Oncogenic MLK3-Pak1 Signaling in ER Negative Breast 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激酶成员，其生物学功能仍然难以捉摸。在剖析通过MLK 3介导的信号通路时，我们观察到酵母Ste 20成员的哺乳动物同源物p21激活激酶（Pak 1）和MLK 3之间的强烈相互作用。最初，我们推测Pak 1可能调节MLK 3激酶活性，类似于其酵母对应物。然而，观察到的结果与我们的推测相反，而是MLK 3直接磷酸化并激活Pak 1，这随后导致 NF-κB活化。这些结果非常有趣，因为Pak 1激活与乳腺增生有关，最近已被确定为乳腺癌的主要癌基因。我们的初步研究表明，Pak 1活性在乳腺癌细胞系中以MLK 3依赖的方式直接调节。我们还观察到在原发性人乳腺肿瘤中，MLK 3活性与Pak 1和NF-κB活化直接相关，仅在ER和PR阴性乳腺肿瘤中，Pak 1在ER- PR-乳腺肿瘤中过表达。如果这些结果是真的，那么可用的MLKs抑制剂CEP-1347/CEP-11004应该诱导ER-乳腺癌细胞中的细胞死亡吗？事实上，用CEP-1347处理ER-而不是ER+乳腺癌细胞系引起细胞死亡。基于我们的观察，我们假设在ER-乳腺癌细胞中，MLK 3激活Pak 1及其下游NF-κB，导致ER-肿瘤发生。因此，靶向MLK 3或其他MLK可以消除ER-乳腺肿瘤。为了实现我们的目标，我们将确定：（1）MLK 3激活Pak 1诱导ER-乳腺细胞肿瘤发生，（2）MLK 3-Pak 1信号级联的破坏减弱ER-乳腺细胞肿瘤发生;和（3）ER-乳腺细胞肿瘤发生中MLK 3、Pak 1和NF-κB激活的机制.预期通过确定MLK 3在Pak 1和NF-κB活化和肿瘤发生中的作用，我们可以通过使用该途径的可用抑制剂来控制/治疗ER-乳腺癌。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。也有人提出MLK抑制剂可用作特定类型癌症的治疗，强调了我们与MLK 3在ER-乳腺癌中的作用相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的ER-乳腺癌。

DESCRIPTION (provided by applicant): Mixed Lineage Kinase 3 (MLK3) is a stress-activated MAP Kinase Kinase Kinase member, whose biological function is still elusive. While dissecting the signaling pathway mediated via MLK3, we observed a strong interaction between mammalian homolog of yeast Ste20 member, p21 activated kinase (Pak1) and MLK3. Initially, we speculated that Pak1 might regulate MLK3 kinase activities similar to its yeast counterparts. However, the observed results were counterintuitive to our speculation, and instead MLK3 directly phosphorylated and activated Pak1, which subsequently lead to NF-κB activation. These results were quite intriguing because Pak1 activation has been implicated in mammary gland hyperplasia, and recently it has been identified as a major oncogene in breast cancer. Our preliminary investigation revealed that Pak1 activity was directly regulated in a MLK3-dependent manner in breast cancer cell lines. We also observed that in primary human breast tumors, the MLK3 activities directly correlated with Pak1 and NF-κB activations, exclusively in ER and PR negative breast tumors and Pak1 was overexpressed in ER- PR- breast tumors. If these results are true then the available MLKs inhibitor, CEP-1347/CEP-11004 should induce cell death in ER- breast cancer cells? Indeed, treatment of ER-, but not ER+ breast cancer cell lines with CEP-1347 caused cell death. Based on our observations, we hypothesize that in ER- breast cancer cells, MLK3 activates Pak1 and its downstream NF-κB, leading to ER- tumorigenesis. Therefore targeting MLK3 or other MLKs could abrogate ER- breast tumors. To achieve our goals, we will determine that: (1) activation of Pak1 by MLK3 induces ER- breast cell tumorigenesis, (2) disruption of MLK3-Pak1 signaling cascade attenuates ER- breast cell tumorigenesis; and (3) the mechanism of MLK3, Pak1 and NF-κB activation in ER- breast cell tumorigenesis. It is expected that by defining the role of MLK3 in Pak1 and NF-κB activation and tumorigenesis, we might control/treat ER- breast 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. 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 ER- breast cancer. Taken together, the present study will lead to the identification of new prognostic factors and specific targeted therapies for difficult to treat ER- breast cancer.