Nanotechnology for targeted therapy and fundamental understanding oftherapeutic resistance in triple negative breast cancer

项目概要/摘要 乳腺癌是美国女性癌症相关死亡的第二大原因。尤其未 靶向治疗在临床上可用于几乎所有三阴性乳腺癌（TNBC）。癌症的出现是一种 是遗传变异累积的结果。因此，开发新的策略来精确地靶向遗传 TNBC的改变可能对对抗恶性疾病有价值。TP 53是一种重要的肿瘤抑制因子 在大多数人类癌症中由于突变或缺失而失活的基因。为了恢复 由TP 53编码的p53蛋白用于癌症治疗的活性。不幸的是，没有基于p53的疗法 由于p53信号传导的复杂性，已经成功地转化为临床。因此，识别 由TP 53缺失而不是恢复p53活性所赋予的脆弱性是一种新的对抗 癌在我们最近发表在《自然》和《自然纳米技术》上的工作中，我们揭示了 TP 53通常伴有半合子（即，部分）丧失细胞所必需的邻近基因POLR 2A 几乎所有53%具有TP 53缺失的TNBC都具有半合子P0 LR 2A缺失（TP 53/P0 LR 2A缺失）。 我们的初步数据表明，通过小干扰RNA干扰抑制POLR 2A表达， 使用低pH激活的纳米球递送的RNA（siRNA）选择性地抑制细胞的增殖、存活和增殖。 TP 53/P0 LR 2Aloss TNBC细胞的致瘤潜力。纳米炸弹保护血液中的siRNA， siRNA的内/溶酶体逃逸进入胞质溶胶，在胞质溶胶中siRNA执行其POLR 2A抑制功能 细胞摄取后。此外，纳米炸弹介导的靶向POLR 2A的siRNA的递送选择性地抑制了P0 LR 2A的表达。 原位TP 53/P0 LR 2A缺失TNBC肿瘤的生长，没有明显的全身毒性， 关于动物体重和血液蛋白（用于肝功能）和细胞因子（用于免疫应答）的数据。 然而，一小部分乳腺癌细胞过度表达CD 44（注意：不是非CD 44）。 正常干细胞上的正常或变异的CD 44）已经显示对临床上使用的抗CD 44抗体特别有抗性。 TNBC的化疗药物如紫杉醇（PTX）。由于POLR 2A对于癌细胞是不可或缺的， 存活，我们假设靶向POLR 2A siRNA和PTX靶向共递送至变体 CD 44+癌细胞可以克服TNBC耐药性。我们将进一步发展上述低 没有主动靶向的pH激活纳米球，能够主动靶向变体CD 44 + 细胞和肿瘤脉管系统。由于癌症转移是癌症相关死亡的主要原因，我们将测试 不仅使用上述原位/原发性TNBC肿瘤而且使用转移性TNBC肿瘤的假设 模型此外，我们还将研究POLR 2A靶向治疗的耐药机制， 不仅是2D和异种移植物，而且还使用我们开发的微流体方法生成3D TNBC模型。 总的来说，这个项目可能会导致一种新的治疗耐药TNBC的机制的理解， 其对于对抗TNBC和可能的许多其它类型的具有TP 53缺失的癌症是非常宝贵的。

Project Summary/Abstract Breast cancer is the second leading cause of cancer-related deaths of American women. In particular, no targeted therapy is clinically available for nearly all triple negative breast cancer (TNBC). Cancer arises as a result of accumulating genetic alterations. Therefore, developing novel strategies to precisely target the genetic alterations of TNBC may be valuable for combating the malignant disease. TP53 is a pivotal tumor suppressor gene inactivated by mutation or deletion in most human cancers. Tremendous effort has been made to restore the activity of the p53 protein encoded by TP53 for cancer treatment. Unfortunately, no p53-based therapy has been successfully translated into the clinic, due to the complexity of p53 signaling. Therefore, identifying vulnerabilities conferred by TP53 deletion instead of restoring the p53 activity is a novel strategy for combating cancer. In our recent work published in Nature and Nature Nanotechnology, we revealed genomic deletion of TP53 is often accompanied by hemizygous (i.e., partial) loss of a neighboring gene POLR2A essential for cell survival, and virtually all 53% TNBCs with TP53 deletion harbor hemizygous POLR2A loss (TP53/POLR2Aloss). Our preliminary data show that suppressing POLR2A expression by RNA interference with small interfering RNA (siRNA) delivered using a low pH-activated nanobomb selectively inhibits the proliferation, survival, and tumorigenic potential of TP53/POLR2Aloss TNBC cells. The nanobomb protects the siRNA in blood and enables endo/lysosomal escape of the siRNA into the cytosol where the siRNA performs its POLR2A inhibition function after cell uptake. Moreover, the nanobomb-mediated delivery of POLR2A-targeting siRNA selectively inhibits the growth of orthotopic TP53/POLR2Aloss TNBC tumors, with no evident systemic toxicity demonstrated by the data on animal body weight and blood proteins (for liver function) and cytokines (for immune responses). However, a small fraction of breast cancer cells overexpressed with the variant CD44 (note: not the non- variant or normal CD44 on normal stem cells) have been shown to be particularly resistant to clinically used chemotherapy drugs of TNBC such as paclitaxel (PTX). Since POLR2A is indispensable for cancer cells to survive, we hypothesize that targeted co-delivery of the POLR2A-targeting siRNA and PTX to the variant CD44+ cancer cells can overcome the TNBC drug resistance. We will further develop the aforementioned low pH-activated nanobomb that has no active targeting, to be capable of actively targeting both the variant CD44+ cells and tumor vasculature. Since cancer metastasis is the major cause of cancer-related death, we will test the hypothesis using not only the aforementioned orthotopic/primary TNBC tumors but also metastatic TNBC model. Furthermore, we will investigate the mechanisms of resistance to the POLR2A-targeted therapy using not only 2D and xenograft but also 3D TNBC models generated using microfluidic approach developed by us. Collectively, this project may result in a novel therapy for drug-resistant TNBC with mechanistic understanding, which is invaluable for combating TNBC and possibly many other types of cancers harboring TP53 deletion.