina may disrupt iron homeostasis and retinal function.

Summary Hemochromatosis is an iron overload disorder with age-dependent oxidative stress and dysfunction in a variety of tissues. Mutations in HFE are responsible for most cases with hemochromatosis. We recently demonstrated that Hfe is expressed exclusively in the basal membrane of retinal pigment epithelium (RPE). Here we used Hfe−/− mice to examine ferritin levels (an indirect readout for iron levels) and morphological changes in retina. We found increased ferritin accumulation in retina in 18-month-old but not in 2-month-old mice with considerable morphological damage compared to age-matched controls. The retinal phenotype included hypertrophy and hyperplasia of RPE. RPE cells isolated from Hfe−/− mice exhibited a hyperproliferative phenotype. We also compared the gene expression profile between wild type and Hfe−/− RPE cells by microarray analysis. These studies showed that many cell cycle-related genes were differentially regulated in Hfe−/− RPE cells. One of the genes upregulated in Hfe−/− RPE cells was Slc7a11 that codes for the ‘transporter proper’ xCT in the heterodimeric cystine/glutamate exchanger (xCT/4F2hc). This transporter plays a critical role in cellular glutathione status and cell cycle progression. We confirmed the microarrray data by monitoring xCT mRNA levels by RT-PCR and also by measuring transport function. We also found increased levels of glutathione and the transcription factor/cell cycle promoter AP1 in Hfe−/− RPE cells. Wild type mouse RPE cells and human RPE cell lines, when loaded with iron by exposure to ferric ammonium citrate, showed increased expression and activity of xCT, reproducing the biochemical phenotype observed with Hfe−/− RPE cells.

MCs express sigmaR1 and demonstrate robust sigmaR1 binding activity, which is inhibited by sigmaR1 ligands and is stimulated during oxidative stress. The potential of Müller cells to bind sigmaR1 ligands may prove beneficial in retinal degenerative diseases such as diabetic retinopathy.