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Evaluation of quantification and normalization strategies for phosphoprotein phosphatase affinity proteomics: application to breast cancer signaling

ABSTRACT: Accurate quantification of proteomics data is essential for correctly characterizing signaling processes. We have recently developed a chemical proteomic strategy, phosphatase inhibitor beads and mass spectrometry (PIB-MS), to investigate endogenous phosphoprotein phosphatase (PPP) dephosphorylation signaling. Here, we compare the robustness and reproducibility of different quantification methods for optimal performance and ease of implementation. We then apply PIB-MS to an array of breast cancer (BC) cell lines to determine differences in PPP signaling between subtypes. BC is a leading cause of cancer death in women. There are three main subtypes: estrogen receptor-positive (ER+), human epidermal growth factor receptor two positive (HER2+), and triple-negative (TNBC). Furthermore, TNBC has few targeted therapies. Therefore, there is a need to identify novel means for treating breast cancers. Using PIB-MS, we distinguished between TNBC and Non-TNBC based on PPP holoenzyme composition. We identified an increase in PPP interactions with Hippo pathway proteins in TNBC. These interactions suggest that phosphatases in TNBC play an inhibitory role on the Hippo pathway and correlate with increased expression of YAP/TAZ target genes both in TNBC cell lines and in TNBC patients.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive Plus

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Arminja Kettenbach

PROVIDER: MSV000090020 | MassIVE | Fri Jul 29 17:54:00 BST 2022

SECONDARY ACCESSION(S): PXD035636

REPOSITORIES: MassIVE

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Project description:Triple negative breast cancer (TNBC) is a highly heterogeneous disease representing the most aggressive breast cancer (BC) subtype. Lack of Estrogen Receptor alpha (ERα), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2/neu) expression makes TNBC immune to common therapies, significantly limiting the treatment options and suggesting the need to identify novel therapeutic targets. It was previously reported that Estrogen Receptor beta (ERβ) is expressed in a fraction of TNBC patients, where its presence correlates with improved patient outcome. Recently, we demonstrated an oncosuppressive ERβ effect in TNBC cell models expressing exogenous ERβ. On the other hand, it was shown that ERβ is involved in miRNA-mediated gene regulation in hormone-responsive BC cells, suggesting similar effect also in TNBC. To verify this hypothesis, we performed small non-coding RNA (sncRNA) sequencing on three engineered cell lines belonging to different TNBC molecular subtypes. ERβ-specific changes of sncRNA profile revealed that the major part of deregulated molecules are subtype specific, with only few commonly regulated ones. In order to validate the obtained results, we performed sncRNA profiling of 12 ERβ positive and 32 ERβ negative TNBC tissues, whose receptor status was assessed by immunohistochemistry in our previous research. Also here, ERβ-specific group of deregulated sncRNAs was identified. Interestingly, comparison of obtained in vitro and in vivo results revealed 2 differentially expressed miRNAs, displaying the same behavior in all three analyzed cell lines and tissues. In concordance with our previous results, IPA signaling pathway analysis performed on genes targeted by deregulated miRNAs highlighted downregulation of cholesterol biosynthesis pathway and upregulation of several signaling processes. Taken together, these findings suggest that ERβ is able to exert its oncosuppressive role in TNBC through miRNA-mediated regulation of gene expression.

Project description:Breast cancer is a heterogeneous disease for which prognosis and treatment strategies are largely governed by the receptor status (estrogen, progesterone and Her2-neu) of the tumor cells. Gene expression profiling of whole breast tumors further stratifies breast cancer into several molecular subtypes which also co-segregate with the receptor status of the tumor cells. We postulated that cancer associated fibroblasts (CAFs) within the tumor stroma may exhibit subtype specific gene expression profiles and thus contribute to the biology of the disease in a subtype specific manner. Several studies have reported gene expression profile differences between CAFs and normal breast fibroblasts but in none of these studies were the results stratified based on tumor subtypes. To address whether gene expression in breast cancer associated fibroblasts varies between breast cancer subtypes, we compared the gene expression profiles of early passage primary CAFs isolated from twenty human breast cancer samples representing three main subtypes; seven ER+, seven triple negative (TNBC) and six Her2+. We observed significant expression differences between CAFs derived from Her2+ breast cancer and CAFs from TNBC and ER+ cancers, particularly in pathways associated with cytoskeleton and integrin signaling. In the case of Her2+ breast cancer, the signaling pathways found to be selectively up regulated in CAFs may contribute to the more invasive properties and unfavorable prognosis of Her2+ breast cancer. These data demonstrate that in addition to the distinct molecular profiles that characterize the neoplastic cells, CAF gene expression is also differentially regulated in distinct subtypes of breast cancer. We isolated CAFs from twenty primary breast cancer samples representing three main subtypes (ER+ (n=7), TNBC (n=7), Her2+ (n=6)) and performed gene expression profile analyses on RNA isolated from these early passage CAFs. Those samples were done in two batches with 4 samples repeated in both batches. One TNBC sample was found to be an outlier and not used in the analysis.

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Evaluation of quantification and normalization strategies for phosphoprotein phosphatase affinity proteomics: application to breast cancer signaling

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