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Background/Hypothesis: The amino acid transporter SLC6A14 is upregulated in estrogen receptor (ER)-positive breast cancer, a subtype that represents 75-80% of all breast cancers. Obesity/overweight increases the risk of breast cancer due to estrogen generated from fat cells that fuel the growth of the ER-positive breast cancer. Previous studies from our lab have demonstrated that blockade of the amino acid transporter SLC6A14 by the small molecule α-methyltryptophan reduces the growth of ER-positive breast cancer in mouse xenografts of human breast cancer cells and in a spontaneous mouse model of ER-positive breast cancer. We have also discovered that α-methyltryptophan functions as a potent weight-loss agent. We believe that this compound would be effective for the treatment of ER-positive breast cancer, especially in obese/overweight women. α-methyltryptophan is metabolized by aromatic amino acid decarboxylase. Therefore, combination of this compound with carbidopa, a well-known inhibitor of this enzyme, might potentiate the anticancer efficacy of α-methyltryptophan. We tested this idea in the present study using a syngeneic mouse model of ER-positive breast cancer using the mouse breast cancer cell line AT3. Results: A syngeneic tumor-cell transplant mouse model was used for ER-positive breast cancer. AT3 cells were injected into the mammary fat pad of C57BL/6 mice. This allows to monitor the growth of AT3 tumor growth in an immunologically competent mammary tissue environment. Drug treatment in drinking water was started after tumor growth was noticeable (100 mm3). There were four groups: (i) control; (ii) α-methyltryptophan at 0.5 mg/ml; (iii) carbidopa at 0.25 mg/ml; (iv) α-methyltryptophan at 0.5 mg/ml + carbidopa at 0.25 mg/ml. Tumor growth was monitored every three days. The mice were killed after 4 weeks of treatment. Tumors were excised and weighed. These studies showed that α-methyltryptophan and carbidopa were independently effective in reducing the tumor (48% reduction, p Conclusion: The combination treatment significantly decreased the tumor size and volume compared to carbidopa and α-methyltryptophan alone. We do not know yet if the improved efficacy of the combination of the two drugs is due to a simple additive effect or due to a synergistic effect. Studies have shown that carbidopa promotes degradation of ER, indicating that carbidopa has its own independent anticancer effect that is relevant to ER-positive breast cancer. Further studies are needed to differentiate between the effect of carbidopa as an independent anticancer agent and its ability to potentiate the anticancer efficacy of α-methyltryptophan by preventing its metabolic degradation. Citation Format: Sirin Falconi, Sabarish Ramachandran, Vadivel Ganapathy. Carbidopa/α-methyltryptophan as an ideal combination therapy for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2785.

Cancer-associated upregulation has been demonstrated for selective glucose transporters and amino acid transporters. The amino acid transporter SN2 (SLC38A5) represents the latest addition to this list. Transcriptomic analysis of tumor tissues has shown SLC38A5 is upregulated in breast and colon cancer. SLC38A5 is an amino acid-dependent Na+/H+ exchanger, induced in cancer, which not only supplies amino acids to cancer cells but also maintains an alkaline intracellular pH. The substrates for SLC38A5 include glutamine, asparagine, histidine, methionine, glycine, and serine, highlighting the role of SLC38A5 in glutamine addiction and one-carbon metabolism, both pathways being essential for cancer cells. In addition, SLC38A5 activates macropinocytosis, a process involved in cellular uptake of proteins in the extracellular fluid to meet amino acid demands in cancer cells. Since the transporter is upregulated in colon cancer, we hypothesized that the multiple functions of SLC38A5 fuel the growth, proliferation, and survival in colon cancer cells and that its induction involves oncogenic mutations in p53 and KRAS that are common mediators of carcinogenesis in colon. Firstly, we used a panel of colon cancer cells and analyzed SLC38A5 expression and its functional characteristics. Next, we compared SLC38A5 expression and function between two isogenic cell lines: SW48 with and without the oncogenic mutation (G12D) in KRAS and HCT116 with and without the tumor suppressor p53. Among the colon cancer cells examined, KM12L4 showed highest expression of SLC38A5; therefore, we characterized the function of the transporter in detail in this cell line. Our studies showed that KM12L4 cells were able to take up serine, one of the substrates for SLC38A5, in a Na+-dependent manner in the presence of 5 mM tryptophan, which is not a substrate for SLC38A5 but is known to block LAT1 transporter, which also transports serine. SLC38A5 exhibited an active serine-uptake when Na+ was replaced with Li+ at a higher pH. Li+ tolerance and a higher activity at an alkaline pH are two unique features of SLC38A5. The oncogenic mutation G12D in KRAS increased SLC38A5 activity in SW48 cells. In contrast, loss of p53 decreased SLC38A5 activity in HCT116 cells. These data demonstrate that SLC38A5 is induced in colon cancer cells and its activity is increased in the presence of oncogenic KRAS mutations and suppressed when p53 is inactivated, thus providing a valuable insight into the molecular mechanisms for the upregulation of the transporter in colon cancer. We conclude that SLC38A5, an amino acids transporter, is upregulated in colon cancer and its functions are uniquely suited to promote cell proliferation and tumor growth. We also conclude that KRAS and p53 functional status play a critical role in the increased SLC38A5 expression and activity in colon cancer. These data suggest that SLC38A5 could be exploited as a drug target for cancer therapy. Citation Format: Nhi Thi Nguyen, Sathish Sivaprakasam, Vadivel Ganapathy. Involvement of p53 and KRAS in the regulation of SLC38A5 in colon cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3699.

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 type–specific 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); 1782–8. ©2015 AACR.

