Novel Role Of Cullin7 In Herceptin Resistance Of Breast Cancer

Background and ObjectiveBreast cancer remains the most common malignant disease in women in the world,given that the overexpression of HER-2 occurs in 20% of breast cancer patients.When HER-2 overexpression and its associated signaling pathways are activated,resulting in an unlimited proliferation of cells,eventually leading to tumor prone invasion and metastasis,with poor prognosis.Herceptin is a humanized HER-2antibody,and the presence of Herceptin has resulted in significant efficacy in many HER-2-positive breast cancer patients,but most patients responsive to the initial herceptin treatment develop resistance within a year,making treatment failed.Although the discovery of the Herceptin resistance mechanism has been going on for more than a decade,it has not yet fully solved Herceptin resistance.Therefore,a thorough elucidation of Herceptin resistance mechanisms will help to improve the survival of HER-2-positive breast cancer patients.Tumor stem cells are cells that have self-renewal ability and can produce heterogeneous cells in the tumor.There is growing evidence that the presence of tumor stem cells is one of the important causes of cancer resistance.Breast cancer has also been confirmed due to the presence of breast cancer stem cells,making breast cancer more easily relapse and become refractory.A large number of studies have shown that activation of the Hedgehog signaling pathway enhances the characteristics of breast cancer stem cells.Liu et al found that the expression of IHH,SMO,Gli1,Gli2 on breast cancer stem cells was significantly higher than that of differentiated tumor cells,which significantly reduced the formation of microspheres after blocking the Hedgehog signaling pathway,confirming that Hedgehog signalpath in breast cancer stem cells is activated [1].Tanaka et al found that SHH and Gli1 were highly expressed in CD44 + CD24-cells from breast cancer cell line MCF-7,and cultured with SMO inhibitor cyclopamine for 72 h,CD44 + CD24-in the proportion of significantly reduced [2].In recent years,there have been reports of TGF-?/Smads signaling pathway activation can also enhance the characteristics of tumor stem cells.TGF-?1 was able to enhance the self-renewal ability and other stem cell characteristics of triple-negative breast cancer stem cells from the stem cell population,stem cell markers,stem cell-like stromal cell phenotype,stem cell self-renewal ability and invasive ability.of triple negative breast cancer CD44 +CD24-[3].A number of important components in the Hedgehog and TGF-?/ Smads signaling pathways can be regulated by ubiquitination.In the activation process of the Hedgehog signaling pathway,the transmembrane receptor PTCH binds to the SHH ligand and internalizes into the cell,while the SMO is enriched in the cilia,which is subjected to ubiquitination in the process of endocytosis,in particular,the single ubiquitination of multiple loci is one of the key signals to promote the transport of membrane receptors.Smurfs is a class of E3 ubiquitin ligase containing the HECT domain during the activation of the TGF-?/Smads signaling pathway,a modulator that specifically mediates the ubiquitination of Smads molecules.CUL family as the main skeleton of ubiquitin-proteasomes(ubiqutin-proteasomesystem,USP),which has the function of regulating cell cycle and apoptosis,as the main skeleton of ubiquitinase E3 in the body,and ubiquitin-activating enzyme E1 and ubiquitin transferase E2,cell proliferation and signal transduction in the protein modification also has important significance,which by the CUL and SKP1,Fbx29(also known as Fbw8),ROC1 composed of SCF-ROC1-like E3 ubiquitin ligase complex is the key structure of the whole ubiquitination process [4].Our previous study found that CUL7 expression was positively correlated with breast cancer malignancy,both in breast cancer and in breast cancer cells,that is,the ability of CUL7 to overexpress breast