Analysis of B-Raf and MPC1 mutations & their correlation with specific DNA methylation patterns using de-identified, FFPE clinically annotated colorectal-specific tumor samples.

我们的长期目标是促进制定新的预防和治疗措施， 通过了解APC突变后最早期的细胞扰动来研究结肠癌的形成。在 在之前的资助期间，我们揭示了一种创新模型，其中APC促进肠上皮细胞 通过控制视黄酸的生物合成来分化。这些发现表明， APC的功能不限于其在调节经典WNT信号传导中的公认作用。 从机制上讲，我们确定了转录辅阻遏物，C-末端结合蛋白（CtBP），作为一个转录辅阻遏物。 一种新的APC调节蛋白，可抑制视黄醇脱氢酶和肠细胞分化 与LEF-1失调有关FAP患者的早期腺瘤中 CtBP和LEF 1与邻近的未受累组织相比。令人惊讶的是，这些相同的 切片显示几乎没有核β-连环蛋白的证据，表明视黄酸和细胞的损失 分化先于β-连环蛋白的核积累。我们进一步证明， KRAS促进细胞核β-catenin的积累和随后的肠细胞增殖。我们 还描述并揭示了APC和一种新的DNA去甲基化酶之间的意外联系 系统这种DNA去甲基化酶在APC和视黄酸缺失时上调， 肠细胞处于祖细胞样状态。这种失败的分化先于K-RAS诱导的增殖 通过β-连环蛋白。目前尚不清楚激活KRAS的信号，因此，β-连环蛋白 这是在APC损失的背景下发生的。有趣的是，成年斑马鱼缺乏视黄酸， 证明了分化缺陷和与细胞增殖相一致的深刻的增殖反应。 炎症基质细胞的活化。我们的初步发现表明成年斑马鱼 缺乏免疫细胞的基因工程显示肠分化缺陷，但没有增殖反应， 当缺乏视黄酸时。我们现在将扩大我们的研究，以检查维甲酸是否 在上皮细胞和肠基质细胞中起作用， 两种细胞群导致结肠腺瘤的发生和发展。我们假设 APC控制着视黄酸的生成维甲酸生产的控制 肠上皮细胞通过重塑表观遗传结构调节细胞分化潜能 景观与此同时，视黄酸可抑制肠道炎症反应， 需要刺激上皮细胞增殖。这个项目将扩大我们对APC如何 和视黄酸有助于肠的发育和分化。这种理解可以 支持一个可检验的临床假设，旨在药理学上重新激活类维生素A信号， 预防和治疗人类结肠癌。

Our long-term goal is to facilitate the development of new prevention and therapeutic measures for colon cancer formation by understanding the earliest cellular perturbations that follow APC mutation. In the previous funding periods, we revealed an innovative model wherein APC promotes enterocyte differentiation by controlling retinoic acid biosynthesis. These findings now demonstrate that the functions of APC are not limited to its well-established role in regulating canonical WNT signaling. Mechanistically, we identified the transcriptional co-repressor, C-terminal binding protein (CtBP), as a novel, APC-regulated protein that suppresses retinol dehydrogenases and intestinal cell differentiation in concert with dysregulated LEF-1. Early adenomas taken from FAP patients showed elevated levels of CtBP and LEF1 in comparison to adjacent, uninvolved tissues. Surprisingly, however, these same sections showed little evidence of nuclear β-catenin indicating that loss of retinoic acid and cell differentiation precedes nuclear accumulation of β-catenin. We further demonstrated that activation of KRAS promoted the accumulation of nuclear β-catenin and subsequent intestinal cell proliferation. We also described and uncovered an unexpected connection between APC and a novel DNA demethylase system. This DNA demethylase is upregulated upon loss of APC and retinoic acid and maintains intestinal cells in progenitor-like state. This failed differentiation precedes K-RAS induced proliferation via β-catenin. It is currently unclear where signals for activating KRAS, and therefore, β-catenin originate in the context of APC loss. Interestingly, adult zebrafish made deficient in retinoic acid demonstrate both differentiation defects and a profound proliferation response that is coincident with activation of inflammatory stromal cells. Our preliminary findings indicate that adult zebrafish engineered to lack immune cells show intestinal differentiation defects, but no proliferative response when made deficient in retinoic acid. We will now expand our studies to examine whether retinoic acid plays roles in both epithelial cells and in intestinal stromal cells and whether interactions between these two cell populations account for both initiation and progression of colon adenomas. We hypothesize that APC controls the production of retinoic acid. Control of retinoic acid production in intestinal epithelial cells regulates cell potential for differentiation by remodeling the epigenetic landscape. In parallel, retinoic acid acts to suppress intestinal inflammatory responses that are need to stimulate epithelial cell proliferation. This project will expand our understanding of how APC and retinoic acid contribute to intestinal development and differentiation. This understanding could support a testable clinical hypothesis aimed at pharmacological re-activation of retinoid signaling in preventing and treating human colon cancers.