mTOR-dependent pathways in skin carcinogenesis

描述（由申请人提供）：哺乳动物雷帕霉素靶点（mTOR）是细胞生长的中心调节剂，可被大量促癌突变激活。mTOR存在于两种多蛋白复合物中：激活蛋白质合成的mTORC1和位于akt依赖性抗凋亡通路上游的mTORC2。mTORC1抑制剂雷帕霉素及其类似物正在临床试验中，用于治疗多种恶性肿瘤。虽然mTOR抑制作为一种化疗策略提供了很大的希望，但mTORC1-和mtorc2依赖性途径在癌变中的不同作用才刚刚开始被探索。最近的临床试验表明，mtor控制通路在非黑色素瘤皮肤癌（NMSC）中的作用表明，与服用钙调磷酸酶抑制剂的患者相比，肾移植患者服用雷帕霉素作为免疫抑制剂的NMSC明显减少。该研究的中心假设是，角质形成细胞干细胞内mTORC1和mTORC2活性对于NMSC的启动和促进至关重要。我们发现，局部应用雷帕霉素在小鼠皮肤上抑制肿瘤启动子TPA的表皮过度增生，并使角化细胞对紫外线照射后的凋亡敏感。众所周知，mTORC2也可以通过激活或抑制对雷帕霉素治疗产生反应，并且mTORC2是依赖于Akt过度激活的前列腺癌发展所必需的。在小鼠NMSC模型中也发现了异常的Akt激活。拟议的实验将使用CreLoxP方法区分NMSC中mTORC1和mTORC2的特异性作用，方法是创建有条件地删除Raptor以去除mTORC1或Rictor以去除皮肤中的mTORC2的小鼠系。许多研究表明，毛囊内的干细胞会产生NMSC。因此，携带有固定raptor或rictor等位基因的小鼠将与K5CreERT2小鼠杂交，允许在局部应用他莫昔芬后在卵泡凸起处出现raptor或rictor缺失。我们将使用这些小鼠来验证mTORC1和mTORC2活性是维持角化细胞干细胞稳态和致癌转化所必需的假设。目的1将使用经典的DMBA/TPA启动-促进模型来研究Raptor或Rictor缺失对皮肤肿瘤发展的影响，并分析干细胞对DMBA的凋亡反应。在Aim 2中，将评估随着Raptor或Rictor缺失而发生的干细胞和祖细胞区室的变化。为了测量对原发性致癌事件的反应，分离的猛禽？？Cre或Rictor / -Cre角化细胞将被v-Ha-ras转导。将评估裸鼠的分化变化和形成乳头瘤的能力。Aim 3将对干细胞富集的Raptor / -Cre和Rictor / -Cre角质形成细胞进行转录分析，从而深入了解NMSC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mTORC1-和mtorc2特异性抑制剂提供强有力的理论依据。

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.