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

项目摘要/摘要 乳腺癌是美国女性癌症相关死亡的第二大原因。尤其是，没有 靶向治疗可用于临床上几乎所有三阴性乳腺癌(TNBC)。癌症的发生是因为 累积基因改变的结果。因此，开发新的策略来精确地针对基因 TNBC的改变可能对抗击恶性疾病有价值。TP53是一种关键的肿瘤抑制因子 大多数人类癌症中因突变或缺失而失活的基因。已经做出了巨大的努力来恢复 TP53编码的P53蛋白用于癌症治疗的活性。不幸的是，没有一种基于p53的疗法 由于P53信号转导的复杂性，已经成功地将其转化到临床上。因此，识别 由TP53缺失而不是恢复P53活性带来的漏洞是一种新的抗击 癌症。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 TP53通常伴随着细胞必需的邻近基因POLR2A的半合子(即部分)丢失 存活率，并且几乎所有具有TP53缺失的TNBCs都存在半合子POLR2A型缺失(TP53/POLR2Aoss)。 我们的初步数据表明，通过RNA干扰和小干扰来抑制POLR2A的表达 使用低pH激活的纳米颗粒传递的RNA(SiRNA)选择性地抑制细胞的增殖、存活和 TP53/POLR2缺失TNBC细胞的致瘤作用纳米炸弹保护血液中的siRNA，并使 SiRNA的内切/溶酶体逃逸到胞浆中，在胞浆中siRNA执行其POLR2A抑制功能 在细胞摄取之后。此外，纳米炸弹介导的以POLR2A为靶向的siRNA选择性地抑制 原位移植的TP53/POLR2无TNBC肿瘤生长，无明显的全身毒性 动物体重和血液蛋白(对肝功能)和细胞因子(对免疫反应)的数据。 然而，一小部分乳腺癌细胞过度表达CD44变体(注：不是非 正常干细胞上的变异或正常CD44)已被证明对临床使用具有特别的抵抗力 紫杉醇(PTX)等TNBC类化疗药物。由于POLR2A对于癌细胞来说是不可缺少的 存活下来，我们假设靶向POLR2A的siRNA和PTX的靶向联合递送到变异体 CD44+肿瘤细胞可克服TNBC耐药。我们将进一步发展前述的低 PH激活的无主动靶向的纳米炸弹，能够主动靶向CD44+变体 细胞和肿瘤血管系统。由于癌症转移是癌症相关死亡的主要原因，我们将测试 假设不仅使用上述原位/原发TNBC肿瘤，还使用转移性TNBC 模特。此外，我们还将研究POLR2A靶向治疗耐药的机制 不仅是2D和异种移植，而且还有我们开发的微流控方法建立的三维TNBC模型。 总的来说，这个项目可能会产生一种新的治疗耐药的TNBC的方法， 这对于对抗TNBC和可能含有TP53缺失的许多其他类型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.