The role of an invasive phenotype in promoting resistance to MAPK-directed therapies in thyroid cancer

甲状腺癌是最常见的内分泌恶性肿瘤，每年新增诊断病例超过52,000例。晚期乳头状甲状腺癌(PTC)和间变性甲状腺癌(ATC)是甲状腺癌的侵袭性亚型，治疗选择有限。因此，PTC和ATC是内分泌癌死亡的主要原因。在PTC和ATC中，MAPK通路的激活突变是常见的，然而，对MAPK定向治疗的耐药性是一个主要问题。作为MAPK通路最下游的节点，抑制ERK1/2有可能减少旁路信号，克服耐药性。我已经在体外和体内证明，联合抑制BRAF和ERK可以阻断通路的重新激活和抑制生长。此外，我已经证明，在获得性耐药(长时间缓慢发生的耐药)的PTC模型和内在耐药(预先发生的耐药)的ATC模型中，BRAF抑制以矛盾的方式增加侵袭性，后者被BRAF和ERK的双重抑制所阻断。一种新出现的靶向治疗耐药机制涉及到细胞表现出更具侵袭性的表型以生存于靶向治疗。我的初步数据显示，抑制BRAF增加了ECM蛋白纤维连接蛋白(FN1)的表达和分泌。此外，补充FN1可以通过增加侵袭力来抑制BRAF的侵袭，这也可以被ERK的抑制所阻断。最后，用BRAF抑制剂处理耐药细胞的条件培养液增加了敏感细胞的侵袭力。综上所述，我假设BRAF的抑制通过ERK的重新激活来增加FN1的产生和分泌，从而促进亲侵袭的自分泌分泌体，使细胞能够入侵和存活。因此，本研究的目的是1)确定ERK和FN1在响应BRAF抑制的侵袭性表型中的作用，2)识别响应BRAF抑制的侵袭性分泌组，以及3)确定侵袭性表型在体内响应BRAF抑制的作用。预期的结果将确定侵袭性表型的关键驱动因素，并将为规避MAPK导向疗法的耐药性和防止侵袭和转移提供新的治疗策略。这项拟议研究的完成将有助于国家癌症研究所履行其支持癌症研究和基础科学培训的使命。

Thyroid cancer is the most common endocrine malignancy, accounting for over 52,000 new diagnoses each year. Advanced papillary thyroid cancer (PTC) and anaplastic thyroid cancer (ATC) are aggressive subtypes of thyroid cancer, and have limited therapeutic options. PTC and ATC are thus the leading causes of endocrine cancer death. Activating mutations of the MAP kinase (MAPK) pathway are common in PTC and ATC, however, drug resistance to MAPK-directed therapies is a major problem. As the most downstream node of the MAPK pathway, inhibition of ERK1/2 has the potential to reduce bypass signaling and overcome resistance. I have shown combined BRAF and ERK inhibition blocks pathway reactivation and inhibits growth in vitro and in vivo. Further, I have shown that BRAF inhibition paradoxically increases invasion in a PTC model of acquired resistance (resistance that occurs slowly over a long period of time) and in ATC models of intrinsic resistance (resistance that occurs upfront), which is blocked by dual inhibition of BRAF and ERK. An emerging mechanism of resistance to targeted therapies involves cells exhibiting a more invasive phenotype to survive targeted therapies. My preliminary data show that BRAF inhibition increases the expression and secretion of the ECM protein fibronectin (FN1). Further, supplementation of FN1 to the media phenocopies BRAF inhibitor treatment by increasing invasion, which can also be blocked by inhibition of ERK. Finally, conditioned media from resistant cells treated with a BRAF inhibitor increases the invasiveness of sensitive cells. Taken together, I hypothesize that BRAF inhibition increases the production and secretion of FN1 through ERK reactivation to promote a pro-invasive autocrine secretome allowing cells to invade and survive. Thus, the goals of this proposal are to 1) determine the role of ERK and FN1 in an invasive phenotype in response to BRAF inhibition, 2) identify the pro-invasive secretome in response to BRAF inhibition, and 3) determine the role of an invasive phenotype in response to BRAF inhibition in vivo. The expected outcomes will identify key drivers of an invasive phenotype and will inform novel treatment strategies to circumvent resistance to MAPK- directed therapies and prevent invasion and metastasis. The completion of the proposed research will help the National Cancer Institute fulfill their mission to support cancer research and training in the fundamental sciences.