Impact Of Hypoxia-inducible Factors Research Articles

Human colorectal cancer: upregulation of the adaptor protein Rai in TILs leads to cell dysfunction by sustaining GSK-3 activation and PD-1 expression

BackgroundThe immunosuppressive tumor microenvironment (TME) of colorectal cancer (CRC) is a major hurdle for immune checkpoint inhibitor-based therapies. Hence characterization of the signaling pathways driving T cell exhaustion within TME is a critical need for the discovery of novel therapeutic targets and the development of effective therapies. We previously showed that (i) the adaptor protein Rai is a negative regulator of T cell receptor signaling and T helper 1 (Th1)/Th17 cell differentiation; and (ii) Rai deficiency is implicated in the hyperactive phenotype of T cells in autoimmune diseases.MethodsThe expression level of Rai was measured by qRT-PCR in paired peripheral blood T cells and T cells infiltrating tumor tissue and the normal adjacent tissue in CRC patients. The impact of hypoxia-inducible factor (HIF)-1α on Rai expression was evaluated in T cells exposed to hypoxia and by performing chromatin immunoprecipitation assays and RNA interference assays. The mechanism by which upregulation of Rai in T cells promotes T cell exhaustion were evaluated by flow cytometric, qRT-PCR and western blot analyses.ResultsWe show that Rai is a novel HIF-1α-responsive gene that is upregulated in tumor infiltrating lymphocytes of CRC patients compared to patient-matched circulating T cells. Rai upregulation in T cells promoted Programmed cell Death protein (PD)-1 expression and impaired antigen-dependent degranulation of CD8+ T cells by inhibiting phospho-inactivation of glycogen synthase kinase (GSK)-3, a central regulator of PD-1 expression and T cell-mediated anti-tumor immunity.ConclusionsOur data identify Rai as a hitherto unknown regulator of the TME-induced exhausted phenotype of human T cells.

The impact of hypoxia-inducible factors in the pathogenesis of kidney diseases: a link through cell metabolism.

Kidneys are sensitive to disturbances in oxygen homeostasis. Hypoxia and activation of the hypoxia-inducible factor (HIF) pathway alter the expression of genes involved in the metabolism of renal and immune cells, interfering with their functioning. Whether the transcriptional activity of HIF protects the kidneys or participates in the pathogenesis of renal diseases is unclear. Several studies have indicated that HIF signaling promotes fibrosis in experimental models of kidney disease. Other reports showed a protective effect of HIF activation on kidney inflammation and injury. In addition to the direct effect of HIF on the kidneys, experimental evidence indicates that HIF-mediated metabolic shift activates inflammatory cells, supporting the HIF cascade as a link between lung or gut damage and worsening of renal disease. Although hypoxia and HIF activation are present in several scenarios of renal diseases, further investigations are needed to clarify whether interfering with the HIF pathway is beneficial in different pathological contexts.

Hypoxia inducible factor signaling in breast tumors controls spontaneous tumor dissemination in a site-specific manner

Hypoxia is a common feature in tumors and induces signaling that promotes tumor cell survival, invasion, and metastasis, but the impact of hypoxia inducible factor (HIF) signaling in the primary tumor on dissemination to bone in particular remains unclear. To better understand the contributions of hypoxia inducible factor 1 alpha (HIF1α), HIF2α, and general HIF pathway activation in metastasis, we employ a PyMT-driven spontaneous murine mammary carcinoma model with mammary specific deletion of Hif1α, Hif2α, or von Hippel-Lindau factor (Vhl) using the Cre-lox system. Here we show that Hif1α or Hif2α deletion in the primary tumor decreases metastatic tumor burden in the bone marrow, while Vhl deletion increases bone tumor burden, as hypothesized. Unexpectedly, Hif1α deletion increases metastatic tumor burden in the lung, while deletion of Hif2α or Vhl does not affect pulmonary metastasis. Mice with Hif1α deleted tumors also exhibit reduced bone volume as measured by micro computed tomography, suggesting that disruption of the osteogenic niche may be involved in the preference for lung dissemination observed in this group. Thus, we reveal that HIF signaling in breast tumors controls tumor dissemination in a site-specific manner.

