mTOR-dependent pathways in skin carcinogenesis

描述(申请人提供)：雷帕霉素的哺乳动物靶标(MTOR)是一种细胞生长的中央调节因子，由大量致癌突变激活。MTOR存在于两个多蛋白质复合体中：激活蛋白质合成的mTORC1和位于Akt依赖的抗凋亡通路上游的mTORC2。MTORC1抑制剂雷帕霉素及其类似物正在对各种恶性肿瘤进行临床试验。虽然mTOR抑制作为一种化疗策略提供了很大的前景，但mTORC1和mTORC2依赖的通路在肿瘤发生中的不同作用才刚刚开始探索。最近的临床试验表明mTOR控制的通路在非黑色素瘤皮肤癌(NMSC)中的作用，表明接受雷帕霉素作为免疫抑制剂的肾移植患者与接受钙调神经磷酸酶抑制剂的患者相比，NMSCs显著减少。拟议工作的中心假设是角质形成干细胞内的mTORC1和mTORC2活性对于NMSC的启动和促进是必不可少的。我们发现，局部应用雷帕霉素在小鼠皮肤上可抑制表皮对肿瘤促进剂TPA的过度增殖，并使角质形成细胞对紫外线照射后的细胞凋亡敏感。众所周知，mTORC2也可以通过激活或抑制雷帕霉素的治疗而产生反应，而mTORC2是依赖Akt过度激活的前列腺癌发生所必需的。在NMSC的小鼠模型中也证实了Akt的异常激活。拟议的实验将使用CreLoxP方法来区分mTORC1和mTORC2在NMSC中的特异性作用，方法是创建小鼠系，条件删除Raptor以消除mTORC1，或使用Rictor来消除皮肤中的mTORC2。许多研究表明，毛囊隆起中的干细胞会产生NMSC。因此，携带Raptor或Rictor等位基因的小鼠将与K5CreERT2小鼠杂交，从而允许局部应用他莫昔芬后在卵泡隆起中发生Raptor或Rictor缺失。我们将使用这些小鼠来解决这样的假设，即mTORC1和mTORC2活性都是维持角质形成细胞干细胞动态平衡和致癌转化所必需的。目的1利用经典的DMBA/TPA启动-促进模型，研究Raptor或Rictor缺失在皮肤肿瘤发生发展中的作用，并分析DMBA对干细胞的凋亡反应。在目标2中，将评估因失去猛禽或Rictor而发生的干细胞室和祖细胞室的变化。为了测量对原发致癌事件的反应，分离的Raptor？？Cre或Rictor/-Cre角质形成细胞将被v-Ha-ras转导。将评估分化变化和在裸鼠身上形成乳头状瘤的能力。AIM 3将对干细胞丰富的Raptor/-Cre和Rictor/-Cre角质形成细胞进行转录图谱分析，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性肿瘤中开发mTORC1和mTORC2特异性抑制物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依赖通路在非黑色素瘤皮肤癌发生中的作用。由于皮肤癌是世界上最常见的恶性肿瘤形式，确定可能的预防和治疗目标与公共健康高度相关。了解mTORC1和mTORC2蛋白复合体的作用也是非常重要的，因为mTOR抑制剂雷帕霉素在多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.