LRIG1 Enhances Cisplatin Induced T24 Bladder Cancer Cells Lesions By Attenuating EGFR Nuclear Import

BACKGROUND AND OBJECTIVESeventy percent of bladder cancer was superficial and can be treated by transurethral resection of bladder tumor (TURBt). The residual 20-30% patients with muscle invasive tumor usually need chemotherapy to enhance survival rate. Cisplatin was one of the most common used DNA damage drugs in patient with advanced bladder cancer. However, as a result from drug resistance, only 15% of these patients received complete remission.Recent studies found that variety of DNA damage stimulations could induce epidermal growth factor receptor (EGFR) nuclear import and promote DNA repair. Attenuation of nuclear import of EGFR might enhance cisplatin DNA damage. Two mainly methods confirmed that inhibition of EGFR activity and tyrosine kinase activity could block EGFR nuclear import. But no research had been conducted on inhibition of EGFR nuclear import in chemotherapy on bladder cancer.LRIG1 was one of the ligands of EGFR and absence or down expressed in bladder cancer tissues. Up regulated of LRIG1 level in BIU-87 bladder cancer cells, could inhibit cell proliferation, reduce cell metastasis and invasion. These made us supposed if LRIG1 could be a new gene for inhibiting EGFR nuclear import after cisplatin induction.We designed this study to investigate whether cisplatin can induce EGFR activation and nuclear import after DNA damage, and if this translocalization could be blocked by LRIG1 and the possible mechanisms in bladder cancer cells. Further studies were conducted to evaluate whether the inhibition can facilitate bladder cancer cell DNA damage, enhance cell apoptosis, arrest cell cycle and reverse cancer cell invasion.MATERIALS AND METHODST24 bladder cancer cell line was exposure by cisplatin for 10,20 and 30 min. Nuclear proteins and total proteins from T24 cells were abstracted. Then EGFR and pEGFR expression levels were evaluated by immunoblotting. LRIG1 was enveloped by adenoviral vector, AdMax system, and transfected T24 bladder cancer cells. After the transfection, T24 cells were exposed under cisplatin for 30 min. Then EGFR and nuclear pEGFR levels were evaluated again to study if LRIG1 could block cisplatin induced pEGFR nuclear import. To identify the mechanism of the phenomenon, cells were treated with proteasome inhibitor MG132 before LRIG1 transfection, and nuclear pEGFR and total EGFR were evaluated.Whether LRIG1 could enhance cisplatin induced bladder cancer DNA damage, cell apoptosis rate and attenuate cell proliferation and invasion abilities were evaluated by single cell gel electrophoresis, cell immunohistochemical staining, flow cytometry (FCM) and matrigel invasion assays, respectively.RESULTSAfter induced by cisplatin for 10,20 and 30 min, nuclear pEGFR expression levels in T24 bladder cancer cells were up regulated with time. But EGFR expression levels were not affected by cisplatin.T24 bladder cancer cells were transfected by Ad-LRIG1, after LRIG1 was successfully enveloped by adenoviral vector. And pEGFR nuclear import in T24 cells was inhibited. Pretreated with proteasome inhibitor MG132, EGFR and nuclear pEGFR expression levels in T24 cells could not be down regulated by LRIG1, compared to the control groups.In addition, cisplatin combined LRIG1 treated T24 bladder cancer cells could increase cancer cell OTM values in comet assay, arrest cancer cell cycle in S phase, enlarge apoptosis rate, humiliate cell growth and reduce matrigel invasive cell numbers.CONCLUSIONCisplatin induced pEGFR nuclear import could be attenuated by LRIG1 transfection. Accelerating total EGFR degradation and attenuating EGFR phosphorylation in bladder cancer cells might be two mechanisms to explain this phenomenon. LRIG1 could enhance cisplatin induced cancer cell DNA damage and inhibit cancer aggressive invasion. This study provides that LRIG1 may represent a new therapeutic approach to improve the response to cisplatin through a novel pathway in bladder cancer.