REGULATORS OF EPITHELIAL TUMOR PROGRESSION

上皮肿瘤进展涉及细胞与细胞外环境的异常相互作用， 肿瘤细胞自身固有的异常皮肤鳞状细胞癌（SCC）起源于 皮肤从紫外线诱变和不成比例地影响美国退伍军人由于阳光照射引起的 服兵役在全国范围内，SCC是第二大常见癌症，每年约有100万新发病例。 SCC可导致毁容，在极少数情况下，致命的转移。最近，这一优点审查确定， 胶原是SCC中最高度突变的基因家族，COL 11 A1基因是SCC中第二大突变基因。 SCC中总的基因突变频率为63%。它还发现了额外的胶原蛋白基因在高水平下突变， 在SCC中的频率（>20%），包括COL 17 A1和COL 4A 4，以及COL 7A 1，其遗传突变 引起具有侵袭性皮肤SCC的大疱性表皮病。SCC胶原突变集中在 Gly-X-Y（X和Y通常为脯氨酸）三螺旋序列中的脯氨酸和甘氨酸，改变 已知产生显性阴性胶原蛋白，增加了肿瘤分泌的突变胶原蛋白 可能会以反式显性的方式引发癌症。与突变体的促癌功能一致 胶原敲入SCC相关的COL 11 A1突变增强体内肿瘤侵袭 与SCC中的同基因COL 11 A1野生型对照和消除内源性突变COL 11 A1相比， 肿瘤在体内损害肿瘤发生，表明突变胶原在功能上促进肿瘤发生。 目的我将测试一个模型，在该模型中，分泌的突变胶原蛋白以非细胞自主的方式起作用 来加速肿瘤的形成。首先，它将确定突变型COL 11 A1是否能促进肿瘤进展 的肿瘤细胞在反式使用马赛克组织模型。第二，将量化对表皮肿瘤的影响 在SCC中经常发生突变的多种其他胶原蛋白的突变进展，包括 COL 17 A1、COL 4A 4和COL 7A 1。目的研究COL 11 A1在表皮肿瘤进展中的作用 并确定其他复发突变的胶原蛋白是否也能促进SCC。 该项目最近还使用了来自一系列人类SCC的47，771个细胞的单细胞RNA测序。 肿瘤，沿着患者和位点匹配的正常对照皮肤，以鉴定肿瘤细胞亚群。 SCC。这定义了一个新的肿瘤特异性角质形成细胞（TSK）群体，在正常组织中没有对应物 表达COL 11 A1和其他与细胞通讯和侵袭相关的基因。此类肿瘤 亚群通过细胞表面相互交流并分泌 然而，蛋白质，即实际上在活体肿瘤中表达的蛋白质组（与RNA相反） 由特定的肿瘤亚群引起的肿瘤转移一直是一个技术上的挑战。为了解决这个问题，我们开发了 一种新的邻近蛋白质组学方法，分泌蛋白质鉴定（SecrID），可以识别细胞表面 和体内异源活肿瘤组织中感兴趣的离散细胞群内的分泌蛋白 以表征肿瘤进展中的细胞亚群蛋白。 目的II是基于一个模型，其中TSK SCC亚群使用特定的细胞通信 导致恶性肿瘤的蛋白质。首先，它将使用SecrID来定义分泌蛋白和细胞表面蛋白的特异性 TSK细胞亚群，基于这些可能介导促肿瘤细胞增殖的前提， 通信第二，它将消除TSK亚群特异性基因，包括那些由 SecrID，以确定它们对肿瘤发生的影响，并寻找新的可获得的治疗靶点。Aim II 将描述肿瘤特异性SCC TSK亚群的特征，并确定其用于影响的分子。 肿瘤进展。 在建议的资助周期结束时，我们计划描述突变胶原蛋白的作用。 以及表皮肿瘤进展中新定义的肿瘤细胞亚群。

Epithelial tumor progression involves abnormal cell interactions with the extracellular environment as well abnormalities intrinsic to tumor cells themselves. Cutaneous squamous cell carcinoma (SCC) arises in skin from ultraviolet mutagenesis and disproportionately affects U.S. Veterans due to sun exposure incurred in military service. Nationally, SCC is the second most common cancer, with ~1 million new cases yearly. SCC can lead to disfigurement and, in rare cases, lethal metastases. This Merit Review recently identified collagens as the most highly mutated gene family in SCC, with the COL11A1 gene the second most frequently mutated gene overall in SCC (63%). It also found additional collagen genes mutated at high frequency (>20%) in SCC, including COL17A1 and COL4A4, as well as COL7A1, whose inherited mutation causes forms of epidermolysis bullosa with aggressive skin SCCs. SCC collagen mutations concentrate at prolines and glycines in the Gly-X-Y (with X and Y commonly proline) triple helical sequence, alterations known to produce dominant-negative collagens, raising the possibility that tumor-secreted mutant collagens may enable cancer in a trans-dominant fashion. Consistent with a pro-oncogenic function for mutant collagens, knocking in an SCC-associated COL11A1 mutation enhanced neoplastic invasion in vivo compared to isogenic COL11A1 wild-type control and ablating endogenously mutant COL11A1 in SCC tumors impaired tumorigenesis in vivo, indicating that mutant collagens functionally promote tumorigenesis. Aim I will test a model in which secreted mutant collagen proteins act in a non-cell autonomous fashion to accelerate tumorigenesis. First, it will determine if mutant COL11A1 can promote neoplastic progression of tumor cells in trans using mosaic tissue models. Second, it will quantify the impacts on epidermal tumor progression of mutations in multiple additional collagens that are frequently mutated in SCC, including COL17A1, COL4A4, and COL7A1. Aim I will study the action of COL11A1 in epidermal tumor progression and determine if other recurrently mutated collagens can also promote SCC. This project also recently used single-cell RNA-sequencing of 47,771 cells from a series of human SCC tumors, along with patient and site-matched normal control skin, to identify tumor cell subpopulations in SCC. This defined a new tumor-specific keratinocyte (TSK) population with no counterpart in normal tissue that expressed COL11A1 and other genes linked to cellular communication and invasion. Such tumor subpopulations enable neoplasia by communicating with each other through cell surface and secreted proteins, however, the sets of proteins – as opposed to RNAs – that are actually expressed in living tumors by specific tumor subpopulations have been a technical challenge to define. To address this, we developed a new proximity proteomics method, Secreted Protein Identification (SecrID) that can identify cell surface and secreted proteins within discrete cell populations of interest in heterogenous living tumor tissues in vivo to characterize cell subpopulation proteins in tumor progression. Aim II is based on a model in which the TSK SCC subpopulation uses specific cellular communication proteins to drive malignancy. First, it will use SecrID to define the secreted and cell surface proteins specific to the TSK cell subpopulation, based on the premise that these may mediate pro-neoplastic cellular communication. Second, it will ablate TSK subpopulation-specific genes, including those identified by SecrID, to define their impact on tumorigenesis and to search for new accessible therapeutic targets. Aim II will characterize the tumor-specific SCC TSK subpopulation and identify the molecules it uses to influence tumor progression. At the end of the proposed funding cycle, we plan to have characterized the actions of mutant collagens and newly defined tumor cell subpopulations in epidermal tumor progression.