Inhibition Of SLC1A5 Sensitizes Colorectal Cancer To Cetuximab

BackgroundCetuximab is.a monoclonal antibody against epidermal growth factor receptor(EGFR).Combined application of cetuximab and conventional chemotherapy reduced the risk of progression of patients with metastatic colorectal cancer(mCRC).However,mCRC patients with KRAS-mutation do not response to cetuximab and benefit from cetuximab treatment,and there is no effective strategy for targeting KRAS mutation to reverse cetuximab resistance so far.Moreover,not all mCRC patients with KRAS-wild type(WT)respond to cetuximab treatment,and even some patients with initial response may acquire resistance later after the treatment.Therefore,it is clinical significant to identify novel biomarkers for predicting the cetuximab response of CRC patients and investigate potential candidates that can be targeted to boost the efficacy of cetuximab.Deregulating cellular energetics is defined as a hallmark of cancer.Glucose and glutamine are the two basic metabolites imported by cancer cells for fueling cellular growth and survival.Studies has shown that deregulating cellular metabolism promoted resistance to anticancer therapy.Blocking of the essential metabolic pathways can effectively increase cancer cell response to many types of cancer treatment.Thus,we wondered whether targeting of the key transporters of glucose(glucose transporter 1,GLUT1)and glutamine(solute carrier 1 family member 5,SLC1A5)would improve the cancer response to cetuximab.Methods and Results1.The expressions of SLC1A5 and GLUT1 in carcinoma tissues of CRC patients with cetuximab treatmentUsing immunohistochemical method,we detected the expressions of SLC1A5 and GLUT1 in carcinoma tissues of CRC patients who treated with cetuximab,and analyzed the correlation between SLC1A5/GLUT1 exprssions and the efficacy of cetuximab.It turned out that the SLC1A5 expression was significantly higher in carcinoma tissues of patients who had no response to cetuximab(non-responders)than in those who responded to cetuximab(responders).No significant differences of GLUT1 expression in carcinoma tissues was detected between the responders and non-responders.These results suggested that SLC1A5 may be involved in CRC response to cetuximab.2.The role of SLC1A5 in CRCTo examine the function of SLC1A5 in CRC proliferation,we performed MTT assay,flow cytometry(FCM)and mouse xenograft model.By targeting of SLC1A5 with shRNA-mediated gene silencing or pharmacological inhibitor L-y-glutamyl-p-nitroanilide(GPNA),we found that in vitro the cell growth of Caco2 and SW480 cells was inhibited,the cell cycle was arrested in G1 phase and the production of autophagy-related protein LC3-? was increased.In vivo,the SW480 cell xenografts in nude mice presented slower growth rate with GPNA treatment in contrast to control treatment.3.The effect of SLC1A5 on the efficacy of cetuximab in CRCTo test the whether SLC1A5 affect the efficacy of cetuximab on CRC,we performed MTT assay,colony formation test,immunofluorescence assay,Western Blot,nude mouse xenograft model.The results came out that the cell proliferation of Caco2 and SW480 was further inhibited when GPNA was added to cetuximab therapy.The generations of yH2AX(the DNA damage marker)and LC3-?(autophagy-related protein)were significantly increased by combined treatment with GPNA and cetuximab.In the SW480 cell xenografts in nude mice,the tumor growth rate was significantly slowed when GPNA was combined to cetuximab therapy.4.Mechanisms about how SLC1A5 affects the efficacy of cetuximab on CRC To examine the potential mechanisms about how SLC1A5 affects the efficacy of cetuximab on CRC,we perforemed Western Blot,immunoprecipitation and immunofluorescence assay.The results we obtained were as follows:(1)by targeting of SLC1A5 with shRNA-mediated gene silencing or pharmacological inhibitor GPNA,we found that the whole expression of EGFR was decreased and the ubiquitinated level of EGFR was increased.Moreover,the reduction of the whole expression of EGFR induced by GPNA could be reversed by the proteasome inhibitor MG 132.These results suggested that inhibition of SLC1A5 promoted the degradation of EGFR through the ubiquitin-proteasome pathway;(2)inhibiting of SLC1A5 induced the reduction of the membrane expression of EGFR and the nuclear expression of EGFR,and resulted in DNA damage of CRC cells.Studies has reported that the sensitivity of cancer cells to cetuxiamb can be improved by increased EGFR degradation.Several studies also declared that the nuclear expression of EGFR was capable of promoting DNA replication and repair,leading to cancer resistance to treatments including anti-EGFR targeted therapies.It has been reported that one of the most important mechanisms of action of cetuximab is competing with endogenous ligands to block the membrane function of EGFR and induce EGFR endocytosis,and the fates of endocytic EGFR include:(1)degrade;(2)transport to nucleus;(3)recycle back to cell surface.Combined with our results,it is reasonable for us to speculate that the affection of SLC1A5 in cetuximab resistance is a result of the intervention of SLC1A5 in the fates of the endocytic EGFR(the enhanced cetuximab efficacy induced by SLC1A5 inhibition could be ascribed to the increased EGFR degradation and nEGFR reduction).5.The effect of SLC1A5 on the efficacy of cetuximab in other types of cancer To checke whether targeting of SLC1A5 could also improve the response of other cancers to cetuximab,we performed Western Blot and MTT assay with head and neck squamous cell carcinoma(HNSCC)cell line CAL27,triple negative breast cancer cell line MDA-MB-231 and non-samll cell lung cancer cell line NCI-H460.The results obtained were as follows:(1)CAL27 cell,MDA-MB-231 cell and NCI-H460 cell were all SLC1A5-positive;(2)the expression of EGFR in these three kinds of cells was also decreased by GPNA treatment;(3)in contrast to monotherapy,the addition of GPNA to cetuximab resulted in higher cell growth inhibition rate.ConclusionsThis study demonstrates that SLC1A5 is an important metabolic molecular which affects the efficacy of cetuximab on CRC using cilinical samples,cell experiments and mouse xenograft model.The underlying mechanism of this effection may be ascribed to the function of SLC1A5 on regulating the fates of the endocytic EGFR(degrade,transport to nucleus or recycle back to cell surface.)This study also showed that interfering with SLC1A5 is an effective strategy for improving the anti-tumor response of cetuximab in multiple malignant cancer cells which expressed SLC1A5.In summary,this study reveals the previously unrecognized role of SLC1A5 in affecting cetuximab response,and offers a promising efficient approach for patients with cetuximab resistance.