SLC6A14 is a Na+/Cl− -dependent amino acid transporter capable of transporting 18 of the 20 proteinogenic amino acids, including arginine, leucine (mTOR activators) and glutamine (necessary for nucleotide biosynthesis). This transporter is expressed at basal levels in normal colon but significantly upregulated in colorectal cancer (CRC). However, the relevance of this upregulation to disease progression remains unknown. We postulated that deletion of SLC6A14 or pharmacological blockade of its function would suppress CRC by depleting amino acids and interfering with mTOR signaling selectively in tumor cells. To test this postulate, we first used Slc6a14-null mice. With two different models of spontaneous CRC (Apcmin/- and DSS/AOM), we found the tumor incidence and tumor growth were much lower in mice with Slc6a14-null background than in mice with Slc6a14. To evaluate the impact of pharmacologic blockade of the transporter on tumor growth we used a syngeneic tumor mouse model with MC-38 cells (a mouse CRC cell line); blockade of Slc6a14 with alpha-methyl tryptophan (α-MT) markedly reduced tumor growth. We then determined the transcriptome profiles of colonic epithelial cells and colonic non-epithelial cells from wild type mice and Slc6a14-null mice by RNAseq. There were hundreds of genes that were either upregulated or downregulated in the null mice. Ingenuity pathway analysis (IPA) of these data revealed predictive activation of canonical AMPK signaling pathway in colonic epithelial of Slc6a14-null mice. AMPK has anticancer activities by modulating multiple pathways including negatively regulating mTOR signaling. Moreover, upstream analysis showed predictive inactivation of two important tumor growth and survival signaling pathways ERK and EGF in the colon of the null mice. Likewise, significant upregulation of APC downregulated 1 (APCDD1) in epithelial cells in null mice implies predictive suppression of canonical Wnt signaling pathway. We also found marked differences in fecal microbiota as a result of Slc6a14 deletion. There was an increase in the relative abundance of beneficial microbiota including those capable of generating short chain fatty acids and lactic acid in null mice. We conclude that deletion of Slc6a14 or its pharmacological blockade protects against CRC by inducing amino acid starvation in tumor cells, thereby causing changes in multiple signaling pathways and in colonic microbiome. These studies identify SLC6A14 as a novel drug target for the treatment of colorectal cancer. Citation Format: Mohd Omar Faruk Sikder, Sathish Sivaprakasam, Vadivel Ganapathy. Amino acid transporter SLC6A14: A novel drug target for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3585.

Cancer cells display a unique phenomenon in which, even in the presence of oxygen, cells switch from oxidative phosphorylation to glycolysis as the primary source of ATP with consequent production of lactic acid. This phenomenon, called the Warburg Effect, is a hallmark of cancer. Lactic acid has long been considered as the necessary end product of this metabolic switch, where lactic acid is effluxed out of tumor cells to prevent intracellular acidification. Recent evidence however suggests that lactate and the excess protons in the tumor microenvironment play an active role in tumor growth. In particular, lactate has been shown to function as an agonist for GPR81, a G-protein-coupled receptor expressed on the surface of tumor cells. This autocrine signaling of lactate promotes tumor growth and metastasis, as well as angiogenesis and immune evasion. The present study assesses whether tumor cell-derived lactate has any paracrine role via its receptor in non-cancer cells present in the tumor microenvironment. We generated MMTV-PyMT-Tg mice, a spontaneous model for breast cancer, on Gpr81+/+ and Gpr81-/- backgrounds. The absence of Gpr81 reduced the mammary tumor incidence, delayed mammary tumor progression, and reduced lung metastasis. These data demonstrate the essential role of GPR81 in breast cancer growth and metastasis, but does not differentiate between Gpr81 in tumor cells versus Gpr81 in the tumor microenvironment. We then used the syngeneic transplant of the mouse mammary tumor cell line AT-3 into the mammary fat pads of wild type and Gpr81-/- mice to assess the involvement of Gpr81 in the microenvironment. AT-3 cells are negative for Gpr81, and therefore our model limits Gpr81 expression to non-tumor cells in the host mouse. We found the growth of transplanted tumor cells was significantly reduced in Gpr81-/- mice than in wild type mice. Preliminary RNA-sequencing transcriptome analysis of AT-3 tumors suggest an immunosuppressive function of Gpr81, where tumors grown in a Gpr81-/- background have much stronger gene expression profiles in T-cell signaling pathways. Specifically, AT-3 tumors grown in Gpr81-/- host express significantly higher levels of genes that are specific for T cells and antigen-presenting cells such as MHC-II complex molecules, T-cell co-stimulatory molecules, and transmembrane CD8. It is well established that tumor-cell derived lactate promotes tumor growth in an autocrine manner via Gpr81 expressed on tumor cells; our studies extend the tumor-promoting role of lactate beyond this autocrine function to include paracrine signaling via Gpr81 expressed on immune cells and possibly other cell types in the tumor microenvironment. Citation Format: Timothy Brown, Sabarish Ramachandran, Stefan Offermanns, Vadivel Ganapathy. The lactate receptor Gpr81 on non-cancer cells promotes an immunosuppressive phenotype in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4357.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the intestinal tract due to disruption of the symbiotic relationship between the host immune system and microbiota. Various factors alter the composition of the gut microbiota which lead to dysbiosis; in particular, diet and dietary fiber constitute important determinants. Dietary fiber protects against IBD; bacteria ferment these dietary fibers in colon and generate short chain fatty acids (SCFAs), which mediate the anti-inflammatory actions of dietary fiber. SLC5A8 is a sodium-coupled transporter in the apical membrane of colonic epithelium which mediates the entry of SCFAs from the lumen into cells. Due to the unique transport kinetics, the function of the transporter becomes important only under conditions of low dietary fiber intake. We have shown previously that deficiency of dietary fiber increased susceptibility to colitis and colon cancer in Slc5a8-null mice. This effect was primarily due to deficiency of regulatory T cells (Treg) and increased pro-inflammatory cytokines. Now we have examined the impact of dietary fiber deficiency on luminal microbial composition and transcriptomic profile in colonic epithelium in wild type (WT) and Slc5a8-null (KO) mice. We fed WT and KO mice with fiber-containing diet (FC) or fiber-free diet (FF) and analyzed the luminal bacterial composition by sequencing 16S rRNA gene in feces. Markedly altered luminal microbiota was observed when fiber was deficient in the diet and also when Slc5a8 was absent. Decreased diversity of microbiota was observed in WT mice fed FF diet; similarly, KO mice showed decreased microbial diversity compared to WT mice even when fed FC diet. Interestingly, the impact of dietary fiber on microbial composition was lost when Slc5a8 was not present. The Principal Coordinate Analysis (PCoA) showed samples were separated into four clusters based on the mouse genotype and dietary fiber condition. These results suggest that there are significant differences in the microbial community depending on dietary fiber content and on the presence or absence of the SCFA transporter Slc5a8. Decreased abundance of Bacteroidetes and Firmicutes was observed in WT mice fed FF-diet and in KO mice fed FC-diet compared to WT mice fed FC-diet. We observed increased abundance of well-known microbial biomarkers of active colitis and also the mucin-degrading Vercomicrobia and Akkermansia and the sulfate-reducing Desulfovibrio in KO mice fed FF-diet. Further, KO mice had decreased abundance of Oscilospira species, which is known to be preset at lower abundance in IBD patients. There was also marked differences in the transcriptomic profile of the colonic epithelium depending on the dietary fiber content and on the presence or absence of Slc5a8. We conclude that absence of fiber in diet in KO mice causes bacterial dysbiosis and promotes inflammation in colon. Citation Format: Sathish Sivaprakasam, Pramodh K. Ganapathy, Sabarish Ramachandran, Kameswara Rao Kottapalli, Ganapathy Vadivel. Deficiency of dietary fiber in Slc5a8-null mice promotes bacterial dysbiosis and inflammatory milieu in colon [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-062.

