Combinatorial Approach for Reducing Invasive Potential of Melanoma Cells

描述（由申请人提供）：黑色素瘤是过去几十年中增长最快的恶性肿瘤之一。它是一种严重的癌症，约占皮肤癌相关死亡的80%。黑色素瘤对目前的治疗方案有抗性，因为它有转移的倾向，并且预后非常差，中位生存率为6个月。研究表明，大多数人黑色素瘤通过BRAF或NRAS中的致癌突变具有组成型活性的丝裂原活化蛋白激酶（MAPK）。在恶性黑色素瘤中，BRAF-MEK-ERK和PI 3 K-AKT-NFkB信号传导途径被RAS信号组成性激活和调节。这些途径的激活导致上皮-间充质转化（EMT）的诱导，从而导致细胞侵袭。EMT是上皮细胞经历从极化分化表型到间充质表型的重编程的分子程序，并且被认为是调节转移进展的初始步骤的关键过程。索拉非尼靶向BRAF导致MAPK信号通路在体外和体内的抑制。然而，作为单药治疗，它在转移性黑色素瘤患者中无效，如II期临床研究所示。这些数据和其他已发表的研究支持这样的假设，即仅抑制单一组成性激活的信号通路不足以治疗黑色素瘤。最近，我们发现非瑟酮治疗通过抑制PI 3 K（p110 a和p85）的蛋白表达和AKT在Ser 473和Thr 308的磷酸化来减少黑素瘤细胞的侵袭。它还降低了转移性黑色素瘤A375细胞在组织工程三维皮肤等同物中的侵袭潜力。此外，发现非瑟酮处理减少A375细胞集落的形成。本提案利用了我们最近未发表的新发现，旨在研究非瑟酮单独使用以及与索拉非尼联合使用通过靶向BRAF和PI 3 K/AKT通路对黑色素瘤细胞侵袭和转移的影响。在这个提议中要检验的中心假设是“非瑟酮与索拉非尼的组合 将比单独使用非瑟酮或索拉非尼更有效地减少黑色素瘤细胞侵袭和转移”。为了检验我们的假设，提出了两个相互关联的具体目标：（I）通过靶向BRAF和PI 3 K/AKT信号传导途径，研究单独的非瑟酮和与索拉非尼组合对黑色素瘤细胞系和组织工程化三维皮肤等同物中的侵袭和EMT的作用，和（II）确定单独的非瑟酮和与索拉非尼组合对无胸腺Crl：NU-Foxn 1 nu裸鼠通过静脉注射GFP标记的黑色素瘤细胞。健康相关性：通过该提议，我们设想鉴定天然无毒膳食类黄酮非瑟酮，其可以与索拉非尼组合用于预防细胞侵袭和转移，大量患者将从中受益。

DESCRIPTION (provided by applicant): Melanoma is one of the fastest-rising malignancies in the past few decades. It is a serious form of cancer responsible for approximately 80% of skin cancer-related deaths. Melanoma is resistant to current therapeutic regimens because for its propensity to metastasize and has a very poor prognosis with a median survival rate of six months. Studies have demonstrated that the majority of human melanomas have constitutively active mitogen activated protein kinases (MAPK) through oncogenic mutation in either BRAF or NRAS. In malignant melanoma the BRAF-MEK-ERK and PI3K-AKT-NFkB signaling pathways are constitutively activated and regulated by RAS signals. Activation of these pathways leads to an induction of epithelial-mesenchymal transition (EMT) resulting in cell invasion. EMT is a molecular program whereby epithelial cells undergo reprogramming from a polarized differentiated phenotype to a mesenchymal phenotype and is considered to be a key process in regulating the initial step of metastatic progression. Targeting BRAF with sorafenib resulted in inhibition of MAPK signaling pathways both in vitro and in vivo. However, as a monotherapy it is not effective in patients with metastatic melanoma as revealed by a phase II clinical study. These data and other published studies support the hypothesis that it is not sufficient to inhibit only a single constitutively activated signaling pathway for the treatment of melanoma. Recently, we found that fisetin treatment reduced melanoma cell invasion by inhibiting the protein expression of PI3K (p110a and p85) and phosphorylation of AKT at Ser473 and Thr308. It also reduced invasive potential of metastatic melanoma A375 cells in tissue-engineered three dimensional skin equivalents. Furthermore, fisetin treatment was found to reduce the formation of A375 cell colonies. The present proposal capitalizes on our recent novel unpublished findings and is designed to investigate the effects of fisetin alone and in combination with sorafenib on invasion and metastasis of melanoma cells by targeting BRAF and PI3K/AKT pathways. The central hypothesis to be tested in this proposal is that the "combination of fisetin with sorafenib will be more effective in reducing melanoma cell invasion and metastasis than with fisetin or sorafenib alone". To test our hypothesis, two inter-related specific aims are proposed: (I) to investigate the effect of fisetin alone and in combination with sorafenib on invasion and EMT in melanoma cell lines and in tissue-engineered three dimensional skin equivalents by targeting BRAF and PI3K/AKT signaling pathways, and (II) to determine the effect of fisetin alone and in combination with sorafenib on the formation of systemic metastases in athymic Crl:NU-Foxn1nu nude mice by an intravenous injection of GFP-tagged melanoma cells. HEALTH RELEVANCE: Through this proposal we envision the identification of a natural nontoxic dietary flavonoid fisetin, which can be exploited in combination with sorafenib for the prevention of cell invasion and metastasis from which a large number of patients would stand to benefit.