Transcriptional regulation of SLIT2 expression in pancreatic cancer cell lines

AbstractBackgroundSLIT2 has been shown to serve as a tumor suppressor in breast, lung, colon, and liver cancers. Additionally, expression of SLIT2 has been shown to be epigenetically regulated in prostate cancer. Therefore, we sought to determine transcriptional regulation of SLIT2 in pancreatic ductal adenocarcinoma.MethodsRNA expression of SLIT2, SLIT3, and ROBO1 was examined in a panel of pancreatic ductal adenocarcinoma cell lines while protein expression of ROBO1 and SLIT2 was examined in tumor tissue. Methylation of the SLIT2 promoter was determined using Sequenom while histone modifications were queried by chromatin immunoprecipitation. Reexpression of SLIT2 was tested by treatment with 5-aza-2’deoxycytidine and Trichostatin A.ResultsPancreatic cancer cell lines fall into three distinct groups based on SLIT2 and ROBO1 expression. The SLIT2 promoter is methylated in pancreatic ductal adenocarcinoma and SLIT2 expression is dependent on the level of methylation at specific CpG sites. Treatment with 5-aza-2’deoxycytidine (but not Trichostatin A) led to SLIT2 reexpression. The SLIT2 promoter is bivalent in pancreatic ductal adenocarcinoma and histone marks around the transcriptional start site are responsible for transcription.ConclusionsLoss of SLIT2 expression modulated by epigenetic silencing may play a role in pancreatic ductal adenocarcinoma progression.

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Inhibition of Sphingosine-1-Phosphate signaling in pancreatic ductal adenocarcinoma leads to downregulation of growth and invasion

10.1101/2020.09.29.318964 ◽

2020 ◽

Author(s):

Brenna A. Rheinheimer ◽

Alex Cardenas ◽

Luis Camacho ◽

Evan S. Ong ◽

Tun Jie ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cell Growth ◽

Pancreatic Ductal Adenocarcinoma ◽

Cell Lines ◽

Cancer Cell ◽

Pancreatic Cancer Cell ◽

Leading Edge ◽

Cancer Cell Lines ◽

Ductal Adenocarcinoma ◽

Sphingosine 1 Phosphate

AbstractBackgroundDeregulated phosphorylation of sphingosine by the sphingosine kinases and signaling through the EDG family of receptors enhances growth and survival in many cell types. Therefore, we sought to elucidate the effect of alterations in the ceramide/sphingosine/S1P rheostat on driving human pancreatic ductal adenocarcinoma towards a malignant phenotype.MethodsPancreatic cancer cell lines were treated with exogenous S1P, FTY720, and siRNA to Sphk1. Migration was evaluated by wound healing assays, cell growth by MTT assays, and invasion by tumorsphere assays. Expression of S1PR1, S1PR3, Sphk1, and Sphk2 were measured by quantitative PCR, western blot, and immunohistochemistry.ResultsS1PR1, S1PR3, and Sphk2 were overexpressed in all pancreatic cancer cell lines. Sphk1 translocated from the cytoplasm to the nucleus in cells located at the leading edge of cell clusters. Exogenous S1P increased cell migration while treatment with FTY720 and Sphk1 siRNA decreased cell growth and invasion.ConclusionsOur results suggest that increased S1PR1 expression may be an early event in pancreatic cancer pathogenesis. Additionally, altered Sphk1 localization may provide a mechanism through which pancreatic ductal adenocarcinoma cells at the leading edge invade into the surrounding matrix. Finally, inhibition of sphingosine-1-phosphate signaling may provide a novel therapeutic target for patients with metastatic disease.

