Protein Kinase D drives the secretion of invasion mediators in triple negative breast cancer cell lines
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ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture
SUBMITTER: 
Thang Pham
LAB HEAD: Connie Jimenez
PROVIDER: PXD041042 | Pride | 2024-01-17
REPOSITORIES: Pride
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Project description:The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope Labelling of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3,505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription and translation. In other cell types PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages.
2014-10-03 | PXD001076 | Pride
Project description:Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in the ADPKD genes PKD1 and PKD2 account for nearly all the cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1, the product of PKD1, regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-1, polycystin-2 can acts as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2. In this study we utilized different kidney cell lines expressing wild-type and mutant PKD2 as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation. Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 activity and consequently had no effect on cell proliferation. On the other hand, expression of mutated PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of STAT-1/p21 pathway. Genome-wide expression analysis in these cells revealed that expression of the cyclin-dependent kinase inhibitor, p57 is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels. Our results indicate the probable involvement of p57 on epithelial cell proliferation in polycystic kidney disease. In addition, it confirms that PKD2-induced proliferation is a multifactorial process. We used microarrays to study the modulation of gene expression by mutated PKD2 in the primary tubular epithelial cells of a transgenic rat mode Keywords: transgene Gene expression in the primary tubular epithelial of a transgenic rat mode with mutated PKD2 was compared with that of SD control rats
2010-05-19 | E-GEOD-11500 | biostudies-arrayexpress
Project description:Oral squamous cell carcinoma (OSCC) is a main reason of oral cancer mortality and morbidity. Cancer of oral cavity in central south Asia, ranks among third most common kinds of cancer. The discovery of candidate markers to differentiate normal from malignant cells in clinical diagnosis of OSCC would be of critical importance because this malignancy has poor prognosis. To improve the clinical outcome in OSCC patients, the present study was aimed at identifying robust candidate biomarkers for early OSCC diagnosis and to enhance understanding of the mechanisms of disease progression and pathogenesis. Of particular interest are proteins that can be found in tissue lysates of OSCC tumor vs normal adjacent mucosa samples and secreted in cell line Secretomes of HNSCC for non-invasive detection. We analysed 17 paired human malignant OSCC tissues and normal adjacent tissue in addition to secretomes of 9 HNSCC cell lines. The proteome dataset of OSCC and normal tissues consisted of 5,123 protein groups, including 299 proteins with strong differential expression (p-value <0.01, fold change barrier to ˃+2 and <-2, 205 upregulated and 94 down regulated) and 134 common proteins were also found out of total dataset of 4473 identified proteins of HNSCC cell line secretomes. Functional data analysis revealed that these differential proteins were significantly associated with multiple biological processes. Myogenesis, Fatty Acid Metabolism and KRAS Signaling DN were associated with the proteins downregulated in cancer tissues, while Protein Secretion, Unfolded Protein Response, Spliceosomal complex assembly, Protein localization to endosome and Interferon Gamma Response were enriched in the set of upregulated proteins and these regulated proteins may be classically or non-classically secreted. Furthermore, we found differential enrichment of Creb3L1, ESRRA, YY, ELF2, STAT1 and XBP transcription factors potentially regulating these major pathways.
2022-04-04 | PXD025701 | Pride
Project description:Most patients with triple negative breast cancer (TNBC) fail to respond to anti-PD1/PDL1 immunotherapy, indicating the necessity to explore immune checkpoint targets. B7H3 is a highly glycosylated protein. However, the mechanisms of B7H3 glycosylation regulation and whether the sugar moiety contributes to immunosuppression remain elusive. Here, we identify aberrant B7H3 glycosylation and found N-glycosylation of B7H3 at NXT motif sites are responsible for its protein stability and immunosuppression in TNBC tumors. Mechanistically, fucosyltransferase FUT8 catalyzes B7H3 core fucosylation at N-glycans to maintain its high expression. Knockdown of FUT8 rescues glycosylated B7H3-mediated immunosuppressive function in TNBC cells. Abnormal B7H3 glycosylation mediated by FUT8 overexpression could be physiologically significant and clinically relevant in TNBC patients. Notably, combination of core fucosylation inhibitor 2F-Fuc and anti-PDL1 results in enhanced therapeutic efficacy in B7H3-positive TNBC tumors. These suggest targeting FUT8-B7H3 axis can be a promising strategy for improving anti-tumor immune responses in TNBC patients. To obtain the direct evidence that B7H3 is N-glycosylated in TNBC cells, we analysed the peptides of purified human B7H3 protein from B7H3-WT re-expressed and B7H3-8NQ re-expressed MDA-MB-231 cell lines by Nanoscale liquid chromatography coupled to tandem MS (nano LC-MS/MS). The result showed that there were eight N-glycosylation sites (Asn positions 91, 104, 189, 215, 309, 322, 407, and 433) in B7H3-WT cells, but not in B7H3-8NQ cells, as determined by Asn to Asp conversion after PNGase F treatment. As B7H3 contains a nearly exact tandem duplication of the IgV-IgC domain, there were four pairs of N-glycosylation sites identified through identical peptide sequence, including N91 and N309, N104 and N322, N189 and N407, and N215 and N433, in each of the IgV-IgC domains. Together, the results indicate that B7H3 is exclusively N-glycosylated at these four pairs of glycosylation sites in TNBC cells.
