Project description:Hypoxia-inducible factor-1 (HIF-1) is a master regulator of glucose metabolism in cancer cells. Here, we demonstrate that a HIF-1α anti-sense lncRNA, HIFAL, is essential for maintaining and enhancing HIF-1α-mediated transactivation and glycolysis. Mechanistically, HIFAL recruits PHD3 to PKM2 to induce its prolyl hydroxylation and introduces the PKM2/PHD3 complex into the nucleus via binding with hnRNPF to enhance HIF-1α transactivation. Reciprocally, HIF-1α induces HIFAL transcription, which forms a positive feed-forward loop to maintain the transactivation activity of HIF-1α. Clinically, high HIFAL expression is associated with aggressive breast cancer phenotype and poor patient outcome. Furthermore, HIFAL overexpression promotes tumor growth in vivo, while targeting both HIFAL and HIF-1α significantly rescues their effect on cancer growth. Overall, our results indicate a critical regulatory role of HIFAL in HIF-1α-driven transactivation and glycolysis, identifying HIFAL as a therapeutic target for cancer treatment.
Project description:Peritoneal metastasis (PM) is diagnosed in almost half of patients with advanced gastric cancer (GCa) and has a very poor prognosis. However, the molecular mechanisms of PM in GCa remain poorly understood. Here, we show that the elevated expression of RAR-related orphan receptor gamma (RORγ) in GCa tumors is a key driver of PM. RORγ drives GCa progression and metastasis by assembling a transcriptional complex with HIF-1α that regulates the expression of HIF-1α targets via recruitment of RNA polymerase II and p300. Mechanistically, RORγ hijacks HIF-1α to disrupt the interaction between HIF-1α and PHD3, leading to decreased HIF-1α hydroxylation, ubiquitylation and increased HIF-1α accumulation, nuclear translocation, and transactivation. RORγ antagonists block tumor growth and PM in multiple xenograft GCa models, and they effectively sensitize GCa tumors to chemotherapy in mice. Thus, our study uncovers a mechanism of RORγ-driven PM and offers a potential therapeutic option against advanced GCa.
Project description:Peritoneal metastasis (PM) is diagnosed in almost half of patients with advanced gastric cancer (GCa) and has a very poor prognosis. However, the molecular mechanisms of PM in GCa remain poorly understood. Here, we show that the elevated expression of RAR-related orphan receptor gamma (RORγ) in GCa tumors is a key driver of PM. RORγ drives GCa progression and metastasis by assembling a transcriptional complex with HIF-1α that regulates the expression of HIF-1α targets via recruitment of RNA polymerase II and p300. Mechanistically, RORγ hijacks HIF-1α to disrupt the interaction between HIF-1α and PHD3, leading to decreased HIF-1α hydroxylation, ubiquitylation and increased HIF-1α accumulation, nuclear translocation, and transactivation. RORγ antagonists block tumor growth and PM in multiple xenograft GCa models, and they effectively sensitize GCa tumors to chemotherapy in mice. Thus, our study uncovers a mechanism of RORγ-driven PM and offers a potential therapeutic option against advanced GCa.
Project description:Hypoxia Inducible Factor (HIF) prolyl hydroxylase domain (PHD) enzymes catalyse the posttranslational hydroxylation of conserved prolyl residues in the alpha-subunit of the HIF transcription factor. These modifications, which promote the degradation of HIF-alpha subunits by the pVHL E3 ligase complex and impart oxygen-dependent regulation of the HIF transcriptional response, have been extensively characterised at the molecular, cellular and organismal level. Since the discovery of the PHDs, a range of less well-characterised non-HIF substrates have been reported with the potential to confer oxygen-sensitivity to a diverse range of cellular processes. We sought to systematically compare all of the reported non-HIF substrates for their ability to support PHD-catalysed hydroxylation. We performed a comprehensive series of in vitro hydroxylation assays reacting synthetic peptides and full-length protein substrates generated by in vitro transcription and translation with purified recombinant enzyme preparations. Prolyl hydroxylation was assayed directly by mass spectrometry and radiochemical assay for hydroxyproline. Both methods enabled quantitative appraisal of enzyme-catalysed hydroxylation, with liquid chromatography mass spectrometry methods employing NMR-quantified peptide standards to calibrate retention time signatures and determine ionisation efficiencies for each target peptide. Using these approaches we assayed hydroxylation on 23 different proteins. Surprisingly, we did not detect measurable hydroxylation on any of the reported non-HIF substrates using either method. In contrast, control assays with HIF1-alpha substrate supported high stoichiometry (typically >90%) hydroxylation. Our findings suggest that PHD substrates are more restricted than has been reported.
