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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.

Observations from the authors' laboratory suggest a physiological role for increased cyclin G1 protein levels in human uterine leiomyoma. The hypothesis of the present study is that the strategic modulation of cyclin G1 by antisense technology will inhibit the survival of in vitro-grown uterine leiomyoma cells. Cultured uterine leiomyoma cells were transfected with cyclin G1 ribbon-type antisense oligonucleotide (cyclin G1 RiAS) to effectively reduce cyclin G1 expression. Cell viability, in situ terminal deoxyuridine nick end-labeling (TUNEL) assay, flow cytometry, DNA fragmentation, and expression of cell cycle regulatory-related proteins were evaluated by Western blot. Antisense oligonucleotides compromised uterine leiomyoma cell viability and inducted apoptosis in a caspase-independent mechanism. In situ TUNEL and DNA fragmentation revealed apoptosis induction, and fluorescent-activated cell sorting analysis showed increased sub-G1-phase cells. Furthermore, abrogation of cyclin G1 enhanced p53 accumulation, phosphorylation of p53 at Ser-15 residue, and increased expression of cyclin-dependent kinase inhibitors p21 and p27. These data imply that cyclin G1 expression is associated with growth promotion and the potential utility and novelty of using ribbon-type antisense oligonucleotides as a gene therapy strategy to treat human uterine leiomyoma.

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

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

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 tumours, 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 tumours 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.

Purpose: Sodium butyrate (NaBT) is principally a histone deacetylase (HDAC) inhibitor, and it has the potential to arrest HPV-positive carcinoma cells at the G1 to S phase transition of the cell cycle. The aim of study was to determine whether phosphatidylinositol 3-kinase (PI3K) inhibition can enhance the inhibitory effect of NaBT on a human cervical cancer cell line (HeLa). Materials and Methods: Cervical cancer cells (HeLa) were treated with NaBT alone or in combination with the PI3K inhibitors wortmannin or LY294002. Cell viability analysis and FACS analysis were carried out. The expressions of the cell cycle related proteins were evaluated by Western-blot analysis. Results: Inhibition of PI3K enhanced NaBT-mediated apoptosis and this decreased the HeLa cell viability. Either wortmannin or LY294002, combined with NaBT, enhanced the activation of caspase 3 and caspase 9, and this enhanced the subsequent cleavage of poly (ADP-ribose) polymerase (PARP). Cervical cancer cells were arrested in the subG1 and G2/M phase, as was detected by FACS analysis. NaBT treatment in combination with PI3K inhibitors showed the increased expression of the CDK inhibitors p21Cip1/Waf1 and p27Kip1, in a p53 dependent manner, and also the increased dephosphorylation of Rb whereas there was a reduction in the expression levels of cyclin A, cyclin D1 and cyclin B1. Conclusion: The results demonstrate that inhibition of PI3K enhances NaBT-mediated cervical cancer cell apoptosis through the activation of the caspase pathway. Moreover, these findings will support future investigation using the PI3K inhibitors in combination with adjuvant treatment for treating carcinoma of the cervix. (Cancer Res Treat. 2006;38:112-117)

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

Accumulation of milk during mammary gland involution induces apoptosis in mammary epithelium, but the underlying molecular mechanism is not known. Slc5a8, a plasma membrane transporter and a candidate tumor suppressor, is silenced in many human cancers, including breast and colon cancers. The tumor-suppressive function of Slc5a8 is believed to be due to its ability to mediate the entry of butyrate and pyruvate into cells in a Na+-coupled manner. Butyrate and pyruvate are inhibitors of histone deacetylases (HDACs), and Slc5a8-mediated concentrative entry of these compounds into cells causes HDAC inhibition. Normal cells possess low HDAC activity, and inhibition of HDACs does not induce apoptosis. In contrast, tumor cells possess high HDAC activity, and these cells are propelled into apoptosis by HDAC inhibition. This probably explains why tumor cells silence this transporter. Breast milk contains significant amounts of butyrate. We postulated that Slc5a8 might play an important role not only in normal mammary gland but also in breast cancer by transporting butyrate into mammary epithelial cells and consequently causing HDAC inhibition. Here we have shown that milk stasis-associated mechanical stretching, which occurs in early onset of mammary gland involution, dramatically induced Slc5a8 expression in luminal epithelium. Deletion of Slc5a8 in mouse is associated with mammary gland hyperplasia, stem cell and progenitor cell proliferation, and delayed mammary gland involution. We hypothesize that the mammary gland hyperplasia and involution delay in Slc5a8-null mice are due to lack of butyrate-induced HDAC inhibition because the absence of the transporter prevents the entry of butyrate into mammary epithelial cells. In wild type mice, mammary gland involution was associated with decreased expression of HDAC1/3, and exogenous administration of butyrate accelerated the involution process. Further, deletion of Slc5a8 predisposed mice to early onset of mammary tumorigenesis in two different mammary tumor models (MMTV-Neu and MMTV-HRas), accelerated lung metastasis, and reduced disease-free survival. Mammary gland-specific overexpression of Slc5a8 (Slc5a8-MMTV-Tg mouse) induced early onset mammary gland involution, reduced mammary tumor incidence, delayed mammary tumor formation, and reduced lung metastasis. Induction of Slc5a8 with a DNA methyltransferase inhibitor (5-azacytidine), coupled with exogenous administration of butyrate, dramatically reduced mammary tumor incidence, delayed mammary tumorigenesis, and increased disease-free survival. We conclude that Slc5a8 functions as a tumor suppressor in mammary gland and that induction of Slc5a8 with DNA methyltransferase inhibitors coupled with butyrate administration might represent a novel approach to breast cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 18. doi:1538-7445.AM2012-18

