mTOR-dependent pathways in skin carcinogenesis

描述（申请人提供）：哺乳动物雷帕霉素靶蛋白（mTOR）是细胞生长的中心调节因子，可被大量促癌突变激活。mTOR存在于两种多蛋白质复合物中：mTORC 1，其激活蛋白质合成，和mTORC 2，其是Akt依赖性抗凋亡途径的上游。mTORC 1抑制剂雷帕霉素及其类似物正在针对各种恶性肿瘤进行临床试验。虽然mTOR抑制作为化疗策略提供了很大的希望，但mTORC 1和mTORC 2依赖性途径在致癌作用中的不同作用才刚刚开始探索。最近的临床试验表明，mTOR控制的途径在非黑色素瘤皮肤癌（NMSC）中的作用，表明给予雷帕霉素作为免疫抑制剂的肾移植患者与服用钙调磷酸酶抑制剂的患者相比，NMSC明显减少。所提出的工作的中心假设是角质形成细胞干细胞内的mTORC 1和mTORC 2活性对于NMSC的启动和促进是必不可少的。我们表明，局部应用雷帕霉素小鼠皮肤抑制表皮过度增殖的肿瘤促进剂TPA和敏化角质形成细胞凋亡后，紫外线照射。已知mTORC 2也可以通过激活或抑制对雷帕霉素治疗作出响应，并且mTORC 2是依赖于Akt超激活的前列腺癌的发展所需的。异常Akt激活也已在NMSC的小鼠模型中得到证实。拟议的实验将使用CreLoxP方法，通过创建具有条件性缺失Raptor以消除皮肤中的mTORC 1或Rictor以消除mTORC 2的小鼠系，来区分NMSC中的mTORC 1和mTORC 2特异性效应。许多研究表明，毛囊隆起内的干细胞产生NMSC。因此，具有floxed raptor或Rictor等位基因的小鼠将与K5 CreERT 2小鼠杂交，允许在局部应用他莫昔芬后在滤泡隆起中发生Raptor或Rictor缺失。我们将使用这些小鼠来解决mTORC 1和mTORC 2活动都需要维持角质形成细胞干细胞的稳态，并成为致癌转化的假设。目的1将使用经典的DMBA/TPA启动-促进模型来检查Raptor或Rictor缺失对皮肤肿瘤发生的影响，并将分析干细胞对DMBA的凋亡反应。在目标2中，将评估Raptor或Rictor缺失时干细胞和祖细胞隔室发生的变化。为了测量对原发性致癌事件的反应，分离的猛禽？? Cre或Rictor / -Cre角质形成细胞将用v-Ha-ras转导。将评估裸鼠的分化变化和形成乳头状瘤的能力。目的3将进行干细胞富集的Raptor / -Cre和Rictor / -Cre角质形成细胞的转录谱分析，从而深入了解NMSC中的其他潜在靶点。如果我们的假设得到证实，这些研究将为在皮肤和其他上皮肿瘤中开发mTORC 1和mTORC 2特异性抑制剂提供强有力的理论基础。 公共卫生相关性：我们的研究研究了mTOR依赖性途径在非黑色素瘤皮肤癌发展中的作用。由于皮肤癌是世界范围内最常见的恶性肿瘤形式，因此确定预防和治疗的可能目标与公共卫生高度相关。了解mTORC 1和mTORC 2蛋白复合物的作用也非常重要，因为mTOR抑制剂雷帕霉素在各种人类癌症的化疗中显示出巨大的前景。

DESCRIPTION (provided by applicant): The mammalian target of rapamycin (mTOR) is a central regulator of cell growth that is activated by a large number of cancer-promoting mutations. mTOR exists in two multi-protein complexes: mTORC1, which activates protein synthesis, and mTORC2, which is upstream of Akt-dependent anti-apoptotic pathways. The mTORC1 inhibitor Rapamycin and its analogues are in clinical trials against a variety of malignancies. While mTOR inhibition offers much promise as a chemotherapeutic strategy, the differential roles of mTORC1- and mTORC2-dependent pathways in carcinogenesis have only begun to be explored. A role for mTOR-controlled pathways in non-melanoma skin cancer (NMSC) has been suggested in recent clinical trials showing that renal transplant patients administered Rapamycin as an immune suppressant suffer from significantly fewer NMSCs compared to patients taking Calcineurin inhibitors. The central hypothesis of the proposed work is that both mTORC1 and mTORC2 activities within keratinocyte stem cells are essential for the initiation and promotion of NMSC. We show that topical application of Rapamycin to mouse skin inhibits epidermal hyperproliferation in response to the tumor promoter TPA and sensitizes keratinocytes to apoptosis following UV irradiation. It is known that mTORC2 can also respond to Rapamycin treatment, either by activation or inhibition, and mTORC2 is required for the development of prostate cancers dependent on Akt hyperactivation. Aberrant Akt activation has also been demonstrated in mouse models of NMSC. The proposed experiments will use a CreLoxP approach to differentiate between mTORC1- and mTORC2-specific effects in NMSC by creating mouse lines with conditional deletion of Raptor to abrogate mTORC1 or Rictor to abrogate mTORC2 in the skin. A number of studies have indicated that stem cells within the hair follicle bulge give rise to NMSC. Thus, mice with a floxed raptor or rictor allele will be crossed with K5CreERT2 mice, allowing Raptor or Rictor deletion to occur in the follicular bulge upon topical Tamoxifen application. We will use these mice to address the hypothesis that both mTORC1 and mTORC2 activities are required to maintain keratinocyte stem cell homeostasis and to become oncogenically transformed. Aim 1 will examine the effect of Raptor or Rictor deletion on the development of skin tumors using the classical DMBA/TPA initiation-promotion model, and will analyze the stem cell apoptotic response to DMBA. In Aim 2, changes in the stem and progenitor cell compartments that occur with loss of Raptor or Rictor will be evaluated. To measure the response to a primary oncogenic event, isolated Raptor ? ?Cre or Rictor / -Cre keratinocytes will be transduced with v-Ha-ras. Differentiation changes and the ability to form papillomas on nude mice will be assessed. Aim 3 will perform transcriptional profiling of stem cell-enriched Raptor / -Cre and Rictor / -Cre keratinocytes, allowing insight into other potential targets in NMSC. If our hypothesis is confirmed, these studies will provide a strong rationale for development of mTORC1- and mTORC2-specific inhibitors in skin and other epithelial tumors. PUBLIC HEALTH RELEVANCE: Our research studies the role of mTOR-dependent pathways in development of non-melanoma skin cancer. Since skin cancers are the most common form of malignancies world-wide, identification of possible targets for prevention and therapy is highly relevant to public health. Understanding the role of the mTORC1 and mTORC2 protein complexes is also of great importance because the mTOR inhibitor Rapamycin has shown great promise in chemotherapy of a variety of human cancers.