The Role of HOXA5 in Breast Cancer

描述（由申请人提供）：我们观察到，与正常乳腺上皮细胞（HMEC）相比，同源基因HOXA5在乳腺癌细胞系和原发性乳腺肿瘤中的表达降低。我们之前已经证明了它调节p53和孕酮受体表达的能力。最近，使用两种不同的技术，我们已经确定了对HOXA5有反应或与之相互作用的基因。1)通过对表达人类HOXA5的酿酒酵母进行微阵列分析，我们发现了可能受HOXA5蛋白调控的新的假定靶基因。酵母中被HOXA5上调的基因之一是DNA错配修复基因，其人类同源基因被鉴定为MutL-1 （hMLH1）。hMLH1的启动子区域包含四个推测的HOXA5结合位点。我们现在已经证明，在乳腺癌细胞系MCF- 7中，HOXA5的过表达上调了hmlh1。我们现在将评估HOXA5对hMLH1的调控及其在维持细胞基因组完整性的关键功能中的作用。2)通过酵母双杂交分析，我们确定了一个抗凋亡基因Twist作为HOXA5蛋白的相互作用因子。我们已经证明：a) Twist在乳腺癌细胞系中的表达导致p27[Kip1]水平下降，同时增加了细胞在血清饥饿期间的生存能力；b)在功能测试中，HOXA5可以部分恢复Twist对p53靶基因的抑制作用。我们将全面描述HOXA5/Twist相互作用的性质。除了研究这些蛋白之间的生化相互作用外，我们还希望阐明HOXA5在乳腺中的生物学作用。60%的乳腺癌发生HOXA5表达缺失。然而，关于其在正常乳腺发育中的表达，或与其他预后因素的关系，我们知之甚少。3)我们将利用免疫组化（IHC）技术表征HOXA5在正常乳腺从青春期前到绝经后发育阶段以及癌症中的表达。通过免疫组化检查乳腺肿瘤阵列，我们想确定HOXA5的缺失是否与高级别乳腺肿瘤的形成相关。因此，我们建议深入研究HOXA5在乳腺癌中的作用。这些研究将对HOXA5的抑瘤作用有新的认识，并进一步阐明HOXA5在乳腺肿瘤发生中的分子功能。

DESCRIPTION (provided by applicant): We observed that the homeotic gene, HOXA5 has decreased expression in breast cancer cell lines and in primary breast tumors, when compared to normal mammary epithelial cells (HMEC). We have previously demonstrated its ability to modulate the expression of p53 and the progesterone receptor. Recently, using two separate technologies, we have identified genes that are responsive to, or interact with HOXA5. 1) By performing microarray analysis of Saccharomyces cerevisiae that express human HOXA5, we have identified novel putative target genes that may be regulated by HOXA5 protein. One of the genes that is up-regulated by HOXA5 in yeast is a DNA mismatch repair gene whose human paralog was identified as MutL-1 (hMLH1). The promoter region of hMLH1 contains four putative HOXA5 binding sites. We have now demonstrated that over-expression of HOXA5 up-regulates hMLH 1 in a breast cancer cell line, MCF- 7. We will now evaluate the regulation of hMLH1 by HOXA5 and its role in the critical function of maintaining the integrity of the cellular genome. 2) Using yeast two-hybrid analysis, we have identified an anti-apoptotic gene, Twist, as an interactor of HOXA5 protein. We have shown that: a) expression of Twist in a breast cancer cell line caused a decrease in p27[Kip1] levels while increasing the cell's ability to survive during serum starvation and, b) in functional assays, HOXA5 can partially restore the suppressive effects of Twist on p53 target genes. We will fully characterize the nature of the HOXA5/Twist interaction. In addition to studying the biochemical interactions between these proteins, we would like to shed light on the biology of HOXA5 in the breast. Loss of expression of HOXA5 occurs in 60% of breast cancers. However, little is known regarding its expression throughout normal breast development, or its relationship with other prognostic factors. 3) We will use immunohistochemistry (IHC) to characterize HOXA5 expression in normal breast during development from pre-pubertal to postmenopausal stages and in cancer. By examining breast tumor arrays by IHC, we would like to determine if loss of HOXA5 correlates with high grade breast tumor formation. Thus, we propose to examine, in depth, the role of HOXA5 in breast cancer. These studies will yield new insights into the tumor suppressor role of HOXA5 and further elucidate the molecular functions of HOXA5 in breast tumorigenesis.