KISS1: Defining Mechanisms for Antimetastatic Therapy

描述(申请人提供)：由于大多数癌症死亡和发病是由转移引起的，当转移得到预防或更有效地治疗时，生存和生活质量将会得到重大改善。确定KISS1转移抑制基因是否能维持播散的肿瘤细胞处于非增殖、休眠状态，可能为癌症治疗提供一种新的途径。背景：KISS1的重新表达阻止了黑色素瘤细胞在多个异位部位定植的能力，同时仍然完成了转移级联的先期步骤。Kiss1被加工成许多多肽，称为Kisspeptins。一些Kispeptins结合并刺激G蛋白偶联受体Gpr54；然而，转移抑制似乎不需要肿瘤细胞Gpr54的表达。假设1：Kiss1的重新表达将阻止已建立的(微)转移瘤的进一步生长。具体目的1：将Tet-诱导型表达载体应用于肺种植GFP黑色素瘤细胞中，当肺内病灶大小不同时，KISS1的表达将被诱导或关闭。进一步的生长、退化或诱导休眠将通过荧光显微镜进行评估。含义：如果KISS1能够阻止进展或逆转已建立的转移，其治疗人类癌症的有效性将显著增加。假设2：选定的Kispeptins负责抑制肿瘤转移。具体目的2：初步数据显示Kiss1-&gt；Kisspeptins的加工发生在细胞外，但Kisspeptin(S)抑制肿瘤转移的作用尚未确定。利用定点突变，KISS1中的KISS1-&gt；kisspeptin加工位点(R-R或R-K)的中断和加工突变体的异位表达将在随后通过Gpr54和转移潜能评估kisspeptin诱导的信号转导。提示：确定哪些Kispeptin(S)负责抑制转移将是未来激动剂开发的重点。假设3：Kiss1通过与基质细胞的旁分泌信号抑制肿瘤转移。具体目的3：初步数据显示，被KISS1重新表达抑制的肿瘤细胞不表达GPR54，这表明肿瘤细胞产生KISS1是通过中间细胞(即旁分泌)起作用的。最初的工作假设是间质成纤维细胞是中间细胞，因为它们在体外表达GPR54。使用RTQ和IHC，我们将确定哪些基质细胞原位表达GPR54。将亲本细胞和表达KISS1的细胞与来自不同组织的成纤维细胞进行2D和3D共培养，将评估组织基质对KISS1的不同反应是否将促进生长或抑制生长的信号返回给肿瘤细胞。这些实验最终测试了KISS1在抑制转移方面的实际靶点是什么。在临床上的应用将根据靶向是肿瘤细胞还是正常细胞而有所不同。来自拟议实验的数据将确定特定Kispeptin(S)的分子和细胞靶点(S)以及抑制转移所需的暴露时间方面。如果KISS1要被开发为抗转移疗法，这些数据将是至关重要的。

DESCRIPTION (provided by applicant): Since the majority of cancer deaths and morbidity are due to metastases, major improvements in survival and quality of life will occur when metastasis are prevented or more effectively treated. Determining whether the KISS1 metastasis suppressor can maintain disseminated tumor cells in a non- proliferating, dormant state may provide a novel approach to cancer therapy. BACKGROUND: Re-expression of KISS1 blocks the ability of melanoma cells to colonize multiple ectopic sites while still completing antecedent steps of the metastatic cascade. KISS1 is processed into numerous peptides, termed kisspeptins. Some kisspeptins bind to and stimulate a G-protein coupled receptor, GPR54; however, metastasis suppression does not appear to require tumor cell GPR54 expression. HYPOTHESIS #1: KISS1 re- expression will halt further growth of established (micro)metastases. Specific Aim 1: Using Tet-inducible expression vectors in GFP-expressing melanoma cells seeding lung, KISS1 expression will be induced or turned off when lung foci are different sizes. Further growth, regression or induced dormancy will be assessed by fluorescence microscopy. Implications: If KISS1 could halt progression or reverse established metastases, its utility for treatment of human cancer would increase significantly. HYPOTHESIS #2: Selected kisspeptins are responsible for metastasis suppression. Specific Aim 2: Preliminary data show that KISS1 -> kisspeptins processing occurs outside the cell, but which kisspeptin(s) suppresses metastasis has not been determined. Using site-directed mutagenesis, disruption of KISS1->kisspeptin processing sites (R-R or R-K) in KISS1 and ectopic expression of the processing mutants will be done followed by assessment of kisspeptin-induced signaling thru GPR54 and metastatic potential. Implications: Identifying which kisspeptin(s) are responsible for metastasis suppression will focus future agonist development. HYPOTHESIS #3: KISS1 suppresses metastasis via paracrine signaling with stromal cells. Specific Aim 3: Preliminary data show that tumor cells suppressed by re-expression of KISS1 do not express GPR54, suggesting that KISS1 production by tumor cells acts via intermediary cells (i.e., paracrine). The initial working hypothesis is that stromal fibroblasts are the intermediary cell because they express GPR54 in vitro. Using RTQ and IHC, we will determine which stromal cells express GPR54 in situ. 2D and 3D co-culture of parental and KISS1-expressing cells with fibro- blasts from different tissues will assess whether differential response of tissue stroma to KISS1 returns growth promoting or growth inhibitory signals to tumor cells. Implications: These experiments ultimately test what the actual target of KISS1 is with regard to metastasis suppression. Utilization in the clinic will vary depending upon whether one is targeting tumor cells or normal cells. Data from the proposed experiments will determine the molecular and cellular target(s) of specific kisspeptin(s) and aspects of the timing of exposure required to suppress metastasis. Those data will be crucial if KISS1 is to be developed as an anti-metastatic therapy.