Type I gamma phosphatidylinositol phosphate kinase modulates invasion and proliferation and its expression correlates with poor prognosis in breast cancer

Abstract

Introduction: The loss of E-cadherin based cell-cell contacts and tumor cell migration to the vasculature and lymphatic system are hallmarks of metastasis of epithelial cancers. Type I gamma phosphatidylinositol phosphate kinase (PIPKIgamma), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PI4,5P2) a lipid messenger and precursor to many additional second messengers, was found to regulate E-cadherin cell-cell contacts and growth factor-stimulated directional cell migration, indicating that PIPKIgamma regulates key steps in metastasis. Here, we assess the expression of PIPKIgamma in breast cancers and have shown that expression correlated with disease progression and outcome.

Methods: Using a tissue microarray, we analyzed 438 breast carcinomas for the levels of PIPKIgamma and investigated the correlation of PIPKIgamma expression with patient survival via Kaplan-Meier survival analysis. Moreover, via knockdown of the expression of PIPKIgamma in cultured breast cancer cells with siRNA, the roles of PIPKIgamma in breast cancer migration, invasion, and proliferation were examined.

Results: Tissue microarray data shows that approximately 18% of the cohort immunostained showed high expression of PIPKIgamma. The Kaplan-Meier survival analysis revealed a significant inverse correlation between strong PIPKIgamma expression and overall patient survival. Expression of PIPKIgamma correlated positively with epidermal growth factor receptor (EGFR) expression, which regulates breast cancer progression and metastasis. In cultured breast cancer cells, PIPKIgamma is required for growth factor stimulated migration, invasion, and proliferation of cells.

Conclusions: The results reveal a significant correlation between PIPKIgamma expression and the progression of breast cancer. This is consistent with PIPKIgamma 's role in breast cancer cell migration, invasion, and proliferation.

Figure 1

Expression of PIPKIγ in breast carcinomas. (a) Shown is H&E, E-cadherin and type I gamma phosphatidylinositol phosphate kinase (PIPKIγ) staining of normal breast tissue (top). Breast carcinomas that show a loss of both E-cadherin and membrane PIPKIγ (middle). A fraction of carcinomas that show both a mistargeting of E-cadherin and a loss of PIPKIγ (bottom). (b) Expression of PIPKIγ in breast carcinomas. (Left panel) Representative breast carcinomas stained negative for PIPKIγ. (Middle panel) Representative breast carcinomas stained weakly positive for PIPKIγ. (Right panel) Representative breast carcinomas stained strong positive for PIPKIγ. (c) Immunohistochemical staining of breast tumor tissue for human epidermal growth factor receptor (HER-1) and PIPKIγ. The panels on the top or on the bottom are from the same tumors. The top panels show strong expression of HER-1 and PIPKIγ, whereas the bottom shows weak staining for both antigens.

Figure 2

Kaplan-Meier survival curves for PIPKIγ staining, with time given in years. (a) The entire cohort of patients. (b) Lymph node negative patients. (c) Lymph node positive patients. PIPKIγ = type I gamma phosphatidylinositol phosphate kinase.

Figure 3

Knockdown of PIPKIγ blocks breast cancer cell line migration and invasion. MDA-MB-231 or MDA-MB-435S cells were transfected with control siRNA, or type I gamma phosphatidylinositol phosphate kinase (PIPKIγ) specific siRNA separately as indicated. (a) Expression of PIPKIγ and actin were detected by their specific antibodies. (b) FBS (1%) induced migration of MDA-MB-231 and MDA-MB-435S cells. (c) FBS (1%) induced invasion of MDA-MB-231 and MDA-MB-435S cells.

Figure 4

PIPKIγ expression is required for rapid cell proliferation in breast cancer cells. Lentiviral vector was used to generate the stable knockdown of type I gamma phosphatidylinositol phosphate kinase (PIPKIγ) in breast cancer cell lines as described in "Material and methods". Decreased expression of PIPKIγ was assessed by using PIPKIγ specific antibody. Cells infected with either Control short-hairpin RNA (shRNA) or PIPKIγ shRNA were seeded into 12-well culture plates at a density of 1000 cells/well. Manual cell counting was performed every two days for the eight days. The cell numbers were counted from at least three wells for each cell type and expressed as mean ± standard deviation from one representative experiment. (a) MDA-MB-231 cells. (b) MDA-MB-435S cells. (c) SKBR3 cells.