Hemochromatosis, an inherited iron-overload disease, causes tissue damage due to deposition of iron in toxic levels in most tissues including the liver, heart, kidney, pancreas and colon. Free iron, both in the inorganic form (i.e, non-transferrin-bound and non-ferritin-bound) and organic form (i.e. heme), facilitates Fenton reaction to generate hydroxyl radicals and to induce cellular oxidative damage that is observed in carcinogenesis. The primary site of iron deposition in hemochromatosis patients is liver, and therefore, liver cancer formation in these patients has been studied extensively. However, little is known about the iron-elicited colonic pathologies, mainly colonic inflammation and colon cancer. Here we postulate that iron overload as observed in hemochromatosis disrupts healthy colonic homeostasis and exacerbates the development of colon inflammation and colon cancer. To test our hypothesis, we examined the progression and severity of colitis and colon cancer in Hfe-/- mouse and the wild type control. Hfe-/- mouse is a model for classical hemochromatosis. Besides serum and liver, high concentrations of iron and heme deposits are found in the colon of Hfe-/- mouse. Experimental colitis was induced by administration of Dextran Sodium Sulfate (DSS) in drinking water. Colitis-associated colon cancer was initiated by the intraperitoneal injection of carcinogen, azoxymethane (AOM) and the carcinogenesis was driven by DSS administration. Colonic inflammation was more severe in the Hfe-/- mouse than in control. In addition, hemochromatosis mouse developed more and larger colonic polyps than the control group. We observed that this high susceptibility to colitis and colon cancer in Hfe-/- but not wild type mouse, lies in the differences in strains regarding the bacterial composition and colonic defense machinery. 16S ribosomal RNA sequencing of fecal bacteria revealed that the microbiota composition of Hfe-/- mouse altered to favor the pathogenic bacteria that belong to phyla Proteobacteria and TM7. In addition, the Hfe-/- proinflammatory bacteria adhered to colon and thus increased the colonic bacterial load. This phenomenon, addressed as bacterial dysbiosis, is considered a hallmark of colon inflammation and colon cancer. Furthermore, we observed that the colonic epithelial cells of Hfe-/- mouse had a lower expression of antimicrobial peptides and thus a defective first line defense against the pathogens. Finally, hemochromatosis mouse colon under inflammation released higher concentration of the pro-inflammatory cytokines that contributed to the creation of the hostile colonic environment. In summary, iron overload as seen in hemochromatosis, impairs the colonic defense machinery and it causes bacterial dysbiosis, thus providing the ideal environment for the colitis and colon cancer development. Citation Format: Bojana Ristic, Sathish Sivaprakasam, Rao Kottapalli, Abdul Hamood, Vadivel Ganapathy. Bacterial dysbiosis in the mouse model of hemochromatosis: Increased risk of colitis and colitis-associated colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1479.

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. Cancer Res; 71(21); 6654–64. ©2011 AACR.

Expression profiling has identified metastasis-associated microRNAs (miRNA) but technical limitations hinder the discovery of metastasis-suppressing miRNAs. In this study, we sought metastasis-suppressing miRNAs by functional screening. Individual miRNAs were lentivirally introduced into metastatic MDA-MB-231 breast cancer cells and analyzed for effects on cell migration, a critical step in cancer metastasis. Among 486 miRNAs screened, 14 were identified that included all of the members of the miRNA-196 family (miR-196a1, miR-196a2, and miR-196b). Enforced expression of miR-196a1/2 or miR-196b abrogated in vitro invasion and in vivo spontaneous metastasis of breast cancer cells, indicating that members of the miR-196 family are potent metastasis suppressors. We found that miR-196 inhibited the expression of transcription factor HOXC8. Functional linkage was implied by small interfering RNA–mediated knockdown of HOXC8, which suppressed cell migration and metastasis, and by ectopic expression of HOXC8, which prevented the effects of miR-196 on cell migration and metastasis. Unlike other metastasis-associated miRNAs that have been described, the expressions of miR-196 were not correlated with breast cancer cell migration or the metastatic status of clinical breast tumor specimens. Instead, we detected an excellent correlation between the ratio of miR-196 to HOXC8 messages and the migratory behavior of breast cancer cell lines as well as the metastatic status of clinical samples. Our findings identify miRNA-196s as potent metastasis suppressors and reveal that the ratio of miR-196s to HOXC8 mRNA might be an indicator of the metastatic capability of breast tumors. Cancer Res; 70(20); 7894–904. ©2010 AACR.

Cancer cells display a unique phenomenon where, even in the presence of oxygen, cells switch from oxidative phosphorylation to glycolysis as the primary source of ATP with the production of lactic acid - called the Warburg Effect. Lactic acid, the end product of this metabolic switch, is effluxed out of tumor cells to prevent intracellular acidification. Recent evidence suggests that lactate and the excess protons in the tumor microenvironment play an active role in tumor growth. In particular, lactate has been shown to function as an agonist for GPR81, a G-protein-coupled receptor expressed on the surface of tumor cells. This autocrine signaling of lactate promotes tumor growth and metastasis, as well as angiogenesis and immune evasion. The present study assesses whether tumor cell-derived lactate has any paracrine role via its receptor in non-cancer cells present in the tumor microenvironment, and also if the lactic acid-induced extracellular acidification has any role in tumor-cell nutrition. To address the first issue, we generated MMTV-PyMT-Tg mice, a spontaneous model for breast cancer, on Gpr81+/+ and Gpr81-/- backgrounds. The absence of Gpr81 reduced the mammary tumor incidence, delayed mammary tumor progression, and reduced lung metastasis. These data demonstrate the essential role of GPR81 in breast cancer growth and metastasis; but does not differentiate between Gpr81 in tumor cells versus Gpr81 in the tumor microenvironment. We then used the syngeneic transplant of the mouse mammary tumor cell line AT-3 into the mammary fat pads of wild type and Gpr81-/- mice to assess the involvement of Gpr81 in the microenvironment. The growth of the transplanted tumor cells was significantly reduced in Gpr81-/- mice than in wild type mice. To address the second issue, we monitored the expression of GPR81 in human breast cancer cell lines and in normal mammary epithelial cell lines. The expression was many-fold higher in cancer cell lines than in normal cell lines. Hypoxic stress and nutritional deprivation further up-regulated GPR81 expression in tumor cells. Treatment of the ER-positive cancer cell line MCF-7 with lactate (endogenous agonist for GPR81) and 3-chloro-5-hydroxy benzoic acid (pharmacological agonist for GPR81), up-regulated the proton-coupled amino acid transporter PAT1 as evident from the increase in PAT1 mRNA. The function of PAT1, assessed by the uptake of glycine in the presence of an acidic extracellular pH, was also increased. We conclude that tumor-cell derived lactic acid functions in two different capacities to promote tumor growth. First, lactate is an autocrine as well as a paracrine signal via GPR81 expressed on tumor cells and on non-cancer cells in the tumor microenvironment to promote tumor growth and metastasis. Second, the lactic acid-induced acidic pH in the external milieu of tumor cells serves as a driving force to enhance amino acid entry into tumor cells via PAT1. Citation Format: Sabarish Ramachandran, Timothy Brown, Vadivel Ganapathy. Autocrine and paracrine role of tumor derived lactic acid in tumor growth and metastasis and in tumor cell nutrition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2402.

