Identification of Genes Differentially Induced by Hypoxia in Pancreatic Cancer Cells

doi:10.1006/bbrc.2001.5867

Biochemical and Biophysical Research Communications

Volume 288, Issue 4, 9 November 2001, Pages 882-886

https://doi.org/10.1006/bbrc.2001.5867 Get rights and content

Abstract

A hypoxic microenvironment is characteristic of many solid tumors, including pancreatic cancer, the fifth leading cause of cancer death in the United States. Hypoxia causes the stabilization of the HIF-1 (hypoxia-inducible factor-1) transcription factor and the induction of many genes that promote angiogenesis, tumor growth, and metastasis. We performed representational difference analysis (RDA) using mRNA extracted from hypoxic and normoxic Capan-2, a human pancreatic cancer cell line. cDNAs corresponding to hypoxia-inducible genes were cloned and sequenced. We identified GPI/NLK/AMF (glucose phosphate isomerase/neuroleukin/autocrine motility factor) as a hypoxic inducible gene. In addition, hexokinase II and DEC1/Stra13, genes known to be hypoxia inducible in other systems, were found to be hypoxia inducible in our pancreatic cancer system. We thus identified three genes that are induced by hypoxia in a human pancreatic cancer, including GPI/NLK/AMF, which was not previously known to be hypoxia inducible in any other system. These genes may provide new targets for diagnosis and treatment of pancreatic cancer.