Abstract IA-017: Chromatin and gene transcription in hypoxia

Abstract Oxygen is essential for the life of most multicellular organism and changes in oxygen availability have implications in physiological and pathophysiological conditions. In response to hypoxia (low oxygen availability) transcriptional changes are induced. The transcriptional response to hypoxia is mediated primarily by the Hypoxia Inducible Factor (HIF) family of transcription factors. HIFs are activated in hypoxia via inhibition of prolyl hydroxylases (PHDs), a group of molecular dioxygenases, which require oxygen for their activity. Whilst the role of HIFs in the transcriptional response to hypoxia is well characterized, little is known with regards to chromatin structure in low oxygen stress and how this coordinates the transcriptional response. It was recently shown that chromatin is sensitive to oxygen levels, with hypoxia altering histone methylation at hypoxia responsive genes via inhibition of specific JmjC histone demethylases. To further investigate chromatin structure and its role in hypoxia induced gene transcription, we have performed ATAC-seq and RNA-seq in human cancer cell lines in acute and prolonged hypoxia. We find that acute hypoxia leads to increased promoter accessibility at a subset of hypoxia inducible genes, and this mostly precedes changes in mRNA expression, with further changes in chromatin structure after prolonged hypoxia. In addition, ChIP-seq data on histone methylation marks (H3K4me and H3K36me3), revealed similar earlier patterns of genomic distribution. Furthermore, we dissected the impact of HIF in the observed changes using a chemical probe and specific HIF1beta depletion analysis of ATAC-seq. This work gives insights into the role of chromatin dynamics in regulating the transcriptional response to hypoxia and elucidating the mechanisms behind these changes may lead to targeted therapies for hypoxia driven diseases. Citation Format: Michael Batie, Julianty Frost, Sonia Rocha. Chromatin and gene transcription in hypoxia [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-017.

Metabolism and Redox in Pulmonary Vascular Physiology and Pathophysiology.

Significance: This review considers how some systems controlling pulmonary vascular function are potentially regulated by redox processes to examine how and why conditions such as prolonged hypoxia, pathological mediators, and other factors promoting vascular remodeling contribute to the development of pulmonary hypertension (PH). Recent Advances and Critical Issues: Aspects of vascular remodeling induction mechanisms described are associated with shifts in glucose metabolism through the pentose phosphate pathway and increased cytosolic NADPH generation by glucose-6-phosphate dehydrogenase, increased glycolysis generation of cytosolic NADH and lactate, mitochondrial dysfunction associated with superoxide dismutase-2 depletion, changes in reactive oxygen species and iron metabolism, and redox signaling. Future Directions: The regulation and impact of hypoxia-inducible factor and the function of cGMP-dependent and redox regulation of protein kinase G are considered for their potential roles as key sensors and coordinators of redox and metabolic processes controlling the progression of vascular pathophysiology in PH, and how modulating aspects of metabolic and redox regulatory systems potentially function in beneficial therapeutic approaches.

Hypoxia-inducible factors in cancer: an overview of major findings from meta-analyses

This paper aims to systematically review the major findings from meta-analyses regarding the impact of hypoxia-inducible factors (HIFs) in various human cancers. A total of 56 eligible meta-analysis papers were identified via the PubMed and EMBASE databases. The associations of HIF-1α gene polymorphism and/or HIF-1α and HIF-2α protein expression with the risk, clinicopathological features, and/or survival were explored in head and neck cancer (n=4), glioma (n=2), oral cancer (n=10), oropharyngeal cancer (n=1), nasopharyngeal cancer (n=1), lung cancer (n=12), breast cancer (n=17), esophageal cancer (n=5), gastric cancer (n=8), colorectal cancer (n=15), pancreatic cancer (n=8), hepatocellular carcinoma (n=5), prostate cancer (n=13), renal cancer (n=13), bladder cancer (n=3), ovarian cancer (n=3), cervical cancer (n=10), endometrial cancer (n=1), and osteosarcoma (n=1). Based on the current evidence, the impact of HIFs should be heterogeneous on various human cancers.

