Determination of molecular pathways regulating LOT1 mediated growth suppressions

（申请人描述）我们之前报道过通过分析正常和恶性转化大鼠卵巢表面上皮（ROSE）细胞（一种体外卵巢癌模型）的基因表达差异，发现LOT1 （Lost On Transformation）基因（GenBank accession #U72620）。与正常祖细胞相比，该基因在独立转化的ROSE细胞系中经常表现出表达减少或缺失。基于这些观察结果，我们认为LOT1基因是参与调节人类卵巢癌的潜在候选基因。为了将模型系统中的这一发现转化为人类疾病，我们克隆了人类同源物LOT1 （GenBank登录#72621）。有趣的是，人类基因定位于6号染色体25带（6q25），这是卵巢癌和许多其他实体肿瘤中杂合性缺失（LOH）的位点。在11个卵巢癌细胞系中，有7个细胞系的LOT1基因表达缺失或减少，这与在大鼠卵巢癌细胞系中的发现一致。推导出的LOT1的氨基酸序列表明它是一个锌指基序，含有转录因子特征结构域的蛋白质。功能分析证实它是一种具有潜在转录因子作用的核蛋白。我们还发现LOT1基因参与表皮生长因子受体（epidermal growth factor receptor， EGFR）信号通路，是卵巢癌细胞生长的负调控因子。本研究的目的是通过确定LOT1的下调或表达/功能缺失如何传导抗增殖信号，并作为肿瘤/生长抑制基因调节其他抑制恶性表型的基因，进一步确定LOT1的临床重要性。为了详细阐述这些机制，我们将研究与LOT蛋白作用相关的上游调节因子和下游事件，并确定这些正常过程在卵巢恶性肿瘤中是如何受到干扰的。希望这些努力将有助于更好地了解卵巢癌的发展和/或进展，并为更有效地诊断、治疗和/或管理卵巢癌提供基础。

DESCRIPTION: (Applicant's Description) We previously reported discovery of the LOT1 (Lost On Transformation) gene (GenBank accession #U72620) through analysis of gene expression differences between normal and malignantly transformed rat ovarian surface epithelial (ROSE) cells, an in vitro ovarian cancer model. The gene frequently shows decreased or lost expression in independently transformed ROSE cell lines compared to the normal progenitor cells. Based on these observations, we considered the LOT1 gene a potential candidate gene involved in regulating human ovarian cancer. In an effort to translate this finding in a model system to human disease, we cloned the human homologue, LOT1 (GenBank accession #72621). Interestingly, the human gene maps to chromosome 6 at band 25 (6q25), which is a site for loss of heterozygosity (LOH) in ovarian cancer and many other solid tumors. Expression of the LOT1 gene was lost or decreased in seven out of eleven ovarian cancer cell lines consistent with the findings in the rat ovarian cancer cell lines. The deduced amino acid sequence of LOT1 indicates that it is a zinc-finger motif containing protein with domains characteristic of transcription factors. Functional assays proved that it is a nuclear protein with a potential role as a transcription factor. We also found that the LOT1 gene is involved in the epidermal growth factor receptor (EGFR) signaling pathway and is a negative regulator of the ovarian cancer cell growth. The goal of this proposal is to further ascertain the clinical importance of LOT1 by determining how down-regulation or lost expression/function of LOT1 transduces anti-proliferative signals and behaves as a tumor/growth suppresser gene regulating other genes, which inhibit the malignant phenotype. To elaborate these mechanisms we will investigate the upstream regulators and downstream events associated with the action of the LOT protein and to determine how these normal processes are perturbed in ovarian malignancies. Hopefully, these endeavors will result in a better understanding of the ovarian cancer development and/or progression and provide a basis for more effective diagnosis, treatment, and/or management of the disease.