Amino Acid Transporter SLC6A14: A Novel Drug Target for Colorectal Cancer & Colitis and Its Transcriptional Regulation by TCF4/β-catenin Pathway Mohd Omar Faruk Sikder, Sathish Sivaprakasam, Vadivel Ganapathy Texas Tech University Health Sciences Center SLC6A14 is a Na+/Cl− -dependent amino acid transporter capable of transporting 18 of the 20 proteinogenic amino acids, including leucine (mTOR activator), glutamine (necessary for nucleotide biosynthesis), and arginine (substrate for iNOS). This transporter is expressed at basal levels in normal colon but significantly upregulated in colorectal cancer (CRC) and colitis. However, the relevance of this upregulation to disease progression and the mechanisms involved in the upregulation remain unknown. We postulated that deletion of SLC6A14 or pharmacological blockade of its function may suppress CRC by depleting amino acids and interfering with mTOR signaling selectively in tumor cells. It may also ameliorate the severity of colitis by diminishing NO synthesis by iNOS in colonic epithelial cells. Since TCF4/β-catenin/Wnt signaling is activated in CRC and colitis, we postulated that TCF4/β-catenin might control the expression of SLC6A14. We tested these hypotheses in the present study. CRC cells treated in vitro with α-methyl tryptophan, a selective blocker of SLC6A14, showed evidence of amino acid deprivation, decreased mTOR activity, and increased autophagy and apoptosis. In nude mouse xenografts with LS174T cells (a CRC cell line), silencing of SLC6A14 by shRNA markedly reduced tumor growth. Accordingly, Slc6a14-/- mice showed reduced tumor incident and tumor size compared to the wild type mice in AOM/DSS-induced colitis-associated colon cancer mouse model. Furthermore, Slc6a14-null mice showed decreased disease progression in an experimental colitis model (Dextran sulfate sodium-induced colitis) compared to wild type mice. In vitro treatment of LS174T cells with calphostin-C, a potent inhibitor of TCF4/β-catenin, dramatically reduced the expression of SLC6A14 mRNA and protein levels, whereas treatment of KM12L4 and KM12C cells with Wnt agonist AMBMP showed the opposite trend. Finally, ChIP assay demonstrated that TCF4/β-catenin complex directly regulates the expression of SLC6A14 in human CRC cells by binding with its promoter. We conclude that the increased β-catenin/Wnt signaling in CRC and colitis is responsible for the upregulation of SLC6A14 under these pathological conditions and that deletion of the transporter or its pharmacological blockade protects against colitis and CRC. These studies identify SLC6A14 as a novel drug target for the treatment of colonic inflammation and CRC. Citation Format: Mohd Omar Sikder, Sathish Sivaprakasam, Ganapathy Vadivel. Amino acid transporter SLC6A14: A novel drug target for colorectal cancer and colitis and its transcriptional regulation by TCF4/beta-catenin pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5735.

The efflux pump ABCG2 is a part of cellular defense. In intestine it is located at the luminal side, and effluxes exogenous/endogenous dysplasia-promoting agents. Since cancer cells exposed to chemotherapeutics overexpress ABCG2 to remove these drugs, ABCG2 is considered a multidrug-resistance protein. However, its physiologic protective role in cancer initiation/progression has not received much attention. In numerous studies, ABCG2 is present in colon at lower levels in patients with colitis and chemotherapy-naïve colon cancer than in controls. We therefore hypothesized that the physiologic role of ABCG2 is to suppress colonic inflammation and colon cancer. To test this, we monitored Abcg2 expression in the intestinal tract in experimental and genetic mouse models of colon cancer and colitis. In normal mice, Abcg2 was expressed throughout the intestinal tract, jejunum being the site with maximal expression. Colon was positive for expression, but the expression was lower than in jejunum. Compared to control colon, Abcg2 was present at lower levels in colonic polyps from experimental and genetic models of colon cancer; the same trend was seen in normal and cancer cell lines of colonic origin. In line with human colitis studies, colons from colitis mouse models had lower expression of Abcg2 compared to control colon. The association of excess iron to colon cancer is well known, but it is not known if excess iron has any role in tumor-associated downregulation of ABCG2 in colon. We used Hfe-/- mice as a model for hemochromatosis, a genetic iron-overload disease, and exploited the ability of Abcg2 to export uric acid as a measure of Abcg2 function. Colons from Hfe-/- mice had lower expression of Abcg2 and higher accumulation of uric acid. This decrease in Abcg2 function indicated that excess iron, directly or indirectly, dictates Abcg2 expression in colon. To better understand this phenomenon, colon cells were treated with ferric ammonium citrate as an iron source. This in vitro iron-overload model demonstrated nuclear depletion of the tumor suppressor p53, and at the same time a decrease in Abcg2 expression. In addition, Abcg2 mRNA was undetectable in p53-null mouse embryonic mouse fibroblasts, suggesting that Abcg2 is a p53 target. Additional in vitro studies included treatment of normal and cancer colon cells with Carbidopa, a drug which was recently shown as an aryl hydrocarbon receptor agonist. Our studies showed that Carbidopa is a potent iron chelator and an inducer of ABCG2 expression. In summary, ABCG2 is silenced in colon cancer and colitis, and the decreased expression of p53 under these conditions might be responsible for the decreased ABCG2 expression. Carbidopa could be used to reduce cellular levels of iron and to reverse iron-induced downregulation of ABCG2 as a prevention/treatment strategy for colitis and colon cancer. Citation Format: Bojana Ristic, Sathish Sivaprakasam, Jiro Ogura, Vadivel Ganapathy. Downregulation of ABCG2 expression in colitis and colon cancer: Relevance to iron overload, hemochromatosis and p53, and therapeutic use of carbidopa to reverse the downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4477.

