The Effects On Biological Characteristics And Mechanism Of HPK1 In Invasive Ductal Breast Carcinoma-not Otherwise Specified

BackgroundBreast cancer is a kind of highly heterogeneous malignant tumor.5-year incidence statistics showed that nearly 11% of breast cancer patients are Chinese in the world.And its incidence trends: the incidence is rising rapidly,the onset is younger,a serious threat to the health of women.Breast cancer was individed into invasive and non-invisive,according to the WHO histological classification method.Invasive ductal carcinoma-not otherwise specified(IDC-NOS)is the most common pathological type of non-invisive ones,also the most common pathological type of breast cancer,demonstrates great intratumoral morphological heterogeneity,compared with lobular carcinoma or tubular,lack of rich characteristics.IDC-NOS patients have obvious curative effect of individual differences in the effects of endocrine therapy,also prognosis.Therefore gaining an insight into the molecular biological characteristics,exploring the IDC-NOS related genes and biomarkers for early detection,monitoring and prognosis judgement,even as new targets of targeted therapy,for IDC-NOS is of great significance.Hematopoietic progenitor kinase 1,HPK1,also called MAP4K1,mitogenactivated protein kinase kinase kinase kinase 1,which gene ID is 11184,location:19q13.1-q13.4,belongs to the mammalian Ste20-like family of serine/threoninekinases of the super family(SLK),a kind of microtubule associated protein.Recent studies have found associations between HPK1 and the tumorigenesis and progression of a varity of malignant tumors in humans.For example,HPK1 inhibited the proliferation of a lung cancer cell line,as well as infiltration and metastasis of non-small-cell lung carcinoma cells;another study found that lack of HPK1 promoted the development and tumorigenesis of pancreatic ductal adenocarcinoma.Although HPK1 plays a critical role in some tumors,HPK1 in IDC-NOS expression and biological functions are not clear.However,no literature reports have discussed a potential association between HPK1 expression and biological functions in breast cancer,particularly IDC-NOS development,either domestically or internationally.This paper did a preliminary attempt to study and discuss biological functions and its mechanism of HPK1 in IDC-NOS through the following three parts: the clinical cases,the cell experiments and animal models,also a progressive layers of the process,for offering a new reference for clinical diagnosis and treatment IDC-NOS.First of all,screen HPK1 expression in IDC-NOS samples and evaluate its relationships with survival factors and overall survival.Secondly,construct and screen of HPK1 stable expression of breast cancer cell lines,clarify the biological functions of HPK1 in IDC-NOS and the change of intracellular signaling pathways.Finally,bulid subcutaneous transplantation tumor animal models by subcutaneous transplant HPK1 lower expression and overexpression of human breast cancer cell lines in the nude mice.Explore the expression of HPK1 in animal models of transplanted tumor growth in vivo.Object:1.To evaluate HPK1 expression in IDC-NOS samples and its relationship with the prognosis related indicators and overall survival.To determine the significance of HPK1 expression in patients with IDC-NOS.2.To culture breast cancer cell lines of MCF-7 and MDA-MB-231,construct slow virus vector p CDH-HPK1-puro,package recombinant lentivirus Lentil-HPK1 and Lentil-con,infect cell lines with recombinant lentivirus,screen of stable expression cell lines.To detect the endogenous HPK1 gene expression in breast cancer cell lines MCF-7 and MDA-MB-231 of stable expression groups and the control ones,respectively.To study the influences of the HPK1 gene expression on biological characteristics of the breast cancer cell lines such as cell proliferation,apoptosis,cell cycle and invasion.To detect the changes of proteins expression,which are involved in tumor biological behavior and HPK1 related molecular MAPK pathway downstream target genes,such as JNK and p53,and also the changes of their proteins.To clear HPK1 genes involved in regulating related molecular mechanism in the IDC-NOS related cell lines.3.To detect the influence of the regulation from HPK1 expression on tumor growth in vivo by constructing subcutaneous transplantation tumor animal models.To make a clear definition of the express of HPK1 in IDC-NOS function and related mechanism of transplanted tumor animal models.Main Content:Part ?: Expression and significance of HPK1 in the tissues of invasive ductal breast carcinoma-not otherwise specifiedMethods:1.Screened cases