Targeting Mixed Lineage Kinase3-Hedgehog Axis in Pancreatic Cancer

描述(由申请人提供)： 混合谱系激酶3(MLK3)是一种应激激活的MAP激酶(MAP3K)成员，其生物学功能尚不清楚。在剖析MLK3介导的信号通路时，我们观察到MLK3对丝氨酸138残基上的一个Pro基异构酶Pin1有很强的磷酸化作用。这些结果意义重大，因为Pin1被认为是一种重要的癌基因，几乎在所有类型的癌症中都过表达。Pin1特异性抑制剂正在开发中，用于治疗癌症。值得注意的是，磷酸化的Pin1(即Pin1S138E突变体)定位在细胞核中，这表明这种依赖磷酸化的Pin1易位可能具有某些生理功能。证实了我们的假设，磷酸化的Pin1在肿瘤中过表达，但在正常匹配的组织中不表达，p-Pin1是细胞周期进展所必需的。因此，我们冒险寻找p-Pin1促进肿瘤发生的靶点。我们发现p-Pin1(即S138E)能够上调Hedgehog途径中的转录因子Gli1的mRNA，Gli1的异常调节与包括胰腺癌在内的癌症有关。此外，我们还观察到趋化因子CXCL5的mRNA在MLK3基因敲除的肝脏中下调，表明MLK3是CXCL5表达所必需的。这些结果非常耐人寻味，因为Gli1激活/失调与胰腺癌的发病机制有关，而且还报道了在动物模型中抑制Hedgehog通路可以克服治疗胰腺癌的常用药物吉西他滨的耐药性。最近，胰腺癌细胞株中CXCL5及其受体的阻断被证明能够抑制胰腺导管癌细胞(PDA)的生长，而胰腺癌细胞株产生的CXCL5能够促进血管生成。我们的初步研究进一步表明，在胰腺癌细胞系中，MLK3的活性受CXCL5的调节，并且MLK3的活性与CXCL5在这些细胞系中的表达水平相关。我们还观察到，在原发的人胰腺肿瘤中，MLK3的活性与肿瘤中Pin1和Gli1的表达水平直接相关。基于这些结果，我们认为现有的MLKs抑制剂CEP1347/CEP-11004或与PIN1抑制剂联合使用应该诱导胰腺癌细胞死亡？根据我们的初步数据，我们假设在胰腺癌细胞中，MLK3激活Pin1及其下游的Gli1，导致PDA。因此，靶向MLK3或其他MLKs的同时抑制Pin1可以根除胰腺肿瘤。为了达到我们的目标，我们将确定：(1)MLK3-Pin1轴激活Gli1促进胰腺细胞肿瘤的发生；(2)破坏MLK3-Pin1轴抑制胰腺癌细胞的发生；(3)我们将确定MLK3、Pin1和Gli1激活在胰腺细胞肿瘤发生中的机制。通过明确MLK3在Pin1和Gli1激活中在胰腺肿瘤发生中的作用，我们有望通过使用这一途径的有效抑制剂来控制/治疗胰腺癌。有趣的是，MLK3家族的特异性抑制剂CEP-1347已经用于治疗神经退行性疾病的临床试验，而特异性Pin1抑制剂正在开发中，用于癌症治疗。也有人建议MLKs抑制剂可能用于治疗特定类型的癌症，强调了我们关于MLK3在胰腺癌中作用的意想不到的新结果。综上所述，本研究将导致识别新的预后因素和针对难治性和致命性胰腺癌的特定靶向治疗。

DESCRIPTION (provided by applicant): Mixed Lineage Kinase 3 (MLK3) is a stress-activated MAP Kinase Kinase Kinase (MAP3K) member, and its biological function is still elusive. While dissecting the signaling pathway mediated via MLK3, we observed a robust phosphorylation of a prolyl isomerase Pin1 by MLK3 on Serine138 residue. These results were significant as Pin1 is considered an important oncogene and is overexpressed in almost all types of cancer. The Pin1 specific inhibitors are under development to treat cancer. Remarkably, the phosphorylated Pin1 (i.e. Pin1S138E mutant) was localized in the nucleus, suggesting that this phosphorylation-dependent translocation of Pin1 could have some physiological function. Corroborating our hypothesis, the phosphorylated Pin1 was overexpressed in tumors but not in normal matching tissues and p-Pin1 was necessary for cell cycle progression. Therefore, we ventured to find the targets of p-Pin1 that could promote tumorigenesis. We identified that p-Pin1 (i.e. S138E) was able to up regulate mRNA of Gli1, a transcription factor in the Hedgehog pathway, whose dysregulation has been linked to cancer, including pancreatic cancer. Furthermore, we also observed that mRNA of a chemokine, CXCL5 was downregulated in MLK3 knockout liver, suggesting that MLK3 is necessary for CXCL5 expression. These results were quite intriguing because Gli1 activation/dysregulation has been implicated in pancreatic cancer pathogenesis and also reported that inhibition of Hedgehog pathway overcomes gemcitabine (common drug for pancreatic cancer) resistance in animal model. More recently, CXCL5 and its receptor blockage in pancreatic cancer cell lines was shown to inhibit the pancreatic ductal carcinoma (PDA) cell growth and CXCL5 produced by pancreatic cancer cell lines was able to promote angiogenesis. Our preliminary investigation further revealed that MLK3 activity was regulated by CXCL5 in pancreatic cancer cell lines and the MLK3 activity correlated with the expression levels of CXCL5 in these cell lines. We also observed that in primary human pancreatic tumors, the MLK3 activities directly correlated with the expression levels of Pin1 and Gli1 in tumors. Based on these results, we conceive that the available MLKs inhibitor, CEP-1347/CEP-11004 or in combination with Pin1 inhibitor, should induce cell death in pancreatic cancer cells? Based on our preliminary data, we hypothesize that in pancreatic cancer cells, MLK3 activates Pin1 and its downstream Gli1, leading PDA. Therefore targeting MLK3 or other MLKs along with Pin1 inhibition could abrogate pancreatic tumors. To achieve our goals, we will determine that: (1) activation of Gli1 by MLK3-Pin1 axis promotes pancreatic cell tumorigenesis, (2) disruption of MLK3- Pin1 axis attenuates pancreatic cancer cell tumorigenesis; and (3) we will determine the mechanism of MLK3, Pin1 and Gli1 activation in pancreatic cell tumorigenesis. It is expected that by defining the role of MLK3 in Pin1 and Gli1 activation in pancreatic tumorigenesis, we might control/treat pancreatic cancers by using available inhibitors of this pathway. Interestingly the specific inhibitor of MLK3 family, CEP-1347 has been used in clinical trials for treating neurodegenerative diseases and specific Pin1 inhibitors are under development for cancer treatment. It has also been suggested that MLKs inhibitors might serve as a treatment for specific type of cancer, underscoring our unexpected novel results related to MLK3 role in pancreatic cancer. Taken together, the present study will lead to the identification of new prognostic factors and specific targeted therapies for difficult to treat and lethal pancreatic cancer.