cancer tissue or breast cancer cells to invade and metastasize significantly enhanced at the same time,in vitro inhibition of CUL7 expression significantly weakened the invasion and metastasis of breast cancer cells.However,tumor resistance and metastasis are not isolated in both processes.It was found that the P-gp and CD44 molecules expressed in the cell membrane could directly affect the drug resistance and metastatic abilityof the tumor cells,indicating that the high expression of P-gp could enhance the survival and metastasis ability of tumor cells.It suggests a close relationship between drug resistance and metastatic invasion,and its complex and diverse regulatory factors [5-6].In our pre-experiment,by comparing the expression of CUL7 in HER-2 positive breast cancer Herceptin resistant cells(SKBR3 / POOL2)and parental cells(SKBR3),it was found that CUL7 was found in resistant cells of HER-2 positive breast cancer showed high expression.Limited studies have confirmed that Herceptin-resistant gastric cancer cells have obtained a tumor stem cell-like phenotype [7].Therefore,we hypothesized that CUL7,as a member of the CUL family,may also promote the production of tumor stem cells by tyrosinoglyculation of Hedgehog,TGF-? /Smads signaling pathways,and induce breast cancer Herceptin resistance.Therefore,this experiment will be done by RT-PCR,Western-Blot,gene interference experiments,immunofluorescence experiments,cell flow cytometryand other techniques,as well as immunohistochemistry and nude mice in vivo experiments,confirm the effect of CUL7 on Herceptin resistant breast cancer from multiple aspects,and to explore its possible molecular mechanism.Methods1.Selection of HER-2 positive breast cancer Herceptin resistant cells(SKBR3 /POOL2)and parental cells(SKBR3),the expression of CUL7 mRNA and protein in SKBR3 and SKBR3 / POOL2 were detected by real-time quantitative RT-PCR and western blot.2.Construction of CUL7 shRNA lentiviral vector,transfection of CUL7 in SKBR3 / POOL2,the cell lines(SKBR3 / POOL2-shRNA)and negative control(SKBR3 / POOL2-NC)that stably interferde with CUL7 expression were screened by puromycin screening.The expression of CUL7 mRNA and protein in SKBR3/POOL2-shRNA and SKBR3 / POOL2-NC were detected by real-time quantitative RT-PCR and western blot.3.The effects of Herceptin on the apoptosis of SKBR3,SKBR3/POOL2,SKBR3/POOL2-shRNA and SKBR3/POOL2-NC were detected by MTT assay and flow cytometry.4.The relationship between the expression of CUL7 and tumor stem cells was detected by western blot,flow cytometry and cell pelvic assay.The expression of thetarget gene in Hidegehog and TGF-?/Smads signaling pathways,such as SHH,PTCH,Gli1,Gli2,TGF-? 1,TGF-? 2,p-smads2 / 3 protein were detected by western blot.5.The expression of CUL7,important molecular proteins of Hedgehog and TGF-?/Smads signaling pathways in sensitive and resistant breast cancer tissues were detected by immunohistochemistry.6.To verify the effect of CUL7 on tumorigenesis in nude mice and the influence on the sensitivity of Herceptin in vivo.Results1.Throgh the MTT test of Herceptin in SKBR3 and SKBR3/POOL2 cells,the IC50 were 3.58ug/ul and 5.48 ug / ulrespectively.RT-PCR and western blot showed that the expression of CUL7 in SKBR3 / POOL2 was significantly higher than that in parental cell SKBR3.2.SKBR3 / POOL2-shCUL7 and its control cell line SKBR3 / POOL2-NC were obtained by shaking the drug-resistant cell line sh-RNA CUL7.Real-time quantitative RT-PCR and western blot showed that the CUL7 gene and protein were down-regulated in SKBR3 / POOL2-shCUL7 cell lines,52% and64 %respectively.The IC50 values of Herceptin in SKBR3 / POOL2-NC and SKBR3/ POOL2-shCUL7 were 5.28 ug / ul and 4.60 ug / ul,respectively,by MTT assay.3.The apoptosis levels of SKBR3,SKBR3/POOL2,SKBR3/POOL2-shRNA and SKBR3/POOL2-NC cells were increased 19.90% to 50.10%,16.51% to 16.94%,15.26% to 18.15%;16.68% to 17.38% respectively,after 12 hours of Herceptin induction by flow