Breast cancer (BC) is most prevalent in females but also accounts for <1% of male cancer cases and 0.2% of male cancer-related deaths. Distribution of histological subtypes, receptor status, and age of diagnosis varies based on sex, and a growing body of evidence supports sex-specific molecular differences in BC. However, this is limited by the smaller number of male cases available for study compared to the thousands of cases of female BC. We combined publicly available male BC gene expression datasets for 195 patients from 4 studies and split randomly into discovery and validation sets. Clustering and gene expression analysis were performed. Two stable clusters were identified initially, confirmed in the validation set. Cluster C1 was enriched for genes associated with MAPK signalling and arylesterase activity. Cluster C2 showed enrichment of genes associated with proliferation, invasion, and metastasis, along with enrichment of gene ontology and pathway terms related to ECM regulation, particularly collagen-containing ECM. Of note, when stratified by ERα and PR status, no enrichment was observed with the predicted PAM50 classification. ERα and MAPK signalling were enriched in both clusters, albeit through different gene sets. Since these pathways were enriched, we investigated the signalling regulation of ERα based on immunohistochemical expression of phosphorylated ERα (S104, S118, S167, S294) and their prognostic roles. This analysis also revealed distinctions from female BC, showing a lack of prognostic outcome for any of these biomarkers. We show that male BC does not align with female BC in the same way that intrinsic subtypes of female BC are not identical. As BC heterogeneity is well recognised, we propose that male BC should be considered as a potentially unique clinical subtype of BC.

Androstenedione driven gene changes post AI therapy (+ months)

Supplementary Figures S1-S4 from Trastuzumab and Pertuzumab Produce Changes in Morphology and Estrogen Receptor Signaling in Ovarian Cancer Xenografts Revealing New Treatment Strategies

Detailed description of the materials and methods

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.

Quicktime file - 7.8MB, Movies of cell behavior

<p>Figure S3 shows generation of Kindlin-1 deficient Met-1 cells</p>

Figure S4 shows supporting data that Kindlin-1 reduces integrin activation and lung colonization

TGFβ and ERα transcriptional activity.

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 .

<p>Figure S5 provides supportiung evidence that tumor cell adhesion to VCAM-1 and endothelial cells is integrin alpha4 dependent</p>

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.

Fulvestrant response in MCF7 with TGFBR2-specific knockdown.

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).

<p>Table S1 provides annotation of the Kindlin-1 dependent secretome</p>

Multivariate analysis of recurrence-free survival regarding prognostic information of SMAD2 phosphorylation.

Table S1. Cell line authentication. Cell line DNA profiling was carried out using ATCC MCF7 DNA profile as reference. Table S2: List of ChIP and mRNA primers used for DNA and mRNA quantification on the Lightcycler (Roche). Table S3: Patient information for endocrine treated patients. Table S4. Over represented TF binding motifs in 666 ER ChIPseq peaks from LetR cells (using the Universe as background, q-value<0.05) Table S5. Enrichment of TF motifs in Veh, Andro peaks separately. Table S6. Associations of EGR3 with patient clinicopathological variables using Fisher's exact test.

High expression of the transmembrane receptor IL6ST (gp130) has been identified as a predictive biomarker of endocrine treatment response in ERα-positive breast cancers. To investigate its function further in this disease, this study evaluated the expression, function and signalling of IL6ST in ERα-positive breast cancer cell lines and investigated crosstalk between ERα and IL6ST. IL6ST was differentially expressed in ERα-positive breast cancer cell lines (low in MCF-7, high in ZR751 and T47D), while multiple soluble isoforms of IL6ST were identified. IL6ST is the common signal transducing receptor component for the IL6ST family of cytokines and the effects of seven IL6ST cytokines on these cell lines were studied. These cytokines caused differential growth and migration effects in these cell lines e.g. MCF-7 cells were growth-stimulated, while ZR751 cells were inhibited by IL6 and OSM.. Activation of the STAT and ERK pathways is associated with these responses. Evidence to support trans-signalling involved in cell growth and migration was obtained in both MCF-7 and ZR751 models. Interaction between cytokines and estrogen on ERα-positive cell lines growth were analysed. High expression of IL6ST (in ZR751) may lead to growth inhibition by interacting cytokines while lower expression (in MCF-7) appears associated with proliferation. High IL6ST expression is consistent with a more beneficial clinical outcome if cytokine action contributes to anti-estrogen action.

Breast cancer stem cells (BCSC) are presumed to be responsible for treatment resistance, tumor recurrence and metastasis of breast tumors. However, development of BCSC-targeting therapies has been held back by their heterogeneity and the lack of BCSC-selective molecular targets. Here, we demonstrate that RAC1B, the only known alternatively spliced variant of the small GTPase RAC1, is expressed in a subset of BCSCs in vivo and its function is required for the maintenance of BCSCs and their chemoresistance to doxorubicin. In human breast cancer cell line MCF7, RAC1B is required for BCSC plasticity and chemoresistance to doxorubicin in vitro and for tumor-initiating abilities in vivo. Unlike Rac1, Rac1b function is dispensable for normal mammary gland development and mammary epithelial stem cell (MaSC) activity. In contrast, loss of Rac1b function in a mouse model of breast cancer hampers the BCSC activity and increases their chemosensitivity to doxorubicin treatment. Collectively, our data suggest that RAC1B is a clinically relevant molecular target for the development of BCSC-targeting therapies that may improve the effectiveness of doxorubicin-mediated chemotherapy.

Multivariate analysis of recurrence-free survival regarding prognostic information of TGFBR2.