The Voltage-dependent Calcium Channel α2δ1 Maintains Stemness In Pancreatic Cancer Tumor-initiating Cell

Objective:Pancreatic cancer is a solid tumor with both high morbidity and mortality,and its 5-year survival rate shows only 10%.Currently,the main treatment for pancreatic cancer includes surgery,chemotherapy,and radiotherapy which can control the growth of pancreatic tumors to a certain extent,but pancreatic cancer cells show high malignancy and often occur recurrence,metastasis,and multidrug resistance,and the poor prognosis for the patients.The cancer stem cells(CSC),also kno wn as tumor initiation cells(TIC)are cell subpopulations with self-renewing,pluripotent differentiation,and high capacity of tumorigenicity,which are associated with tumor formation,recurrence,metastasis,and therapy resistance.Thus,finding identification specific molecular markers for pancreatic cancer TIC and exploring the molecular mechanism of regulating TIC function are the hot points which the researchers concerned about,that can provide a novel strategy for pancreatic cancer treatment.We found that the voltage-dependent calcium channel subunit CACNA2D1(also known asα2δ1)could recognize TIC in many solid tumors,including gastritis carcinoma,lung cancer,and breast cancer.However,we have not systemically deep studied whether α2δ1 can be used as a specific functional marker for pancreatic cancer TIC subpopulations,and how molecular mechanism of regulating stemness properties.Therefore,this project further study effect ofα2δ1 on identifying pancreatic cancer TIC subpopulations,explore the molecular mechanism that α2δ1 influx active status of protein kinase 2 D(CaMK2D)via calcium,and regulate phosphorylation of pyruvate kinase 2(PKM2)and further clarified potential therapeutic effect of targeting α2δ1 regulating signaling.Methods:We prepared monoclonal antibody Mab 1B50-1 against α2δ1.Fluorescence labeling technology was used to label Mab 1B50-1,which can be used for flow cytometry analysis and sorting;flow cytometry was used to analyze α2δ1 expression in pancreatic cancer cell lines(PANC-1,BxPC-3,Aspc-1,and MIA-PaCaⅡ)and tumor tissues;fluorescence-activated flow cytometry sorting(FACS)was used to sort α2δ1 positive and negative cell populations,and those cells were done for sphere formation,Real-time quantitative polymerase chain reaction(RT-PCR),western blot and animal experiments et al.RT-PCR and western blot were used to detect the expression of TIC associated genes in α2δ1 positive and negative pancreatic cancer cells.Knock-down and overexpression techniques were used to verify α2δ1 et al.genes on TIC characteristics.74 cases of specimens from pancreatic cancer with surgical resection were selected.Immunofluorescence was used to detect α2δ1 expression in pancreatic cancer and adjacent tissues,and positive expression rate of α2δ1 and the relationship of clinical physiological characteristics was analyzed.Sequencing and bioinformatics analysis showed that CaMK2D is a downstream target of α2δ1.Mass spectrometry and co-immunoprecipitation(Co-IP)was used to screen PKM2 as an interacting protein with CaMK2D.Knock-down of PKM2 and PKM2-Y10 site mutation as the active status of phosphorylation or inactive status of dephosphorylation was performed to validate the regulation effect of phosphorylated Y105 on TIC properties.Immunofluorescence assay was used to detect the localization of phosphorylated PKM2.NOD/SCID mice were used to establish pancreatic cancer cell-derived xenograft(CDX)model and patient tumor tissues-derived xenograft(PDX)model.1B501,gemcitabine or combinational these two reagents were used to treat CDX and PDX mice models,and an inhibitory effect on pancreatic cancer growth was observed.Results:α2δ1 located on the cell membrane surface with a positive rate of α2δ1 expression ranged from 1.3 to 5%in pancreatic tumor tissues and cell lines.Positive α2δ1 cells shower higher sphere formation and tumor formation rates than negative α2δ1 cells,and an even higher rate of secondary sphere formation and tumor formation for the positive cells,that α2δ1 positive cells possess self-renewal ability.1B50-1 or knock-down of α2δ1 could significantly inhibit self-renewal ability for the α2δ1 positive pancreatic cancer cells.The expression level of stemness genes was higher in the positive cells than those in the negative subpopulations.After the positive cells were cultivated in a serum-containing medium for two weeks,the expression level of α2δ1 in the cultivated cells was similar to the parental cells,which showing differentiation ability.Knockdown of α2δ1 decreased sphere formation,tumor formation and expression level of stemness associated genes,and overexpression of α2δ1 increased sphere formation,tumorigenicity,and expression level of stemness associated genes.α2δ1 positive cells demonstrated higher efficiencies of sphere formation and tumor formation than the other reported stem cell surface markers including EpCAM,CD44,and DCLK.The positive rate ofα2δ1 in pancreatic tumor tissues(66/74)was higher than that in para cancer tissues(44/74),positive expression was associated with lymph node metastasis,nerve infiltration,and shorter survival,that is an independent factor for pancreatic cancer.In addition,high-throughput sequencing analysis showed that α2δ1 could effectively regulate the protein expression and activity of CaMK2D,and the expression of CaMK2D in α2δ1-positive cells was significantly higher than that in negative cells.CaMK2D overexpression enhanced sphere formation,tumorigenicity ability,and expression level of stemness associated genes,and knockdown of this gene inhibited these TIC properties.Furthermore,the results showed that CaMK2D and PKM2 could form complex,PKM2 phosphorylation at Y105 promoted PKM2 nuclear translocation and active TIC stemness genes.PKM2 nucleation was inhibited and the stemness properties of pancreatic cancer were reduced by blocking phosphorylation at Y105.Compared with the monotherapy group and control group,α2δ1 antibody 1B50-1 combined with gemcitabine significantly inhibited tumor growth,and prolonged the mice survival of CDX and PDX mice.α2δ1 positive cells were significantly decreased in the tumor tissues of the combinational group.Conclusion:α2δ1 maintained pancreatic cancer TIC stemness by activating calciumassociated protein CaMK2D,and promoting phosphorylation of PKM2 at the Y105 site.α2δ1 can be used as a functional surface marker for identifying TIC in pancreatic cancer,and targeting α2δ1 has a therapeutic effect for pancreatic cancer.Our study further enriches the regulatory mechanism of TIC stemness and provides a new strategy for pancreatic treatment.