Regulation of Ovarian Cancer Multidrug Resistance by MAP Kinases

描述(申请人提供)：许多卵巢肿瘤对最初的药物治疗有反应，但复发后对药物治疗变得无效。因此，明确耐药机制对现有和新的癌症治疗方法具有重要意义。选择性的信使核糖核酸剪接影响74%的人类基因组。选择性mRNA剪接因子表达的改变发生在癌症中，并能强烈影响细胞凋亡反应。剪接因子SPF45在卵巢癌细胞中的过度表达导致对他莫昔芬敏感的耐药表型。SPF45介导的耐药机制尚不清楚。许多化疗药物和细胞刺激可诱导丝裂原活化蛋白激酶(MAP)ERK、JNK和p38的激活，所有这些都与通过其蛋白底物的磷酸化来调节细胞生存反应有关。目前，Sam68是唯一已知的受MAP激酶磷酸化影响的剪接因子。我们已经确定SPF45是所有三种MAP激酶的新底物，并确定了SPF45上的两个磷酸化位点。我们产生了磷酸特异性抗体，并在人类腹水和细胞系中显示了SPF45的磷酸化，以响应化疗药物、氧化应激、佛波酯和紫外线照射。最重要的是，丝氨酸氨基转移酶使Ser222上的SPF45磷酸化，增加了其选择性剪接活性。这些数据有力地证明了MAP激酶、SPF45磷酸化、选择性剪接活性和耐药性之间的直接联系。这项应用的总体目标是确定SPF45介导耐药性的机制，以及SPF45的MAP激酶磷酸化如何调节耐药性。这些研究将有助于确定治疗干预的新靶点，以对抗SPF45过表达肿瘤的耐药性。我们假设，SPF45对化疗药物的反应中，MAP激酶的磷酸化通过刺激SPF45的选择性剪接活性来增强耐药表型。我们将通过以下特定目标来验证这一假设：特定目标1.产生针对SPF45的磷酸化特异性抗体。确定MAP激酶特异性SPF45磷酸化模式在化疗药物反应中的变化，并确定参与其中的激酶(S)。具体目的2.用Fas基因分析法检测MAPK的磷酸化对SPF45剪接活性的影响。通过外显子阵列分析确定SPF45的新剪接靶点，并确定这些靶点在SPF45介导的耐药性中的作用机制。具体目的3.阐明SPF45及其磷酸化在卵巢癌前细胞株、肿瘤细胞株、裸鼠肿瘤和人恶性腹水化疗反应中的作用。明确雌激素受体在SPF45介导的耐药性中的作用。公共卫生相关性：卵巢肿瘤经常对化疗药物产生耐药性，但其机制尚不清楚。我们假设，这种现象部分是由细胞内信号通路的能力调节的，该通路在癌细胞中经常被过度激活，以化学方式修饰具有已知耐药作用的蛋白质。阐明这种协同耐药机制将为卵巢癌耐药治疗提供重要信息。

DESCRIPTION (provided by applicant): Many ovarian tumors respond to initial drug therapy, but become refractory to drug treatment upon recurrence. Therefore, defining the mechanisms of drug resistance is important for existing and new cancer therapies. Alternative mRNA splicing affects 74% of the human genome. Alterations in alternative mRNA splicing factor expression occur in cancer and can strongly influence apoptotic responses. Over-expression of the splicing factor SPF45 in ovarian cancer cells results in a tamoxifen sensitive drug-resistant phenotype. The mechanism of SPF45-mediated drug resistance is unknown. Numerous chemotherapeutic agents and cellular stimuli induce activation the mitogen-activated protein kinases (MAP kinases) ERK, JNK and p38, all of which are associated with modulating cell survival responses through phosphorylation of their protein substrates. Currently, Sam68 is the only known splicing factor affected by MAP kinase phosphorylation. We have identified SPF45 as a novel substrate of all three MAP kinases and identified two phosphorylation sites on SPF45. We generated phospho-specific antibodies and have shown SPF45 phosphorylation in human ascites and in cell lines in response to chemotherapeutic drugs, oxidative stress, phorbol esters and UV irradiation. Most importantly, phosphorylation of SPF45 on Ser222 by MAP kinases increases its alternative splicing activity. These data strongly demonstrate a direct link between MAP kinases, SPF45 phosphorylation, alternative splicing activity and drug resistance. The overall objective of this application is to define the mechanism by which SPF45 mediates drug resistance and how resistance is regulated by MAP kinase phosphorylation of SPF45. These studies will enable the identification of novel targets for therapeutic intervention to combat drug resistance in SPF45 over-expressing tumors. We hypothesize that MAP kinase phosphorylation of SPF45 in response to chemotherapeutic agents enhances the drug resistant phenotype by stimulating SPF45 alternative splicing activity. We will test this hypothesis through the following Specific Aims: Specific Aim 1. Generate phospho-specific antibodies to SPF45. Identify the changes in MAP kinase- specific SPF45 phosphorylation patterns in response to chemotherapeutic agents and identify the kinase(s) involved. Specific Aim 2. Determine the effect of MAP kinase phosphorylation on SPF45 splicing activity using the ?fas minigene assay. Identify novel splicing targets of SPF45 by exon array analysis and identify the mechanism by which these targets contribute to SPF45-mediated drug resistance. Specific Aim 3. Elucidate the role of SPF45 and its phosphorylation in the response to chemotherapeutic agent treatment in pre-neoplastic and neoplastic ovarian cell lines, tumors in nude mice and in malignant human ascites. Define the role o estrogen receptors in SPF45-mediated drug resistance. PUBLIC HEALTH RELEVANCE: ovarian tumors often develop resistance to chemotherapeutics, but the mechanisms are poorly understood. We hypothesize that this phenomenon is regulated in part by the ability of an intracellular signaling pathway that is often hyper-activated in cancer cells to chemically modify a protein with a known role in drug resistance. Elucidation of this synergistic mechanism of drug resistance would provide important information for treating drug-resistant ovarian cancer.