Apoptotic resistance is often associated with metastatic phenotype in tumor cells and is considered a hallmark of tumor progression. In this study, IFN regulatory factor 8 (IRF8) expression was found to be inversely correlated with an apoptotic-resistant and metastatic phenotype in human colon carcinoma cell lines in vitro. This inverse correlation was further extended to spontaneously arising primary mammary carcinoma and lung metastases in a mouse tumor model in vivo. Exogenous expression of IRF8 in the metastatic tumor cell line restored, at least partially, the sensitivity of the tumor cells to Fas-mediated apoptosis, and disruption of IRF8 function conferred the poorly metastatic tumors with enhanced apoptotic resistance and metastatic capability. DNA demethylation restored IRF8 expression and sensitized the metastatic tumor cells to Fas-mediated apoptosis. Analysis of genomic DNA isolated from both primary and metastatic tumor cells with methylation-sensitive PCR revealed hypermethylation of the IRF8 promoter in metastatic tumor cells but not in primary tumor cells. Taken together, our data suggest that IRF8 is both an essential regulator in Fas-mediated apoptosis pathway and a metastasis suppressor in solid tumors and that metastatic tumor cells use DNA hypermethylation to repress IRF8 expression to evade apoptotic cell death and to acquire a metastatic phenotype. [Cancer Res 2007;67(7):3301–9]

Recently, impressive technical advancements have been made in the isolation and validation of mammary stem cells and cancer stem cells (CSC), but the signaling pathways that regulate stem cell self-renewal are largely unknown. Furthermore, 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. Cancer Res; 76(11); 3224–35. ©2016 AACR.

In recent years, impressive technical advancements have been made in the isolation and validation of the mammary stem cells (MaSCs) and cancer stem cells (CSCs). However, the signaling pathways that regulate stem cell self-renewal are largely unknown. Further, CSCs are believed to contribute to resistance to chemotherapy and radiation therapy. However, an effective therapeutic strategy to overcome this resistance is yet to be identified. We have recently discovered that the DNA methyltransferases, especially DNMT1, play critical role in MaSCs and CSCs self-renewal and targeted deletion of this gene impaired mammary tumor formation by inhibiting CSCs formation. However, the molecular mechanism(s) by which DNMTs control CSCs and the therapeutic relevance of DNMTs inhibitors in regulation of CSCs and overcome drug resistance are also largely unknown. In this study, using MMTV-Neu-Tg mouse mammary tumor model, we found that both luminal progenitor and basal stem cells are susceptible to genetic and epigenetic modifications, which leads to activation of un-activated Neu-Tg into transformed tumor forming phenotype. Combination of 5-Azacytidine, a DNMT inhibitor, and butyrate, a HDAC inhibitor, markedly reduces CSCs and consequently increases the overall survival of the animal. RNA-seq analysis of the CSCs treated with 5-AzaC+butyrate provides evidence that combined inhibition of DNMTs and HDACs reduces CSCs pool in the mammary gland by blocking growth-promoting signaling molecules like RAD51AP1 and SPC25. RAD51AP1 and SPC25, which are known to play a key role in DNA damage repair and kinetochore assembly, are significantly overexpressed in breast tumor tissues and associated with decreased overall patients’ survival. Further, these two genes are overexpressed in Tamoxifen and Taxol resistant human breast cancer cell lines. Functional inactivation of these genes in breast caner cells facilitates chemotherapy-induced apoptosis and reduces tumor growth. Overall, our studies provide strong evidence that breast CSCs (both basal stem cells and luminal progenitor cells) are susceptible for genetic and epigenetic modifications and associated with resistance to chemo- and radiotherapy. Thus, combination of DNMT and HDAC inhibitors can serve as an effective therapeutic strategy to block mammary tumor growth and to overcome drug resistance by inhibiting CSCs. Citation Format: Rajneesh Pathania, Ravindra B. Kolhe, Sabarish Ramachandran, Gurusamy Mariappan, Priyanka Thakur, Puttur D. Prasad, Vadivel Ganapathy, Muthusamy Thangaraju. Combination of DNMT and HDAC inhibitors reprogram cancer stem cell signaling to overcome drug resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3325.

Carbidopa is used in combination with L-DOPA to treat Parkinson's disease; it does not have any therapeutic use by itself in Parkinson's disease, but when used along with L-DOPA prevents the conversion of the latter into dopamine in the periphery by inhibiting aromatic amino acid decarboxylase. Carbidopa however does not cross the blood-brain barrier; thus does not impact on the conversion of L-DOPA into dopamine in the brain. We hypothesized that carbidopa might have potential as an anticancer drug with the following rationale: (a) carbidopa is an amino acid derivative and therefore might block the entry of amino acids into cancer cells via certain amino acid transporters; (b) the stress hormones epinephrine and norepinephrine are known to promote cancer progression, and carbidopa as an inhibitor of aromatic amino acid decarboxylase might interfere with the generation of these tumor-promoting hormones; (c) carbidopa is also an analog of phenylhydrazine, which is an inhibitor of the immunosuppressive enzyme indoleamine-2,3-dioxygenase, a drug target for cancer treatment; carbidopa might inhibit this enzyme and thus enhance the ability of the immune system to recognize cancer cells as foreign and fight against them. Based on this rationale, we examined the efficacy of carbidopa to treat pancreatic and breast cancer. We found carbidopa to be effective in blocking the proliferation of pancreatic cancer cell in vitro. We then examined its efficacy in vivo using xenografts of pancreatic cancer cells in nude mice; again, the drug was effective in decreasing the growth of the xenografted tumor cells into tumors. We also tested its efficacy on proliferation of breast cancer cell lines; we used ZR-75.1, MB-231, and HCC-1937 breast cancer cell lines as models for ER-positive, ER-negative and BRCA-1 mutant breast cancers, respectively. Carbidopa decreased the proliferation of all three cell lines. We then examined its in vivo efficacy against breast cancer using the MMTV-PyMT-transgenic mouse as a model of spontaneous breast cancer. In this model, breast cancer develops initially as an ER-positive subtype but then turns into an ER-negative subtype. Carbidopa markedly decreased the growth of breast cancer in this mouse model. Based on these in vitro and in vivo data, we conclude that carbidopa has promise for use as an anticancer drug. As the drug potentially elicits its anticancer effects by targeting multiple pathways, the anticancer efficacy of the drug is likely to be broad against different types of human cancers. For in vivo studies, we used the drug intraperitoneally at a dose of 1 mg/mouse that approximately translates to a human dose of 300-400 mg/day. As the drug has been shown to have no detectable side effects in humans at doses as high as 400 mg/day, it can be taken to clinical trials readily to test its efficacy in humans as an anticancer drug. Citation Format: Vadivel Ganapathy, Ellappan Babu, Sabarish Ramachandran, Yangzom D. Bhutia. Repurposing the FDA-approved drug carbidopa to treat human cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1015.

