ABSTRACT
Annexin-1 (ANXA1) is known to be involved in important cellular processes and implicated in cancer. Our previous study showed its roles in cell migration and DNA-damage response processes in breast cancer initiation. In order to understand its roles in tumorigenesis, we extended our studies to analyze tumors derived from polyomavirus middle T-antigen ANXA1 heterozygous (ANXA1(+/-) ) and ANXA1 null (ANXA1(-/-) ) mice. We performed quantitative comparison of ANXA1(+/-) and ANXA1(-/-) tumors employing reductive dimethyl labeling quantitative proteomics. We observed 253 differentially expressed proteins (DEPs) with high statistical significance among over 5000 quantified proteins. Combinatorial use of pathway and network-based computational analyses of the DEPs revealed that ANXA1 primarily modulates processes related to cytoskeletal remodeling and immune responses in these mammary tumors. Of particular note, ANXA1(-/-) tumor showed reduced expression of a known epithelial-to-mesenchymal transition (EMT) marker vimentin, as well as myosin light-chain kinase, which has been reported to induce Rho-kinase mediated assembly of stress fibers known to be implicated in EMT. Integrative network analysis of established interactome of ANXA1 alongside with DEPs further highlights the involvement of ANXA1 in EMT. Functional role of ANXA1 in tumorigenesis was established in invasion assay where knocking down ANXA1 in murine mammary tumor cell line 168FARN showed lower invasive capability. Altogether, this study emphasizes that ANXA1 plays modulating roles contributing to invasion-metastasis in mammary tumorigenesis, distinctive to its roles in cancer initiation.
Subject(s)
Annexin A1/metabolism , Carcinogenesis/metabolism , Mammary Glands, Animal/pathology , Mammary Neoplasms, Animal/metabolism , Mammary Neoplasms, Animal/pathology , Protein Interaction Maps , Animals , Annexin A1/genetics , Carcinogenesis/genetics , Carcinogenesis/pathology , Epithelial-Mesenchymal Transition , Female , Gene Knockdown Techniques , Mammary Glands, Animal/metabolism , Mammary Neoplasms, Animal/genetics , Mass Spectrometry , Mice , Mice, Inbred BALB C , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , ProteomicsABSTRACT
The core LATS kinases of the Hippo tumor suppressor pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Recent studies have identified various E3 ubiquitin ligases that negatively regulate the Hippo pathway via ubiquitination, yet few deubiquitinating enzymes (DUB) have been implicated. In this study, we report the DUB USP9X is an important regulator of the core kinases of this pathway. USP9X interacted strongly with LATS kinase and to a lesser extent with WW45, KIBRA, and Angiomotin, and LATS co-migrated exclusively with USP9X during gel filtration chromatography analysis. Knockdown of USP9X significantly downregulated and destabilized LATS and resulted in enhanced nuclear translocation of YAP and TAZ, accompanied with activation of their target genes. In the absence of USP9X, cells exhibited an epithelial-to-mesenchymal transition phenotype, acquired anchorage-independent growth in soft agar, and led to enlarged, disorganized, three-dimensional acini. YAP/TAZ target gene activation in response to USP9X knockdown was suppressed by knockdown of YAP, TAZ, and TEAD2. Deletion of USP9X in mouse embryonic fibroblasts resulted in significant downregulation of LATS. Furthermore, USP9X protein expression correlated positively with LATS but negatively with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. Overall, these results strongly indicate that USP9X potentiates LATS kinase to suppress tumor growth. Cancer Res; 77(18); 4921-33. ©2017 AACR.
Subject(s)
Breast Neoplasms/pathology , Endopeptidases/physiology , Pancreatic Neoplasms/pathology , Protein Serine-Threonine Kinases/metabolism , Ubiquitin Thiolesterase/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Angiomotins , Animals , Apoptosis , Biomarkers, Tumor , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Proliferation , Cells, Cultured , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Follow-Up Studies , Hippo Signaling Pathway , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Microfilament Proteins , Neoplasm Staging , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation , Prognosis , Protein Serine-Threonine Kinases/genetics , Signal Transduction , Survival Rate , Transcription Factors/genetics , Transcription Factors/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitination , YAP-Signaling ProteinsABSTRACT
Despite efforts in the last decade, signaling aberrations associated with obesity remain poorly understood. To dissect molecular mechanisms that define this complex metabolic disorder, we carried out global phosphoproteomic analysis of white adipose tissue (WAT) from mice fed on low-fat diet (LFD) and high-fat diet (HFD). We quantified phosphorylation levels on 7696 peptides, and found significant differential phosphorylation levels in 282 phosphosites from 191 proteins, including various insulin-responsive proteins and metabolic enzymes involved in lipid homeostasis in response to high-fat feeding. Kinase-substrate prediction and integrated network analysis of the altered phosphoproteins revealed underlying signaling modulations during HFD-induced obesity, and suggested deregulation of lipogenic and lipolytic pathways. Mutation of the differentially-regulated novel phosphosite on cytoplasmic acetyl-coA forming enzyme ACSS2 (S263A) upon HFD-induced obesity led to accumulation of serum triglycerides and reduced insulin-responsive AKT phosphorylation as compared to wild type ACSS2, thus highlighting its role in obesity. Altogether, our study presents a comprehensive map of adipose tissue phosphoproteome in obesity and reveals many previously unknown candidate phosphorylation sites for future functional investigation.