Impact of hypoxia inducible factors on estrogen receptor expression in breast cancer cells

In women breast cancer is still the most commonly diagnosed cancer. This type of cancer is classified as a hormone-dependent tumor. Estrogen receptor (ER) expression and functional status contribute to breast cancer development and progression. Another important factor associated with cancer is hypoxia which is defined as the state of reduced oxygen availability in tissues. Intratumoral hypoxia results in the activation of the hypoxia inducible factors (HIFs). HIFs are heterodimeric transcription factors involved in the regulation of many cellular processes, such as angiogenesis, anaerobic metabolism, cell proliferation/survival, and promotion of metastasis. In this study we evaluated the interplay between hypoxia, HIF stabilization and the ER-α/β-ratio in several ER-positive breast cancer cell lines. Hypoxia was shown to inhibit ER expression in ER-positive breast cancer cells. Further experiments using the hypoxia mimetic CoCl2 and HIF-1α knockdown cells indicated that the influence of hypoxia on breast cancer cells involves other pathways than the molecular oxygen sensing pathway. Moreover, we demonstrated that MCF-7 cells in long-term culture lost part of their ability to respond to hypoxic incubation. Understanding the relationships between HIF, ER-α and ER-β expression holds the promise of the development of new therapeutic agents and may provide future advances in prognosis.

Abstract 533: S100A10 as a novel biomarker in hepatocellular carcinoma

Abstract Background and Objective: Hepatocellular carcinoma (HCC) is the dominant form of primary liver tumor and the second most lethal malignancy in worldwide. From transcriptome sequencing analysis, we observed that S100A10 was overexpressed in our cohort of 16 HCC patients. S100A10 is a calcium binding protein and accumulating studies have indicated that annexin A2- S100A10 heterotetramer on the cell surface is important for the conversion of plasminogen to plasmin, which promotes activation of matrix metalloproteinases and degradation of extracellular matrix. This enhances cancer invasion and migration. While annexin A2 overexpression has been documented in various malignancies and shows significant association with local invasion and metastasis, information regarding the expression of S100A10 in human cancers is scarce. Furthermore, how S100A10 was regulated remains unexplored. Hypoxia is a common phenomenon in HCC microenvironment and stabilizies transcription factors, hypoxia inducible factor (HIF)-1α and -2α, to initiate transcription of a wide repertoire of genes for cancer progression. The major aim of this study is to evaluate the possible clinical relevance and diagnostic value of S100A10 in HCC and study the molecular mechanism that drives S100A10 overexpression under the influence of hypoxia in HCC. Experimental Procedures: We evaluated S100A10 expression in human HCC and the corresponding non-tumorous liver (NT) tissues in a cohort of 67 HCC patients by RT-PCR. S100A10 expression was correlated with various HCC clinico-pathological features by Student's t-test and survival outcome of HCC patients by Kaplan-Meier plot followed by log rank test. Promoter of S100A10 was analyzed for potential hypoxia responsive elements -A/GCGTG- and confirmed by ChIP assay with HIF antibodies. To evaluate the impact of HIF on S100A10 expression, we knocked-down (shRNA) and knocked-out (TALEN) HIF-1/2α in a HCC cell line, MHCC97L and evaluated S100A10 mRNA and protein expression. Results: S100A10 was significantly upregulated (P = 0.001, Wilcoxon signed rank test) in HCC as compared to NT tissues. Overexpression of S100A10 (≥ 2 fold) was found in 50.746% (34/67) of HCC patients and was closely correlated with direct liver invasion (P = 0.038), tumor microsatellite formation (P = 0.050) and absence of tumor encapsulation (P = 0.048). More importantly, S100A10 overexpression was associated with poorer 5-year survival rate in HCC patients (P = 0.062). ROC analysis suggested that S100A10 could be a potential diagnostic marker for HCC detection (Area Under Curve = 0.685, P = 0.001). Hypoxia induced S100A10 mRNA and protein expression levels in multiple HCC cell lines and this was abolished in HIF-1/2α knockdown or knockout HCC cells. Conclusion: Our current data have presented evidence that S100A10 can be a potential diagnostic and prognostic marker in HCC and S100A10 is a transcriptional target of HIF. Citation Format: David Kung-Chun Chiu, Carmen Chak-Lui Wong, Irene Oi-Lin Ng, Aki Pui-Wah Tse. S100A10 as a novel biomarker in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 533. doi:10.1158/1538-7445.AM2015-533