Project description:Interferon regulatory factor 3 (IRF3) functions as a key transcription factor in the innate antiviral immune response, which is depended on its nuclear localization. However, its function in the cytoplasm during non-infection states remains largely unknown. In this study, we found that resting cytoplasmic IRF3 interacts with HIF-1α and HIF-2α, two master regulators of hypoxia signaling. This interaction retains HIF-α in the cytoplasm under hypoxic conditions, preventing it from exerting their transcription factor function and attenuating hypoxia signaling. Disruption of IRF3 in both mice and zebrafish resulted in increased expression of hypoxia response genes and enhanced tolerance to hypoxia. These findings suggest that, in the absence of viral infection, cytoplasmic IRF3 modulates hypoxia signaling by retaining HIF-α in the cytoplasm under hypoxic conditions.
Project description:The identification of predictive markers to determine the premetastatic phase before metastasis is critical for developing effective strategies for early detection and prevention. By applying the dynamic network biomarker (DNB) approach to analyze time-series transcriptomic data from a pulmonary metastasis HCC mouse model, we revealed that the premetastatic phase occurred during the fourth week after implantation. A total of 142 DNB genes were identified as functionally important biomarkers of this critical phase, among which 60S ribosomal protein L6 (RPL6) was a core DNB member. RPL6 was significantly upregulated in HCC tissues with extrahepatic metastasis and was strongly correlated with poor prognosis in HCC patients. RPL6 promoted the invasion and metastasis of HCC cells, both in vitro and in vivo. Mechanistically, RPL6 directly bound to the HMGCS1 mRNA 3’UTR, a rate-limiting enzyme in cholesterol biosynthesis, thus increasing HMGCS1 mRNA stability and protein expression and subsequently elevating intracellular cholesterol level. Elevated cholesterol inhibited of the ubiquitin-dependent degradation of HIF-1α by binding to PHD2 and reducing PHD2-mediated hydroxylation of HIF-1α, which further resulted in HIF-1α signaling pathway activation. Moreover, RPL6, HMGCS1 and HIF-1α were coexpressed in HCC tissues with extrahepatic metastasis. Taken together, this study provides new insights into the dynamic transcriptome profiles of HCC pulmonary metastasis and establishes an important role for the RPL6-HMGCS1-HIF-1α axis in HCC metastasis, suggesting potential prognostic biomarkers and therapeutic targets in HCC.
Project description:The identification of predictive markers to determine the premetastatic phase before metastasis is critical for developing effective strategies for early detection and prevention. By applying the dynamic network biomarker (DNB) approach to analyze time-series transcriptomic data from a pulmonary metastasis HCC mouse model, we revealed that the premetastatic phase occurred during the fourth week after implantation. A total of 142 DNB genes were identified as functionally important biomarkers of this critical phase, among which 60S ribosomal protein L6 (RPL6) was a core DNB member. RPL6 was significantly upregulated in HCC tissues with extrahepatic metastasis and was strongly correlated with poor prognosis in HCC patients. RPL6 promoted the invasion and metastasis of HCC cells, both in vitro and in vivo. Mechanistically, RPL6 directly bound to the HMGCS1 mRNA 3’UTR, a rate-limiting enzyme in cholesterol biosynthesis, thus increasing HMGCS1 mRNA stability and protein expression and subsequently elevating intracellular cholesterol level. Elevated cholesterol inhibited of the ubiquitin-dependent degradation of HIF-1α by binding to PHD2 and reducing PHD2-mediated hydroxylation of HIF-1α, which further resulted in HIF-1α signaling pathway activation. Moreover, RPL6, HMGCS1 and HIF-1α were coexpressed in HCC tissues with extrahepatic metastasis. Taken together, this study provides new insights into the dynamic transcriptome profiles of HCC pulmonary metastasis and establishes an important role for the RPL6-HMGCS1-HIF-1α axis in HCC metastasis, suggesting potential prognostic biomarkers and therapeutic targets in HCC.