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Histone chaperone FACT complex coordinates with HIF to mediate an expeditious transcription program to adapt to poorly oxygenated cancers
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Citation Excerpt :
For instance, HIF-1 activates the transcription of glucose transporters (GLUT1 and GLUT3) to increase the uptake of glucose (Chen et al., 2001; Royer et al., 2000). HIF-1 also stimulates the activation of glycolytic enzymes, such as glucose-6-phosphate isomerase (GPI), Aldolase C (ALDOC), and pyruvate kinase (PKM), to accelerate glucose metabolism (Figure 4A; Wang et al., 2005; Yoon et al., 2001). LDHA then converts pyruvate into lactate to prevent pyruvate to be diverted into the TCA cycle, as well as speeding up glycolysis by regenerating NAD+ (Figure 5A; Fantin et al., 2006; Semenza et al., 1996).
Cancer cells adapt to hypoxia through HIFs (hypoxia-inducible factors), which initiate the transcription of numerous genes for cancer cell survival in the hypoxia microenvironment. In this study, we find that the FACT (facilitates chromatin transcription) complex works cooperatively with HIFs to facilitate the expeditious expression of HIF targets for hypoxia adaptation. Knockout (KO) of the FACT complex abolishes HIF-mediated transcription by impeding transcription elongation in hypoxic cancer cells. Interestingly, the FACT complex is post-translationally regulated by PHD/VHL-mediated hydroxylation and proteasomal degradation, in similar fashion to HIF-1/2α. Metabolic tracing confirms that FACT KO suppresses glycolytic flux and impairs lactate extrusion, leading to intracellular acidification and apoptosis in cancer cells. Therapeutically, hepatic artery ligation and anti-angiogenic inhibitors adversely induce intratumoral hypoxia, while co-treatment with FACT inhibitor curaxin remarkably hinders the growth of hypoxic tumors. In summary, our findings suggest that the FACT complex is a critical component of hypoxia adaptation and a therapeutic target for hypoxic tumors.
Stra13 and Sharp-1, the non-grouchy regulators of development and disease
2014, Current Topics in Developmental Biology
Citation Excerpt :
Target genes of HIF-1 can be categorized into angiogenesis-related factors, glucose transporters and glycolytic enzymes, tumor invasion and metastasis-related factors, and cell proliferation and apoptosis-related factors (Semenza, 2009; Yang, Sun, Wang, & Jiao, 2013). Stra13 and Sharp-1 are target genes of HIF-1 and are induced by hypoxia (Feige et al., 2011; Koukourakis et al., 2006; Ma et al., 2013; Miyazaki et al., 2002; Olbryt et al., 2006; Yoon et al., 2001; Zheng et al., 2012). Under normoxic conditions, Stra13 expression is downregulated by von Hippel–Lindau (VHL), a component of VHL/E3 ligase complex which ubiquitinates HIF-1 and targets it for degradation (Ivanova, Ivanov, Danilkovitch-Miagkova, & Lerman, 2001; Wykoff, Pugh, Maxwell, Harris, & Ratcliffe, 2000).
Stra13 and Sharp-1 are transcriptional repressors that share domain structure similarity with members of the basic helix-loop-helix-Orange subfamily. In contrast to other members that include Hes and Hey proteins, transcriptional repression mediated by Stra13 and Sharp-1 does not involve recruitment of the corepressor Groucho. Both proteins undergo sumoylation at evolutionarily conserved sites, and this posttranslational modification serves as a platform for association with chromatin-modifying enzymes including histone deacetylases and histone methyltransferases. In addition to being widely expressed during embryonic development and in adult tissues, the expression of both genes is induced by a number of stimuli. Loss-of-function and gain-of-function studies have demonstrated their function in cellular differentiation and regeneration, in regulation of circadian rhythms, immune homeostasis, and metabolism. Given their diverse physiological functions in several tissues, it is not surprising that deregulated expression of Stra13 and Sharp-1 is apparent in human pathologies. Here, we review our current understanding of their cellular functions that suggest a requirement in maintenance of tissue homeostasis.
Hypoxia inducible BHLHB2 is a novel and independent prognostic marker in pancreatic ductal adenocarcinoma
2010, Biochemical and Biophysical Research Communications
The cyclic adenosine monophosphate-inducible basic helix–loop–helix (bHLH) domain containing class-B2 transcriptional factor BHLHB2 is differentially expressed in a number of human malignancies. In the present study, the expression, regulation, functions and prognostic impact of BHLHB2 in pancreatic cancer were investigated.
Expression analyses were carried out in tissues of the normal pancreas (n = 10) and pancreatic ductal adenocarcinoma (n = 77) as well as in eight pancreatic cancer cell lines using quantitative RT-PCR, semiquantitative immunohistochemistry, and immunoblot analyses. In vitro functional experiments were conducted using siRNA transfection, hypoxia, serum starvation, apoptosis induction with gemcitabine and actinomycin-D, and invasion assays. Survival analysis was performed using the Kaplan–Meier method. Prognostic factors were determined in a multivariable analysis using a Cox proportional hazards model.
