Activin A signaling regulates head and neck squamous cell carcinoma invasion and metastasis

 描述（由申请人提供）：头颈部鳞状细胞癌（HNSCC）是全球第六大常见癌症。虽然这组癌症在美国不是最普遍的，但在过去十年中，HNSCC的发病率稳步上升，而5年生存率保持不变。虽然对HNSCC启动的风险因素有了普遍的了解，如吸烟和饮酒，但对HNSCC进展的机制，特别是侵袭和转移的模式知之甚少。对激活素A（Act A）信号传导（TGFβ超家族的一个分支）的应答丧失最近已涉及癌症进展和转移。已经发现特征性Act A信号传导促进细胞凋亡和生长停滞，以及有效地抑制血管生成。然而，与正常组织相比，Act A表达在肿瘤中通常上调。该临床证据表明，癌细胞可以获得对Act A信号传导的抗性，例如通过改变参与信号传导级联的蛋白质，因此使细胞对Act A的生长抑制作用不敏感。肿瘤细胞对Act A信号传导应答的丧失可能是Act A信号传导级联的一种或多种组分下调的结果。来自乳腺癌和结直肠癌患者样本的证据表明，I型激活素受体表达减少，同时Act A分泌增加，以阶段和等级依赖性方式发生。此外，具有IB型激活素受体（ALK 4）缺失的胰腺癌细胞系对Act A刺激无反应，而具有完整信号传导的细胞经历生长停滞。我们实验室的初步数据表明，与对照组相比，当与稳定过表达Act A的成纤维细胞共培养时，具有完整Act A信号传导的发育不良食管鳞状细胞显示出较少的迁移、侵袭和增殖。然而，当具有未改变的Act A信号传导的食管鳞状细胞癌（ESCC）细胞与稳定过表达Act A的成纤维细胞长期共培养时，癌细胞响应于连续Act A刺激而下调ALK 4。由于ESCC和HNSCC的模型在生理学上相似，我们将我们获得的关于Act A在ESCC中的作用的结果外推到HNSCC。因此，我们假设，在HNSCC中，通过改变Act A信号级联的组分（例如通过ALK 4的下调）而丧失对Act A的细胞响应性使得细胞对Act A的生长抑制作用不敏感，从而促进侵袭性癌症表型。我们将首先使用CRISPR介导的ALK 4缺失来特异性靶向Act A信号传导，确定细胞对Act A信号传导的反应性丧失对体外HNSCC细胞迁移和侵袭的功能后果。由于肿瘤微环境对癌症进展至关重要，我们将研究体内Act A信号传导的破坏如何影响癌细胞侵袭和转移。完成这些实验后，我们将确定HNSCC中Act A信号传导的性质及其对癌症进展的贡献。最终，这些结果将有助于发现更好的HNSCC诊断和预后生物标志物。

 DESCRIPTION (provided by applicant): Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Though this group of cancers is not among the most prevalent in the United States, the incidence rate of HNSCC has steadily increased over the last decade while the 5-year survival rate has remained static. Though there is a general understanding of the risk factors for HNSCC initiation, such as cigarette smoking and alcohol consumption, there is little knowledge regarding the mechanisms of HNSCC progression, particularly modes of invasion and metastasis. Loss of response to Activin A (Act A) signaling, an arm of the TGFβ superfamily, has been recently implicated in cancer progression and metastasis. Characteristic Act A signaling has been found to promote apoptosis and growth arrest, as well as potently inhibit angiogenesis. However, Act A expression is commonly upregulated in tumors, compared to normal tissue. This clinical evidence suggests that the cancer cells may acquire resistance to Act A signaling, such as through alterations of proteins involved in the signaling cascade, therefore rendering the cells insensitive to the growth inhibitory effects of Act A. Loss of tumor cell response to Act A signaling may be a result of a downregulation of one or multiple components of the Act A signaling cascade. Evidence from breast and colorectal cancer patient samples have demonstrated a decrease in expression of activin receptor type I, with a concurrent increase in Act A secretion, occurring in a stage- and grade-dependent manner. Additionally, pancreatic cancer cell lines with loss of activin receptor type IB (ALK4) are unresponsive to Act A stimulation, while cells that have intact signaling undergo growth arrest. Preliminary data from our laboratory has demonstrated that dysplastic esophageal squamous cells with intact Act A signaling show less migration, invasion, and proliferation when co-cultured with fibroblasts that stably overexpress Act A, compared to control. However, when esophageal squamous cell carcinoma (ESCC) cells with unaltered Act A signaling are co-cultured long-term with fibroblasts that stably overexpress Act A, the carcinoma cells downregulate ALK4 in response to continious Act A stimulation. As models of ESCC and HNSCC are physiologically similar, we have extrapolated the results we attained regarding the action of Act A in ESCC to HNSCC. Therefore, we hypothesize that loss of cellular responsiveness to Act A through alteration of constituents of the Act A signaling cascade, such as through the downregulation of ALK4, in HNSCC renders the cells insensitive to the growth inhibitory effects of Act A, promoting an invasive cancer phenotype. We will first determine the functional consequences of loss of cellular responsiveness to Act A signaling on HNSCC cell migration and invasion in vitro, using CRISPR-mediated deletion of ALK4 to specifically target Act A signaling. As the tumor microenvironment is critical for cancer progression, we will investigate how disruption of Act A signaling in vivo impacts cancer cell invasion and metastasis. Upon completion of these experiments, we will have determined the nature of Act A signaling in HNSCC and its contribution to cancer progression. Ultimately, these results will aid on the discovery of better diagnostic and prognostic biomarkers for HNSCC.