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Phosphatidylinositol-5-phosphate (PtdIns5P) 4-kinase β (PIP4K2B) directly regulates the levels of two important phosphoinositide second messengers, PtdIns5P and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P2]. PIP4K2B has been linked to the regulation of gene transcription, to TP53 and AKT activation, and to the regulation of cellular reactive oxygen accumulation. However, its role in human tumor development and on patient survival is not known. Here, we have interrogated the expression of PIP4K2B in a cohort (489) of patients with breast tumor using immunohistochemical staining and by a meta-analysis of gene expression profiles from 2,999 breast tumors, both with associated clinical outcome data. Low PIP4K2B expression was associated with increased tumor size, high Nottingham histological grade, Ki67 expression, and distant metastasis, whereas high PIP4K2B expression strongly associated with ERBB2 expression. Kaplan–Meier curves showed that both high and low PIP4K2B expression correlated with poorer patient survival compared with intermediate expression. In normal (MCF10A) and tumor (MCF7) breast epithelial cell lines, mimicking low PIP4K2B expression, using short hairpin RNA interference-mediated knockdown, led to a decrease in the transcription and expression of the tumor suppressor protein E-cadherin (CDH1). In MCF10A cells, knockdown of PIP4K2B enhanced TGF-β–induced epithelial to mesenchymal transition (EMT), a process required during the development of metastasis. Analysis of gene expression datasets confirmed the association between low PIP4K2B and low CDH1expression. Decreased CDH1 expression and enhancement of TGF-β–induced EMT by reduced PIP4K2B expression might, in part, explain the association between low PIP4K2B expression and poor patient survival. Cancer Res; 73(23); 6913–25. ©2013 AACR.

PDF file - 60K, A. The gene expression profiles of PIP4K2B in normal and tumour tissues were extracted (GSE10780) and compared. PIP4K2B levels were significantly lower in tumor samples (P=0.04) B. Breast tumour samples from 17 data sets representing 2999 tumours were clustered into the molecular subtypes shown. The PIP4K2B expressions are shown as box plots. Significant differences were determined between different subtyps and the p values for the comparisons are shown in the inset.

PDF file - 89K, A. Gene expression profiles of widely used breast tumour cell lines were extracted from three different data sets, normalised and clustered into the designated molecular subtypes. The expression of PIP4K2A, 2B and 2C are graphically shown below. B. Box plots representing the expression of PIP4K2B in the three molecular subtypes determine by cluster analysis. C. Expression of PIP4K2B in normal cells (MCF10A) and luminal cells (MCF7) and ERBB2 positive cells (ZR-75).

PDF file - 274K, A. Expression profiles from 17 data sets representing 2999 tumors were extracted, normalised and hierarchically clustered according to tumour subtype. The heat map shows the relative transcript expression of genes in order (named on the right) along chromosome surrounding the ERBB2 gene. The ERBB2 and PIP4K2B are labelled in red. B. ACGH array data from the Chin et al study are shown for genes in order on chromosome 17 surrounding the ERBB2 gene. ERBB2 and PIP4K2B are marked in red on the right side.

PDF file - 65K, A. Lysates from control (pLKO1) or PIP4k2B (shPIP4k2B) knockdown MCF10A cells were immunoblotted using the PIP4K2BP6 antibody. B. The PIP4K2BP6 antibody was immunodepleted by passing it through a column to which the P6 antigenic peptide was coupled. The antibody before or after, as indicated, was used to probe western blots of increasing levels of PIP4K2B .

PDF file - 46K, A. Lysates from MCF10A cells after 2 and 4 days of growth were analysed by western blotting using the antibodies shown. B. Lysates from cells treated as controls or with MG132 were analysed by western blotting with an anti-ubiquitin antibody to demonstrate that MG132 increases polyubiquitination of proteins.

PDF file - 67K, Control or PIP4K2B knockdown MCF10A cells were labelled with 32P-orthophosphate for two hours after which lipids were extracted, deacetylated and the lipid head groups were separated by HPLC. Radioactivity was quantified using an online detector and the data are presented as counts in each head group divided by the total counts + the SD. B. Lipids were extracted from control or PIP4K2B knockdown MCF10A cells and the abundance of PtdIns5P was measured. The data are presented as the pmoles of PtdIns5P/nmol of total lipid phosphate +the SD.

PDF file - 77K

Phosphatidylinositol-5-phosphate (PtdIns5P) 4-kinase β (PIP4K2B) directly regulates the levels of two important phosphoinositide second messengers, PtdIns5P and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P2]. PIP4K2B has been linked to the regulation of gene transcription, to TP53 and AKT activation, and to the regulation of cellular reactive oxygen accumulation. However, its role in human tumor development and on patient survival is not known. Here, we have interrogated the expression of PIP4K2B in a cohort (489) of patients with breast tumor using immunohistochemical staining and by a meta-analysis of gene expression profiles from 2,999 breast tumors, both with associated clinical outcome data. Low PIP4K2B expression was associated with increased tumor size, high Nottingham histological grade, Ki67 expression, and distant metastasis, whereas high PIP4K2B expression strongly associated with ERBB2 expression. Kaplan–Meier curves showed that both high and low PIP4K2B expression correlated with poorer patient survival compared with intermediate expression. In normal (MCF10A) and tumor (MCF7) breast epithelial cell lines, mimicking low PIP4K2B expression, using short hairpin RNA interference-mediated knockdown, led to a decrease in the transcription and expression of the tumor suppressor protein E-cadherin (CDH1). In MCF10A cells, knockdown of PIP4K2B enhanced TGF-β–induced epithelial to mesenchymal transition (EMT), a process required during the development of metastasis. Analysis of gene expression datasets confirmed the association between low PIP4K2B and low CDH1expression. Decreased CDH1 expression and enhancement of TGF-β–induced EMT by reduced PIP4K2B expression might, in part, explain the association between low PIP4K2B expression and poor patient survival. Cancer Res; 73(23); 6913–25. ©2013 AACR.