Targeting Mixed Lineage Kinase3-Hedgehog Axis in Pancreatic Cancer

描述（由申请人提供）： 混合谱系激酶3（MLK 3）是应激激活的MAP激酶（MAP 3 K）成员，其生物学功能尚不清楚。在剖析通过MLK 3介导的信号通路时，我们观察到MLK 3在丝氨酸138残基上对脯氨酰异构酶Pin 1的强烈磷酸化。这些结果是重要的，因为Pin 1被认为是一种重要的癌基因，并且在几乎所有类型的癌症中过表达。Pin 1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Pin 1（即Pin 1 S138 E突变体）位于细胞核中，这表明Pin 1的磷酸化依赖性易位可能具有某些生理功能。证实了我们的假设，磷酸化Pin 1在肿瘤中过表达，但在正常匹配组织中不表达，并且p-Pin 1是细胞周期进展所必需的。因此，我们尝试寻找p-Pin 1的靶点，以促进肿瘤的发生。我们发现p-Pin 1（即S138 E）能够上调Gli 1的mRNA，Gli 1是Hedgehog通路中的一种转录因子，其失调与癌症有关，包括胰腺癌。此外，我们还观察到趋化因子CXCL 5的mRNA在MLK 3敲除的肝脏中下调，表明MLK 3对于CXCL 5的表达是必需的。这些结果非常有趣，因为Gli 1激活/失调与胰腺癌发病机制有关，并且还报道了Hedgehog途径的抑制克服了动物模型中吉西他滨（胰腺癌常用药物）的耐药性。最近，胰腺癌细胞系中的CXCL 5及其受体阻断显示出抑制胰腺导管癌（PDA）细胞生长，并且胰腺癌细胞系产生的CXCL 5能够促进血管生成。我们的初步研究进一步揭示了MLK 3活性在胰腺癌细胞系中受到CXCL 5的调节，并且MLK 3活性与这些细胞系中CXCL 5的表达水平相关。我们还观察到，在原发性人类胰腺肿瘤中，MLK 3活性与肿瘤中Pin 1和Gli 1的表达水平直接相关。基于这些结果，我们设想可用的MLKs抑制剂CEP-1347/CEP-11004或与Pin 1抑制剂组合，应该诱导胰腺癌细胞的细胞死亡？基于我们的初步数据，我们假设在胰腺癌细胞中，MLK 3激活Pin 1及其下游Gli 1，导致PDA。因此，靶向MLK 3或其他MLK沿着Pin 1抑制可以消除胰腺肿瘤。为了实现我们的目标，我们将确定：（1）MLK 3-Pin 1轴激活Gli 1促进胰腺细胞肿瘤发生，（2）MLK 3-Pin 1轴的破坏减弱胰腺癌细胞肿瘤发生;（3）我们将确定MLK 3，Pin 1和Gli 1激活胰腺细胞肿瘤发生的机制。预期通过确定MLK 3在胰腺肿瘤发生中Pin 1和Gli 1活化中的作用，我们可以通过使用该途径的可用抑制剂来控制/治疗胰腺癌。有趣的是，MLK 3家族的特异性抑制剂CEP-1347已用于治疗神经退行性疾病的临床试验，而特异性Pin 1抑制剂正在开发用于癌症治疗。也有人提出MLKs抑制剂可能作为特定类型癌症的治疗，强调了我们与MLK 3在胰腺癌中的作用有关的意想不到的新结果。总之，本研究将导致识别新的预后因素和特定的靶向治疗难以治疗和致命的胰腺癌。

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.