Smad4-independent TGF-b signaling in p16-negative HNSCC

描述（由申请人提供）：头颈部鳞状细胞癌（HNSCC）是全球第六大最常见的癌症，发生的风险因素包括吸烟、饮酒和人乳头瘤病毒（HPV）感染。HPV阳性（+）/p16（+）肿瘤患者预后良好[3年总生存率（OS）：~80%]，而HPV阴性（-）肿瘤患者的相似分期，通常为p16阴性（-），OS较差（~50%）。因此，非常需要改善HPV（-）/p16（-）患者的治疗和临床反应。Smad 4（或DPC 4）在转化生长因子-β（TGF-β）信号传导中起核心作用。最近，小鼠口腔中的条件性Smad 4缺失（Smad 4-/-）显示出引起自发性肿瘤，其组织学使人联想到人类疾病。在我们的初步研究中，发现Smad 4表达状态在一组HNSCC细胞系中差异表达，并与上皮向间充质转化（EMT）相关。此外，Smad 4的稳定敲低（KD）诱导EMT样表型和增加的侵袭。在用TGF-β 1刺激后，SCC 25-Smad 4 KD和FaDu（Smad 4-无效HNSCC细胞系）显示出与SCC 25-乱序对照相比增强的Smad 2活化。我们还确定了14/77例（18%）来自HNSCC患者的肿瘤中Smad 4丢失明显，证明与p16（-）肿瘤显著相关[13/53，25%的p16（-）vs. 1/24，4%的p16（+）]。由于p16阳性是一个强有力的替代标记， HPV感染，绝大多数Smad 4（-）肿瘤是HPV（-）。Smad 4（-）肿瘤患者的生存率较低，转移发生率增加。在本申请中，我们提出：1）确定HNSCC中Smad 4非依赖性Smad 2活化的机制及其对TGF-β抑制剂敏感性的影响，和2）使用允许无偏评价肿瘤-微环境相互作用的体内原位小鼠模型确定Smad 4非依赖性Smad 2活化在转移过程中的作用。新的治疗靶点和反应的预测性生物标志物的鉴定将允许在未来的临床试验中选择适当的患者，并可能影响临床结果特别差的HNSCC患者的生存。

DESCRIPTION (provided by applicant): Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide, and risk factors for development include tobacco use, alcohol consumption, and human papillomavirus (HPV) infection. Patients with HPV positive (+)/p16 (+) tumors have a favorable prognosis [3-year overall survival (OS): ~80%] while patients with HPV-negative (-) tumors of similar stage, typically p16-negative (-), experience a worse OS (~50%). Consequently, there is a great need to improve the treatment and clinical response of HPV (-)/p16 (-) patients. Smad4 (or DPC4) plays a central role in transforming growth factor-beta (TGF-ß) signaling. Recently, conditional Smad4 loss (Smad4-/-) in the mouse oral cavity was shown to cause spontaneous tumors with histology reminiscent of the human disease. In our preliminary investigations, Smad4 expression status was found to be differentially expressed across a panel of HNSCC cell lines and associated with epithelial-to- mesenchymal transition (EMT). Additionally, stable knockdown (KD) of Smad4 induced EMT-like phenotypes and increased invasion. After stimulation with TGF-ß1, SCC25-Smad4 KD and FaDu (Smad4-null HNSCC cell line) demonstrated enhanced activation of Smad2 compared to SCC25-scramble control. We also determined Smad4 loss was evident in 14/77 (18%) tumors obtained from HNSCC patients demonstrating a significant association with p16 (-) tumors [13/53, 25% of p16 (-) vs. 1/24, 4% of p16 (+)]. As p16 positivity is a strong surrogate marker for HPV infection, the vast majority of Smad4 (-) tumors are HPV (-). Patients with Smad4 (-) tumors experienced poor survival and an increased incidence of metastasis. In this application, we propose to; 1) determine the mechanism of Smad4-independent Smad2 activation and its effect on TGF-ß inhibitor sensitivity in HNSCC, and 2) determine the role of Smad4-independent Smad2 activation in the metastatic process using an in vivo orthotopic mouse model which allows for an unbiased evaluation of tumor- microenvironment interactions. Identification of novel therapeutic targets and predictive biomarkers of response will allow for appropriate patient selection in future clinical trials, and potentially impact the survival of HNSCC patients with particularly poor clinical outcome.