BHLHB2 mRNA and protein expressions were strongly induced by hypoxia and by serum starvation in pancreatic cancer cell lines. BHLHB2 silencing with RNAi had no significant effects on growth and invasion but increased apoptosis resistance against gemcitabine by reducing caspace-3 cleavage. In BHLHB2 silenced cells the ED50 of gemcitabine increased from 13.95 ± 1.353 to 38.70 ± 5.262 nM (p < 0.05). Ex vivo, the weak/absent nuclear staining in normal pancreatic ducts and acinar cells was replaced by moderate to strong nuclear/cytoplasmic staining in PanIN lesions and pancreatic cancer cells. Patients with weak/absent nuclear BHLHB2 staining had significantly worse median survival compared to those with strong staining (13 months vs. 27 months, p = 0.03). In a multivariable analysis, BHLHB2 staining was an independent prognostic factor (Hazard-Ratio = 2.348, 95% CI = 1.250–4.411, p = 0.008).
Hypoxia-inducible BHLHB2 expression is a novel independent prognostic marker in pancreatic cancer patients and indicates increased chemosensitivity towards gemcitabine.
Genomic response of hypoxic Müller cells involves the very low density lipoprotein receptor as part of an angiogenic network
2009, Experimental Eye Research
Citation Excerpt :
Expression of anti-apoptosis genes BCL2/adenovirus E1B 19 kDa-interacting protein 3 (bnip3) (Sowter et al., 2001)and BCL2/adenovirus E1B 19 kDa-interacting protein 3-like (bnip3l) was also increased by 4.3 (p = 6.3E-07) and 3.6-fold (p = 0.0007), respectively. Glycolysis genes hexokinase 2 (Hk2, p = 0.0003, 4.1-fold) (Mathupala et al., 2001), aldolase C/fructose-biphosphate (aldoc, p = 0.0003, 3.5-fold), (Jean et al., 2006) enolase 2 (eno2, p = 0.0001, 2.7-fold) (Celtik et al., 2004; Mizukami et al., 2004) and glucose phosphate isomerase (gpi, p = 0.0004, 2.1-fold) (Yoon et al., 2001) were upregulated as well. Expression levels measured with real-time PCR from the second RNA isolation matched those seen in the microarray assay: vldlr was upregulated 3.0-fold and trib3 5.1-fold.
Müller cells have recently been found to produce select angiogenic substances. In choosing a more comprehensive approach, we wanted to study the genomic response of Müller cells to hypoxia to identify novel angiogenic genes. An established Müller cell line (rMC-1) was exposed to standard or hypoxic conditions. We analyzed gene expression with three independent microarrays and determined differential expression levels compared to normoxia. Selected genes were confirmed by real-time PCR (RTPCR). Subcellular localization of proteins was examined by immunocytochemistry. A network-based pathway analysis was performed to investigate how those genes may contribute to angiogenesis. We found 19 004 of 28 000 known rat genes expressed in Müller cells. 211 genes were upregulated by hypoxia 1.5 to 14.9-fold (p < 0.001, FDR ≤ 5%) and 220 genes were downregulated 1.5–4.6-fold (p < 0.001, FDR ≤ 5%). Unexpectedly, expression patterns of cell proliferation, differentiation and organogenesis were increased besides predictable declines in cell function. Very low density lipoprotein receptor (VLDLR) and tribbles 3 (TRIB3) were further analyzed because of recent implication in retinal neovascularization and macular degeneration (VLDLR) and in ocular mesodermal development and differentiation (TRIB3), respectively. VLDLR was upregulated 3.1-fold (p = 0.001, RTPCR 3.0-fold) and TRIB3 2.8-fold (p = 0.025, RTPCR 5.1-fold). VEGF was increased 3.1-fold (p = 0.003, RTPCR 8.3-fold) and apelin, a novel factor of retinal angiogenesis, 5.6-fold (p = 0.006, RTPCR 8.7-fold). A network of interacting angiogenic genes was identified in silico that included VLDLR as a surface receptor. VLDLR protein localized to the perinucleus, cytoplasm and cell membrane, while TRIB3 was found in nucleoli, the nucleus and cytoplasm. We conclude that hypoxia triggers an angiogenic network response in Müller cells with VLDLR as a novel node and gene expression patterns of proliferation, differentiation and organogenesis.
The effect of HIF on metabolism and immunity
2022, Nature Reviews Nephrology
A potential signaling axis between RON kinase receptor and hypoxia-inducible factor-1 alpha in pancreatic cancer
2021, Molecular Carcinogenesis

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¹: To whom correspondence and reprint requests should be addressed at UCLA Department of Pathology and Laboratory Medicine, 10833 Le Conte Avenue, P.O. Box 951732, Los Angeles, CA 90095-1732. Fax: (310) 794-9272. E-mail: [email protected].

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Regular ArticleIdentification of Genes Differentially Induced by Hypoxia in Pancreatic Cancer Cells

Abstract

Int. J. Radiat. Oncol. Biol. Phys.

Gastroenterology

Mutat. Res.

Dev. Biol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Cancer statistics, 2000

CA Cancer J. Clin.

Cancer Facts and Figures 1999

Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours

Nature

Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix

Cancer Res.

Regular Article
Identification of Genes Differentially Induced by Hypoxia in Pancreatic Cancer Cells