CAPER, a New Regulator of DNA Damage and Repair in Triple Negative Breast Cancer

摘要 在所有乳腺癌亚型中，三阴性乳腺癌(TNBC)的临床预后最差。 TNBC缺乏在其他亚型中发现的三种主要受体(雌激素受体[ER]， 孕激素受体和/或激素表皮生长因子受体-2)使这种癌症特别 在治疗方式方面具有挑战性。可杀死TNBC细胞或致敏的新型靶向治疗 他们非常渴望接受化疗和放射治疗，以提高这些患者的存活率。由于它的速度很快 增殖率，TNBC的生存在很大程度上依赖于DNA修复机制和参与其中的蛋白质 重要的检查站是癌症治疗的有吸引力的目标。CAPER(Rbm39)蛋白最近被发现 与正常乳腺组织相比，乳腺癌组织中高表达。当跳跃击倒时 抑制乳腺癌细胞生长及其在乳腺癌DNA损伤和修复机制中的作用 在TNBC中，化疗和放射治疗的进展和反应仍然完全未知。我们的 初步数据显示，在TNBC细胞中抑制CAPER的表达会增加DNA损伤，因为 表现为H_2AX和ATM的磷酸化增加。细胞总数减少，细胞总数增加 在MDA-MB-231和BT549 TNBC细胞中观察到CAPER基因敲除后caspase-3/7的切割 暗示无法克服的DNA损伤会导致细胞程序性死亡(细胞凋亡)。跳跃的效果 对TNBC细胞DNA损伤的抑制是不依赖于细胞周期的，对癌细胞具有选择性，因为非 成瘤细胞缺乏CAPER的表达，在慢病毒CAPER的传递后保持不受影响 ShRNA。我们的初步结果还显示，DNA修复蛋白RAD-51、C-Alb和Rb 在Caper被击倒后，TNBC下调了监管。我们假设CAPER在TNBC中的过度表达 通过优化DNA修复途径，在防止DNA损伤方面发挥重要作用。目前的提案 以我们以前的工作和初步结果为基础，旨在描述CAPER在基础DNA中的作用 损伤/修复途径及其在TNBC生长中的临床意义 (异种移植)和(同种)免疫活性(同种)原位小鼠模型(目标1)。重要的是，我们还将使用 这些TNBC细胞和小鼠模型来测试CAPER在DNA损伤反应中的未知作用 体外和体内环境下的化疗和放射治疗(目标2)。目前的提案将验证 CAPER作为一种重要的信号分子在TNBC的发生和对DNA的应答中的作用 使用与临床相关的模型进行损伤，该模型也包含免疫监视，并将在 进一步开发治疗TNBC的靶向治疗方法。

ABSTRACT Triple negative breast cancer (TNBC) has the poorest clinical outcome amongst all breast cancer subtypes. TNBC lacks the expression of the three major receptors found in other subtypes (estrogen receptor [ER], progesterone receptor, and/or hormone epidermal growth factor receptor-2) making this cancer particularly challenging with regards to treatment modalities. Novel targeted treatments that could kill TNBC cells or sensitize them to chemo- and radiation therapies are highly coveted to increase survival of these patients. Due to its fast proliferative rate, TNBC relies heavily on DNA repair mechanisms for its survival and proteins involved in this important checkpoint are attractive targets for cancer treatment. CAPER (Rbm39) protein was recently shown to be overexpressed in breast cancer specimens compared to normal breast tissues. While CAPER knockdown inhibits breast cancer cell growth, its role on DNA damage and repair mechanisms in breast cancer and its role in TNBC progression and response to chemo- and radiation therapies remain completely unexplored. Our preliminary data demonstrate that knockdown of CAPER expression in TNBC cells increases DNA damage as reflected by increased phosphorylation of H2AX and ATM. The decreased total cell number and increased caspase-3/7 cleavage observed following CAPER knockdown in MDA-MB-231 and BT549 TNBC cells is suggestive of insurmountable DNA damage leading to programmed cell death (apoptosis). The effect of CAPER knockdown on DNA damage in TNBC cells was cell cycle-independent and selective to cancer cells, as non- tumorigenic cells lack the expression of CAPER and remain unaffected following delivery of lentiviral CAPER shRNAs. Our preliminary results also revealed that DNA repair proteins RAD-51, C-Alb and RB were significantly downregulated in TNBC following CAPER knockdown. We posit that CAPER overexpression in TNBC plays an important role in protection against DNA damage by optimizing DNA repair pathways. The current proposal builds on our previous work and preliminary results and aims to delineate the roles of CAPER in basal DNA damage/repair pathways and to determine its clinical relevance in TNBC growth using both immunocompromised (xenograft) and immunocompetent (syngeneic) orthotopic mouse models (Aim 1). Importantly, we will also use these TNBC cells and mouse models to test the unexplored role of CAPER in the response to DNA damaging chemo- and radiation therapies in both in vitro and in vivo settings (Aim 2). The current proposal will validate the role of CAPER as an important signaling molecule in the progression of TNBC as well as response to DNA damage using clinically relevant models that also incorporate immune surveillance and will be insightful in the further development of targeted therapies for the treatment of TNBC.