Dataset Information

Loss of HIF1A From Pancreatic Cancer Cells Increases Expression of PPP1R1B and Degradation of p53 to Promote Invasion and Metastasis

ABSTRACT: Here we utilized a mouse model of PDAC and human pancreatic cancer cell lines to investigate the role of HIF1alpha in pancreatic cancer. Contrary to the conventional notion that HIF1alpha plays a tumor promoting role in several cancers, our findings indicate a tumor suppressive role of HIF1alpha in PDAC. Ablation of Hif1alpha in mice resulted in drastically increased metastasis of the pancreatic cancer and substantially reduced survival. We traced HIF1alpha's metastasis-suppression to the regulation of Protein Phosphatase 1 Regulatory Inhibitor subunit 1B (PPP1R1B), a dual kinase/phosphatase inhibited by HIF1alpha. We show a mechanistic link between loss of HIF1alpha, increased expression of PPP1R1B and degradation of the p53 protein that promotes invasion and metastasis in both mouse and human PDACs. Increased expression of PPP1R1B correlates with higher metastasis and poor survival in human pancreatic cancer patients. Since the inhibition of PPP1R1B reduced the metastatic potential of pancreatic cancer cells, we propose PPP1R1B as a therapeutic target to improve the prognosis of pancreatic cancer patients. _____ LC-MS/MS methodology _____ Dried peptide samples were reconstituted with 2% ACN-0.1% FA and quantified by NanoDropTM spectrophometer (ThermoFisher) prior to LC-MS/MS analysis using a nanoACQUITY system (Waters) coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Peptides were separated using a 200 cm micro-chip pillar array C18 column (PharmaFluidics) at a flow rate of 500 nl/min using a 123-min gradient: 1% to 5% B in 1 min, 5% to 10% B in 22 min, 10% to 18% B in 53 min, 18% to 28% B in 39 min, and 28% to 38% B in 8 min (A= FA 0.1%; B=100% ACN: 0.1% FA). The mass spectrometer was operated in positive data-dependent acquisition mode. MS1 spectra were measured in the Orbitrap with a resolution of 60000 (AGC target: 4e5; maximum injection time:50 ms; mass range: from 375 to 1500 m/z). The instrument was set to run in top speed mode with 2 s cycles for the survey and the MS/MS scans. After a survey scan, the most abundant precursors (with charge state between +2 and +7) were isolated in the quadrupole (isolation window: 1.6 m/z) and fragmented in the Ion Routing Multipole HCD-Cell (collision energy: 30%). fragmented precursors were detected in the ion trap cell with a rapid scan (First mass: 110 m/z; AGC target for MS/MS: 1e4; maximum injection time: 35 ms). The dynamic exclusion was set to 60 s with a 10 ppm mass tolerance around the precursor.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Anindya Bagchi

PROVIDER: MSV000084223 | MassIVE | Wed Aug 21 22:48:00 BST 2019

SECONDARY ACCESSION(S): PXD015120

REPOSITORIES: MassIVE

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Publications

Loss of HIF1A From Pancreatic Cancer Cells Increases Expression of PPP1R1B and Degradation of p53 to Promote Invasion and Metastasis.

Tiwari Ashutosh A Tashiro Kojiro K Dixit Ajay A Soni Aditi A Vogel Keianna K Hall Bryan B Shafqat Iram I Slaughter Joseph J Param Nesteen N Le An A Saunders Emily E Paithane Utkarsha U Garcia Guillermina G Campos Alexandre Rosa AR Zettervall Jon J Carlson Marjorie M Starr Timothy K TK Marahrens York Y Deshpande Aniruddha J AJ Commisso Cosimo C Provenzano Paolo P PP Bagchi Anindya A

Gastroenterology 20200805 5

<h4>Background & aims</h4>Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice.<h4>Methods</h4>We performed studies with Kras<sup>LSL-G12D/+</sup>;Trp53<sup>LSL-R172H/+</sup>;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with ...[more]

PMID: 32768595

Similar Datasets

Project description:Pancreatic ductal adenocarcinoma (PDAC) is a heterogeneous cancer in which differences in survival rates might be related to a variety in gene expression profiles. Although the molecular biology of PDAC begins to be revealed, genes or pathways that specifically drive tumour progression or metastasis are not well understood. Therefore, we performed microarray analyses on whole-tumour samples of 2 human PDAC subpopulations with similar clinicopathological features, but extremely distinct survival rates after potentially curative surgery, i.e., good outcome (OS and DFSM-bM-^@M-^I>M-bM-^@M-^I50M-bM-^@M-^Imonths) versus bad outcome (OSM-bM-^@M-^I<M-bM-^@M-^I19M-bM-^@M-^Imonths and DFSM-bM-^@M-^I<M-bM-^@M-^I7M-bM-^@M-^Imonths). Additionally, liver- and peritoneal metastases were analysed and compared to primary cancer tissue. The integrin and ephrin receptor families were upregulated in all PDAC samples, irrespective of outcome, supporting an important role of the interaction between pancreatic cancer cells and the surrounding desmoplastic reaction in tumorigenesis and cancer progression. Moreover, some components, such as ITGB1 and EPHA2, were upregulated in PDAC samples with a poor outcome, Additionally, overexpression of the non-canonical Wnt/M-NM-2-catenin pathway and EMT genes in PDAC samples with bad versus good outcome suggests their contribution to the invasiveness of pancreatic cancer, with M-NM-2-catenin being also highly upregulated in metastatic tissue. Thus, we conclude that components of the integrin and ephrin pathways and EMT-related genes might serve as molecular markers in pancreatic cancer as their expression seems to be related with prognosis. Microarray analysis was performed on 'Good' and 'Bad' patient samples (samples with similar pathological characteristics were chosen based on the definition of the 2 diverse survival outcome groups and the required RIN values above 7.1), on surrounding non-tumoural pancreatic control samples, liver metastasis (LM) and peritoneal metastasis (PM). Comparisons between good vs. control, bad vs. control, good vs. bad, and primary pancreatic cancer versus metastasis were performed.