Effect of hypoxia inducible factor-1α gene silencing on glycolysis correlated gene expression in BxPC-3 cell line

Objective To investigate the impact of hypoxia inducible factor (HIF)-1α gene silencing on glycolysis correlated gene expression in human pancreatic cancer cell line (BxPC-3) under hypoxic conditions.Methods The siRNA was synthesized to silence the expression of HIF-1α in BxPC-3 cell line.The transfected cells were divided into three groups:blank control group (BxPC-3),empty plasmid vector transfection group (GFP) and HIF-1α gene silencing group (sh-HIF-1α).All cells were cultured under either normoxic (21.0％ O2) or hypoxic (0.5％-1.0％ O2) conditions for 48 h.Then pyruvate dehydrogenase kinase 1 (PDK-1),lactate dehydrogenase A (LDH-A),and citrate synthase (CS) expression and the lactate acid level were analyzed respectively.Results Under hypoxic conditions,the expression of HIF-lα mRNA was increased by 12.4％ and HIF-1α protein increased by 1.6 folds correspondingly,which were significantly lower than in BxPC-3 and GFP groups (P ＜ 0.05).PDK-1 and LDH-A expression levels were also lower in sh-HIF-1α group than the other two control groups.Accordingly,the final product of glycolysis,lactate acid level in sh-HIF-lα group [(4.8 ±0.3) nmol/106 cells] was less than in BxPC-3 group [(9.1 ± 0.5) nmol/106 cells] and GFP group [(9.2 ± 0.5) nmol/106 cells] (P ＜ 0.05).In contrast,the expression levels of CS mRNA and protein in BxPC-3 and GFP groups were repressed and lower than in sh-HIF-1α group (P ＜ 0.05).Conclusion Hypoxia would induce HIF-1α expression in BxPC-3 cell line,which upregulated the glycolysis correlated PDK-1 and LDH-A expression.Gene silencing of HIF-1α could inhibit PDK-1 and LDH-A,and promote the CS expression.As a result,the glycolysis pathway was greatly attenuated under hypoxic conditions. Key words: Hypoxia inducible factor-1α;  Glycolysis;  Pyruvate dehydrogenase kinase 1; Lactate dehydrogenase A ;  Citrate synthase

Expressions of hypoxia-inducible factor-1α and hexokinase-II in gastric adenocarcinoma: the impact on prognosis and correlation to clinicopathologic features

The impact of hypoxia-inducible factor (HIF)-1α and hexokinase-II (HK-II) expression on prognosis of gastric adenocarcinoma patients has not been clearly established. We identified all patients in Cancer Center of Sun Yat-Sen University who were diagnosed as gastric adenocarcinoma and underwent radical gastrectomy between January 1999 and December 2001. We used immunohistochemistry to determine the expressions of HIF-1α protein and HK-II in the surgical sections. We identified 188 patients with gastric adenocarcinoma for the final analysis. The positive rate of HIF-1α and HK-II were 110/188 (54.6%) and 40/188 (21.3%), respectively. Both HIF-1α and HK-II were all positively correlated with tumor size, lower differentiation, and tumor stage. Univariate analysis showed that advanced tumor stages (P < 0.001), tumor size (P = 0.003), HIF-1α expression (P < 0.001), and HK-II expression (P < 0.001) were all significantly associated with shorter survival. The multivariate Cox analysis revealed that tumor stage (P < 0.001), HIF-1α expression (P < 0.001), and HK-II expression (P = 0.002) remained independent prognostic variables for survival. In addition, there was a positive correlation of HIF-1α protein expression and HK-II (P = 0.022). Both HIF-1α and HK-II were overexpressed in gastric adenocarcinoma. The multivariate Cox analysis revealed that both of them were independent factors on survival of gastric adenocarcinoma patients.