Fig 1: 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.
Fig 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.
Fig 3: 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.
Fig 4: 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.
Fig 5: 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-ß-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.
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