Cell Signaling Pathway Analysis Induced By PIK3CA-HER2 Co-occuring And Functional Investigation Of Novel Oncogene EGFL9 In Breast Cancer

Breast cancer is one of the most malignancy in the world.Its morbidity still increases by years in most countries.According to data from the American Cancer Society,more than 255,000 breast cancer patients were diagnosed in 2017,and over 40,000 patients died of breast cancer.In China,the incidence and mortality of breast cancer were increasing in recent years.According to the latest statistics from the National Cancer Center,more than 278,000 women in mainland China were diagnosed with breast cancer,and the death records were 64,000.At present,the survival rate of patients with breast cancer is greatly upward,due to the improvement of early detection methods and the comprehensive treatment strategies.However,the 5-year survival rate of patients with metastatic breast cancer remains at the similar level.The main cause of breast cancer invasion and metastasis is the genetic heterogeneity of breast cancer.For example,abnormal amplification of oncogenes and evolving gene mutations can lead to differences in clinical outcomes of breast cancer.As genomic technology improved,multiple oncogenes have been found to be closely related to the development of breast cancer.Some of target therapies were initially effective when administered in clinical trials.However,due to the specific signaling mechanism of co-activation of the oncogenes combination that leads to the progress of breast cancer has not been properly studied;the overall efficacies of treatments are limited as the development of drug resistance.Therefore,to explore novel oncogenes associated with breast cancer,and elucidate mechanisms of co-currence and advance progress against breast cancer that especially the signal regulation mechanism under the condition of co-activating oncogene is necessary.By inhibiting these oncogenes and reversing the activation of signal pathway to overcome the drug resistance and tumor metastasis that are the primary strategies in clinical breast cancer research.Part 1:Pathway analysis for breast cancer with HER2 amplification and PIK3CA mutationPurpose:HER2 is one of the major receptors for the development of breast cancer.Approximately 20%of amplification of the HER2 gene in breast cancer patients can be detected,and 40%of these patients encounted with the mutation of phosphatidylin-3-kinase(PI3K).PI3K is a lipid kinase,which generates PIP3 at the plasma membrane.When RTK receptors are stimulated by extracellular growth factors and other stimuli;they can raise PI3K to activate the involved signaling.The PIK3CA gene encodes the p110? catalytic subunit of PI3K,and its mutation,such as the H1047R site,can result in the production of a variety of cancers including breast cancer.PI3K mutation is the main pathogenesis of cancer.Clinically,HER2 amplification with PIK3CA mutations and the following activation of the PI3K/AKT signaling pathway is considered the main determinant of resistance to breast cancer.In order to explore the PIK3CA mutation and/or HER2 amplification's effect that induce cancer and the mechanism of cell signaling regulation,we employed experiments to detect the relationship between PIK3CA mutation and HER2.At the same time,IPA was used to analyze the changes of related genes and pathways in breast cancer cells with both abnormal HER2 and PIK3CA genes.Methods:In this experiment,bioinformatic analysis revealed coexistence of mutated PIK3CA and amplified HER2 in tumor cells.In vitro experiments,cellular models with both PIK3CA mutation and/or HER2 amplification were reconstructed in the normal-like breast cell line MCF10A.The growth of cancer cells was observed by using medium containing different nutrients respectively to understand the effects of different nutrients on the growth of cancer cells.In migration and invasion assay,transwell chambers were used to observe the transmembrane changes of MCF10A cells with different genotypes.By treating cells with inhibitors of PI3K and HER2,we observed the growth inhibition of cancer cells and the synergy between two oncogenetic inhibitors.We used Affymetrix microarray to compare gene expression of MCF10A cells with different genotypes in case to find common changes in cell signaling regulations in the presence of all three genotypes,as well as the single presence of MCF10A/PIK3CA or MCF10A/HER2 gene.Results:(1)Using comparative genomic hybridization(aCGH)bioinformatics technology to detect the mutations in PIK3CA mutant cells.There were multiple gene amplification sites on different chromosomes,the most significant amplification sites HER2 was among them.(2)Through growth curve,migration,invasion and drug inhibition experiments,we found that the growth and metastasis ability of breast cancer cells that contain two gene changes were more obvious than the single gene.The changes were not affected by culturing in different medium that contained different nutrients.This indicated that the MCF10A/HER2/PIK3CA cells are not affected by growth factors and insulin.The synergistic use of drugs was more effective in inhibiting cell growth and proliferation.Therefore,the combination of anti-HER2 and PI3K should be one of the important targets for the growth and metastasis of breast cancer.