Mechanisms of non-classical multidrug resistance in cancer

Division Of Basic Sciences - Nci

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Abstract

Three major approaches have been taken to define non-classical multidrug resistance in cancer. In the first, we isolate cancer cells resistant to increasing levels of cisplatin (CP-r) and histone deacetylase (HDAC) inhibitors and demonstrate multidrug resistance to other cytotoxic agents. We have undertaken 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 alterations in cisplatin-resistant cells that contribute to drug resistance. Comparing gene expression in cisplatin-sensitive 1A9 ovarian cancer cells, cisplatin-resistant 1A9CP80 cells and partially revertant 1A9CP80R cells, we noted increased expression of TPPP3 (tubulin polymerization promoting protein 3) in the resistant cells, with lower levels in the revertant cells and no expression observed in the parental line. Additionally, we noted that cisplatin treatment destabilizes microtubule ends and reduces microtubule length and hypothesized that TPPP3 might mitigate these effects. Interestingly, the ability of TPPP3 to counteract the effects of cisplatin treatment were most effective in tubulin purified from 1A9CP80 cells and least effective in 1A9 cells and appeared to correlate with changes in expression of tubulin isoforms in the cell lines. Deletion of TPPP3 via CRISPR knockout partially resensitized the 1A9CP80 cells to cisplatin. High expression of TPPP3 in tumors from patients treated with cisplatin correlated with worse survival probability, suggesting a possible clinical role for this protein. In addition, cells exposed to cisplatin (which we have shown destabilizes microtubules) are more resistant to paclitaxel, an antimicrobial drug that stabilizes microtubules. These results have implications both for the neurotoxicity of cisplatin, and for strategies that employ combinations of cisplatin and paclitaxel to treat ovarian cancer and other cancers. To understand more about non-classical mechanisms of multidrug resistance in cancer, we are undertaking CRISPR screens in cells exposed to various drugs including cisplatin, oxaliplatin, and paclitaxel. These screens involve using gRNAs in combination with CRISPR-cas constructs that can activate, inhibit, or knock out target genes. Pancreatic cancer cells exposed to the paclitaxel-albumi complex Abraxane survive when the spindle accessory checkpoint is inactivated. Cells exposed to platinum compounds or other drugs undergo cell death and surviving cells overexpress gRNAs which turn on genes which can independently confer resistance, or under express genes whose expression is needed for sensitivity to cisplatin. We are identifying genes whose over- or under-expression affects drug resistance with the goal of defining clinically relevant molecular changes. We have also used an expression library of 1800 transcription factors to study the developmental pathways in neuroendocrine types of small cell lung cancer to determine which factors and pathways predispose to cisplatin resistance and have identified several candidate transcription factors. Recent studies using CRISPR screens to determine the basis of oxaliplatin resistance in colon cancer cells has identified an amino acid transporter as playing an important role in sensitivity to this drug. This transporter, known as LAT3 or SLC43a1, when overexpressed in several cell types results in increased sensitivity specifically to oxaliplatin and increased intracellular accumulation compared to other platinum compounds. When knocked out, the cells become resistant to oxaliplatin and have reduced cellular accumulation. The mechanism by which LAT3 regulates oxaliplatin levels is currently under investigation. In another screen selecting for resistance to the CHEK1/2 inhibitor, prexasertib (a drug in clinical trials for treatment of cisplatin/paclitaxel resistant ovarian cancer) in OVCAR8 cells, we found several genes involved directly in the toxicity pathway of prexasertib, some genes which seemed to modify sensitivity to prexaserib, as well as three transporters which affect its accumulation in cells. Several of these genes are potential targets to reduce resistance to prexasertib which occurs clinically. In the case of solid tumors, histone deacetylase inhibitors (HDIs) have not been effective, suggesting intrinsic resistance mechanisms to these drugs. Resistance to HDI's such as romidepsin can occur in cultured cells owing to overexpression of P-glycoprotein, but in clinical cancers resistance does not appear to be due to this mechanism. To identify non-P-gp mechanisms of resistance, we selected MCF-7 breast cancer cells with romidepsin and verapamil to yield the MCF-7 DpVp300 line which is about 200-fold more resistant to romidepsin than the parental cells. Knockout of METTL7A from DpVp300 cells resensitized the cells to romidepsin as well as other thiol-based HDIs such as KD5170 and largazole. Interestingly, HEK293 cells transfected with METTL7A were resistant to all of the thiol-based HDIs, but METTL7B overexpression conferred less resistance to largazole and KD5170 than METTL7A and no resistance to romidepsin. METTL7A and METTL7B thus appear to be methyltransferases with somewhat different specificity that confer resistance to thiol-based HDIs by inactivating these drugs. Recently, we have also determined that inhibitors of farnesyl transferase and geranylgeranyl transferases can be methylated and inactivated by METTLA and METTL7B, reinforcing our work showing that these methyltransferases methylate thiol groups. To determine if animal models could be used to elucidate the normal function of these methyltransferases, we examined homologs of METTL7A from different species. We found that METTL7A is conserved across vertebrates while METTL7B is not. To determine if the ability of METTL7A to methylate thiols is conserved, we transfected HEK293 cells to express mouse, rat, chicken, or zebrafish METTL7A. We found that expression of any of the METTL7A isoforms could confer resistance to all of the thiol-containing HDACis tested, suggesting that the function of METTL7A is conserved across species. These results have led us to create a transgenic zebrafish where METTL7A is deleted. We will characterize the knockout fish in hopes of finding a physiological role for METTL7A. 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 via diffusion. 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.