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. Reexpression 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 down-regulation of Bcl-2, Bcl-xL, and cyclin D1 and up-regulation of death receptor pathway. In addition, GPR109A/butyrate suppresses nuclear factor-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. [Cancer Res 2009;69(7):2826–32]

Hemochromatosis is a highly prevalent genetic disease associated with excessive iron accumulation in a variety of tissues in an age-dependent manner. In a majority of patients (>85%) with hemochromatosis, mutations in the iron-regulatory gene HFE are the cause. Iron, when present in excess, is an inducer of oxidative stress and suppresses mitochondrial function. Patients with hemochromatosis show evidence of colonic inflammation. Further, some studies have shown increased risk for colon cancer associated with genetic mutations known to cause hemochromatosis. Based on these findings in the literature, we hypothesized that hemochromatosis is an important determinant of disease progression in patients with colitis and colon cancer. We tested this hypothesis by comparing progression of experimentally induced colitis and colon cancer between wild type mice and Hfe-null mice, a model for hemochromatosis. We also compared the transcriptome profile between wild type and Hfe-null colonic epithelial cells. In addition, we analyzed fecal bacteria in wild type mice and Hfe-null mice because colonic microbiome is an important determinant of colonic inflammation and colon cancer. With dextran sulfate sodium-induced colitis, Hfe-null mice suffered more weight loss and exhibited more severe bleeding and diarrhea scores than wild type mice. With ApcMin-driven colon and intestinal cancer, Hfe-null mice had more polyps in the small intestine and colon than wild type mice. Transcriptome analysis showed that Hfe-null colonic epithelial cells, compared to wild type cells, had increased expression of the cytokines Ccl3 and Ccl5 and the interferon-stimulated gene 15 (ISG15 or Usp18), which are all known to promote inflammation and cancer. Hfe-null colonic epithelial cells also had decreased expression of Erdr1 (erythroid differentiation regulator 1), whose expression is known to suppress tumor cell proliferation, invasion, migration and metastasis. Analysis of fecal microbiome indicated that the prevalence of Bacteroidetes and Firmicutes decreased while that of Proteobacteria increased in Hfe-null mice compared to wild type mice, a finding particularly striking in male mice. These studies demonstrate that hemochromatosis enhances the progression of colonic inflammation and colon carcinogenesis. This conclusion is further supported by xenograft studies using the colon cancer cell line HCT116 with and without shRNA-induced downregulation of HFE. When HFE was silenced, there was a significant increase in the growth of HCT116 cells in mouse xenografts, demonstrating that inactivation of HFE promotes colon cancer progression. Based on these data, we conclude that the iron-overload disease hemochromatosis is a promoter of disease progression in colitis and colon cancer, and that use of iron chelators might have a logical basis for inclusion in therapeutic modalities in the treatment of colonic inflammation and colon cancer. Citation Format: Vadivel Ganapathy, Ashish Gurav, Jaya P. Gnanaprakasam, Ellappan Babu, Yangzom D. Bhutia, Cynthia Reinoso Webb, Matthew B. Grisham. The iron-overload genetic disease hemochromatosis potentiates colonic inflammation and colon carcinogenesis. [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 1557. doi:10.1158/1538-7445.AM2015-1557

Tumor propagation is the hallmark feature of the cancer stem/tumor propagating cells. Several genetic and epigenetic components are involved in regulation of this process; however, DNA methylation provides a potential epigenetic mechanism for the cellular memory, which needed to preserve the tumorigenic potential through repeated cell divisions. Further, DNA methylation plays a critical role in stem/progenitor cell maintenance wherein the DNMT proteins get enriched in undifferentiated cells and thereby it retains the regenerative capacity while suppressing differentiation. However, the precise role of DNMTs in maintaining stem/progenitor and tumorigenic phenotype in constantly replenished organ, like mammary glands and mammary tumor is not yet known. Here we show that Dnmt1 is required for mammary gland outgrowth and terminal end bud development and that mammary-gland specific Dnmt1 deletion in mice leads to significant reduction in mammary stem/progenitor cell formation. Interestingly, Dnmt1 deletion almost completely abolishes Neu-Tg- and C3(1)-SV40-Tg- driven mammary tumor formation and metastasis. This phenomenon is associated with significant reduction in cancer stem cell (CSC) formation. Similar observations were also recapitulated using pharmacological inhibitors of Dnmts in Neu-Tg mice. To unravel the cause of tumorigenicity of tumor propagating cells, we used genome-wide methylation and RNA sequence approach and find that DNA methylation plays a vital role in regulation of abnormal self-renewal by hypermethylating genes that are involved in development and cell commitment pathways; thereby leading to immortality and autonomous growth to the tumor propagating cells. Overall, our studies provide the first in vivo evidence that DNMT1 is indispensable for mammary stem, progenitor and cancer stem cell formation and that functional inactivation of this gene drastically reduces mammary tumor formation even in the aggressive triple-negative breast cancer subtype. Furthermore, we identified ISL1 as a functional target of DNMT1 in tumor progenitor cells, and stable expression of ISL1 induces apoptosis in cancer stem cells. Thus, DNMT1 specific inhibitors could have a great impact on eradication of cancer stem cells and associated disease recurrence, and ISL1 hypermethylation status could be used as a prognostic marker for early breast cancer diagnosis. Citation Format: Rajneesh Pathania, Sabarish Ramachandran, Puttur Prasad, Vadivel Ganapathy, Muthusamy Thangaraju. Functional role of DNA methyltransferase1 (DNMT1) in regulation of mammary stem/progenitor and cancer stem cells. [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 LB-142. doi:10.1158/1538-7445.AM2015-LB-142