Emerging evidences on the role of iron-regulatory proteins in tumor growth provide new insights into the role of iron in cancer. Iron, though essential for normal cellular function, is toxic if present in excess. Hemochromatosis is an autosomal recessive disease of iron overload linked to mutations in HFE gene [Histocompatability leukocyte antigen class I-like protein involved in iron (FE) homeostasis]. HFE interacts with transferrin receptors, TfR1 and TfR2, and inhibits cellular iron uptake. The prevalence of the disease is very high with homozygosity incidence of ∼1 in 300 and heterozygosity incidence of ∼1 in 10. The disease manifests with liver cirrhosis/cancer, cardiomyopathy, diabetes, and arthritis. Several studies have shown increased prevalence of HFE mutations in breast cancer patients across all races. There is also evidence of increased levels of iron in breast cancer tissue. Although excessive iron and oxidative stress have been postulated to play a role in breast cancer, the expression/function of HFE in mammary gland iron homeostasis and how mutations in HFE affect breast cancer progression have not been studied directly. Here we demonstrate the expression of HFE mRNA in mouse mammary gland and primary mammary epithelial cells. The mRNA was absent in HFE-/- mice. Immunohistochemistry showed localization of HFE protein in luminal and myoepithelial cells. Similar staining was also seen in primary mammary epithelial cells from wild type mice but not from HFE-/- mice. We then examined the physiological role of HFE in mammary gland. Ferritin is a cytoplasmic protein involved in iron storage, and increase in intracellular iron levels results in increased level of ferritin. Recently, ferritin has also been shown to promote angiogenesis. Western blot and immunohistochemical analysis revealed that mammary tissue from HFE-/- mice had increased ferritin levels in an age-dependent manner. Thus, for the first time we show that HFE-/- mice have increased iron accumulation in the mammary gland. Immunostaining with antibody against 4-hydroxynonenal, one of the byproducts of lipid peroxidation, showed that HFE-/- mammary tissue have oxidative stress mediated by iron accumulation. MTT assay and thymidine incorporation showed that primary mammary epithelial cells from HFE-/- mice proliferate faster than the cells from HFE+/+ mice. Increase in histone deacetylase (HDAC) activity has been implicated in many cancers. We found that mammary tissue from HFE-/- mice has increased HDAC activity compared to wild type tissue. This shows that functional loss of HFE in mammary tissue can also promote cell cycle progression via epigenetic mechanisms. Based on these data, we hypothesize that tissue iron is an important modifier of breast cancer progression and that mutations in HFE (heterozygosity as well as homozygosity) may enhance the incidence and/or progression of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3599. doi:1538-7445.AM2012-3599

SIRT1, a NAD+-dependent deacetylase, first emerged as a potential anti-aging factor by controlling senescence, but now it is also implicated in tumor development and cancer cell survival. SIRT1 is overexpressed and/or catalytically activated in a variety of human cancers, suggesting a potential role for SIRT1 in tumor promotion. SIRT1 promotes oncogenic signaling, in part, by deacetylating the tumor suppressor protein p53 resulting in its inactivation. Similarly, estrogen receptor alpha (ERα) also promotes the development and progression of hormone-dependent cancers such as breast cancer, in part, by inactivating p53 function. It is not known whether ERα and SIRT1 functionally cooperate to elicit their biologic effects. Thus, we hypothesized that there could be a functional link between ERα and SIRT1 and that this link could play a role in cellular immortalization and tumor formation in mammary epithelial cells. Using a series of coimmunoprecipitation and immunofluorescence assays, we demonstrated that ERα physically interacts and functionally cooperates with SIRT1 in breast cancer cells and that SIRT1 is necessary for ERα-induced p53 inactivation. This interaction requires active estrogen-ERα (E2-ERα) complex and catalytically active SIRT1. Functional disruption of either E2-ERα or SIRT1 dramatically reduces this interaction. Further, ERα binds to SIRT1 promoter and increases SIRT1 transcription whereas SIRT1 does not affect the expression and acetylation status of ERα. In breast cancer cells, SIRT1 inactivation or knockdown decreases E2-induced cell growth, and this process is associated with prevention of ERα-induced p53 inactivation and induction of antioxidant enzymes. Finally, mouse xenograft studies of human ER-positive breast cancer cells provide in vivo evidence for the importance of the ERα-SIRT1 complex in breast cancer growth in which deletion of SIRT1 abrogates the tumor-promoting activity of estrogen completely. These studies show that SIRT1 is essential for the promotion of E2/ERα-associated breast cancer growth and that a combination of SIRT1 inhibitors and anti-estrogens could offer a novel approach for breast cancer management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4032. doi:10.1158/1538-7445.AM2011-4032

