Functional Analysis of Chemoresistance Genes: a Continuity of Genome-Wide Search for Cisplatin Resistance

  • Chao, Chuck C.-K. (PI)

Project: National Science and Technology CouncilNational Science and Technology Council Academic Grants

Project Details

Abstract

Chemoresistance causes a major obstacle in cancer chemotherapeutic treatment. The mechanism of cancer cell resistance to cisplatin-based chemotherapy is multifactorial. The cell response to anticancer drugs depends on the genetic make-up of the cells and the concentration and frequency of drugs applied to the cells. Genome-wide analysis has been applied to explore dysregulated genes in chemoresistant cell models as well as clinical tumors. Using this strategy, we have previously identified a panel of “cisplatin resistance genes” (CPR genes) by cDNA microarray, confirmed by quantitative-PCR (Q-PCR) of the mRNA levels. The effect of these CPR genes on drug sensitivity is associated DNA damage, primary stress that causes apoptosis by cisplatin, independent of mitotic damages. To unravel the molecular basis of cisplatin resistance (we called hereafter “chemoresistance”), functional studies of these candidate genes in drug response will be assessed in cell-based model, and under certain circumstances in mouse model. The CPR genes will be overexpressed (gain-of- function) or inhibited by RNAi, shRNA (loss-of- function) in cells and the drug sensitivity measured by MTT assay and apoptosis assay (caspase activity protein markers plus sub-G1 cell induction). The action mechanism, including upstream signal and downstream proteins, of these CPR gene products will be investigated in cell lines. Importantly, the modulatory effect of these CPR genes on tumor response to chemotherapy will be explored in immune-deficient nude or SCID mice and immune-competent BALB/c mice. Furthermore, we will very much like to have these laboratory results confirmed by clinical specimens (depending on the possibility of collecting the specimens from Chang Gung Memorial Hospital). Three CPR genes will be focused on, but not limited to, in this study: NAPA, CITED2 and PTPN21 because the preliminary results suggest that their drug modulatory effects are dramatic or the associated informations are available. NAPA is initially identified as a universal “protein transport” required protein which has not been known to be involved in the anticancer drug response. CITED2 is known as a regulator of transcriptional control, and PTPN21 is a cytosolic nonreceptor tyrosine phosphatase. Neither one is reported about their role in the drug response. A reason to start with NAPA and CITED2 is that proapoptotic p53 is regulated by these proteins (see preliminary data). The aim of this study is to investigate the mechanism of these CPR gene products and to overcome their chemoresistance. First, we will study the action mechanism ofCITED2 in cisplatin resistance, including the mechanism of p53 accumulation and p21 reduction following CITED2 knockdown by shRNA. Second, we will study the action mechanism of NAPA in cisplatin resistance, including the mechanism of p53 accumulation following NAPA knockdown by shRNA through ER-associated degradation (ERAD)-mediated p53 degradation complex. We will also study the translocation of NAPA associated proapoptotic BNIP-1 to mitochondia where it participates in caspase-9 activation. To assess the functional role of NAPA and CITED2 in chemoresponse, we will simultaneously investigate the post-translational modification of p53 (including phosphorylation, acetylation and ubiquitination) and the alterations of downstream proteins by gene knockdown alone and in combination with cisplatin. Third but not the last, we will study the action mechanism ofPTPN21 in cisplatin resistance. Importantly, combined cisplatin/shCPR treatment suppressed tumor growth in vivo in xenograph experiments will be performed in nude mice. The final goal of this research is to construct a network of cisplatin resistance and CPR protein converging pathways. Hopefully, the assessment of sensitizing chemotherapy in this study will provide a niche for better understanding of chemoresistance and a plausible way to develop new regimen for cancer therapy.

Project IDs

Project ID:PC10101-1999
External Project ID:NSC100-2320-B182-026-MY3
StatusFinished
Effective start/end date01/08/1231/07/13

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