The Expression Of CARM1in Breast Cancer And Its Modulation To MDR1-Mediated Multidrug Resistance

[Background]Worldwide, invasive breast cancer is the most common cancer in female and accounts for23%of female cancers. the incidence rate is still increasing year by year. Chemotherapy is one of the primary means in breast cancer treatment. However, the effectiveness of chemotherapy drugs is decreased in case of the multidrug resistance (MDR) of cancer cells. The multidrug resistance refers to that when a kind of tumor become resistant to one drug, it would also become resistant to other anti-cancer agents which even have different structures,target sites, or mechanisms.The P-glycoprotein (P-gp), encoded by multidrug resistance gene1(MDR1), is the most important drug transporters. P-gp is a major cause of primary drug resistance, and is closely related with drug concentration and drug resistant degree in cancer cells. It is reported that many anticancer drugs, such as doxorubicin and paclitaxel, are all substrates of P-gp. MDR1gene is widely expressed in human malignancies and covers about50%of multidrug resistances in breast cancer. Even tumors with low P-pg expression would produce a high level of P-glycoprotein after treatment. P-glycoprotein expression rate is up to88%after chemotherapy in malignant tumors. MDR is one of the major reasons which causes failure of chemotherapy, tumor metastasis, shortened survival, and increased costs of cancer treatment. Chemical syntheses of inhibitors are used in P-gp overexpression malignancies in order to increase the sensitivity of chemotherapeutic drugs in tumor cells and prolong clinical remission. However, chemical drugs at therapeutic concentrations often causeundesired toxicity.It is necessary to furtherresearchthe mechanism of MDR1/P-gp expression and regulation.Recent studies have revealed that the nuclear receptor, steroid and xenobiotic receptor (SXR), is the master transcriptional regulators of MDR1gene expression.In the transcriptional regulation of nuclear receptors, manycoregulators are needed and the coregulators play critical roles in regulating chromatin conformation and regulating the recruitment and activation ofRNApolymerase Ⅱ. The nuclear receptors can recruit several different coactivators to the promoter region of target genes after binding with ligands. Some coregulators function by protein-protein interactions, forexampleby facilitating or inhibiting recruitment of other coregulators or specific components of the transcription machinery. Other coregulators are enzymes that catalyze posttranslational modifications to histones, nuclear receptors, other coregulators, and components of signal transduction pathways. Thus, posttranslational modifications of many components of the basal and regulatory transcription machinery also play major roles in nuclear receptor-mediated transcriptional regulation. As for SXR, after binding to a variety of xenobiotics, it can activate the xenobiotic-response elements (XREs), which locate in the MDR1gene promoter, and stimulates transcription. P160family is involved in this process andcan bind directly to SXR. P160family functions as a scaffoldto recruit other coactivators. However, it still remainsunknownwhatelse factors are involved in activation and regulation of SXR/MDR1pathway.Methylation of histones by protein arginine methyltransferases (PRMTs) is increasingly being acknowledged as an important aspect for the dynamic regulation of gene expression. The11mammalian PRMTs fall into two predominant classes, based on the types of methylarginine products they produce. Type Ⅰ enzymes (PRMT1, PRMT3, PRMT4/CARM1, PRMT6, and PRMT8) form monomethylarginine and asymmetric dimethylarginine, and type Ⅱ enzymes (PRMT5, PRMT7, and FBXO11) form monomethylarginine and symmetric dimethylarginine. PRMT4, namely coactivator-associated arginine methyltransferase1(CARM1) is initially identified as a SRC-2bindingprotein in a yeast two-hybrid screening. There are increasing evidences indicate CARM1plays pleiotropic roles in cell proliferation and survival. Some researchers had investigated the expression of CARM1in many kinds of malignant tumors. Aberrant expression of CARM1has been linked to human breast cancer tissue in a few reports; however, current studies are contradictory and incomplete. The mRNA level of CARM1was found to be elevated in grade3breast tumors in a cohort of81human breast carcinomas of various types. While another study demonstrated there was inverse correlation between CARM1expression and tumor grade in ER+and LN-breast cancer cases. Kim YR et al reported CARM1overexpression was noted only in small number of breast cancer patients (27%). All these reports suggest CARM1is an important factor involved in progression and may affect prognostication of breast cancer. However, many of these studies were limited either by low n values of breast cancer patients or by a special tumor type. It still remains unclear whether CARM1expression is correlated with clinicopathological features, molecular subtype and prognosis.CARM1functions as a coactivator for many nuclear receptors, such as the estrogen receptor (ER), androgen receptor (AR), the glucocorticoid receptor, and the thyroid receptor. However, it is still unknown whether CARM1is involved in the transcriptional regulation of MDR1. The regulation of SXR on MDR1requires the anticipation of p160protein family. While CARM1has been showed to be a coactivator of p160family, all members of p160family are natural substrates of CARM1. In summary, because CARM1activation plays an important role in the transcriptional regulation of nuclear receptor and p160, we presume that CARM1might be involved in the transcriptional activation of MDR1; probably through SXRpathway. It is necessary to investigate the correlation between CARM1, SXR and MDR1and the possible mechanism. This might contribute to understand the reversal of multidrug resistance and might provide new potential therapeutic targets.In this study, we first examined expression of