Project description:The identification of predictive markers to determine the premetastatic phase before metastasis is critical for developing effective strategies for early detection and prevention. By applying the dynamic network biomarker (DNB) approach to analyze time-series transcriptomic data from a pulmonary metastasis HCC mouse model, we revealed that the premetastatic phase occurred during the fourth week after implantation. A total of 142 DNB genes were identified as functionally important biomarkers of this critical phase, among which 60S ribosomal protein L6 (RPL6) was a core DNB member. RPL6 was significantly upregulated in HCC tissues with extrahepatic metastasis and was strongly correlated with poor prognosis in HCC patients. RPL6 promoted the invasion and metastasis of HCC cells, both in vitro and in vivo. Mechanistically, RPL6 directly bound to the HMGCS1 mRNA 3’UTR, a rate-limiting enzyme in cholesterol biosynthesis, thus increasing HMGCS1 mRNA stability and protein expression and subsequently elevating intracellular cholesterol level. Elevated cholesterol inhibited of the ubiquitin-dependent degradation of HIF-1α by binding to PHD2 and reducing PHD2-mediated hydroxylation of HIF-1α, which further resulted in HIF-1α signaling pathway activation. Moreover, RPL6, HMGCS1 and HIF-1α were coexpressed in HCC tissues with extrahepatic metastasis. Taken together, this study provides new insights into the dynamic transcriptome profiles of HCC pulmonary metastasis and establishes an important role for the RPL6-HMGCS1-HIF-1α axis in HCC metastasis, suggesting potential prognostic biomarkers and therapeutic targets in HCC.
Project description:The identification of predictive markers to determine the premetastatic phase before metastasis is critical for developing effective strategies for early detection and prevention. By applying the dynamic network biomarker (DNB) approach to analyze time-series transcriptomic data from a pulmonary metastasis HCC mouse model, we revealed that the premetastatic phase occurred during the fourth week after implantation. A total of 142 DNB genes were identified as functionally important biomarkers of this critical phase, among which 60S ribosomal protein L6 (RPL6) was a core DNB member. RPL6 was significantly upregulated in HCC tissues with extrahepatic metastasis and was strongly correlated with poor prognosis in HCC patients. RPL6 promoted the invasion and metastasis of HCC cells, both in vitro and in vivo. Mechanistically, RPL6 directly bound to the HMGCS1 mRNA 3’UTR, a rate-limiting enzyme in cholesterol biosynthesis, thus increasing HMGCS1 mRNA stability and protein expression and subsequently elevating intracellular cholesterol level. Elevated cholesterol inhibited of the ubiquitin-dependent degradation of HIF-1α by binding to PHD2 and reducing PHD2-mediated hydroxylation of HIF-1α, which further resulted in HIF-1α signaling pathway activation. Moreover, RPL6, HMGCS1 and HIF-1α were coexpressed in HCC tissues with extrahepatic metastasis. Taken together, this study provides new insights into the dynamic transcriptome profiles of HCC pulmonary metastasis and establishes an important role for the RPL6-HMGCS1-HIF-1α axis in HCC metastasis, suggesting potential prognostic biomarkers and therapeutic targets in HCC.
Project description:Mutational inactivation of VHL is the earliest genetic event in the majority of ccRCCs, leading to activation of the HIF-1α and HIF-2α transcription factors. While correlative studies of human ccRCCs and functional studies using human ccRCC cell lines have implicated HIF-1α as an inhibitor and HIF-2α as a promoter of aggressive tumour behaviours, their roles in tumour onset have not been functionally addressed. Using an autochthonous ccRCC model, we show genetically that Hif1a is necessary for tumour formation whereas Hif2a deletion has only minor effects on tumour initiation and growth. Both HIF-1α and HIF-2α are necessary for the clear cell phenotype. Transcriptomic and proteomic analyses revealed that HIF-1α regulates glycolysis while HIF-2α regulates genes associated with lipoprotein metabolism, ribosome biogenesis and E2F and MYC transcriptional activities. Deficiency of HIF-2α increased CD8+ T cell infiltration and activation. These studies reveal different functions of HIF-1α and HIF-2α in ccRCC. SIGNIFICANCE The roles of HIF-1α and HIF-2α in ccRCC pathogenesis remain unclear. Using a mouse genetic approach we show that HIF-1α but not HIF-2α is important for tumour formation, contrary to predictions from studies of human ccRCC. We show that HIF-1α and HIF-2α transcriptionally regulate different aspects of metabolism and identify HIF-2α as a suppressor of immune cell infiltration and activation.