Tumor-Suppressive Function of SLC5A8 in Mammary Gland and its Relevance to Breast

项目描述（由申请人提供）：本项目旨在研究SLC5A8在乳腺中的抑瘤功能，评价其在乳腺癌治疗中的治疗潜力。SLC5A8是一种质膜转运蛋白，最初被认为是结肠中的肿瘤抑制因子，但其转运功能的身份和如何引起肿瘤抑制尚不清楚。我们最近的研究不仅确定了该蛋白的转运功能，而且还确定了该转运蛋白促进肿瘤细胞特异性凋亡的机制。SLC5A8是单羧酸盐（如丙酸盐、丁酸盐、乳酸盐、丙酮酸盐）的Na+偶联转运体。丁酸盐是一种众所周知的组蛋白去乙酰化酶（HDACs）抑制剂，在结肠中由细菌发酵产生。HDAC抑制剂优先杀死肿瘤细胞。我们最近表明，丙酮酸也是一种有效的HDAC抑制剂，丙酮酸和丁酸盐以slc5a8依赖的方式诱导肿瘤细胞特异性凋亡。因此，SLC5A8介导HDAC抑制剂丙酮酸盐和/或丁酸盐进入细胞的能力是该转运体肿瘤抑制功能的基础。SLC5A8在正常乳腺中表达，在乳腺癌中通过DNA甲基化沉默。该转运体在乳腺肿瘤细胞系中的外源性表达以丙酮酸/丁酸依赖的方式导致细胞死亡。SLC5A8基因在乳腺上皮细胞中的甲基化涉及DNA甲基转移酶DNMT1；癌基因Ras的激活在这一过程中起着关键作用。用DNA去甲基化剂治疗乳腺肿瘤细胞可重新激活SLC5A8的表达。这种现象见于雌激素受体（ER）阳性和ER阴性细胞。重要的是，在丙酮酸盐或丁酸盐存在的情况下，SLC5A8的重新表达会导致er阳性和er阴性细胞死亡。为了更好地了解乳腺癌细胞是如何下调SLC5A8的，以及转运体表达的再激活是如何导致肿瘤细胞特异性凋亡和阻断乳腺肿瘤发生的，我们提出了三个目标。目的1：探讨SLC5A8在乳腺癌中的下调机制。假设：SLC5A8甲基化相关的沉默是继发于ras相关的MAPK信号通路的激活。目的2是确认SLC5A8在体内阻断乳腺肿瘤发生的能力。假设：乳腺癌中SLC5A8的再激活或正常乳腺中SLC5A8的过表达将有效阻断乳腺肿瘤的形成。目的3是评估SLC5A8在er阴性乳腺癌中的治疗潜力。假设：在er阴性乳腺癌中激活SLC5A8表达会诱导丙酮酸/丁酸介导的肿瘤细胞特异性凋亡。这些研究将产生重要的和临床/治疗相关的信息，这将有助于制定预防和/或治疗乳腺癌的新策略。

DESCRIPTION (provided by applicant): The purpose of this project is to investigate the tumor-suppressive function of SLC5A8 in mammary gland and to evaluate its therapeutic potential in the treatment of breast cancer. SLC5A8 is a plasma membrane transporter originally identified as a tumor suppressor in colon, but neither the identity of its transport function nor how it elicits tumor suppression was known. Our recent studies have not only established the identity of the transport function of this protein but also the mechanism by which the transporter promotes tumor cell-specific apoptosis. SLC5A8 is a Na+coupled transporter for monocarboxylates (e.g., propionate, butyrate, lactate, pyruvate). Butyrate is a well known inhibitor of histone deacetylases (HDACs) and is generated in the colon by bacterial fermentation. HDAC inhibitors preferentially kill tumor cells. We have shown recently that pyruvate is also a potent HDAC inhibitor and that pyruvate and butyrate induce tumor cell-specific apoptosis in a SLC5A8-dependent manner. Thus, the ability of SLC5A8 to mediate the concentrative entry of the HDAC inhibitors pyruvate and/or butyrate into cells underlies the tumor-suppressive function of this transporter. SLC5A8 is expressed in normal mammary gland and the gene is silenced in breast cancer by DNA methylation. Exogenous expression of the transporter in mammary tumor cell lines leads to cell death in a pyruvate/butyrate-dependent manner. Methylation of SLC5A8 gene in mammary epithelial cells involves the DNA methyltransferase DNMT1; activation of the oncogene Ras plays a critical role in this process. Treatment of mammary tumor cells with DNA demethylating agents re-activates SLC5A8 expression. This phenomenon is seen in estrogen receptor (ER)-positive and in ER-negative cells. Importantly, re-expression of SLC5A8 leads to cell death both in ER-positive and ER-negative cells in the presence of pyruvate or butyrate. To achieve a better understanding of how breast cancer cells down-regulate SLC5A8 and how re-activation of the transporter expression leads to tumor cell-specific apoptosis and blockade of mammary tumorigenesis, we propose three aims. Aim 1 is to investigate the mechanism of down-regulation of SLC5A8 in breast cancer. Hypothesis: Methylation-associated silencing of SLC5A8 is secondary to the activation of Ras-associated MAPK signaling pathway. Aim 2 is to confirm the ability of SLC5A8 to block mammary tumorigenesis in vivo. Hypothesis: Re-activation of SLC5A8 in breast cancer or over-expression of SLC5A8 in normal mammary gland will effectively block mammary tumor formation. Aim 3 is to evaluate the therapeutic potential of SLC5A8 in ER-negative breast cancer. Hypothesis: Activation of SLC5A8 expression in ER-negative breast cancer will induce pyruvate/butyrate-mediated tumor cell-specific apoptosis. These studies will generate important and clinically/therapeutically relevant information which will aid in the development of novel strategies for the prevention and/or treatment of breast cancer. PUBLIC HEALTH RELEVANCE: The purpose of this project is to investigate the potential of a newly discovered tumor suppressor as a drug target for the treatment of breast cancer. This tumor suppressor is a plasma membrane transporter which mediates the entry of histone deacetylase (HDAC) inhibitors pyruvate and butyrate into cells. HDAC inhibition causes tumor cell-specific apoptosis and cell death. Breast cancer cells shut off the expression of the transporter to avoid this cell death. In the current project, we will test the hypothesis that re-activation of the expression of the transporter gene in breast cancer cells would induce tumor cell-specific apoptosis through transporter-mediated entry of pyruvate or butyrate. This mode of therapy may be effective not only for the estrogen receptor-positive breast cancer but also for the estrogen receptor-negative breast cancer.