Introduction: SLC6A14 is an amino acid transporter that transports all essential amino acids as well as glutamine and arginine, and is energized by Na+ gradient, Cl- gradient, and membrane potential. The expression of SLC6A14 is upregulated in ER-positive breast cancer cells but not in ER-negative breast cancer cells. Since cancer cells have an increased need for essential amino acids (protein synthesis), Gln (nucleotide synthesis/glutaminolysis), and Arg (essential amino acid in cancer), we hypothesize that cancer cells satisfy their need for these amino acids by upregulating SLC6A14, and that blockade of SLC6A14 offers a rational strategy for treatment of ER-positive breast cancer. Methods: The transport of Leu, Gln, and Arg via SLC6A14 was studied in X. laevis oocyte expression system. SLC6A14 expression was monitored by RT-PCR and immunohistochemistry in normal and tumors (ER-positive and ER-negative) from primary breast cancer specimens. The influence of estrogen on SLC6A14 expression was investigated in ER-positive BT474 breast cancer cells. α-MT was used as the blocker of SLC6A14. The differential effects of α-MT on MCF7 (ER-positive), MB231 (ER-negative) and MCF10A (non-malignant) cells were investigated in terms of mTOR activity, autophagy, and apoptosis. The ability of α-MT to inhibit the growth of breast cancer cells was evaluated by xenograft in nude mice. α-MT was administered to mice in drinking water (2 mg/ml). Plasma levels of α-MT were measured by HPLC. Results: The transport of Leu, Gln, and Arg via SLC6A14 was dependent on Na+ and Cl- with a Na+:Cl-:amino acid stoichiometry of 2:1:1, and was blocked by α-MT. SLC6A14 expression was upregulated in ER-positive breast cancer. SLC6A14 was not expressed in ER-negative breast cancer. Estrogen induced SLC6A14 expression in ER-positive breast cancer cells. Exposure of ER-positive MCF7 breast cancer cells to α-MT induced the expression of asparagine synthetase and CHOP, inhibited mTOR activity, and induced autophagy and apoptosis. When combined with an autophagy inhibitor, α-MT induced apoptosis in MCF7 cells more potently. These effects were specific for ER-positive breast cancer cells, and were not seen in ER-negative cells or in non-malignant cells. In mouse xenografts, α-MT reduced the growth of ER-positive ZR-75-1 breast cancer cells, but did not have any effect on the growth of ER-negative MB231 cells. MCF7 cells did not grow in nude mice unless estrogen was administered, and under these conditions, α-MT did not block the growth of the cells. Plasma concentration of α-MT in treated mice was 8.5 ± 1.5 μM. α-MT was not detectable in the plasma of untreated mice. Conclusions: SLC6A14 is an ideal and effective drug target for treatment of ER-positive breast cancer. α-MT has potential for use as an anticancer drug, and may also serve as a lead compound in the design/development of newer blockers of SLC6A14 for use in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4459. doi:10.1158/1538-7445.AM2011-4459

