Role for DKC1 in cell proliferation and transformation

描述（由申请人提供）：c-MYC是吸烟相关癌症（包括头颈部癌症）中最常见的过表达基因之一。c-MYC的异位表达是原代口腔角质形成细胞转化所需的五种不同分子改变之一，但c-MYC诱导细胞转化的分子机制仍知之甚少。先天性角化不良1（DKC 1）基因是由c-MYC调控的两个分子途径的组成部分。我们在此描述的初步数据表明：（1）DKC 1是c-MYC的直接和保守的转录靶点;（2）DKC 1在口腔鳞状细胞癌中经常过表达;并且c-MYC和DKC 1表达在肿瘤和细胞系的子集中相关;（3）DKC 1突变细胞表现出过早衰老和细胞死亡。这使我们假设DKC 1在癌症形成中起重要作用; DKC 1是c-MYC介导的肿瘤发生的重要效应子。本R 03提案的目的是：（1）确定DKC 1在c-MYC诱导的细胞增殖和转化中的作用;（2）确定DKC 1的过表达是否可以重现易感细胞中的c-MYC功能。c-MYC的过表达刺激增殖并诱导细胞转化。相比之下，c-Myc-null细胞生长缓慢，并表现出通过G2期的延迟进展。我们认为c-MYC需要DKC 1来维持细胞增殖和诱导转化。为了验证我们在目标1中的假设，我们将在DKC 1突变体或野生型人淋巴母细胞中过表达c-MYC。在另一个实验中，我们将使用短发夹RNA沉默表达诱导型c-MYC-雌激素受体（MYC-ER）融合蛋白的原代口腔角质形成细胞中的DKC 1。然后，我们将测量衰老相关的-2-半乳糖苷酶的生产，并使用标准的测定和流式细胞术，以调查角质形成细胞和淋巴母细胞的增殖和细胞周期分布的变化。然后，我们将敲低野生型大鼠1a-MYC-ER成纤维细胞中的Dkc 1;并研究MYC-ER刺激软琼脂中锚定非依赖性生长的能力。在目标2中，我们将研究DKC 1过表达对c-Myc缺失和野生型Rat 1a细胞的影响。我们认为DKC 1的异位表达将缓解G2期细胞周期缺陷，并增加c-Myc-null细胞的增殖率。我们进一步假设DKC 1过表达也会增加野生型细胞的增殖并诱导其转化。为了验证我们的假设，我们将在c-Myc-null细胞中过表达DKC 1，然后测量对细胞增殖和DNA合成的影响。将使用流式细胞术评估细胞周期分布的变化;特别是G2期细胞的状态。然后，我们将在野生型细胞中过表达DKC 1，并确定对锚定非依赖性生长的影响。本文概述的实验研究了DKC 1功能的丧失和获得对细胞增殖和转化的影响。通过采用这种方法，拟议的研究将使人们更深入地了解DKC 1在癌症发展中的作用;以及作为c-MYC介导的肿瘤发生的效应子。 叙述：阐明DKC 1在肿瘤发生中的重要作用可能会导致研究吸烟相关癌症发展机制的新途径。确定DKC 1的作用也可能导致新的治疗策略的开发，从而可能提高吸烟相关癌症患者的生活质量和长期生存率。将DKC 1鉴定为c-MYC介导的肿瘤发生的重要效应子也可能导致c-MYC阳性肿瘤的新靶向治疗。

DESCRIPTION (provided by applicant): c-MYC is among the most commonly overexpressed genes in smoking-related cancers, including those of the head and neck. Ectopic expression of c-MYC is one of only five distinct molecular alterations needed for transformation of primary oral keratinocytes.Yet, the molecular mechanisms by which c-MYC induces cellular transformation remain poorly understood. The dyskeratosis congenita 1 (DKC1) gene is a component of two molecular pathways that are regulated by c-MYC. Our preliminary data described herein indicate that (1) DKC1 is a direct and conserved transcriptional target of c-MYC, (2) DKC1 is frequently overexpressed in oral squamous cell carcinomas; and c-MYC and DKC1 expression correlate in a subset of tumors and cell lines, and (3) DKC1-mutant cells exhibit premature senescence and cell death. This has led us to hypothesize that DKC1 plays an important role in cancer formation; and DKC1 is an essential effector of c-MYC-mediated tumorigenesis. The objectives of this R03 proposal are: (1) Determine the role of DKC1 in c-MYC-induced cellular proliferation and transformation; and (2) Determine if overexpression of DKC1 can recapitulate c-MYC function in susceptible cells. Overexpression of c-MYC stimulates proliferation and induces cellular transformation. In contrast, c-Myc-null cells grow slowly and exhibit delayed progression through G2 phase. We propose that c-MYC requires DKC1 to sustain cellular proliferation and induce transformation. To test our hypotheses in Aim 1, we will overexpress c-MYC in DKC1-mutant or wild-type human lymphoblasts. In another experiment, we will use short hairpin RNA to silence DKC1 in primary oral keratinocytes that express an inducible c-MYC-estrogen receptor (MYC-ER) fusion protein. We will then measure senescence-associated-2- galactosidase production; and use standard assays and flow cytometry to investigate changes in proliferation and cell cycle distribution of the keratinocytes and lymphoblasts. Then, we will knockdown Dkc1 in wild-type Rat 1a-MYC-ER fibroblasts; and investigate the ability of MYC-ER to stimulate anchorage-independent growth in soft agar. In Aim 2, we will investigate the effects of DKC1 overexpression in c-Myc-null and wild type Rat 1a cells. We propose that ectopic expression of DKC1 will alleviate the G2 cell cycle defect and increase the proliferation rate of the c-Myc-null cells. We further hypothesize that DKC1 overexpression will also increase proliferation and induce transformation of the wild-type cells. To test our hypotheses, we will overexpress DKC1 in the c-Myc-null cells, and then measure the effects on cell proliferation and DNA synthesis. Flow cytometry will be used to assess changes in cell cycle distribution; and, in particular, the status of the cells in G2. We will then overexpress DKC1 in wild-type cells and determine the effect on anchorage-independent growth. The experiments outlined here investigate the effects of the loss and gain of DKC1 function on cell proliferation and transformation. By taking this approach, the proposed studies will lead to greater insight into the role of DKC1 in cancer development; and as an effector of c-MYC-mediated tumorigenesis. Narrative: Elucidating a significant role for DKC1 in tumorigenesis may lead to new avenues of study into the mechanisms that underlie the development of smoking-related cancers. Identifying a role for DKC1 may also lead to the development of new therapeutic strategies, thereby potentially increasing the quality of life and the long-term survival rate of affected individuals with smoking-associated cancers. Identifying DKC1 as an essential effector of c-MYC-mediated tumorigenesis may also lead to new targeted therapies for c-MYC-positive tumors.