Characterization of AMH as part of a novel TGF-beta signaling loop

项目概要/摘要。 肺癌每年在美国造成约16万人死亡，使其成为最致命的癌症类型，诊断病例的5年生存率仅为15.9%。热休克蛋白90（HSP 90）作为一种分子伴侣蛋白，对许多参与肿瘤发生的蛋白质的活性起重要作用，其过表达与不良预后相关。虽然早期的HSP 90抑制剂由于毒副作用在临床上并不成功，但第二代化合物，如ganetespib，在后期临床试验中显示出相当大的前景，包括一些肺癌。在广泛的初步短干扰（si）RNA筛选后，我发现抗苗勒管激素（AMH）和AMH II型受体（AMHRII）的耗竭显著使三种KRAS突变的肺癌细胞系对ganetespib敏感。重要的是，AMH是转化生长因子（TGF）-β超家族的成员，并且通过共享效应级联的部分而与TGF-β信号传导相关。TGF-β信号传导经常参与肺癌增殖、存活和转移，后者是这种恶性肿瘤最致命的方面。TGF-β已被广泛研究，但 其在侵袭性癌症中的功能方面仍然不清楚。我们的初步数据表明，一种新的自分泌信号环涉及AMH和AMHRII，代表这些蛋白质首次被确定为在非性腺组织癌症背景下发挥作用。有趣的是，Taipale等人最近发现AMHRII的稳定性最依赖于314种测试激酶中的HSP 90，而在线数据库和我自己的研究已经在肺癌细胞系的一个子集以及许多原发性肺癌中建立了AMH和AMHRII的显着表达。因此，我们认为AMH和AMHRII在肺癌亚群中起着关键作用，可能是可利用的靶点，以增强当前和新型疗法的疗效，或至少用作生物标志物。 我们广泛的长期目标是进一步阐明TGF-β在癌症增殖和转移中的作用，以及如何利用这些信息改善肺癌患者的现状。我们的三个目标将更好地描述AMH作为与TGF-β信号传导相关的促生存配体，并探索我们发现的转化可能性。对于我们的第一个目标，我们将使用siRNA和过表达实验来探测AMH和TGF-β效应级联的相互作用，并探索AMH与上皮-间充质转化的联系。第二个目的将在改变的免疫应答的情况下，使用ganetespib与AMHIIR靶向抗体或AMH靶向融合蛋白的组合的体外测试。 AMH功能和异种移植小鼠模型将用于研究AMH的短发夹RNA（shRNA）敲低与ganetespib治疗组合的体内作用。最后，作为临床前评估的一部分，我们将对AMH、AMHRII、HSP 90和其他几种蛋白质的人肺癌组织微阵列进行染色。

Project Summary/Abstract. Lung cancer kills approximately 160,000 people in the US annually, making it the deadliest type of cancer, with a 5-year survival rate of only 15.9% of diagnosed cases. As a chaperone protein, heat shock protein 90 (HSP90) is essential to support activity of many proteins involved in oncogenesis, and its overexpression correlates with worse outcomes. Although early HSP90 inhibitors were not clinically successful due to toxic side effects, second-generation compounds, such as ganetespib, are showing considerable promise in late stage clinical trials, including some for lung cancer. Following extensive preliminary short interfering (si)RNA screening, I found that depletion of anti-Müllerian hormone (AMH) and the AMH type II receptor (AMHRII) significantly sensitized three KRAS-mutated lung cancer cell lines to ganetespib. Importantly, AMH is a member of the transforming growth factor (TGF)-β superfamily and relates to TGF-β signaling by sharing parts of an effector cascade. TGF-β signaling is frequently involved in lung cancer proliferation, survival and metastasis, the last being the deadliest aspect of this malignancy. TGF-β has been studied extensively, but aspects of its function in aggressive cancer remain opaque. Our preliminary data suggests a novel autocrine-signaling loop involving AMH and AMHRII, representing the first time these proteins have been identified as functioning in a non-gonadal tissue cancer context. Intriguingly, Taipale et al. recently discovered that AMHRII stability is most dependent on HSP90 out of 314 tested kinases, while online databases and my own investigations have established significant expression of AMH and AMHRII in a subset of lung cancer cell lines as well as in many primary lung cancers. Thus, we propose that AMH and AMHRII play a critical role in a subpopulation of lung cancers, and may be exploitable targets to potentiate the efficacy of current and novel therapies or at minimum be utilized as biomarkers. Our broad, long-term objectives are to further elucidate the role TGF-β plays in cancer proliferation and metastasis and how this information can be used to improve the status quo of patients afflicted with lung cancer. Our three aims will better delineate AMH as a pro-survival ligand linked to TGF-β signaling and explore the translational possibilities of our findings. For our first aim, we will use siRNA and overexpression experiments to probe interactions of AMH and the TGF-β effector cascades and explore links of AMH to epithelial-mesenchymal transition. The second aim will use in vitro testing of combinations of ganetespib with AMHIIR-targeting antibodies or an AMH-targeting fusion protein, in the context of altered AMH function, and xenograft mouse models will be used to study the effects of short hairpin RNA (shRNA) knockdown of AMH in combination with ganetespib treatment in vivo. Lastly, we will stain tissue microarrays of human lung cancer for AMH, AMHRII, HSP90 and several other proteins as part of a preclinical assessment.