CARM1, ER, PR, HER2, p53, Ki-67index and MDR1in247cases of untreated primary invasive ductal carcinoma by immunohistochemistry. Furthermore, We detected the expression of CARM1in the breast cancer cell lines MCF-7and the multidrug resistant subline MCF-7/ADM. The results showed a higher expression of CARM1and MDR1/P-gp on both mRNA and protein levels in MCF-7/ADM cells, compared with the sensitive MCF-7cell lines.These results suggested the potential roles of CARM1in the development of multidrugresistance in breast cancer cells.Therefore, in this study, we proposed that the silencing of CARM1by plasmid-mediated expression of small interference RNA (siRNA) might result in the down-regulation of MDRl/P-gp, and thus reverse multidrug resistance in MDR cells. Anda full-length cDNA of CARM1mediated by plasmids was also transfected into MCF-7to investigate whetherexogenous overexpression of CARM1would improve drug resistance.[Methods]1. We examined the expression of CARM1, ER, PR, HER2, p53, Ki-67index and MDR1by immunohistochemistry of streptavidin peroxidase complex method. Then, we analyzed the relationship between CARM1, the clinical pathological characters, universal molecular markers and molecular subtypes respectively, as well as the correlation between CARM1and MDRl/P-gp.2. Using RT-PCR and Western blot, we examined the expression of CARMl in breast cancer cell lines MCF-7and the multidrug resistant subline MCF-7/ADM.3. Small hairpin siRNA sequences targeted at CARM1and negative controls, non-targeting siRNAs sequences, were designed and synthesized as64oligonucleotides. After annealed, they were cloned into the pSUPER. neo+GFP expression vector respectively. After comfirmed by enzyme digestion and DNA sequencing, the recombinant plasmids were amplified in bacteria and extracted subsequently.4. The mammalian expression plasmid of pSG5.HA-CARM1was generously provided by Michael R. Stallcup (University of Southern California). We constructed pEGFP-C1-CARM1by insertion of CARM1which was cut out from pSG5.HA-CARM1with EcoRIinto pEGFP-C1plasmids. After comfirmed by enzyme digestion and DNA sequencing, the recombinant plasmids were amplified in bacteria and extracted subsequently.5. The MDR cells ADM were transfected with pSUPER-siFZDl and pSUPER-siNotarget respectively. And sensitive cells MCF-7were transfected with pEGFP-C1-CARM1.After another48h culture, mRNA and protein expression of CARM1and MDRl/P-gp were examined by RT-PCR and Western blot. MTT assay was used to assess the effect of CARM1silencing and exogenous overexpression of CARM1on ADM and MCF-7cells respectively to doxorubicin.[Results]1. In invasive breast cancer tissues, CARM1expression was increased in invasive breast cancer cells compared with adjacent benign epithelium.The increased expression was observed in the cytoplasm and/or the nucleus Nuclear CARM1expression was associated with a younger age at diagnosis; multi-center origin or multiple tumors; a higher tumor grade; a higher rate of HER2, p53, and Ki-67expression; and a lower rate of ER and PR expression. We also found that the rate of CARM1expression was significantly different among different molecular subtypes of breast cancer. We showed that in the HER2subtype, the nuclear expression of CARM1was the highest (69.6%), followed by the luminal B (59.6%) and basal type (57.1%). The luminal A type showed the lowest percentage of cells with CARM1nuclear expression (41.3%). As to MDR1, the percentage of P-Gpexpressing was45.7%, and the expression of CARM1was positively related to that of P-gpexpression.2. In MCF/ADM cells, CARM1was found to havehigher mRNA and protein levels in relation to MDRl/P-gp expression, compared with the sensitive cell line MCF-7.3. The effects of CARM1interferencein ADM were verified by semi-quantitative RT-PCR. The level of CARM1and MDR1mRNA was down-regulated significantly in comparison with the control.Western blotanalysesindicated that CARM1and MDRl/P-gp were down-regulated in response to CARM1interference. MTT assay showed thatIC50value of doxorubicin wasdecreased remarkably after CARM1interferencein ADM cells.4. The effects of CARM1exogenous overexpression in MCF-7cells were verified by semi-quantitative RT-PCR. The level of CARM1mRNA was up-regulated significantly in comparison with the control, while MDR1mRNA was notalterednotably.Western blotanalysesindicated that CARM1wasup-regulated afterexogenous overexpression of CARM1, whileMDR1/P-gp was not altered significantly. MTT assay also showed MCF-7IC50valueof doxorubicin did not changeremarkably in response to CARM1exogenous overexpression.[Conclusions]1. CARM1expression was increased in invasive breast cancer cells compared with adjacent benign epithelium. Nuclear CARM1expression was associated with a younger age at diagnosis; multi-center origin or multiple tumors; a higher tumor grade; a higher rate of HER2, p53, and Ki-67expression; and a lower rate of ER and PR expression. All of these predictors were clinicpathologic parameters that correlate with poor prognosis. We also found that the rate of CARM1expression was significantly different among different molecular subtypes of breast cancer.2. In invasive breast cancer tissues, the expression of CARM1was positively related to that of MDR1. CARM1was also overexpressed in the multidrug resistant breast cancer cells, consistent with MDR1/P-gp expression.3. In the multidrug resistant breast cancer cells, the silencing of CARM1decreased MDR1expression and reversed the multidrug resistance. It was indicated that CARM1was involved in the regulation of MDRl/P-gp.4. The exogenous overexpressionofCARMl in breast cancer sensitive cells did not change MDR1/P-gp expression and function.These resultssupportedthe hypothesis that CARM1might be a secondary coactivator in SXR/MDR1pathway.5. CARM1can modulate multidrug resistance through regulating the MDR1,probably through SXRpathway.CARM1is possible to be identified as a new molecular marker to predict multidrug resistance for personalized treatment and new latent target for MDR reversing in breast cancer cells.