The Mechanism Of Promoting Triple Negative Breast Cancer Metastasis Via Type I? Phosphatidylinositol Phosphate Kinase

Significance:Type I ? phosphatidylinositol phosphate kinase(PIPKI)has been associated with poor prognosis in breast cancer patients by promoting metastasis.The 5-year or 10-year survival rate of breast cancer patients with positive lymph node metastasis is higher in weakly or negative expressed PIPKI? group than that in positive expressed PIPKI? group.Moreover,expression of PIPKI? in invasive breast cancer is also high.PIPKI? regulates cell migration in cancers,which is an initial step of metastasis.Thus,we predict PIPKI? could promote metastasis.Among the six alternative-splicing isoforms of PIPKI,PIPKI_i2 specifically targets to focal adhesions and regulates focal adhesion turnover.Based on that,PIPKI_i2 might be the major isoform which contributes to tumor metastasis.Purpose:This research aims to confirm the role of PIPKI_i2 on metastasis in order to provide more specific biomarker to predict the prognosis of triple negative breast cancer.Methods:(1)Cell biology experiments: Human triple breast cancer cell line MDA-MB-231 and murine triple breast cancer cell line 4T1 were depleted pan-PIPKI? and PIPKI_i2 relatively.And several characteristics were measured.MTT assay was preformed to evaluate the capacity of proliferation in cancer cells.Would-healing assay and transwell migration assay wereexploited to examine tumor cell migration.Matrix degradation assay was used for invasion of tumor cell.(2)Biochemical experiments: We used western blotting to test phosphorylation of AKT and ERK1/2 which is associated with tumor growth and invasion in both pan-PIPKI? and PIPKI?_i2 depleted 4T1 cells and MDA-MB-231 cells.MMP9 and some EMT markers were examined as well.(3)In-vivo experiments: To explore whether the impact of pan-PIPKI? and PIPKI?_i2 depleted on 4T1 cells is the same,we exploited Balb/c mice to build up the tumor model.Control cells,transfected PIPKI? or PIPKI?_i2 4T1 cells were injected into mammary fat pads.At the end point,we collected the growth curve of tumor and tumor volumn and lung metastasis.(4)Histochemistry experiments: we used immunohistochemistry and immunofluorescence to evaluate hypoxia,tumor associated macrophage infiltration,angiogenesis and PD-L1 expression in tumor tissues.Results:In vitro we specifically depleted PIPKI?_i2 in murine triple negative breast cancer 4T1 cells and human triple negative breast cancer MDA-MB-231 and analysed their biological characteristics.As expected,loss of PIPKI?_i2 can decrease tumor proliferation,migration and invasion to a comparable level to depletion of pan-PIPKI?.Depletion of PIPKI?_i2 and pan-PIPKI? in MDA-MB-231 cells and 4T1 cells both can downregulated phosphorylation of AKT and ERK1/2,so as MMP9.The phosphorylation of AKT and ERK1/2 usually increased in triple negative breast cancer,which could promote tumor growth and migration.MMP9 was considered as one of major enzyme to degrade matrix surrounding tumor cells during invasion.High expression of MMP9 is associated with poor prognosis.However,depletion of PIPKI?_i2 had no effect on suppressing 4T1 tumor progression in vivo.Compared with control group,there is no difference withdepletion of PIPKI?_i2 in tumor growth,hypoxia,macrophage infiltration,angiogenesis and PD-L1 expression in tumor microenvironment.Nevertheless,loss of pan-PIPKI? will significantly decrease all the levels mentioned above.Furthermore,it suggested that depletion of PIPKI?_i2 had no impact on expression of EMT markers and anchorage-independent growth unlike depletion of pan-PIPKI?.In this research,we examined epithelial markers ZO-1 and E-cadherin and mesenchemal marker vimentin and snail.In pan-PIPKI? depleted 4T1 cells,expression of ZO-1 and E-cadherin were upregulated and vimentin was slightly downregulated.One of the most important EMT transcriptional factor snail was downregulated obviously.But depletion of PIPKI?_i2 in 4T1 cells has not aroused the consistent results with depletion pan-PIPKI?.Meanwhile,expression of snail was at similar status in MDA-MB-231 cells to 4T1 cells.Snail has more impact on breast cancer metastasis than TWIST or ZEB.Overexpression or knockdown of snail could increase or decrease the number of lung metastasis nodes in breast cancer bearing mice.Thus,while PIPKI_i2 indeed is required for cell migration and invasion,these characteristics are not decisive for metastasis.Tumor metastasis involves lots of aspects.In this research,we mainly tested hypoxia,macrophage infiltration,angiogenesis and EMT markers.According to the consequences above,we speculated depletion of PIPKI?_i2 is insufficient to inhibit tumor metastasis and other isoforms of PIPKI? may work on metastasis together.Conclusion:(1)Loss of PIPKI?_i2 can decrease tumor proliferation and migration to a comparable level to depletion of pan-PIPKI?.Depletion of PIPKI?_i2 and pan-PIPKI? in MDA-MB-231 cells and 4T1 cells both can downregulated phosphorylation of AKT and ERK1/2,suggesting pan-PIPKI? and PIPKI?_i2may affect tumor proliferation and migration via AKT and ERK1/2 signaling.(2)Depletion of pan-PIPKI? or PIPKI?_i2 can decrease the invasion of4T1 cells.Moreover,the expression of MMP9 decreased in both group of cells.(3)In tumor bearing mice which expressed sh NC,sh PIPKI?,sh PIPKI?_i2,depleted pan-PIPKI? can reduce tumor growth rate and lung metastasis,while only PIPKI?_i2 depleted has no difference with control group.(4)The mechanism of that depletion of PIPKI?_i2 cannot inhibit tumor growth and lung metastasis is because of unchanged tumor microenvironment and EMT.