Mechanisms of non-classical multidrug resistance in cancer

三种主要的方法被用来定义癌症中的非经典多药耐药。首先，我们分离出对顺铂（CP-r）耐药的KB细胞和卵巢癌细胞，并证明了对许多其他细胞毒性药物的多药耐药。在某些情况下，这种交叉抗性模式是由于每种药物的摄取减少，因为它们的受体已经从细胞表面重新定位到细胞的细胞质中。表面转运蛋白的这种重新定位似乎是由于顺铂耐药细胞中细胞骨架的改变影响了内吞循环区，从而改变了这些转运蛋白的再循环。最近对来自顺铂耐药人类癌症的顺铂耐药细胞系的研究表明，顺铂积累减少并不是耐药肿瘤的必然特征。我们正在使用RNA-seq、ATAC-seq和Pro-seq技术进行完整的基因组分析，以确定顺铂选择细胞系中伴随耐药发展的基因表达改变，并对顺铂耐药细胞中导致耐药的改变进行编目录。这些变化将与浆液性卵巢癌和小细胞肺癌临床样本中的基因表达变化进行比较，其中主要治疗包括顺铂作为细胞毒性药物。初步结果表明，除了参与DNA损伤修复的基因外，许多基因与卵巢癌细胞顺铂耐药的进化有关。我们还在暴露于多种不同药物（包括顺铂）的细胞中进行CRISPR阳性筛选，以确定与顺铂敏感性相关的基因。暴露于顺铂或其他药物的细胞会发生细胞死亡，存活的细胞会过度表达grna，这些grna会开启能够独立赋予耐药性的基因。组蛋白去乙酰化酶抑制剂（hdi）在临床上用于治疗皮肤和周围t细胞淋巴瘤，其中3种hdi已被FDA批准为单药治疗。在实体瘤的情况下，hdi没有效果，这表明对这些药物的内在耐药机制。基于与Susan Bates博士合作的早期发现，表明信号通路的激活可以增强对HDI罗米地辛的耐药性，我们发现HDI和MAPK和PI3K信号通路抑制剂可以在含有Ras突变的细胞中实现协同杀伤。我们还发现，当与HDI联合使用时，双重ERK/PI3K抑制剂可以取代单独的MAPK和PI3K抑制剂。进一步的研究表明，双BRD4/PI3K抑制剂SF2523与HDI联合使用时对Ras突变细胞具有协同毒性。与Mari Yohe博士合作，我们证明SF2523单独对儿童横纹肌肉瘤细胞系模型特别有效，并且可以通过添加HDI罗米地新来提高其疗效。美国高级临床前研究中心（CAPR）已同意研究SF2523/罗米地辛联合疗法在横纹肌肉瘤患者来源异种移植模型中的疗效。验证这些结果，表明耐多药在临床癌症中是复杂和多因素的，将需要开发可靠的体外培养模型。为了实现这一目标，我们开发了一种生物反应器，可以模拟在3D悬浮液中生长的细胞中氧气的毛细管输送（通过硅水凝胶和聚合物PTMS）。我们已经证明了生理氧梯度和癌细胞的改变生长更接近体内表型。通过详细的RNAseq分析获得了氧气梯度实质上改变基因表达模式的证据。通过人工毛细血管将生理浓度的3%氧气直接输送到细胞中，模拟了通过扩散输送20%氧气的基因表达模式。该生物反应器可按比例放大，用于原发癌细胞或培养癌细胞的多种培养物的生长，以确定生长条件和氧气输送模式是否在影响耐药模式中起主要作用。

Three major approaches have been taken to define non-classical multidrug resistance in cancer. In the first, we isolate KB cells and ovarian cancer cells resistant to increasing levels of cisplatin (CP-r) and demonstrate multidrug resistance to many other cytotoxic agents. In some cases, this cross-resistance pattern is due to reduced uptake of each of these agents because their receptors have been relocalized from the cell surface into the cytoplasm of the cell. This relocalization of surface transporters appears to be due to altered recycling of these transporters due to alterations in the cytoskeleton that affect endocytic recycling compartments in cisplatin-resistant cells. Recent studies on cisplatin-resistant cell lines derived from cisplatin-resistant human cancers indicate that reduced cisplatin accumulation is not an obligatory characteristic of resistant tumors. We are undertaking a complete genomic analysis using RNA-seq, ATAC-seq and Pro-seq technologies to define the alterations in gene expression that accompany the development of drug resistance in cisplatin-selected cell lines and one cataloguing alteration in cisplatin-resistant cells that contribute to drug resistance. These changes will be compared to gene expression changes in clinical samples of serous ovarian cancer and small cell lung cancers for which the primary treatment involves cisplatin as a cytotoxic agent. Preliminary results indicate that a number of genes in addition to those involved in DNA damage repair are associated with the evolution of cisplatin resistance in ovarian cancer cells. We are also undertaking a positive CRISPR screen in cells exposed to multiple different drugs including cisplatin, in order to identify genes associated with cisplatin sensitivity. Cells exposed to cisplatin or other drugs undergo cell death and surviving cells overexpress gRNAs which turn on genes which can independently confer resistance. Histone deacetylase inhibitors (HDIs) are used clinically to treat cutaneous and peripheral T-cell lymphomas, diseases for which 3 HDIs have been FDA approved as single-agent therapies. In the case of solid tumors, the HDIs have not been effective, suggesting intrinsic resistance mechanisms to these drugs. Based on earlier findings in collaboration with Dr. Susan Bates demonstrating that activation of signaling pathways can potentiate resistance to the HDI romidepsin, we found that synergistic killing can be achieved with HDIs and inhibitors of the MAPK and PI3K signaling pathways in cells that harbor Ras mutations. We also found that a dual ERK/PI3K inhibitor could take the place of separate MAPK and PI3K inhibitors when combined with an HDI. Further studies have shown that the dual BRD4/PI3K inhibitor SF2523 is synergistically toxic to Ras mutant cells when combined with an HDI. In collaboration with Dr. Mari Yohe, we demonstrated that SF2523 alone is particularly effective in childhood rhabdomyosarcoma cell line models and its efficacy can be increased by the addition of the HDI romidepsin. The Center for Advanced Preclinical Research (CAPR) has agreed to examine the efficacy of the SF2523/romidepsin combination in patient-derived xenograft models of rhabdomyosarcoma. Validation of these results, indicating that MDR is complex and multifactorial in clinical cancers, will require the development of reliable in vitro culture models. Towards this goal, we have developed a bioreactor that mimics capillary delivery (through silicon hydrogels and the polymer PTMS) of oxygen to cells grown in 3D suspension. We have demonstrated physiological oxygen gradients and altered growth of cancer cells more closely approximating in vivo phenotypes. Evidence that oxygen gradients substantially change gene expression patterns has been obtained by detailed RNAseq analysis. Delivery of physiological concentrations of 3% oxygen directly to cells via artificial capillaries mimics the gene expression patterns of 20% oxygen delivered viadiffusion. The bioreactor can be scaled up for growth of multiple cultures of primary cancer cells or cultured cancer cells to determine whether growth conditions and mode of oxygen delivery play a primary role in affecting patterns of drug resistance.