(3)Using affymetrix microarray analysis to detected the regulatory changes of multiple genes.The results of biometrics analysis of these genes showed that most of the significantly regulated genes in breast cancer cells that contain both genetic alterations were mainly enriched in four signaling pathways.They were aromatic compounds(AhR)pathway,the AMP-dependent protein kinase(AMPK)signaling,the protein ubiquitination pathway,and the hereditary breast cancer signaling pathway.Importantly,in co-existence of PIK3CA mutations and HER2 amplification cell line,genes mostly enriched in cell cycle control pathway,DNA damage response and more significant in DNA replication stress.This study,for the first time,found that co-activation of the oncogenes PIK3CA and HER2 not only led to breast cancer progression,but also resulted in further gene duplication stress in signal regulation.These findings provided a new insight in the future diagnosis and treatment of breast cancer.Part 2:Novel Oncogene EGFL9 Promotes Breast Cancer Metastasis and StemnessPurpose:EGFR and c-MET are widely studied as members of the RTKs receptor family.Single use of two drug inhibitors is still effective for some breast cancer subtypes.However,the evolving tumor metastasis and complex drug resistance not only trap the scientists to overwhelm breast cancer,but also frustrated the faith of survival among breast cancer patients.In recent years,a growing number of studies have found that co-activation of c-MET and EGFR play a common role in the development of tumors.This effect can be dominate by extracellular ligand such as HGF or adjusts by receptor internal autocrine and paracrine that eventrally induces breast cancer metastasis and drug resistance.In the courses of clinical application,researchers found that adding MET-TKI to EGFR-TKI resistant breast cancer can effectively increase the drug sensitivity of breast cancer.This part of the study will answer these questions and provide a more complete mechanism of RTKs receptor regulation.This contributes to a promising treatment of breast cancer.Methods:In this study,gene expression analysis was performed in 14 breast cancer cell lines by performing quantitative PCR which validated EGFL family members in 14 breast cancer cell lines.The results also confirmed by western blotting that the expression of EGFL9 in breast cancer cell lines.Tumor growth and metastasis were observed by MTT and transwell experiments in vitro and implantation of tumors in nude mice in vivo.Using of MRI and TMA that observed distant metastasis and acquainted the relationship between EGFL9 and metastasis.The human phosphor-RTK antibody array and confocal microscopy were used to observe the signal regulation in EGFL9 overexpressing breast cancer cells.Finally,a series of experiments on the characteristics of cancer stem cells that uncovered the relationship between EGFL9 and pluripotency of breast cancer,and the correlation between breast cancer metastasis and sternness.Results:(1)The expression of whole epidermal growth factor-like domain(EGFL)family in different breast cancer subtypes was detected by PCR.The result showed that EGFL9 is a novel oncogene,which mainly expressed in basal-like breast carcinoma.(2)The overexpression of EGFL9 had little effect on the growth of breast cancer cells through in vivo experiments including TMA,MRI and in vitro experiments(growth curve,migration and invasion).The results demonstrated that breast cancer can invade into adjacent tissues and/or disseminat to lungs in vivo.(3)Human RTK phosphorylation antibody array analysis showed that EGFL9 overexpression can co-activate EGFR and c-MET phosphorylation.Western blot and Immunofluorescence assay not only validated EGFR9 regulated the two receptors in breast cancer cell lines,but also verified its effect on downstream regulators such as FAK and ERK1/2.(4)The result of confocal microscope showed that EGFL9 could regulate the co-activation of EGFR and c-MET through binding of c-MET receptor directly,but there was no such direct interaction between EGFL9 and EGFR.Drug inhibition of c-MET prevented EGFL9 mediating downstream signaling pathways and reversed breast cancer cell metastasis.(5)Expression analysis of 44 cancer cell markers by real-time quantitative PCR showed that EGFL9 could significantly increase the expression of Nanog,Oct-4 and c-KIT which associated to multipotency of cancer.The results were also verified by western blotting.Meanwhile,we observed the tumorsphere of cells and the population changes of ALDH+andCD44+/CD24-that related to cancer stemness.We found that EGFL9 overexpression can cause breast cancer cells to form larger and denser stem cell spheres,ALDH+cells increased but CD44+/CD24-cells remained unchanged.Summary and innovative discoveries:This dissertation explores the unique regulation of signal pathways under the co-occurring of two genes that play a crucial role in the development of breast cancer.A new type of oncogene was discovered and its signal pathway was further explored.Following are the aspects of innovative discoveries:(1)It was found that in breast cancer cells with both PIK3CA mutation and HER2 amplification,ten major signaling pathways and related genes were explored.Most of these ten pathways were related to the cell cycle checkpoint and DNA damage repair.(2)In PIK3CA mutations and/or HER2 amplification of breast cancer cells,the AhR pathway,AMPK pathway,the protein ubiquitin and hereditary breast cancer related signaling is the most prominent four pathways by mutual regulation.This finding can be used to guide the targeting therapy of breast cancer subtypes.(3)Epidermal growth factor-like domain,multiple 9(EGFL9)was found to be highly expressed in basal-like breast cancer.It is a brand-new oncogene.(4)EGFL9 overexpression is closely related to breast cancer metastasis.This can lead to breast cancer invade to surrounding tissues and distant lung area.EGFL9 knockdown reverses the activation of these signaling,effectively inhibit cancer cell migration and invasion.(5)EGFL9 induced breast cancer metastasis initiates the phosphorylation of c-MET and EGFR by directly activating the c-MET receptor.Drug inhibition of c-MET reverses EGFL9induced activation of signaling pathways and cell motility,thereby reversing tumor formation.(6)EGFL9 leads to cancer stem cell markers aberrant increasing and tumorsphere formation,providing a theoretical basis for the link between breast cancer metastasis and stemness.