KISS1: Defining Mechanisms for Antimetastatic Therapy

描述（由申请人提供）：由于大多数癌症死亡和发病是由于转移，当转移被预防或更有效地治疗时，生存和生活质量将得到重大改善。确定KISS 1转移抑制因子是否可以将播散的肿瘤细胞维持在非增殖的休眠状态，可能为癌症治疗提供新的方法。背景技术背景：KISS 1的再表达阻断了黑色素瘤细胞在多个异位部位定殖的能力，同时仍然完成转移级联的前期步骤。KISS 1被加工成许多肽，称为kisspeptins。一些kisspeptins结合并刺激G蛋白偶联受体GPR 54;然而，转移抑制似乎不需要肿瘤细胞GPR 54表达。假设#1：KISS 1再表达将阻止已建立的（微）转移的进一步生长。具体目标1：在表达GFP的黑色素瘤细胞接种肺中使用Tet-inducible表达载体，当肺病灶大小不同时，KISS 1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KISS 1可以阻止进展或逆转已建立的转移，那么其用于治疗人类癌症的效用将显着增加。假设#2：选择的kisspeptins负责转移抑制。具体目标二：初步数据显示，KISS 1-> kisspeptin的加工发生在细胞外，但哪种kisspeptin抑制转移尚未确定。使用定点诱变，将进行KISS 1中KISS 1->kisspeptin加工位点（R-R或R-K）的破坏和加工突变体的异位表达，随后评估kisspeptin诱导的通过GPR 54的信号传导和转移潜力。含义：确定哪些kisspeptin负责转移抑制将成为未来激动剂开发的重点。假设#3：KISS 1通过基质细胞的旁分泌信号传导抑制转移。具体目标3：初步数据显示，被KISS 1的再表达抑制的肿瘤细胞不表达GPR 54，表明肿瘤细胞产生KISS 1通过中间细胞（即，旁分泌）。最初的工作假设是基质成纤维细胞是中间细胞，因为它们在体外表达GPR 54。使用RTQ和IHC，我们将确定哪些基质细胞原位表达GPR 54。亲本和表达KISS 1的细胞与来自不同组织的成神经细胞的2D和3D共培养将评估组织基质对KISS 1的差异应答是否向肿瘤细胞返回生长促进或生长抑制信号。这些实验最终测试KISS 1在转移抑制方面的实际靶点是什么。在临床上的应用将取决于是否靶向肿瘤细胞或正常细胞。来自所提出的实验的数据将确定特异性kisspeptin的分子和细胞靶点以及抑制转移所需的暴露时间的方面。如果KISS 1被开发为抗转移疗法，这些数据将是至关重要的。公共卫生相关性：由于大多数癌症死亡和发病是由于转移，当转移被预防或更有效地治疗时，生存和生活质量将得到重大改善。我们提出了一个基于机制的研究，这将使我们能够开发一种新的抗转移策略来治疗癌症。该方法是使用KISS 1转移抑制剂（或衍生的kisspeptin）来维持播散的肿瘤细胞处于非增殖、休眠状态（使转移成为慢性、可控制的疾病而不是急性情况），同时确定特定kisspeptin的分子和细胞靶点以及抑制转移所需的暴露时间方面。

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. PUBLIC HEALTH RELEVANCE: 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. We propose a mechanism-based study that will allow us to develop a novel anti-metastasis strategy to treat cancer. The approach is to use the KISS1 metastasis suppressor (or derivative kisspeptins) to maintain disseminated tumor cells in a non-proliferating, dormant state (rendering metastases a chronic, controllable disease rather than an acute situation) while simultaneously determining the molecular and cellular target(s) of specific kisspeptin(s) and aspects of the timing of exposure required to suppress metastasis.