following the standard in IDC-NOS from our hospital library.Collected related data and the corresponding paraffin specimens of these cases.HPK1 protein expression in samples from included patients was detected using immunohistochemistry(IHC).2.HPK1 protein and m RNA expression in samples from 148 IDC-NOS patients was detected by western blotting and Quantitative Real-time PCR(RT-q PCR),respectively.Clear the correlation between HPK1 expression and tumor stage,classic index(ER,PR and human epidermalgrowth factor receptor-2,Her-2protein),histological classification of the clinical pathological characteristics.Explored HPK1 significance to the prediction of the prognosis of patients with IDC-NOS through the survival analysis and risk regression model.Results:1.The enrolled cases were 148 consecutive female patients,age range: 29-78 years.Follow-up durations range: 1-80months(the median follow-up durations:62±1).Fifty-four(36.5%)samples of m RNA and protein were HPK1-positive,and 100(67.6%)were ER-positive;of the latter,28%(28/100)were HPK1-positive,and a significant negative association of HPK1 expression with ER positivity was observed(P = 0.002,r =-0.254).Among IDC-NOS tissues,43.2% and 32.4% were PR-and Her-2-positive,respectively;neither indicator correlated with HPK1(P = 0.109,0.558,respectively),no obvious correlation.2.There were no significant correlation between HPK1 expression and prognosis in the ER negative group.HPK1 expression,lymph node metastasis,and TNM stage were independent factors of overall survival(OS)in the ER-positive group(P <0.05);HPK1 positivity was associated with longer OS(P = 0.03<0.05).HPK1 m RNA levels did not differ between IDC-NOS and normal breast tissues,whereas HPK1 protein levels were lower in IDC-NOS(P <0.05).Part ?: The construction of HPK1 stable over expression in breast cancer cell lines,the determination of the related biological behavior and the change of intracellular signaling pathwaysMethods:1.HPK1 gene over expression lentiviral vector pCDH-HPK1-puro was transformed and constructed by using p CDH-CMV-MCS-EF1-puro.The positive vector clones were identified by colony PCR,enzyme digestion and DNA sequencing analysis.Transfered plasmid p CDH-HPK1-puro,packaging plasmid p CMV-d R8.2dvpr and p CMV-VSVG into HEK293 T cell by using lipofectamine2000,and got lentiviral virus Lenti-con and Lenti-HPK1.And then stable over expression cell lines MCF-7-HPK1 and MDA-MB-231-HPK1 generated from screening.2.Identification of MCF-7-HPK1 and MDA-MB-231-HPK1 stable transfected cell lines.Detected the expression of HPK1 in the stable transfected cell lines by using real-time fluorescent quantitative PCR,western blotting and immunocytochemistry staining.3.The influence of HPK1 on the multiplication capacity in breast cancer cell lines MDA-MB-231 and MCF-7 was detected by MTT experiment.4.The influence of HPK1 on the clonogenesis ability in breast cancer cell lines MDA-MB-231 and MCF-7 was detected by soft agarose assay.5.The influence of HPK1 on the apoptosis and cell cycle in breast cancer cell lines MDA-MB-231 and MCF-7 was detected by flow cytometry.6.The influence of HPK1 on the apoptosis in breast cancer cell lines MDA-MB-231 and MCF-7 was detected by TUNEL test.7.The influence of HPK1 on the invasiveness in breast cancer cell lines MDA-MB-231 and MCF-7 was detected by Wounding healing assay and Transwell test.8.The influence of HPK1 on the proteins involved in tumor biological behavior in breast cancer cell lines,such as caspase3,PTEN,Ki-67,MMP9 and Fbxw8,also the downstream target protein in the MAPK pathway.Results:1.Restriction enzyme digestion and sequencing showed p CDH-HPK1-puro was constructed successfully.The enzyme digestion product of lentivirus expression vector was a single strip,2500 bp,which was in line with expectations.Sequencing results are consistent with the NCBI reference sequence,which showed HPK1 over expression lentivirus vectorp CDH-HPK1-puro was constructed successfully.2.HPK1 m RNA was detected by real-time quantitative PCR(RT-qPCR)and western blot.And the results showed there was no difference between blank groups and control groups of MDA-MB-231 and MCF-7 cell lines.HPK1 m RNA levels in p CDH-HPK1-puro groups were significantly increased compared with blank ones and control ones.The protein of HPK1 was examined by immunocytochemical staining.The results were similar with m RNA test.HPK1 protein levels in p CDH-HPK1-puro groups were significantly higher than blank ones and control ones(P<0.05).3.MTT test showed cell proliferation was significantly inhibited in HPK1 over expression groups of breast cancer cell lines MDA-MB-231 and MCF-7(P<0.05).4.The influence of