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DNAJA1 dysregulates metabolism promoting an anti-apoptotic phenotype in pancreatic ductal adenocarcinoma

10.21203/rs.3.rs-76631/v1 ◽

2020 ◽

Author(s):

Heidi Roth ◽

Fatema Bhinderwala ◽

Rodrigo Franco ◽

You Zhou ◽

Robert Powers

Keyword(s):

Pancreatic Cancer ◽

Pancreatic Ductal Adenocarcinoma ◽

Cancer Cells ◽

Cell Lines ◽

Cancer Cell ◽

Pancreatic Cancer Cell ◽

Cancer Cell Lines ◽

Ductal Adenocarcinoma ◽

Pancreatic Cancer Cells ◽

The Impact

Abstract BackgroundAt less than 7%, pancreatic ductal adenocarcinoma (PDAC) has one of the poorest 5-year cancer survival rates and is set to be the leading cause of cancer related deaths by 2030. The co-chaperone protein DNAJA1 (HSP40) is downregulated four-fold in pancreatic cancer cells, but its impact on pancreatic ductal adenocarcinoma (PDAC) progression remains unclear.MethodsDNAJA1 was overexpressed in pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2, through retroviral transfection. The impact of overexpressing DNAJA1 was investigated using a combination of untargeted metabolomics, stable isotope resolved metabolomics (SIRM), confocal microscopy, flow-cytometry, and cell-based assays.ResultsPancreatic cancer cells overexpressing DNAJA1 exhibited a global metabolomic change. Specifically, differential output from Warburg glycolysis, an increase in redox currency, and an alteration in amino acid levels were observed in both overexpression cell lines. DNAJA1 overexpression also led to mitochondrial fusion, an increase in the expression of Bcl-2, a modest protection from redox induced cell death, a loss of structural integrity due to the loss of actin fibers, and an increase in cell invasiveness in BxPC-3. These differences were more pronounced in BxPC-3, which contains a loss-of-function mutation in the tumor suppressing gene SMAD4.ConclusionsThe overexpression of DNAJA1 promoted cellular proliferation, redox tolerance, invasiveness, and anti-apoptosis, which suggests DNAJA1 has numerous regulatory roles. Overall, our findings suggest a proto-oncogenic role of DNAJA1 in PDAC progression and suggests DNAJA1 may function synergistically with other proteins with altered activity in pancreatic cancer cell lines.

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Distribution of Characteristic Mutations in Native Ductal Adenocarcinoma of the Pancreas and Pancreatic Cancer Cell Lines

Cell Biology Research & Therapy ◽

10.4172/2324-9293.1000104 ◽

2013 ◽

Vol 02 (02) ◽

Author(s):

Saeger HD Rückert F

Keyword(s):

Pancreatic Cancer ◽

Cell Lines ◽

Cancer Cell ◽

Pancreatic Cancer Cell ◽

Cancer Cell Lines ◽

Ductal Adenocarcinoma ◽

Adenocarcinoma Of The Pancreas

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Synergistic killing in pancreatic cancer cell lines of the combination of a MEK inhibitor and DNA methylation inhibitor.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e15025 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e15025-e15025

Author(s):

Matlock Arizona Jeffries ◽

Carla Kurkjian

Keyword(s):

