Transmembrane TNF-α Mediates Resistance To The Chemotherapeutic Drug Adryiamycin In Human Breast Cancer Cells

Tumor Necrosis Factor-alpha (TNF-α) is a pleiotropic cytokine which exists in two bioactive forms. One is transmembrane TNF-α(tmTNF-α) anchored in the cell plasma membrane as a trimer; another form is soluble TNF-α(sTNF-α) cleaved extracellularly from the membrane molecule by TNF-αconverting enzyme (TACE). tmTNF-αand sTNF-αare different in molecular structure, receptor binding ability and action manner, leading to a significant difference in their biological function. Cumulating reports are concerning the biologic function of sTNF-α, while reports on tmTNF-αare relatively rare. tmTNF-αcould not only function as a ligand mediating forward signaling but also as a receptor inducing reverse signaling.Recently, endogenous TNF-αwhich was locally produced within the tumor microenvironment functioning as a key inflammatory factor involved in the initiation and development of cancer are under the spotlight. Elevated TNF-αlevel in serum has been linked to poor prognosis of breast cancer. Blocking TNF-αcontribute to inhibition of tumor growth in HER2 knock in mice. Our previously observation show that Elevated tmTNF-αexpression could be found in 90% invasive breast cancer and overexpression of TNF-LS confer breast cancer cell resistance to the apoptosis mediated by sTNF-α, indicating that tmTNF-a could render resistance to apoptosis via reverse signaling. However, it is still little known about the role of tmTNF-αin chemoresistance of breast cancer.In the present study, we investigate the relationship of tmTNF-αand the Adriamycin (ADM) resistance in vitro, and also illustrate its possible mechanism. Using the shRNA technology in vivo, we further uncover the role of tmTNF--αin inducing drug resistance, which could provide evidence for the enhancement of the therapeutic efficacy of breast cancer and provide a new target for clinical treatment for breast cancer. PartⅠ. The mechanism of ADM resistance mediated by tmTNF-αin vitro1 tmTNF-αis linked to the ADM resistance in breast cancer cellsUsing MTT and DNA fragmentation for apoptosis, the high level of tmTNF-αin MDA-MB-231 is more resistance to ADM treatment compared with the low level of tmTNF-αin MCF, indicating that tmTNF-αis correlative with the ADM resistance in breast cancer cells.2 Down-regulated tmTNF-αenhance apoptosis induced by ADMDown-regulated tmTNF-αby RNAi technology resulting in decreased expression of tmTNF-αfrom 76.3% to 21.5%,leading to increase apoptosis in MDA-MB-231. These finding show that tmTNF-a could confer to the resistance of ADM treatment in beast cancer.3. Overexpression of TNF-LS confer MCF-7 resistance to the cytotoxicity of ADMOverexpression of TNF-LS render sensitive MCF-7 resistance to the apoptosis induced by ADM, indicating that reverse signaling through tmTNF-αmediates resistance to the chemotherapeutic drug ADM in human breast cancer cells4 tmTNF-mediated ADM resistance is independent of transcription expression of MDR1,BCRP,MRP1Realtime PCR show that no obviously changes was found in MDR1, MRP1, BCRP mRNA expression in tmTNF-αtransfected MCF-7 and control cell regardless of ADM treatment. Similarly, no difference in the transcription levels of MDR1,MRP 1,BCRP was observed in knockdown MDA-MB-231 and control cell. These result suggest that induction of ADM resistance by tmTNF is independent of the transcription expression of MDR1,MRP1,BCRP.in breast cancer cells6. ADM resistance induced by tmTNF-αis independent of expression of HER2FCM show that no obviously changes was found in HER2 expression in TNF-LS transfected MCF-7 and control cell regardless of ADM treatment. The similar result was also found in down-regulated tmTNF-αin MDA-MB-231. These results indicate that induction of ADM resistance by tmTNF is independent of the expression of HER2.in breast cancer cells.7.ERK1/2 pathway is required for tmTNF-induced Adriamycin resistanceOverexpression of TNF-LS lead to constitutively increased activity of ERK1/2 whereas knockdown of tmTNF-αresult in constitutively decreased activity of ERK1/2.The specific ERK1/2 blocker could reverse the resistance to cytotoxic effect induced by ADM. Together, these results indicate that ERK1/2 activity play a critical role in the tmTNF-α-mediated ADM resistance.8. NF-κB pathway is indispensable in ADM resistance mediated by tmTNF-αOverexpression of TNF-LS result in constitutively increased activity of NF-κB whereas knockdown of tmTNF-αlead to constitutively decreased activity of NF-κB.The specific NF-κB blocker could reverse the resistance to cytotoxic effect mediated by ADM. Together, these results indicate that NF-κB activity is required for tmTNF-cc-induced ADM resistance9. BCL-xl, cIAP1,xIAP is associated with tmTNF-a-dependent Adriamycin resistanceUp-regulation of TNF-LS contribute to constitutively increased expression of BCL-xl, cIAP1,xIAP, whereas down-regulation of tmTNF-αlead to constitutively decreased expression of BCL-xl, cIAP1,xIAP.These results indicate that BCL-xl, cIAP1,xIAP is required for tmTNF- -induced ADM resistancePartⅡ. The effect of Knockdown tmTNF-αand combination ADM treatment in vivo1. Effect of tmTNF-αknockdown on tumor incidence of MDA-MB-231 in vivoThe 100% tumor was 20 days in knockdown of tmTNF-αwhereas it was 10 days in parental tumor, indicating that Knockdown tmTNF-αcould contribute to decreased tumor incidence.2. Knockdown of tmTNF-αcombination ADM treatment inhibit tumor growth in nude miceTumor growth curve show that knockdown of tmTNF-αalone could result in decreased tumor volume and weight. Knockdown of tmTNF-αcombination ADM treatment is more significantly decreased tumor volume and weight. These results suggest that knockdown of tmTNF-αcombination ADM could enhance the efficacy of ADM treatment in vivo3. Down-regulated tmTNF-αcombination with ADM treatment increase apoptosis of MDA-MB-231 in vivoThe activity of caspase 3 analysis show that down-regulated tmTNF-αcombination with ADM treatment could increase the activity of caspase 3,suggest down-regulated tmTNF-αcombination with ADM treatment could enhance the apoptosis in vivo.4. Effect of Knockdown tmTNF-αcombined ADM treatment on the survival curve of MDA-MB-231The tumor survival curve show that the longest median survival days was 60.5 days in knockdown tmTNF-αcombined ADM treatment, compared with 31 days in parental tumor without ADM treatment, indicating knockdown tmTNF-αcombined ADM treatment could extend the survival time for tumor in vivo.On the whole, our findings suggest that reverse signaling through tmTNF-αmediates resistance to the chemotherapeutic drug ADM in human breast cancer cells through constitutively activated ERK1/2 and NF-κB, resulting in up-regulation expression ofBCL-xl, cIAP1,xIAP. Our finding also show that knockdown tmTNF-αcombined ADM treatment could inhibition growth and enhance the efficacy of ADM treatment in vivo indicating that tmTNF-αis a new potential target for clinical treatment for breast cancer.