clonogenesis ability did no statistical differ between HPK1 over expression group and control groups in breast cancer cell lines MDA-MB-231 by soft agarose assay(P>0.05).While the clonogenesis ability of HPK1 over expression group in MCF-7 was significantly inhibited,compared with the control groups(P<0.05).5.The influence of apoptosis did no statistical differ between HPK1 over expression groups and control groups of breast cancer cell lines MDA-MB-231 and MCF-7 by flow cytometry(P>0.05).Similarly as the influence of cell cycle in HPK1 over expression groups of cell line MDA-MB-231.The influence of cell cycle was significative in HPK1 over expression groups of cell line MCF-7(P<0.001).The cell count of S phase and G2 phase in MCF-7 cell line was significantly decreased compared with control groups,which showedthe cell line MCF-7 cell cycle was inhibited in G1 phase when HPK1 was over expression.6.TUNEL test showed the influence of HPK1 over expression on the apoptosis in breast cancer cell lines MDA-MB-231 and MCF-7 was statistical.7.Wound healing assay and Transwell test showed the invasiveness of breast cell lines MDA-MB-231 and MCF-7 was inhibited by HPK1 over expression(P<0.05).8.The differences between before and after transfection were statistical,for ER,Caspase3,Ki-67,PTEN and MMP9 in cell line MCF-7 and Caspase3,Ki-67,PTEN,Fbxw8 and MMP9 in cell line MDA-MB-231(P<0.01),respectively.Above all confirmed HPK1 can affect cell proliferation,invation and metastasis.Contrasted before and after HPK1 was transfected,HPK1 over expression influenced on the downstream gene JNK and p53 in MAPK pathway,for JNK in cell line MCF-7 and for JNK and p53 in MDA-MB-231,respectively.HPK1 influenced on breast cancer cell line biological behavior may be through targeted regulating JNK pathway for MCF-7,pathways of JNK and p53 for MDA-MB-231,respectively.Part ?: HPK1 overexpression in breast cancer MCF-7 and MDA-MB-231 cells in nude mice transplantation tumor growth in vivoMethods:1.Constructed nude mice transplantation tumor model by suspension inoculation of breast cancer cells in abdominal breast fat pads in the lower left quadrant.Divided these nude mice into 6 groups according to the inoculation cell types,MDA-MB-231,MDA-MB-231-con,MDA-MB-231-HPK1,MCF-7,MCF-7-con,MCF-7-HPK1,respectively.Each inoculation of 107/200?l.There were 15 in each group.5 from each group were randomly selected to be put to death,and the transplanted tumors were anatomied for the determination of transplanted tumor size and weight.The other 10 mice in each group for survival analysis experiment.2.Detected HPK1 expression regulation effected on breast cancer tumor growth in vivo.Measuremented size of tumor nude mice in HPK1 different expression groups on time,drawed tumor growth curve.3.Determined nude mice survival in HPK1 different expression groups of transplantation tumor.4.Detected the protein expression of HPK1 and the downstream gene JNK and p53 in MAPK-p53-JNK pathway by IHC.Results:1.Nude mice transplantation tumor modesl were contructed successfully in both experiments.2.Volumes and weights of tumors in groups of MCF-7-HPK1 and MDA-MB-231-HPK1 were significantly lower(P<0.01).The growth of tumors in nude mice could be inhibited by HPK1 overexperssion.3.Survival rates were increased by HPK1 overexperssion.At the end of experiment,The survival rate of MDA-MB-231-HPK1(66%)was significantly higher than the ones of MDA-MB-231(35.049%)and MDA-MB-231-con(35.294%).Similarly,the survival rate of MCF-7-HPK1(71.296%)was significantly higher than the ones of MCF-7(30.720%)and MCF-7-con(33.083%).4.IHC results showed that JNK and P53 expression were up-regulated,accompanied by up-regulated HPK1 expression,which showed a positive correlation trend.Conclusions:1.HPK1 gene was positive expressed in tissue adjacent to carcinoma significantly higher than that of IDC-NOS in the clinical enrolled cases,and a significant negative association of HPK1 expression with ER positivity was observed.The loss of HPK1 protein expression may have a close association with tumorigenesis and progression in ER-positive IDC-NOS.2.Up-regulated HPK1 expression inhibited the ability of proliferation,clonogenesis and invasion,and promoted apoptosis in both the breast cancer stable overexpression cell lines MDA-MB-231-HPK1 and MCF-7-HPK1,also blocked MCF-7 cells in G1 phase.3.In animal vivo experiment,up-regulated HPK1 expression inhibited tumorigenic ability and improved the nude mice survival rate,which confirmed HPK1 takes anticancer effect on breast cancer in vivo.The role of HPK1 in breast cancer may be associated with regulating key sites JNK gene and p53 gene in MAPK pathway.