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. 2018 Nov 20;9(91):36358-36370.
doi: 10.18632/oncotarget.26357.

The Phosphorylation Status of PIP5K1C at Serine 448 Can Be Predictive for Invasive Ductal Carcinoma of the Breast

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Free PMC article

The Phosphorylation Status of PIP5K1C at Serine 448 Can Be Predictive for Invasive Ductal Carcinoma of the Breast

Nisha Durand et al. Oncotarget. .
Free PMC article

Abstract

Phosphatidylinositol-4-phosphate 5-kinase type-1C (PIP5K1C) is a lipid kinase that regulates focal adhesion dynamics and cell attachment through site-specific formation of phosphatidylinositol-4,5-bisphosphate (PI4,5P2). By comparing normal breast tissue to carcinoma in situ and invasive ductal carcinoma subtypes, we here show that the phosphorylation status of PIP5K1C at serine residue 448 (S448) can be predictive for breast cancer progression to an aggressive phenotype, while PIP5K1C expression levels are not indicative for this event. PIP5K1C phosphorylation at S448 is downregulated in invasive ductal carcinoma, and similarly, the expression levels of PKD1, the kinase that phosphorylates PIP5K1C at this site, are decreased. Overall, since PKD1 is a negative regulator of cell migration and invasion in breast cancer, the phosphorylation status of this residue may serve as an indicator of aggressiveness of breast tumors.

Keywords: PIP5K1C; breast cancer; invasive phenotype; phosphorylation.

Conflict of interest statement

CONFLICTS OF INTEREST All authors have no conflicts of interest.

Figures

Figure 1
Figure 1. The expression of PIP5K1C is not predictive for breast cancer survival or subtype
(A) Percent alteration frequency (mutations or alterations in expression) of PIP5K1A, PIP5K1B and PIP5K1C in 3 studies: breast cancer (BC; n = 2509 samples; [42]), breast invasive carcinoma (BIC; n = 1105 samples; TCGA) and mutational profiles of metastatic breast cancers (MBC; n=216 samples; [43]). The analysis was performed using cBioPortal (http://www.cbioportal.org/public-portal/index.do). (B) Relative expression of PIP5K1C in breast cancer cell lines (n=51) grouped into basal or luminal subtypes (left side) or grouped into TN, HER2+ or HR+ subtypes (right side). The analysis was performed using GOBO from Lund University (http://co.bmc.lu.se/gobo/). (C) Tissue microarrays with indicated groups of samples were immunohistochemically-stained for PIP5K1C expression. Relative expression was determined and rated from 0-6 (0 = no expression; 6 = strongest expression). (D) Distant metastases-free survival (DMFS) of breast cancer patients with high or low expression of PIP5K1C over time. The analysis was performed with the Kaplan-Meier Plotter (http://kmplot.com/analysis/index.php?p=service&cancer=breast) using standard settings. Patient samples (n=1746) were split by median, the follow up threshold was set 10.
Figure 2
Figure 2. Phosphorylation of PIP5K1C at S448 is decreased in invasive ductal carcinoma of the breast
(A) Antibody specificity control. Immunohistochemical staining of normal breast tissue. Samples were stained for pS448-PIP5K1C alone or in the presence of blocking phospho-peptides (at 1:100) to demonstrate antibody specificity for this serine phosphorylation site. The bar indicates 100 μm. (B) Indicated groups of samples were immunohistochemically-stained for pS448-PIP5K1C. Relative expression was determined and rated from 0-6 (0 = no expression; 6 = strongest expression). The asterisk indicates statistical significance n < 0.0001, when compared to normal tissue. (C) Representative pictures of pS448-PIP5K1C expression in normal/benign breast tissue, DCIS, ILC and different IDC subgroups. The bar indicates 100 μm. (D) Tissue microarrays with indicated groups of samples were immunohistochemically-stained for pS448-PIP5K1C. Relative expression was determined and rated from 0-6 (0 = no expression; 6 = strongest expression). The asterisk indicates statistical significance when compared to normal tissue; ns = not significant as compared to normal tissue.
Figure 3
Figure 3. Phosphorylation of PIP5K1C at S448 in BC cells is mediated by PKD1
(A) Indicated cell lines were analyzed by SDS-PAGE and immunoblotting for endogenous expression of PKD1 (anti-PKD1), PKD2 (anti-PKD2) or PKD3 (anti-PKD3). Immunoblotting with anti-β-actin served as a loading control. (B) Indicated cell lines were treated with DMSO control or PMA (100 nM) for 10 min. Cells were lysed, endogenous PIP5K1C was immunoprecipitated (anti-PIP5K1C), and immunoprecipitates were analyzed by SDS-PAGE and immunoblotting for phosphorylation of PIP5K1C at S448 (anti-pS448-PIP5K1C). Samples were re-probed for total PIP5K1C. (C) MCF-7 cells were transfected with tagged constitutively-active versions of PKD1, PKD2 or PKD3 together with HA-tagged PIP5K1C. Cells were lysed, overexpressed PIP5K1C was immunoprecipitated (anti-HA), and immunoprecipitates were analyzed by SDS-PAGE and immunoblotting for phosphorylation of PIP5K1C at S448 (anti-pS448-PIP5K1C). Samples were re-probed for total PIP5K1C by staining with anti-HA. In addition expression of active PKD isoforms was determined by Western blotting of lysates with TAG-specific antibodies as indicated.
Figure 4
Figure 4. The PKD1 expression status indicates invasive ductal carcinoma
(A) Relative expression of PRKD1 (PKD1) in breast cancer cell lines (n=51) grouped into basal or luminal subtypes. The analysis was performed using GOBO from Lund University (http://co.bmc.lu.se/gobo/). (B) Tissue microarrays with indicated groups of samples were immunohistochemically-stained for PKD1. Relative expression was determined and rated from 0-6 (0 = no expression; 6 = strongest expression). The asterisk indicates statistical significance when compared to normal tissue; ns = not significant as compared to normal tissue.
Figure 5
Figure 5. PKD1 regulates phosphorylation of PIP5K1C at S448 in orthotopic tumors in vivo
(A, B) Analyses of 5 week old primary tumors of MDA-MB-231 cells stably expressing vector control, wildtype PKD1 or a kinase-dead version (PKD1.KW) orthotopically-injected into the mfp of mice (experiment described in [29]). (A) Shown are IHC analyses of a representative tumor area for PKD1 (anti-PKD1 antibody) and for PIP5K1C phosphorylated at S448 (anti-pS448-PIP5K1C) as well as H&E staining. (B) Shown is an immunofluorescence-IHC analyses of a representative tumor area for co-occurrence of PKD1 expression (red; anti-PKD1 antibody) and PIP5K1C phosphorylated at S448 (green; anti-pS448-PIP5K1C) in tumor cells. In A and B the scale bar indicates 50 μm.
Figure 6
Figure 6. PKD1 expression status and phosphorylation of PIP5K1C at S448 correlate in patient samples of TNBC
(A) Relative expression of PKD1, pS448-PIP5K1C and PIP5K1C in patient samples. Shown are a representative normal sample and two different representative IDC patient samples stained by H&E or by IHC using anti-PKD1, anti-pS448-PIP5K1C and anti-PIP5K1C antibodies. (B) Relative expression of PKD1 and pS448-PIP5K1C in benign and TN IDC. The quantification analysis is described in Materials and Methods.

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