The Discovery Of The Breast Cancer Drug Resistance Associated Membrane Proteins Ck8 Resistant Tumor Therapeutic Targets

Multidrug resistance (MDR) is a phenomenon whereby tumor cells acquire cross-resistance to a variety of structurally and functionally unrelated drugs. Following the cytotoxic chemotherapy, MDR occurs almost universally in various tumors and becomes a major obstacle to successful cancer treatment. The mechanisms involved in MDR have been extensively investigated.Indeed, accumulating evidence suggests that multiple genes are activated concomitantly during cytotoxic drug exposure, and various molecular mechanisms may contribute to MDR phenotypes simultaneously and cooperatively. The fact that drug resistance in diverse cancer cells overexpressing ABC transporters could not be reversed completely by specific chemical inhibitors or gene-specific siRNAs against the corresponding ABC transporters suggests the existence of additional mechanisms in MDR. These mechanisms remain, however, to be fully elucidated. Moreover, a variety of modulators or reversal agents that focus on specific individual MDR pathways have been developed and tested in various clinical trials, but with rather disappointing outcome. Thus, searching for potential membrane-associated molecules related to ABC-mediated MDR phenotype may contribute to better understanding of the molecular mechanisms underlying cancer MDR, which is essential to develop novel and more effective molecular targets for MDR diagnosis and therapy.Using MS-based proteomics, we have attempted to identify the changes in membrane components in multidrug-resistant cancer cells. However, it is difficulty to analyze membrane proteins, by traditional proteomics approaches, because of their inherently hydrophobic nature and low abundance. Thus, we provide an alternative strategy combining comparative screening of antibody with identification of the target antigens by immunoprecipitation and MS to identify additional MDR-related membrane proteins that may contribute to the diverse and/or redundant mechanisms of drug resistance. MCF-7/MX, a mitoxantrone-selected MDR human breast tumor cell line with overexpression of breast cancer resistant protein (BCRP), was used as a multifactorial model system. Using MCF-7/MX cells as the immunogen, we isolated a mouse monoclonal antibody, 9C6, that preferentially reacts with MCF-7/MX cells over its parental counterpart by comparative selection. With immnoprecipitation /Mass spectrometry approach, the molecular target for 9C6 was identified as cytokeratin 8 (CK8). As further evidence to confirm the reliability of proteomic identification, the preferentially binding of 9C6 to the surface of MCF-7/MX cells over its parental MCF-7 cells was observed under confocal immunofluorescence microscopy, thus providing direct evidence of cell surface over-expression of CK8 in the MDR cells.What are the exact physiological and pathological roles of cell surface-expressed CK8 remains to be clearly elucidated. Here we have reported the positive correlation between membrane CK8 and MDR in cancer, adding further comprehensive information to the function of membrane CK8. We observed that that CK8 plays a direct role in drug resistance in tumor cells and may serve as an independent indicator of MDR. SiRNA specific to CK8 was able to partially reverse drug resistance of CK8-overexpressing MCF-7/MX cells to all the 3 chemotherapeutic agents tested, clearly supporting the notion that CK8 directly contributes to the emergence and/or maintenance of MDR.To investigate the possible mechanisms by which overexpressed membrane CK8 contributes to MDR in cancer cells, we assessed the role of membrane CK8 in cell adhesion because there is much evidence to implicate that cell-cell and cell-extracellular matrix adhesion could mediate drug resistance through different mechanisms in cancer cells. As expected, our further experiments demonstrated that increased membrane CK8, in fact, plays an important role in enhanced adhesion in drug-resistive cells. Taken together these previous observations with those of ours, it is, in our opinion, likely that CK8 mediates drug resistance via its increased surface expression and, as a result, enhanced adhesion between cells and cell- extracellular matrix proteins. Our results in this study suggest that overexpression of CK8 may be independent of other MDR-related molecular alternations including increased expression of BCRP. The observation that transfection with siRNA to CK8 did not alter the expression, and perhaps function of BCRP, and vice versa, suggest that CK8 expression and function may represent an independent indicator of MDR phenotype. It is intriguing that, unlike the anti-BCRP siRNAs, anti-CK8 siRNAs did not seem to significantly affect cellular accumulation of cytotoxic agents. Moreover, forced expression of CK8 by gene transfection in NIH3T3 cells resulted in significantly increased resistance to mitoxantrone. When co-expressed in BCRP-transfected cells, CK8 led to further enhanced drug resistance over the cells that only express BCRP. While knockdown expression by siRNA of CK8 or BCRP individually significantly reduced cancer cell drug resistance, simultaneous inhibition of both protein expressions demonstrated stronger degree of reversal of the MDR phenotype. Taken together, these results suggests that CK8 and BCRP likely represent two independent mechanisms conferring MDR to MCF-7/MX cells, as two contingent events of vital response triggered by selection pressure.Up-regulation of CK8 and BCRP may be cooperatively responsible for drug resistance in MCF-7/MX cells, and thus represents potential targets for multi-targeting diagnostic and therapeutic applications in the management of MDR cancers. Oligonuceotides target to CK8 were inserted into the pGE-1 vectors that can produce short hairpin RNA in vivo, and then were transfected into MCF-7/MX cells in combination with psiRNA/BCRP-B. After selected by G418 and/or Zeocin, cell lines producing shRNA-CK8 and/or shRNA-BCRP were developed. The inhibition efficiency of CK8 and BCRP were determined by western blot. The sensitivity to drug was determined by SRB method and the results showed that while knockdown expression by shRNA of CK8 or BCRP individually significantly reduced cancer cell drug resistance, simultaneous inhibition of both protein expressions demonstrated stronger degree of reversal of the MDR phenotype. Therefore, combinational approaches that target multiple drug-resistance-related molecules/pathways in cancer cells may represent more efficacious strategies to overcome MDR.In summary, by using a rational combination of MS-based proteomic identification and antibody-based screening techniques, we identified up-regulated membrane CK8, a potential clinically useful biomarker for MDR breast cancer that could be pursued in larger studies for clinical relevant validation purposes. This approach could be extended to most MDR cancers for investigating a number of MDR associated biomarkers for both diagnostic and therapeutic applications. Alterations in the level of expression and cellular localization of CK8 may represent a novel MDR-related marker for both diagnostic and therapeutic applications.