ZIA BC 010830 (ZIA) | |||
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Title | Mechanisms of non-classical multidrug resistance in cancer | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Gottesman, Michael | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $1,007,541 | Project Dates | 00/00/0000 - 00/00/0000 |
Fiscal Year | 2017 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Bioengineering (15.0%) Cancer (100.0%) Chemotherapy (30.0%) Childhood Cancers (5.0%) Digestive Diseases (20.0%) Taxol (30.0%) |
Breast (30.0%) Cervical Cancer (10.0%) Colon/Rectum (10.0%) Kidney Cancer (5.0%) Kidney Disease (5.0%) Liver Cancer (10.0%) Ovarian Cancer (15.0%) Prostate (10.0%) Urinary System (5.0%) |
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Research Type | |||
Systemic Therapies - Discovery and Development Development and Characterization of Model Systems |
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Abstract | |||
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. These 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. Our interest in the checkpoint kinases and their role in resistance led us to the protein phosphatase 2A (PP2A) inhibitor LB100, currently in Phase I clinical trials for breast cancer. As PP2A controls the phosphorylation status of a number of DNA-damage response (DDR) genes, we hypothesized that LB100 would sensitize ovarian cancer cells to cisplatin. We demonstrated that inhibition of PP2A by LB100 sensitized cells (OVCAR8 and SKOV3) to cisplatin, and that LB100 induces hyperphosphorylation of Chk1 and other genes in the DNA-damage response pathway, preventing cisplatin-induced G2 arrest and forcing cells into mitosis, resulting in apoptosis. We showed that mice injected intraperitoneally with SKOV3-luciferase cells were sensitized to cisplatin (3 mg/kg) when treated with LB100 compared with control. We recently also completed an RNAi screen in cells exposed to cisplatin, in order to identify genes associated with cisplatin sensitivity. If cells exposed to sub-toxic cisplatin undergo cell death when a particular gene is deleted, one can hypothesize that inhibition of this gene target might prove to be a useful adjuvant for platinum chemotherapy. The strongest sensitizing effects were observed when DNA damage repair genes (including a phosphoprotein phosphatase) were silenced, and several of these are now being investigated for their role in cisplatin tolerance. In this screening context, we found a need to identify a solvent appropriate for dissolving cisplatin for screening. We recently showed that DMSO inactivated the biological activity of all clinical and experimental platinum complexes tested. Furthermore, a review of the cisplatin literature revealed that about a third of all research papers have used cisplatin dissolved in DMSO, calling into question the data and conclusions of those papers. This has important implications for the reliability of a significant portion of the literature and points the way for appropriate use of platinum drugs in research. In another approach, we have developed a Taqman Low Density Array (TLDA) microfluidic chip to detect mRNA expression of 380 different putative drug resistance genes and demonstrated that it is a sensitive, accurate, reproducible, and robust way to measure mRNA levels in tumor samples. Previous work from our laboratory indicates that mRNA measurements of levels of drug-resistance genes are, to a first approximation, predictive of functional expression of drug-resistance mechanisms. This drug-resistance chip has been applied to analysis of human cancers. One result from this analysis is that existing cancer cell lines do not mimic the expression patterns of actual human cancers for the 380 |