Dietary fiber has long been known to protect against colonic inflammation and colon cancer. Short-chain fatty acids (SCFA) such as butyrate generated in colonic lumen by bacterial fermentation of dietary fiber mediate most of these protective effects, but the underlying molecular mechanisms are poorly understood. We examined the role of the plasma membrane transporter Slc5a8 in the beneficial effects of these bacterial metabolites against colitis and colon cancer. Slc5a8 is expressed on the luminal membrane of colonic epithelial cells and also in mucosal immune cells including the antigen-presenting dendritic cells. The transporter mediates the entry of SCFAs into colonic epithelium from the lumen and also into dendritic cells in the lamina propria. We interrogated the role of the transporter in colon by comparing the function of dendritic cells and progression of experimentally induced colitis and colon cancer between wild type mice and Slc5a8-null mice. Since the amount of SCFAs generated in colonic lumen depends on the fiber content in the diet, we used two different diets, one with optimal fiber content (FC diet) and the other with no fiber (FF diet). These studies have shown that Slc5a8 is obligatory for butyrate-dependent inhibition of histone deacetylases in colonic epithelium and dendritic cells and also for the maintenance of the intestinal barrier function; but this obligatory need for the transporter is evident only when the mice are fed FF diet. The transporter is dispensable with FC diet. Compared to wild type mice, Slc5a8-null mice exhibit increased susceptibility to dextran sulfate sodium-induced colitis and azoxymethane/dextran sulfate sodium-induced colon cancer, again only with FF diet and not with FC diet. Butyrate induces the immunosuppressive enzymes indoleamine dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2) in dendritic cells in an Slc5a8-dependent manner. This is corroborated by the decreased expression of these two enzymes in the colon of germ-free mice compared to conventional mice. IDO1 and Aldh1A2 play a critical role in dendritic cells to maintain an immunosuppressive phenotype. Butyrate, which induces the expression of both enzymes, promotes the ability of dendritic cells to convert naive T cells into FoxP3-positive immunosuppressive Tregs and also their ability to suppress interferon-gamma-secreting pro-inflammatory T cells. As such, Slc5a8-null mice have decreased Tregs and increased IFN-gamma-positive T cells in colon. We conclude that Slc5a8 may be dispensable in colon under dietary conditions associated with high fiber content when the luminal concentrations of SCFAs are high, but the transporter is indispensable for protection against colonic inflammation and colon cancer with a diet containing suboptimal fiber which results in decreased concentrations of SCFAs in the lumen. Thus, Slc5a8 is a conditional tumor suppressor in colon linked to dietary fiber content. Citation Format: Vadivel Ganapathy, Ashish Gurav, Nagendra Singh. The butyrate transporter SLC5A8 is a tumor suppressor in colon linked to dietary fiber content. [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 2037. doi:10.1158/1538-7445.AM2015-2037

Pancreatic cancer is the deadliest of all cancers. Currently, gemcitabine is the only drug of choice to treat this cancer but the results are modest as the disease itself is diagnosed at an advanced stage and often times develops resistance to gemcitabine via silencing of the influx transporters for the drug. We hypothesized that the delivery of gemcitabine into pancreatic cancer cells in a different form via some other transporters would provide a logical alternative approach. It would even be better if we can exploit for this purpose the transporters that are selectively upregulated in pancreatic cancer. Several nucleoside drugs are transportable substrates for the amino acid transporter SLC6A14 if used in the form of amino acid-based prodrugs. Such strategy would work for gemcitabine if SLC6A14 is upregulated in pancreatic cancer. With this in mind, we analyzed publically available microarray datasets for the amino acid transporters that are upregulated in pancreatic cancer. We found four transporters (SLC1A5, SLC7A5, SLC7A11, and SLC6A14) overexpressed in tumors compared to normal tissue. The fold-increase in expression is the highest for SLC6A14 in all datasets (range, 1.3 - 163.3; p, 0.05 - 2×10−14). The fold-increase for the other three transporters is 2 at the maximum. We confirmed these findings using primary pancreatic cancer tissues and cancer cell lines. First, we used pancreatic cancer tissues and compared with the normal tissues. SLC6A14 mRNA is increased at least by 7-fold in tumors versus normal tissue. We then evaluated the expression of SLC6A14 mRNA and protein in normal and pancreatic cancer cell lines. The expression is higher in all cancer cell lines than in a normal cell line. This was further confirmed with a tissue microarray containing pancreatic tumor tissues from 52 patients. The delivery of gemcitabine in the form of amino acid-based prodrugs via SLC6A14, an exceptionally energy-coupled transporter, as a means to overcome gemcitabine resistance is a novel idea that has never been tested. As a proof of principle, we have shown that SLC6A14 can transport several nucleoside drugs (acyclovir, ganciclovir, and cytarabine) in the form of amino acid-based prodrugs. The parent nucleosides are not substrates for the transporter. To specifically assess the interaction of amino acid-based prodrugs of gemcitabine with SLC6A14, we synthesized the valyl ester of gemcitabine and investigated its transport via SLC6A14 using two different heterologous expression systems: a mammalian cell expression system and the Xenopus oocyte expression system. In both experimental approaches, we found that the valyl ester of gemcitabine, but not the parent drug, is a transportable substrate for SLC6A14. The transport process is Na/Cl-coupled and electrogenic. We conclude that amino acid-based prodrugs of gemcitabine represent a viable alternative to overcome resistance to gemcitabine in the treatment of pancreatic cancer. Citation Format: Yangzom D. Bhutia, Ellappan Babu, Pankaj K. Singh, Vadivel Ganapathy. Amino acid-based prodrugs of gemcitabine - a therapeutic option to overcome chemoresistance in pancreatic cancer. [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 348. doi:10.1158/1538-7445.AM2015-348

Consumption of dietary fiber is positively correlated with lower incidence of inflammation and carcinogenesis in intestine. In colon dietary fibers are fermented by gut microbiota into short chain fatty acids (SCFA, acetate, propionate and butyrate). Several studies demonstrate that SCFA play a key role in protection of intestine from inflammation and carcinogenesis. However, detail molecular mechanisms underlying the biological effects of SCFA on intestine health are not fully understood. Gpr43 is cell surface receptor for SCFA and recent evidences suggest that it regulates colonic inflammation in a context dependent manner. Intestinal inflammation is one of the risk factor for development of cancer in gut. Based on these findings, role of Gpr43 in regulation of colon carcinogenesis was evaluated. Colitis associated colon cancer in wild type (WT) and Gpr43-/- mice was induced by single intraperitoneal injection of azoxymethane (AOM) and repeated cycles of dextran sulfate sodium (DSS) in drinking water. Gpr43-/- mice showed drastically reduced survival, severe weight loss and increased diarrhea and rectal bleeding than WT mice. At the end of treatment, colons of Gpr43-/- mice exhibited increased number of polyps than that of WT mice. Reciprocal bone marrow chimera experiments revealed that Gpr43 expression in non-hematopoietic cells play a major role in suppression of colonic inflammation and carcinogenesis. The data presented here suggest a model in which Gpr43 plays a critical role in facilitating positive effects of dietary fiber and gut microbiota on intestinal health. Citation Format: Sathish Sivaprakasam, Ashish Gurav, Ganapathy Vadivel, Nagendra Singh. Role of Gpr43 in intestinal inflammation and carcinogenesis. [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 986. doi:10.1158/1538-7445.AM2015-986