2021-03-15 | PXD024672 | Pride
Project description:Background: Autosomal dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide. Mutations in PKD1 and PKD2 cause cyst formation through unknown mechanisms. To unravel the pathogenic mechanisms in ADPKD, multiple studies have investigated transcriptional mis-regulation in cystic kidneys from patients and mouse models, and numerous dysregulated genes and pathways have been described. Yet, the concordance between studies has been rather limited. Furthermore, the cellular and genetic diversity in cystic kidneys has hampered the identification of mis-expressed genes in kidney epithelial cells with homozygous PKD mutations, which are critical to identify polycystin-dependent pathways. Methods: Autosomal dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide. Mutations in PKD1 and PKD2 cause cyst formation through unknown mechanisms. To unravel the pathogenic mechanisms in ADPKD, multiple studies have investigated transcriptional mis-regulation in cystic kidneys from patients and mouse models, and numerous dysregulated genes and pathways have been described. Yet, the concordance between studies has been rather limited. Furthermore, the cellular and genetic diversity in cystic kidneys has hampered the identification of mis-expressed genes in kidney epithelial cells with homozygous PKD mutations, which are critical to identify polycystin-dependent pathways. Results: Autosomal dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide. Mutations in PKD1 and PKD2 cause cyst formation through unknown mechanisms. To unravel the pathogenic mechanisms in ADPKD, multiple studies have investigated transcriptional mis-regulation in cystic kidneys from patients and mouse models, and numerous dysregulated genes and pathways have been described. Yet, the concordance between studies has been rather limited. Furthermore, the cellular and genetic diversity in cystic kidneys has hampered the identification of mis-expressed genes in kidney epithelial cells with homozygous PKD mutations, which are critical to identify polycystin-dependent pathways. Conclusions: Autosomal dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide. Mutations in PKD1 and PKD2 cause cyst formation through unknown mechanisms. To unravel the pathogenic mechanisms in ADPKD, multiple studies have investigated transcriptional mis-regulation in cystic kidneys from patients and mouse models, and numerous dysregulated genes and pathways have been described. Yet, the concordance between studies has been rather limited. Furthermore, the cellular and genetic diversity in cystic kidneys has hampered the identification of mis-expressed genes in kidney epithelial cells with homozygous PKD mutations, which are critical to identify polycystin-dependent pathways.
2021-07-15 | GSE179947 | GEO
Project description:Based on the dosage effect hypothesis, renal cysts could arise in transgenic murine models overexpressing either PKD1 or PKD2, which are causal genes responsible for autosomal dominant polycystic kidney disease (ADPKD). To prove whether PKD genes overexpression is a universal mechanism driving cystogenesis or is merely restricted to rodents, other animal models are required. Previously, we failed to observe any renal cysts in a PKD2 overexpression transgenic pig model partially due to epigenetic silencing of transgene. Thus, to explore the feasibility of pig models and identification potential affected genes/pathways related to ADPKD, LLC-PK1 cells with high PKD2 expression were generated. RNA-seq was performed and MYC, IER3, ADM were found to be commonly upregulated genes in different PKD2 overexpression cell models. MYC is a well-characterized factor contributing to cystogenesis, and ADM is a biomarker for chronic kidney disease. Thus, theses genes might be indicators of disease progression. Additionally, some ADPKD associated pathways, e.g., MAPK, were also enriched in the cells. Besides, GO analysis demonstrated proliferation, apoptosis, cell cycle regulation, which are hall marks of ADPKD were disturbed. Therefore, our experiment not only identified some biomarkers or indictors regarding ADPKD, but also demonstrated high PKD2 expression would like to drive cystogenesis in future porcine models.
