Molecular Impact of Platinum Drugs on the Proteasome and SQSTM1_P62 Complexes_A Paradigm Shift in Resistance

与肺癌相关的死亡主要是由于疾病复发、耐药和转移。白金 顺铂(CDDP)和卡铂(CBDCA)及其衍生物等化合物广泛应用于 肺癌的治疗。虽然肿瘤最初对铂类药物有反应，但它们会熟练地发展。 抵抗力，从而逃避治疗。因此，了解癌细胞逃避的机制 治疗和产生耐药性是开发肺癌新的治疗方法的关键。 该应用程序解决了高度创新和高影响力的翻译研究领域，其重点是 研究铂类药物对蛋白酶体和隔离小体(SQSTM1)/p62功能的影响 癌细胞在肺癌中产生抗药性。此外，以纳米递送方法为目标的 建议将蛋白酶体和SQSTM1/p62与顺铂联合用于克服抗性。 我们对检测蛋白酶体和SQSTM1/p62对化疗耐药性的兴趣源于一次偶然的 在实验室里所做的观察。我们观察到β5(β5)的表达，这是大26S的一个亚单位 与顺铂耐药(CDDPR)癌细胞株相比，蛋白酶体复合体显著减少 其同基因顺铂敏感(CDDPS)细胞系。β5是蛋白酶体的胰凝乳酶成分，即 降解泛素化蛋白质和回收氨基酸以合成新蛋白质所必需的 手机。β-5在CDDPR细胞中的表达减少与细胞内蛋白质的积累有关。 细胞内蛋白质积累引起的细胞应激是如何被克服的 细胞揭示了SQSTM1/p62的作用。SQSTM1/p62是一种支架蛋白，其主要功能是 在细胞应激反应中被激活，是通过聚集细胞内积累的物质来防止细胞死亡 多泛素化蛋白进入侵袭体并引导自噬，从而促进细胞存活。 此外，对人类肺癌组织亚群中p62表达的分析表明，化疗经历了 肺肿瘤p62的表达高于化疗后的肿瘤。尽管减少了蛋白酶体 以前在癌细胞和干细胞中已经报道了SQSTM1/p62的功能和增加的表达 细胞，化疗药物对这些细胞机制的影响以及它们在促进 阻力以前没有被调查过，是这项创新提案的基础。 根据我们的初步结果，我们假设蛋白酶体和SQSTM1/p62功能的改变 癌细胞中对铂的耐药性也有贡献。为了验证我们的假设，我们确定了三个具体目标。 目的1.研究CDDPR和CDDPS癌细胞中蛋白酶体和SQSTM1/p62的调控 在体外，在正常细胞中改变对铂类药物的治疗反应。 为此，蛋白酶体的β-5亚基和SQSTM1/p62对铂敏感性的要求 对其他抗癌药物的交叉耐药性将使用同基因的人类癌细胞株和 与正常细胞相比。 目的2.证明用同时击倒的方法恢复蛋白酶体的β-5亚单位 使用多功能纳米颗粒的SQSTM1/p62在体内逆转CDDPR肺癌对顺铂的敏感性。 为此，我们将使用肺肿瘤异种移植和患者来源的异种移植模型(Pdx)来测试a)是否 在CDDPR肿瘤中恢复β-5并同时沉默p62可恢复铂敏感性和b) CDDPS肿瘤中β-5基因的下调伴随着p62的过表达导致对铂类药物耐药。 目的3.确定β-5和SQSTM1/p62蛋白在化疗中的生物学意义 并对化疗后的人肺肿瘤组织标本进行临床应用。 在这个目标中，代表原发和转移肿瘤的档案肺肿瘤标本将被检查 β-5和SQSTM1/p62的表达与临床疗效相关。

Lung cancer-related death is primarily due to disease recurrence, drug resistance, and metastasis. Platinum compounds such as cisplatin (CDDP) and carboplatin (CBDCA) and their derivatives are widely used in the treatment of lung cancer. Although the tumors initially respond to platinum drugs, they adeptly develop resistance thereby escaping therapy. Therefore, understanding the mechanisms by which cancer cells evade therapy and develop resistance is essential for developing new therapeutic approaches for lung cancer. This application addresses a highly innovative and high-impact area of translational research that focuses on investigating how platinum-based drugs impact the proteasome and sequestosome (SQSTM1)/P62 function in cancer cells to produce drug resistance in lung cancer. Further, a nanodelivery approach targeted towards the proteasome and SQSTM1/P62 in combination with CDDP for overcoming resistance is proposed. Our interest in testing the proteasome and SQSTM1/P62 in chemoresistance stems from a serendipitous observation made in the laboratory. We observed beta 5 (β5) expression, a subunit of the large 26S proteasome complex was markedly reduced in cisplatin-resistant (CDDPR) cancer cell lines when compared to its isogenic cisplatin-sensitive (CDDPS) cell lines. β5 is the chymotryptic component of the proteasome that is required for degrading ubiquitinated proteins and recycling of amino-acids for synthesis of new proteins in the cell. Associated with reduced β5 expression in the CDDPR cells was the intracellular accumulation of proteins. Investigation into how the cellular stress induced by intracellular accumulation of proteins is overcome by the cells revealed a role for SQSTM1/P62. The primary function of SQSTM1/P62, a scaffolding protein that is activated in response to cellular stress, is to prevent cell death by aggregating intracellular accumulated polyubiquitinated proteins into aggresomes and directing towards autophagy, thereby promoting cell survival. Further, analysis for P62 expression in a subset of human lung tumor tissues showed that chemoexperienced lung tumors had higher P62 expression compared to chemonaive tumors. Although, reduced proteasome function and increased SQSTM1/P62 expression have previously been reported in cancer cells and in stem cells, the impact of chemotherapy drugs on these cellular machineries and their role in contributing to resistance has not been previously investigated and is the basis of this innovative proposal. Based on our preliminary results, we hypothesize that alterations in the proteasome and SQSTM1/p62 function in cancer cells contributes to platinum resistance. To test our hypothesis we have identified three specific aims. Aim 1. Investigate how modulating the proteasome and SQSTM1/P62 in CDDPR and CDDPS cancer cells and in normal cells alters the therapeutic response to platinum drugs in vitro. In this aim, the requirement of beta-5 subunit of the proteasome and SQSTM1/P62 to platinum sensitivity and cross-resistance to other anticancer drugs will be investigated using isogenic human cancer cell lines and compared to normal cells. Aim 2. Demonstrate restoring beta-5 subunit of the proteasome with simultaneous knock-down of SQSTM1/p62 using multifunctional nanoparticle reverts CDDPR lung tumor sensitivity to cisplatin in vivo. In this aim, we will use lung tumor xenograft and patient-derived xenograft models (PDX) to test a) whether restoration of beta-5 with simultaneous silencing of P62 in CDDPR tumors restores platinum sensitivity and b) knock-down of beta-5 with concomitant overexpression of P62 in CDDPS tumors results in platinum resistance. Aim 3. Determine the biological significance of beta-5 and SQSTM1/P62 protein expression in chemonaïve and chemotreated human lung tumor tissue specimens with clinical benefit. In this aim, archival lung tumor specimens representing primary and metastatic tumor will be examined for beta-5 and SQSTM1/P62 expression and correlate with clinical benefit.