Differential contribution of n-and c-terminal regions of hif1α and hif2α to their target gene selectivity

Cellular response to hypoxia is controlled by the hypoxia-inducible transcription factors HIF1α and HIF2α. Some genes are preferentially induced by HIF1α or HIF2α, as has been explored in some cell models and for particular sets of genes. Here we have extended this analysis to other HIF-dependent genes using in vitro WT8 renal carcinoma cells and in vivo conditional Vhl-deficient mice models. Moreover, we generated chimeric HIF1/2 transcription factors to study the contribution of the HIF1α and HIF2α DNA binding/heterodimerization and transactivation domains to HIF target specificity. We show that the induction of HIF1α-dependent genes in WT8 cells, such as CAIX (CAR9) and BNIP3, requires both halves of HIF, whereas the HIF2α transactivation domain is more relevant for the induction of HIF2 target genes like the amino acid carrier SLC7A5. The HIF selectivity for some genes in WT8 cells is conserved in Vhl-deficient lung and liver tissue, whereas other genes like Glut1 (Slc2a1) behave distinctly in these tissues. Therefore the relative contribution of the DNA binding/heterodimerization and transactivation domains for HIF target selectivity can be different when comparing HIF1α or HIF2α isoforms, and that HIF target gene specificity is conserved in human and mouse cells for some of the genes analyzed.This work was supported by grants from Ministerio de Economia y Competitividad (SAF2016-76815-R and SAF2017-90794-REDT), Ministerio de Ciencia e Innovación (PID2019-106371RB-I00) and Fundació La Marató de TV3 (534/C/2016). A.A.U is supported by the CAM “Atracción de Talento” program and Universidad Autónoma de Madrid, grant SI1/PJI/2019-0039

Lat-1 and glut-1 carrier expression and its prognostic value in gastroenteropancreatic neuroendocrine tumors

Cancer cells develop mechanisms that increase nutrient uptake, including key nutrient carriers, such as amino acid transporter 1 (LAT-1) and glucose transporter 1 (GLUT-1), regulated by the oxygen-sensing Von Hippel Lindau-hypoxia-inducible factor (VHL-HIF) transcriptional pathway. We aimed to analyze these metabolic players in gastroenteropancreatic neuroendocrine tumors (GEP-NET) and correlate them with tumor malignancy and progression. LAT-1, GLUT-1, and pVHL expression was analyzed in 116 GEP-NETs and 48 peritumoral tissue samples by immunohistochemistry. LAT-1 was stably silenced using specific shRNA in the human NET BON cell line. LAT-1 expression was significantly increased in tumor tissue compared to non-tumor tissue in both gastrointestinal (67% vs. 44%) and pancreatic NETs (54% vs. 31%). Similarly, GLUT-1 was substantially elevated in gastrointestinal (74% vs. 19%) and pancreatic (58% vs. 4%) NETs. In contrast, pVHL expression was decreased (85% vs. 58%) in pancreatic NETs. Tumors with metastases at diagnosis displayed increased LAT-1 and GLUT-1 and decreased pVHL expression (p < 0.001). In accordance with these data, silencing LAT-1 curtailed cell proliferation in BON cells. These findings suggest that specific mechanisms that increase nutrient uptake, such as LAT-1 and GLUT-1, are increased in GEP-NETs, whereas pVHL is decreased. These markers might be related to the proliferation and metastatic capacity of these tumors.This work was supported by the following grants: Proyectos de Investigación en Salud (FIS) PIE13-0041, PI16-02091 and PI19-00584 (funded by Instituto de Salud Carlos III), TIRONET2-CM, B2017/BMD-3724 (funded by Comunidad de Madrid), GETNE G1707 and GCI1901 (funded by Grupo Español de Tumores Neuroendocrinos y Endocrinos) and cofinanced by FEDER funds to M.M. Proyectos de Investigación en Salud (FIS) PI19/01316-FEDER (funded by Instituto de Salud Carlos III), given to J.C.T. Grants from the Ministerio de Economia y Competitividad (SAF2016-76815 and SAF2017-90794-REDT), and Fundació La Marato de TV3 (534/C/2016) ceded to J.A

Differential Contribution of N- and C-Terminal Regions of HIF1α and HIF2α to Their Target Gene Selectivity

Cellular response to hypoxia is controlled by the hypoxia-inducible transcription factors HIF1α and HIF2α. Some genes are preferentially induced by HIF1α or HIF2α, as has been explored in some cell models and for particular sets of genes. Here we have extended this analysis to other HIF-dependent genes using in vitro WT8 renal carcinoma cells and in vivo conditional Vhl-deficient mice models. Moreover, we generated chimeric HIF1/2 transcription factors to study the contribution of the HIF1α and HIF2α DNA binding/heterodimerization and transactivation domains to HIF target specificity. We show that the induction of HIF1α-dependent genes in WT8 cells, such as CAIX (CAR9) and BNIP3, requires both halves of HIF, whereas the HIF2α transactivation domain is more relevant for the induction of HIF2 target genes like the amino acid carrier SLC7A5. The HIF selectivity for some genes in WT8 cells is conserved in Vhl-deficient lung and liver tissue, whereas other genes like Glut1 (Slc2a1) behave distinctly in these tissues. Therefore the relative contribution of the DNA binding/heterodimerization and transactivation domains for HIF target selectivity can be different when comparing HIF1α or HIF2α isoforms, and that HIF target gene specificity is conserved in human and mouse cells for some of the genes analyzed

Papel del transportador de aminoácidos LAT1 en eritropoyesis y su regulación por el factor de respuesta a hipoxia HIF2α

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 12-11-2021Esta tesis tiene embargado el acceso al texto completo hasta el 12-05-2023Cellular response to hypoxia is largely controlled by the hypoxia-inducible transcription factors HIF1α and HIF2α. In particular, HIF2α - but not HIF1α - isoform drives cellular proliferation in some particular biological settings when oxygen becomes limited. The amino acid carrier LAT1 (SLC7A5) has been involved in this proliferative response. This amino acid carrier is composed by LAT1 - that is transcriptionally induced by the HIF2α isoform - and the CD98 subunit. In order to investigate which HIF2α region is required to explain its specific effect on SLC7A5 we generated chimeric proteins of the HIF transcription factors where HIF1α (or HIF2α) DNA binding/heterodimerization domain were fused to HIF2α (or HIF1α) transactivation domain. We show that HIF2α transactivation domain is more relevant to promote HIF2α-dependent gene transcription including the induction of SLC7A5 (LAT1). In contrast HIF1α-dependent specific induction of genes such as CAIX or BNIP3 is more dependent of both binding/heterodimerization and transactivation domains of HIF1α. In addition to this molecular characterization, we also assessed the possible role of HIF2α-LAT1 molecular axis in erythropoiesis taking into account that this biological process requires the proliferation of erythroid precursors to increase tissue oxygenation in hypoxic conditions. In this study we first demonstrate that LAT1/CD98 heterodimer is expressed in splenic erythroid precursors as well as in circulating reticulocytes, but not in mature erythrocytes. Moreover, surface LAT1/CD98 expression in erythroid precursors is further induced in anemic conditions induced by phenylhidrazine treatment or phlebotomy. