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

 描述(申请人提供)：头颈部鳞状细胞癌(HNSCC)是全球第六大最常见的癌症。尽管这一组癌症在美国并不是最常见的，但在过去的十年里，HNSCC的发病率一直在稳步上升，而五年生存率保持不变。虽然人们对HNSCC发生的危险因素，如吸烟和饮酒已有了普遍的了解，但对HNSCC进展的机制，特别是侵袭和转移模式，知之甚少。作为转化生长因子β超家族的一个分支，对激活素A(A)信号的反应缺失最近被认为与癌症的进展和转移有关。特征性的Act A信号已被发现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在肿瘤中的表达通常上调。这一临床证据表明，癌细胞可能对ActA信号产生抗性，例如通过改变参与信号级联反应的蛋白质，从而使细胞对ActA的生长抑制效应不敏感。肿瘤细胞对ActA信号反应的丧失可能是ActA信号级联的一个或多个成分下调的结果。来自乳腺癌和结直肠癌患者样本的证据表明，激活素受体I型的表达减少，同时ACTA分泌增加，并以一种分期和分级依赖的方式发生。此外，失去激活素受体IB(ALK4)的胰腺癌细胞株对A幕刺激没有反应，而具有完整信号的细胞则经历生长停滞。我们实验室的初步数据显示，与对照组相比，与稳定高表达Act A的成纤维细胞共培养时，具有完整Act A信号的发育不良的食道鳞状细胞表现出较少的迁移、侵袭和增殖。然而，当ACTA信号未改变的食管鳞状细胞癌(ESCC)细胞与稳定高表达ACTA的成纤维细胞长期共培养时，癌细胞对ACTA持续刺激下调ALK4的表达。由于ESCC和HNSCC的模型在生理上相似，我们将我们在ESCC中A行为的作用外推到HNSCC。因此，我们假设，在HNSCC中，通过改变ActA信号级联的成分，例如通过下调ALK4，使细胞对ActA的生长抑制作用不敏感，从而促进侵袭性癌症表型，从而失去对ActA的细胞反应性。我们将首先利用CRISPR介导的ALK4的缺失来特异性靶向ActA信号，在体外确定细胞对ActA信号失去反应性在HNSCC细胞迁移和侵袭中的功能后果。由于肿瘤微环境对肿瘤的进展至关重要，我们将研究体内ACTA信号的中断如何影响癌细胞的侵袭和转移。在完成这些实验后，我们将确定在HNSCC中ACTA信号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.