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

Tumor cells have an increased need for amino acids. Mammalian cells cannot synthesize essential amino acids; they must obtain these amino acids via specific transporters. Glutamine, though a non-essential amino acid, is critical for tumor cells (glutamine addiction). Entry of amino acids into tumor cells is enhanced by upregulation of specific transporters. If we can interfere with the entry of amino acids into tumor cells, we should be able to starve these cells to death. If the transporters that are specifically induced in tumor cells are identified, blockade of the induced transporters would constitute a logical strategy for cancer treatment. Mammalian cells express ∼40 amino acid transporters, expressed in different combinations and in a cell type-specific manner. Among them, SLC6A14 is unique in that it transports all essential amino acids as well as glutamine, and is expressed only at low levels in normal tissues, but induced in colon cancer and in ER+ breast cancer. Tumor cells in these cancers upregulate SLC6A14 to meet their increased demand for essential amino acids and glutamine, indicating that SLC6A14 drives their “glutamine addiction.” We have now established the potential of this transporter as a drug target for breast cancer treatment using genetic and pharmacologic approaches. For this, we first generated Slc6a14-/- mouse. The Slc6a14 gene is located on X chromosome. Deletion of the gene is not lethal and there is no overt phenotype in hemizygous males (-/y) or in homozygous females (-/-). We then examined the progression of breast cancer in Polyoma middle T antigen (Py-MT) Tg mouse on Slc6a14+/+ and Slc6a14-/- background. Deletion of Slc6a14 markedly suppressed breast cancer and lung metastasis induced by the Py-MT oncogene. We have also identified -methyl-L-tryptophan (-MLT) as a selective blocker of Slc6a14. We investigated the consequences of pharmacologic blockade of Slc6a14 on Py-MT-induced breast cancer with oral administration of -MLT (1 mg/ml in drinking water). The blocker was able to suppress breast cancer to a marked extent. Py-MT-induced breast tumors showed robust upregulation of Slc6a14; however, the tumors also showed upregulation of Slc7a5/Slc3a2, another amino acid transporter. Therefore, we examined the interaction of -MLT with Slc7a5/Slc3a2 and found that while -MLT is a blocker of Slc6a14, it is a transportable substrate for Slc7a5/Slc3a2, demonstrating that only the function of Slc6a14 is selectively blocked by -MLT. We conclude that blockade of Slc6a14 is a viable strategy for treatment of certain specific subtypes of breast cancer (e.g., ER-positive) that are associated with upregulation of the transporter. Citation Format: Babu Ellappan, Yangzom D. Bhutia, Muthusamy Thangaraju, Puttur D. Prasad, Vadivel Ganapathy. Genetic deletion or pharmacologic blockade of the amino acid transporter Slc6a14 in mice suppresses breast cancer induced by Polyoma middle T oncogene. [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 3928. doi:10.1158/1538-7445.AM2014-3928

Pancreatic cancer is the deadliest of all cancers with worst outcome and poor survival rate. Chemotherapy with gemcitabine works well for early stage cancer, but becomes ineffective for advanced-stage cancer. As such, there is a dire need for new approaches to treat this cancer. The metabolism of tumor cells is very different from that of normal cells. In particular, the differences in amino acid metabolism are gaining increasing attention in cancer biology. Selective amino acid transporters are upregulated in cancer in response to the increased demands for amino acids in tumor cells. Such tumor-selective amino acid transporters are logical druggable targets for cancer therapy. As such, pharmacologic blockade of such upregulated transporters would lead to cell death selectively in tumor cells by depriving the tumor cells of essential nutrients. With this in mind, we analyzed 8 different publically available microarray datasets in Gene Expression Omnibus for the amino acid transporters that are upregulated in pancreatic cancer. This led to the identification of three transporters (SLC1A5, SLC7A5, and SLC6A14) that are overexpressed in tumors compared to normal tissue. Among these, the fold-increase in expression was the highest for SLC6A14 in all datasets (range for fold-increase, 1.3 - 163.3; range for p value, 0.05 - 2x10-14). The fold-increase for the other two transporters is 2 at the maximum. We confirmed these findings using primary pancreatic cancer tissues and cancer cell lines. First, we used paired samples of pancreatic cancer tissues and the corresponding adjacent normal tissues. SLC6A14 mRNA was increased at least by 7-fold in tumor versus normal tissue. The same was true with three additional unpaired cancer and normal pancreatic tissues. We then evaluated the expression of the transporter at mRNA and protein level in a normal pancreatic cell line (HPNE) and five different pancreatic cancer cell lines. The transporter expression was higher in cancer cell lines compared to the normal cell line. This was confirmed by immunofluorescence by comparing the transporter expression between the normal cell line HPNE and the cancer cell line BxPC-3. Blockade of the transporter function in BxPC-3 cells with α-methyltryptophan (-MT) induced amino acid starvation, activated autophagy, altered cell cycle distribution and inhibited mTOR and colony formation. Decreased invasion and migration observed with α-MT in tumor cells was associated with a decrease in stabilization of HIF-1α. Xenograft studies in nude mice showed that administration of α-MT decreased the growth of BxPC-3 and Mia PaCa-2 cells into tumor. To conclude, SLC6A14 is upregulated in pancreatic cancer and blockade of the transporter with α-MT shows differential and selective detrimental effects on pancreatic tumor cells, both in vitro and in vivo. These studies identify SLC6A14 as a novel and effective drug target for pancreatic cancer treatment. Citation Format: Yangzom D. Bhutia, Veena Coothankandaswamy, Sha Cao, Ying Xu, Vadivel Ganapathy. The amino acid transporter SLC6A14 is an effective drug target for treatment of pancreatic 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 4340. doi:10.1158/1538-7445.AM2014-4340

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