Project description:Pancreatic ductal adenocarcinoma (PDAC) is known by its aggressiveness and lack of effective therapeutic options. Thus, improvement in current knowledge of molecular changes associated with pancreatic cancer is urgently needed to explore novel venues of diagnostics and treatment of this dismal disease. While there is mounting evidence that long noncoding RNAs (ncRNAs) transcribed from intronic and intergenic regions of the human genome may play different roles in the regulation of gene expression in normal and cancer cells, their expression pattern and biological relevance in pancreatic cancer is currently unknown. In the present work we investigated the relative abundance of a collection of ncRNAs in patients’ pancreatic tissue samples aiming at identifying gene expression profiles correlated to pancreatic cancer and metastasis. To investigate the relative abundance, we used a custom 3,355-element spotted cDNA microarray interrogating protein-coding genes and putative ncRNA were used to obtain expression profiles from 38 clinical samples of tumor and non-tumor pancreatic tissues. Bioinformatics analyses were performed to characterize structure and conservation of ncRNAs expressed in pancreatic tissues, as well as to identify expression signatures correlated to tissue histology. Strand-specific reverse transcription followed by PCR and qRT-PCR were employed to determine strandedness of ncRNAs and to validate microarray results, respectively. We found that long intronic/intergenic ncRNAs are ubiquitously expressed across tumor and non-tumor pancreatic tissue samples. Enrichment of promoter-H3K4me3 associated chromatin marks and over-representation of conserved DNA elements and stable secondary structure predictions suggest that these transcripts are generated from independent transcriptional units and that at least a fraction is under evolutionary selection, and thus potentially functional. Statistically significant expression signatures comprising protein-coding mRNAs and long ncRNAs that correlate to PDAC or to pancreatic cancer metastasis were identified. Interestingly, loci harboring intronic ncRNAs differentially expressed in PDAC metastases were enriched in genes associated to the MAPK pathway. Orientation-specific RT-PCR documented that intronic transcripts are expressed in sense, antisense or both orientations relative to protein-coding mRNAs. Differential expression of a subset of intronic ncRNAs (PPP3CB, MAP3K14 and DAPK1 loci) in metastatic samples was confirmed by Real-Time PCR. All this results reveal sets of long intronic ncRNAs expressed in pancreatic tissues whose abundance is correlated to PDAC or metastasis, thus pointing to the potential relevance of this class of transcripts in biological processes related to malignant transformation and metastasis in pancreatic cancer. Fluorescent labeled cDNA targets derived from pancreatic tumor and non-tumor tissue from each patient were combined and hybridized to spotted cDNA microarrays. For each patient, a replicate hybridization was performed. For each sample, there are 4 replicate measurements for each probe.

Project description:Background Pancreatic ductal adenocarcinoma (PDAC) tends to undergo distant metastasis, especially liver metastasis, leading to a poor prognosis. Metabolic remodelling and epigenetic reprogramming are two important hallmarks of malignant tumours and participate in regulating PDAC tumorigenesis and metastasis. However, the interaction between these two processes during PDAC metastasis has not been fully elucidated. Methods We performed metabolomics analysis to identify the critical metabolites associated with PDAC liver metastasis and focused on guanidinoacetic acid (GAA). Intracellular GAA content was significantly increased in liver metastatic PDAC cells compared to primary cancer cells in mouse xenograft tumour models. The effects of GAA supplementation and glycine amidinotransferase (GATM) knockdown on PDAC metastasis were assessed by analysing cell migration, filopodia formation, epithelial-mesenchymal transition (EMT), and in vivo metastasis in different cell and animal models. Next, ChIP‒qPCR, 3C‒qPCR, and CRISPRi/dCas9-KRAB experiments were used to validate the “epigenome-metabolome" mechanism. Finally, the results of in vitro approaches, including RNA-seq, CUT&RUN, RT‒qPCR, and western blot analyses, as well as luciferase reporter gene assay and transwell assay, revealed the GAA-c-Myc-HMGA axis and transcription-activating histone modifications reprogramming. Results A high level of intracellular GAA was associated with PDAC liver metastasis. GAA could promote the migration, EMT, and liver metastasis of pancreatic cancer cells in vitro and in vivo. Next, we explored the role of GATM-mediated de novo GAA synthesis in pancreatic cancer metastasis. High expression of GATM was positively correlated with advanced N stage in PDAC. Knockdown of GATM significantly reduced the intracellular level of GAA, suppressed EMT, and inhibited PDAC liver metastasis, and these effects were attenuated by GAA supplementation. Mechanistically, we identified the active enhancers looped to the GATM gene locus that promoted GATM expression and PDAC liver metastasis. Furthermore, we found that GAA promoted cell migration and EMT by regulating c-Myc-mediated high mobility group AT-hook protein expression. Moreover, GAA increased the H3K4me3 modification level by upregulating histone methyltransferases, which induced the transcription of metastasis-related genes, including MYC. Conclusions These findings revealed the critical role of the epigenome-metabolome interaction in regulating PDAC liver metastasis and suggested potential therapeutic strategies targeting GAA metabolism and epigenetic regulatory mechanisms.