2019-12-04 | GSE141355 | GEO
Project description:Polycystic Kidney Disease (PKD) is a genetic disease of the kidney characterized by the gradual replacement of normal kidney parenchyma by fluid-filled cysts and fibrotic tissue. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is caused by mutations in the PKD1 or PKD2 gene. Here we present an RNASeq experiment designed to investigate the effect of a kidney specific and Tamoxifen inducible knockout of the Pkd1 gene in mice. The Pkd1cko mice were harvested at different time points 2-weeks, 3-weeks, 5-weeks, 10.5-weeks, 11-weeks and 15-weeks after gene inactivation.
2019-12-24 | E-MTAB-8086 | biostudies-arrayexpress
Project description:To provide preliminary insights into metabolic and lipidomic characteristics in radioresistant triple-negative breast cancer (TNBC) cells and suggest potential therapeutic targets, we performed a comprehensive metabolic and lipidomic profiling of radioresistant MDA-MB-231 (MDA-MB-231/RR) TNBC cells and their parental cells using gas chromatography-mass spectrometry and nano electrospray ionization-mass spectrometry, followed by multivariate statistical analysis. Buthionine sulfoximine (BSO) and radiation were co-treated to radioresistant TNBC cells. The level of glutathione (GSH) was significantly increased, and the levels of GSH synthesis-related metabolites, such as cysteine, glycine, and glutamine were also increased in MDA-MB-231/RR cells. In contrast, the level of lactic acid was significantly reduced. In addition, reactive oxygen species (ROS) level was decreased in MDA-MB-231/RR cells. In the lipidomic profiles of MDA-MB-231/RR cells, the levels of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were significantly increased, whereas those of most of the phosphatidylinositol species were significantly decreased. BSO sensitized MDA-MB-231/RR cells to radiotherapy, which resulted in decreased GSH level and increased ROS level and apoptosis. Radioresistant TNBC cells showed distinct metabolic and lipidomic characteristics compared to their parental cells. We suggested activated GSH, PC, and PE biosynthesis pathways as potential targets for treating radioresistant TNBC cells. Particularly, enhanced radiosensitivity was achieved by inhibition of GSH biosynthesis in MDA-MB-231/RR cells.
2021-09-22 | ST001920 | MetabolomicsWorkbench
Project description:Polycystic Kidney Disease is characterized by the formation of large fluid-filled cysts that eventually destroy the renal parenchyma leading to end-stage renal failure. Although remarkable progress has been made in understanding the pathologic mechanism of the disease, the precise orchestration of the early events leading to cyst formation is still unclear. Abnormal cellular proliferation was traditionally considered to be one of the primary irregularities leading to cyst initiation and growth. Consequently, many therapeutic interventions have focused on targeting this abnormal proliferation, and some have even progressed to clinical trials. However, the role of proliferation in cyst development was primarily examined at stages where cysts are already visible in the kidneys and therefore at later stages of disease development. In this study we focused on the cystic phenotype since birth in an attempt to clarify the temporal contribution of cellular proliferation in cyst development. Using a PKD2 transgenic rat model (PKD2 (1-703)) of different ages (0-60 days after birth) we performed gene expression profiling and phenotype analysis by measuring various kidney parameters. Phenotype analysis demonstrated that renal cysts appear immediately after birth in the PKD2 transgenic rat model (PKD2 (1-703)). On the other hand, abnormal proliferation occurs at later stages of the disease as identified by gene expression profiling. Interestingly, other pathways appear to be deregulated at early stages of the disease in this PKD model. Our data suggest that cystogenesis precedes deregulation of proliferation-related pathways, suggesting that proliferation abnormalities may contribute in cyst growth rather than cyst formation. In this study we focused on the cystic phenotype since birth in an attempt to clarify the temporal contribution of cellular proliferation in cyst development. Using a PKD2 transgenic rat model (PKD2 (1-703)) of different ages (0,6 and 24 days after birth) we performed gene expression profiling of kidney cells.
2010-01-31 | E-GEOD-19460 | biostudies-arrayexpress
Project description:Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in the ADPKD genes PKD1 and PKD2 account for nearly all the cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1, the product of PKD1, regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-1, polycystin-2 can acts as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2. In this study we utilized different kidney cell lines expressing wild-type and mutant PKD2 as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation. Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 activity and consequently had no effect on cell proliferation. On the other hand, expression of mutated PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of STAT-1/p21 pathway. Genome-wide expression analysis in these cells revealed that expression of the cyclin-dependent kinase inhibitor, p57 is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels. Our results indicate the probable involvement of p57 on epithelial cell proliferation in polycystic kidney disease. In addition, it confirms that PKD2-induced proliferation is a multifactorial process. We used microarrays to study the modulation of gene expression by mutated PKD2 in the primary tubular epithelial cells of a transgenic rat mode Keywords: transgene
2009-04-30 | GSE11500 | GEO