STAT3 (Signal Transducer and Activator of Transcription 3) is persistently activated in many human primary tumors as well as in cancer cell lines, including breast cancer. This activated STAT3 is associated with cell survival and proliferation, inhibition of apoptosis, activation of angiogenesis and suppression of immune surveillance. Thus, STAT3 is generally considered as a growth-promoting factor. However, STAT3 activation also plays an important role in initiation of apoptotic signaling, especially, in mammary epithelial cells during mammary gland involution. Conditional deletion of STAT3 is associated with delayed involution mainly due to inadequate induction of apoptosis in mammary epithelial cells. Additionally, STAT3 activation is associated with induction of growth arrest and apoptosis in mouse mammary epithelial cells, myeloid cells and granulocyte. Thus, STAT3 plays dual role as a growth promoter as well as a growth inhibitor. However, the molecular switch that is responsible for switching the STAT3- associated pro-oncogenic signal to pro-apoptotic signal is not known. In order to identify this molecular switch we screened for signaling molecules that regulate STAT3 as well as those that are regulated by STAT3 during mammary gland involution, in human primary breast tumor tissues, and in human breast cancer cell lines. Our results show that early in the onset of mammary gland involution there is a significant up regulation of STAT3 and SLC5A8, a Na+-coupled transporter for monocarboxylates, including butyrate an inhibitor of histone deacetylases (HDACs). Further, STAT3 expression during mammary gland involution was found to be predominantly acetylated and phosphorylated. However, in human breast cancer cell lines we found an increase in phosphorylated STAT3 with almost undetectable expression of SLC5A8 and significantly decreased expression of acetylated STAT3 expression. Heterologous expression of SLC5A8 in human mammary tumor cell lines resulted in a butyrate-dependent induction of apoptosis with an increase in acetylated forms of STAT3. These results suggest that SLC5A8 is the molecular switch that converts the pro-oncogenic STAT3 signal to a pro-apoptotic signal by mediating the butyrate entry into mammary epithelial cells and thus it activates death receptor signaling by inhibiting HDACs and activation of STAT3 acetylation during mammary gland involution. However, in cancer cells the expression of SLC5A8 is silenced and thus it no longer able to inhibits HDACs and activates Ac-Stat3 and death receptor signaling. Thus, our studies provide a molecular link between the STAT3-associated pro-apoptotic and pro-oncogenic signaling that observed in normal mammary epithelium and in human breast cancer, respectively. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-236.

Objective: To determine the anti-tumor effect of isoliquiritigenin (ISL) on endometrial cancer cell and to evaluate its effect on apoptosis in Hec1A endometrial cancer cell lines. Methods: Human endometrial cancer cell lines (Hec1A) and Ishikawa, and normal endometrial cell line (T-HESCS) were cultured in vitro. The viabilities of three cell lines on ISL were measured. Cell cycle distribution and induction of apoptosis were measured in Hec1A cells after ISL treatment. Results: ISL significantly reduced cell viabilities of endometrial cancer cell lines but not normal cell line in a dose-dependent manner. Cell cycle analysis indicated that ISL treatment increased the proportion of cells in the sub-G0/G1 phase. DNA frag- mentation and fluorometric TUNEL assays also revealed apoptotic cell death after ISL incubation. ISL treatment markedly up-regulated the expression of cyclin-dependent kinase inhibitor, p21 Cip1/Waf1 in a p53 independent manner and down regulated the expressions of cyclins and CDKs, with concomitant increase in FAS and cleavage of caspase 7, caspase 8, and caspase 9. In addition, elevation of caspase 3 activity also observed in a dose and time dependent manner. Conclusion: ISL inhibited cell proliferation and triggered apoptosis in human endometrial cancer cell line Hec1A. Hence, ISL can be used as a potentially potent clinical chemotherapeutic agent for treating endometrial cancer.

Objective：Hibiscus protocatechuic acid (PCA) is a food-derived polyphenol antioxidants used as a food additive and a traditional herbal medicine. In this study, PCA was to determine its effect on cell proliferation and cell cycle progression in primary cultured human uterine leiomyoma cells. Methods：The effect of PCA on cell proliferation and cell cycle progression was examined in the primary cultured human uterine leiomyoma cells. MTT reduction assay was carried out to determine the viability of uterine leiomyoma cells. Cell cycle analysis for Hibiscus protocatechuic acid treated leiomyoma cells was done by FACS analysis. DNA fragmentation assay was performed to determine fragmentation rate by PCA in leiomyoma cells. Western blot analysis was done using anti pRB, anti-p21cip1/waf1, anti-p53, anti-p27kip1, anti-cyclinE, anti CDK2 antibodies to detect the presence and expression of these proteins in PCA treated myoma cells. Results：PCA induced growth inhibition in a dose dependent manner, treatment with 5 mmol/L PCA blocked 80% cell growth. FACS results showed that there was increased the percentage of cells in sub G1. DNA fragmentation assay by ELISA was done to find the rate of apoptosis. Apoptosis took place but in a dose dependent manner. From Western blot analysis it revealed PCA induced the expression of p21cip1/waf1 and p27kip1 increasingly and was not mediated by p53. Caspase-7 pathway was activated and dephosphorylation of pRB took place. Conclusion：In Conclusions, PCA, a polyphenol antioxidant, inhibited cell proliferation and induced cell cycle arrest at sub G1 phase by enhancing the production of p21cip1/waf1 and p27kip1. These results indicate that PCA will be a promising agent for use in chemopreventive or therapeutics against human uterine leiomyoma.