Pancreatic Cancer ◽

Dna Methylation ◽

Valproic Acid ◽

Cell Lines ◽

Cancer Cell ◽

Pancreatic Cancer Cell ◽

Trichostatin A ◽

Mek Inhibitor ◽

Cancer Cell Lines ◽

Synergistic Cytotoxicity

e15025 Background: A growing body of evidence implicates aberrant DNA methylation in the etiology of a number of cancers, including pancreatic. Previous reports link defective ERK signaling with reduced DNA methylation via DNMT1. Here, we examined the ability of the MEK inhibitor U0126 to alter expression of DNMTs in k-RAS mutant and wild type pancreatic cancer cell lines and assess additive cytotoxicity of U0126, the hypomethylating agent 5-Azacytidine and two HDAC inhibitors. Methods: DNMT levels were quantified in AsPC1 and BxPC3 lines 2 days after adding 25 umolar U0126 using real-time RT-PCR. Significance was calculated using a Student t-test. For survival, agents were added singly and in combination: U0126, 25 umol; 5-Aza, 10 umol; Valproic acid 1 mmol and Trichostatin-A 5 umol. Survival was assessed using a colorimetric MTS method at 1, 2, 3, and 4 days. Expected cell survival and synergism were calculated via relative risk ratios, and significance via a Student t-test. Results: U0126 treatment reduced expression of DNMT1 significantly in AsPC1: 2.06 ± 0.3 vs. 1.09 ± 0.08, p = 0.004 (mean ± S.D., arbitrary units). No change was seen in BxPC3. Increased expression of de novo DNMT3a and DNMT3b was seen: AsPC1 1.28 ± 0.3 vs. 3.13 ± 0.8, p = 0.02; BxPC3 1.78 ± 0.3 vs. 4.14 ± 1, p = 0.02; AsPC1 1.26 ± 0.3 vs. 2.66 ± 0.4, p = 0.006, respectively. DNMT3b in BxPC3 was not altered significantly. U0126 and 5-Aza were individually cytotoxic in both cell lines, and synergism was observed in combination. In AsPC1, the observed vs. expected combination survival was 13% ± 3% vs. 29% ± 2%, p = 0.001; for BxPC3, 7% ± 2% vs. 62% ± 3%, p = 0.002, both at 4d. No synergism was found when adding Valproic acid or Trichostatin-A. Conclusions:Treatment of k-RAS mutant pancreatic cancer cell lines with the MEK inhibitor U0126 results in significantly reduced expression of DNMT1 and increased DNMT3a and DNMT3b. U0126 combined with 5-Aza produced synergistic cytotoxicity, which may be due to enhanced hypomethylation activity. Although no significant change in expression of DNMT1 was found in the BxPC3 cells, we noted synergistic cytotoxicity when treated with both U0126 and 5-Aza.

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Gastrin regulates growth of human pancreatic cancer in a tonic and autocrine fashion

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1996.270.5.r1078 ◽

1996 ◽

Vol 270 (5) ◽

pp. R1078-R1084 ◽

Cited By ~ 12

Author(s):

J. P. Smith ◽

A. Shih ◽

Y. Wu ◽

P. J. McLaughlin ◽

I. S. Zagon

Keyword(s):

Gene Expression ◽

Pancreatic Cancer ◽

Cell Lines ◽

Cancer Cell ◽

Pancreatic Cancer Cell ◽

Cancer Cell Lines ◽

Human Pancreatic Cancer ◽

Growth Media ◽

Human Pancreatic Cancer Cell ◽

Cell Extracts

The gastrointestinal peptides gastrin and cholecystokinin (CCK) stimulate growth of human pancreatic cancer through a CCK-B/gastrin- like receptor. In the present study we evaluated whether growth of human pancreatic cancer is endogenously regulated by gastrin. Immunohistomical examination of BxPC-3 cells and tumor xenografts revealed specifc gastrin immunoreactivity. Gastrin was detected by radioimmunoassay in pancreatic cancer cell extracts and in pancreatic cancer cell extracts and in the growth media. With use of reverse-transcriptase polymerase chain reaction gastrin gene expression was detected in both cultured BxPC-3 cancer cells and transplanted tumors, as well as seven addition human pancreatic cancer cell lines. Growth of BxPC-3 human pancreatic cancer cell in serum-free medium was inhibited by the addition of the CCK-B/gastrin receptor antagonist L-365,260, and gastrin treatment reversed the inhibitory effect of the antagonist. A selective gastrin antibody (Ab repressed growth of BxPC-3 cells. Gastrin immunoreactivity was detected in fresh human pancreatic cancer specimens but not in normal human pancreatic tissue. These data provide the first evidence that growth of a human pancreatic cancer is tonically stimulated by the autocrine production of gastrin. Evidence for the ubiquity of this system was provided by the detection of gastrin gene expression in multiple human pancreatic cancer cell lines and detection of gastrin in cell lines and fresh pancreatic tumors.

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