Dataset Information

Hypoxia-inducible factor 1alpha determines gastric cancer chemosensitivity via modulation of p53 and NF-kappaB.

ABSTRACT:

Background

Reduced chemosensitivity of solid cancer cells represents a pivotal obstacle in clinical oncology. Hence, the molecular characterization of pathways regulating chemosensitivity is a central prerequisite to improve cancer therapy. The hypoxia-inducible factor HIF-1alpha has been linked to chemosensitivity while the underlying molecular mechanisms remain largely elusive. Therefore, we comprehensively analysed HIF-1alpha's role in determining chemosensitivity focussing on responsible molecular pathways.

Methodology and principal findings

RNA interference was applied to inactivate HIF-1alpha or p53 in the human gastric cancer cell lines AGS and MKN28. The chemotherapeutic agents 5-fluorouracil and cisplatin were used and chemosensitivity was assessed by cell proliferation assays as well as determination of cell cycle distribution and apoptosis. Expression of p53 and p53 target proteins was analyzed by western blot. NF-kappaB activity was characterized by means of electrophoretic mobility shift assay. Inactivation of HIF-1alpha in gastric cancer cells resulted in robust elevation of chemosensitivity. Accordingly, HIF-1alpha-competent cells displayed a significant reduction of chemotherapy-induced senescence and apoptosis. Remarkably, this phenotype was completely absent in p53 mutant cells while inactivation of p53 per se did not affect chemosensitivity. HIF-1alpha markedly suppressed chemotherapy-induced activation of p53 and p21 as well as the retinoblastoma protein, eventually resulting in cell cycle arrest. Reduced formation of reactive oxygen species in HIF-1alpha-competent cells was identified as the molecular mechanism of HIF-1alpha-mediated inhibition of p53. Furthermore, loss of HIF-1alpha abrogated, in a p53-dependent manner, chemotherapy-induced DNA-binding of NF-kappaB and expression of anti-apoptotic NF-kappaB target genes. Accordingly, reconstitution of the NF-kappaB subunit p65 reversed the increased chemosensitivity of HIF-1alpha-deficient cells.

Conclusion and significance

In summary, we identified HIF-1alpha as a potent regulator of p53 and NF-kappaB activity under conditions of genotoxic stress. We conclude that p53 mutations in human tumors hold the potential to confound the efficacy of HIF-1-inhibitors in cancer therapy.

SUBMITTER: Rohwer N

PROVIDER: S-EPMC2919384 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Publications

Hypoxia-inducible factor 1alpha determines gastric cancer chemosensitivity via modulation of p53 and NF-kappaB.

Rohwer Nadine N Dame Christof C Haugstetter Anja A Wiedenmann Bertram B Detjen Katharina K Schmitt Clemens A CA Cramer Thorsten T

PloS one 20100810 8

<h4>Background</h4>Reduced chemosensitivity of solid cancer cells represents a pivotal obstacle in clinical oncology. Hence, the molecular characterization of pathways regulating chemosensitivity is a central prerequisite to improve cancer therapy. The hypoxia-inducible factor HIF-1alpha has been linked to chemosensitivity while the underlying molecular mechanisms remain largely elusive. Therefore, we comprehensively analysed HIF-1alpha's role in determining chemosensitivity focussing on respons ...[more]

PMID: 20706634

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Project description:In this work we present evidence that the p53 tumor suppressor protein and NF-kappaB transcription factors could be related through common descent from a family of ancestral transcription factors regulating cellular proliferation and apoptosis. P53 is a homotetrameric transcription factor known to interact with the ankyrin protein 53BP2 (a fragment of the ASPP2 protein). NF-kappaB is also regulated by ankyrin proteins, the prototype of which is the IkappaB family. The DNA binding sequences of the two transcription factors are similar, sharing 8 out of 10 nucleotides. Interactions between the two proteins, both direct and indirect, have been noted previously and the two proteins play central roles in the control of proliferation and apoptosis.Using previously published structure data, we noted a significant degree of structural alignment between p53 and NF-kappaB p65. We also determined that IkappaBalpha and p53 bind in vitro through a specific interaction in part involving the DNA binding region of p53, or a region proximal to it, and the amino terminus of IkappaBalpha independently or cooperatively with the ankyrin 3 domain of IkappaBalpha In cotransfection experiments, kappaBalpha could significantly inhibit the transcriptional activity of p53. Inhibition of p53-mediated transcription was increased by deletion of the ankyrin 2, 4, or 5 domains of IkappaBalpha Co-precipitation experiments using the stably transfected ankyrin 5 deletion mutant of kappaBalpha and endogenous wild-type p53 further support the hypothesis that p53 and IkappaBalpha can physically interact in vivo.The aggregate results obtained using bacterially produced IkappaBalpha and p53 as well as reticulocyte lysate produced proteins suggest a correlation between in vitro co-precipitation in at least one of the systems and in vivo p53 inhibitory activity. These observations argue for a mechanism involving direct binding of IkappaBalpha to p53 in the inhibition of p53 transcriptional activity, analogous to the inhibition of NF-kappaB by kappaBalpha and p53 by 53BP2/ASPP2. These data furthermore suggest a role for ankyrin proteins in the regulation of p53 activity. Taken together, the NFkappaB and p53 proteins share similarities in structure, DNA binding sites and binding and regulation by ankyrin proteins in support of our hypothesis that the two proteins share common descent from an ancestral transcriptional factor.

Project description:BACKGROUND: Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of genes regulating oxygen homeostasis. The HIF-1 protein is composed of two HIF-1alpha and HIF-1beta/aryl hydrocarbon receptor nuclear translocator (ARNT) subunits. The prognostic relevance of HIF-1alpha protein overexpression has been shown in breast cancer. The impact of HIF-1alpha alternative splice variant expression on breast cancer prognosis in terms of metastasis risk is not well known. METHODS: Using real-time quantitative reverse transcription PCR assays, we measured mRNA concentrations of total HIF-1alpha and 4 variants in breast tissue specimens in a series of 29 normal tissues or benign lesions (normal/benign) and 53 primary carcinomas. In breast cancers HIF-1alpha splice variant levels were compared to clinicopathological parameters including tumour microvessel density and metastasis-free survival. RESULTS: HIF-1alpha isoforms containing a three base pairs TAG insertion between exon 1 and exon 2 (designated HIF-1alphaTAG) and HIF-1alpha736 mRNAs were found expressed at higher levels in oestrogen receptor (OR)-negative carcinomas compared to normal/benign tissues (P = 0.009 and P = 0.004 respectively). In breast carcinoma specimens, lymph node status was significantly associated with HIF-1alphaTAG mRNA levels (P = 0.037). Significant statistical association was found between tumour grade and HIF-1alphaTAG (P = 0.048), and total HIF-1alpha (P = 0.048) mRNA levels. HIF-1alphaTAG mRNA levels were also inversely correlated with both oestrogen and progesterone receptor status (P = 0.005 and P = 0.033 respectively). Univariate analysis showed that high HIF-1alphaTAG mRNA levels correlated with shortened metastasis free survival (P = 0.01). CONCLUSIONS: Our results show for the first time that mRNA expression of a HIF-1alphaTAG splice variant reflects a stage of breast cancer progression and is associated with a worse prognosis.See commentary: http://www.biomedcentral.com/1741-7015/8/45.

Dataset Information

Hypoxia-inducible factor 1alpha determines gastric cancer chemosensitivity via modulation of p53 and NF-kappaB.

Background

Methodology and principal findings

Conclusion and significance

Publications

Hypoxia-inducible factor 1alpha determines gastric cancer chemosensitivity via modulation of p53 and NF-kappaB.

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