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

肺癌相关死亡主要是由于疾病复发、耐药性和转移。铂 诸如顺铂（CDDP）和卡铂（CBDCA）的化合物及其衍生物广泛用于治疗癌症。 肺癌的治疗尽管肿瘤最初对铂类药物有反应， 从而逃避治疗。因此，了解癌细胞逃避的机制， 治疗和产生耐药性对于开发肺癌的新治疗方法至关重要。 该应用程序解决了一个高度创新和高影响力的转化研究领域，重点是 研究铂类药物如何影响蛋白酶体和螯合体（SQSTM 1）/P62功能， 癌细胞对肺癌产生抗药性。此外，一种靶向靶向药物的纳米递送方法， 蛋白酶体和SQSTM 1/P62与CDDP组合用于克服抗性。 我们对检测蛋白酶体和SQSTM 1/P62在化疗耐药性中的作用的兴趣源于一个偶然的发现。 在实验室进行的观察。我们观察到β 5（β5）的表达，β 5是大26 S 顺铂耐药（CDDPR）癌细胞系中的蛋白酶体复合物显著减少， 其等基因顺铂敏感（CDDPS）细胞系。β5是蛋白酶体的胰凝乳蛋白酶组分， 所需的降解泛素化的蛋白质和回收的氨基酸合成新的蛋白质， cell.与CDDPR细胞中β5表达减少相关的是蛋白质的细胞内积累。 研究了细胞内蛋白质积累引起的细胞应激如何被细胞因子克服。 细胞揭示了SQSTM 1/P62的作用。SQSTM 1/P62是一种支架蛋白， 在细胞应激反应中被激活，是通过聚集细胞内积累的 多聚泛素化蛋白进入侵袭体并导向自噬，从而促进细胞存活。 此外，对人肺肿瘤组织亚组中P62表达的分析表明，化疗后P62表达增加， 肺癌P62表达高于化疗组。虽然，蛋白酶体减少 先前已经报道了在癌细胞和干细胞中SQSTM 1/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 抗性以前没有被研究过，并且是该创新建议的基础。 基于我们的初步结果，我们假设蛋白酶体和SQSTM 1/p62功能的改变， 导致了铂耐药性。为了验证我们的假设，我们确定了三个具体目标。 目标1。研究如何在CDDPR和CDDPS癌细胞中调节蛋白酶体和SQSTM 1/P62， 在正常细胞中改变了体外对铂类药物的治疗反应。 在这个目标中，蛋白酶体β-5亚基和SQSTM 1/P62对铂敏感性的要求是 和对其他抗癌药物的交叉耐药性将使用同基因人类癌细胞系进行研究， 与正常细胞相比。 目标2.证明恢复蛋白酶体的β-5亚基，同时敲低 使用多功能纳米颗粒的SQSTM 1/p62在体内逆转CDDPR肺肿瘤对顺铂的敏感性 为此，我们将使用肺肿瘤异种移植物和患者来源的异种移植物模型（PDX）来测试a）是否 CDDPR肿瘤中β-5的恢复与P62的同时沉默恢复了铂敏感性和B） CDDPS肿瘤中β-5的敲低伴随P62的过表达导致铂抗性。 目标3：确定β-5和SQSTM 1/P62蛋白表达在化疗中的生物学意义 和具有临床益处的化学处理的人肺肿瘤组织标本。 为此，将检查代表原发性和转移性肿瘤的存档肺肿瘤标本， β 5和SQSTM 1/P62表达与临床获益相关。

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.