cytometry.The results suggest that SKBR3/POOL2 has a significant increase in Herceptin sensitivity after down-regulation of CUL7.4.The clone formation rate of CUL7 overexpressing cells(SKBR3 / POOL2 and SKBR3 / POOL2-NC)was significantly higher than that of CUL7 cells(SKBR3and SKBR3 / POOL2-shRNA)by plate clone formation assay.The percentage of S phase of sensitive SKBRS decreased from 16.62% to 2.66% after flow cytometry,and the number of SKBR3 / POOL2 decreased from 13.84% to 10.98% SKBR3 /POOL2-shRNA cells decreased from 24.64% to 10.04%,while SKBR3 /POOL2-NC in the control group decreased from 17.63% to 11.55%.Suggesting that down-regulation of CUL7 can block cells into the cell cycle and confirm that down-regulation of CUL7 results in reduced proliferation of drug-resistant cells.5.By scratch and transwell experiments,compared with SKBR3 / POOL2-NC cells in control group,the migration and invasion ability of SKBR3 /POOL2-shRNA cells were decreased.These results suggest that CUL7 can promote the migration and invasion of breast cancer cells.6.The percentage of cells with CD44 + CD24-phenotype in SKBR3,SKBR3 /POOL2,SKBR3 / POOL2-shRAN,SKBR3 / POOL2-NC cells was 0.607%,4.73%,2.44%,respectively,by flow cytometry.The volume and the number of breast ball in CUL7 low expression group(SKBR3,SKBR3 / POOL2-shRNA)was significantly decreased.Western blot showed that the level of NANOG protein in CUL7 overexpression group(SKBR3/ POOL2,SKBR3 / POOL2-NC)was significantly higher.The above experiments have proved that CUL7 can promote the formation of breast cancer stem cell.7.The expression of target gene in the Hedgehog signaling pathway,CUL7 low expression group(SKBR3?SKBR3 / POOL2-shRNA)was significantly lower than that in CUL7 overexpression group(SKBR3 / POOL2,SKBR3 / POOL2-NC)by Western blot,while the level of PTCH2 was significantly higher.There was no significant difference between the CUL7 overexpression group(SKBR3/ POOL2,SKBR3 / POOL2-NC)and in CUL7 low expression group(SKBR3,SKBR3 /POOL2-shRNA)in the important molecular protein level of TGF-? /Smads signaling pathway.The results suggest that CUL7 may affect the dryness of breast cancer cells through the Hedgehog signaling pathway.8.Immunohistochemical detection of breast cancer tissue,found that CUL7 high expression in sensitive and resistant breast cancer,respectively,accounted for 30%,66.67%.The expression of PTCH2,Gli1 and Gli2 were 60%,10% and 30% in sensitive tissues,respectively,and 22.22%,55.56% and 77.78% respectively in resistant tissues.The expression of p-smad2 in TGF-? / smads signaling pathway was 40% and 44.44%,respectively,in sensitive and resistant breast cancer tissues.The results showed that the expression of SHH,Gli1 and Gli2 in CUL7 and hedgehog signaling pathogens were significantly higher in resistant breast cancer tissues,while PTCH2 showed low expression.There is no significant difference in TGF-? / smads signaling pathway in sensitive and resistant breast cancer.Suggesting that CUL7 may affect the sensitivity of the Hedgehog signaling pathway to the breast cancer Herceptin.9.From the plot of the tumor growth curve and the weight of the tumor,we can see that the tumorigenic ability of SKBR3 and SKBR3 / POOL2-shRNA cells with low expression of CUL7 decreased significantly(P <0.05).After treatment with Herceptin and saline,it was found that SKBR3/POOL2 and SKBR3/POOL2-NC were significantly increased in the CUL7 overexpression group,whether or not given Herceptin treatment.In the CUL7 low expression of SKBR3 and SKBR3/POOL2-shRNA regardless of whether to give Herceptin treatment,tumor size was significantly reduced.The results suggest that cells with high expression of CUL7 exhibit stronger tumorigenic ability.Conclusions1.CUL7 may be one of the markers of Herceptin resistance in breast cancer.2.CUL7 may induce drug resistance by affecting breast cancer stem cells.3.CUL7 may activate HH signaling pathway to affecting breast cancer stem cell characteristics.