MiR-152/PKM2/SLC7A5 axis in breast cancer development, chemo- and radiation-treatment response

乳腺癌(BCA)是全球女性癌症死亡的主要原因，三重阴性乳腺癌 癌细胞(TNBC)占BCA的15%-20%。然而，较高的治疗耐药性和较低的存活率 的患者仍然是BCA治疗的主要障碍。阿霉素(Dox)和放射治疗是常见的 用于TNBC治疗。在我们的初步研究中，我们发现TNBC的表达要低得多 MiR-152水平。ZEST同源物2(EZH2)和DNA高甲基化的表达水平较高 参与抑制TNBC细胞miR-152的表达。为了解miR-152抑制的机制，以及 MiR-152在调控肿瘤发生、阿霉素和放射诱导中的作用及其机制 ，我们进行了大量的初步实验，以确定哪些分子是潜在的 MIR-152的直接靶点以及在耐药的TNBC或PDX肿瘤组织中上调的靶点。我们 发现丙酮酸激酶2(PKM2)，溶质载体家族7成员5(SLC7A5)，以及 多嘧啶结合蛋白(PTBP1)作为miR-152的直接靶标具有潜在的逆转作用 DOX介导的抗性。当miR-152表达时，PKM2、SLC7A5和PTBP1水平增加 在TNBC细胞和PDX模型中被抑制。我们发现miR-152的抑制发挥了重要的作用 在介导TNBC代谢重编程、Warburg效应转换、肿瘤生长和多药耐药中的作用 通过PKM2/SLC7A5/PTBP1。我们发现miR-152或PKM2基因敲除的过表达呈现 TNBC细胞对放射治疗更为敏感。这些新发现为调查创造了新的机会 TNBC耐药机制的研究。我们假设对化疗的抗药性 MiR-152诱导放射治疗、代谢重编程和TNBC的发展 抑制和上调PKM2、SLC7A5和PTBP1。为了检验这一中心假设，我们将 通过三个目标进行实验。在目标1中，我们将研究miR-152的作用和机制 抑制多柔比星和放射诱导的耐药对TNBC细胞的影响 PKM2开关和通过miR-152抑制选择性剪接诱导诱导Warburg效应， 治疗耐药和肿瘤生长。在目标2中，我们将确定miR-的作用和机制。 152/SLC7A5/PTBP1通路在调节代谢重编程和Warburg效应开关中的作用 介导阿霉素和辐射诱导的治疗耐药。在目标3中，我们将确定是否 MIR-152抑制和PKM2诱导通过CXCL8表达调节肿瘤血管生成 人源化嵌合肿瘤模型；以及miR-152、PKM2、HIF-1α、hnRNPA3、SLC7A5和 PTBP1相互关联，并与治疗反应和癌症分期相关 和生存。这个R01项目将确定治疗耐药的机制，以及代谢 重新规划；并为今后开发新的TNBC治疗方案提供信息。

Breast cancer (BCa) is the leading cause of women cancer mortality worldwide, and triple negative breast cancer (TNBC) cells accounts for 15-20% BCa. However, higher therapeutic resistance and lower survival of TNBC patients remain the major hinder of BCa treatment. Doxorubicin (Dox) and radiation are commonly used for TNBC treatment. In our preliminary study, we found that TNBC showed much lower expression levels of miR-152. Higher expression levels of zeste homologue 2 (EZH2) and DNA hypermethylation were involved in miR-152 suppression in TNBC cells. To understand mechanism of miR-152 suppression, and role and mechanism of miR-152 in regulating cancer development and Dox- and radiation-induced resistance, we performed a lot of preliminary experiments to identify what molecules that were potential miR-152 direct targets as well as that were upregulated in Dox-resistant TNBC or PDX tumor tissues. We found that pyruvate kinase muscle 2 (PKM2), solute Carrier Family 7 Member 5 (SLC7A5), and polypyrimidine tract-binding protein (PTBP1) as direct targets of miR-152 with potential ability to converse Dox-mediated resistance. PKM2, SLC7A5 and PTBP1 levels were increased when miR-152 expression was suppressed in TNBC cells and PDX model. We found that miR-152 suppression played an important role in mediating TNBC metabolic reprogramming, Warburg effect switch, tumor growth and Dox resistance through PKM2/SLC7A5/PTBP1. We showed that overexpression of miR-152 or PKM2 knockdown rendered TNBC cells more sensitive to radiation treatment. These new findings create new opportunity to investigate mechanism of TNBC therapeutic resistance. We hypothesize that resistance to chemotherapy and radiation treatment, metabolic reprogramming, and TNBC development are induced by miR-152 suppression and upregulation of PKM2, SLC7A5, and PTBP1. To test this central hypothesis, we will perform experiments through three aims. In Aim 1, we will investigate role and mechanism of miR-152 suppression in TNBC cells in doxorubicin- and radiation-induced resistance; and role and mechanism of PKM2 switch and induction via alternative splicing by miR-152 suppression to induce Warburg effect, therapeutic resistance, and tumor growth. In Aim 2, we will determine role and mechanism of miR- 152/SLC7A5/PTBP1 pathway in regulating metabolic reprogramming and Warburg effect switch, and in mediating doxorubicin- and radiation-induced therapeutic resistance. In Aim 3, we will determine whether miR-152 suppression and PKM2 induction regulate tumor angiogenesis through CXCL8 expression using a humanized chimeric tumor model; and whether levels of miR-152, PKM2, HIF-1α, hnRNPA3, SLC7A5, and PTBP1 are correlated each other, and are correlated with therapeutic responses and with the cancer stages and survival. This R01 project will identify mechanisms of therapeutic resistance, and metabolic reprogramming; and provide information for developing new treatment option of TNBC in the future.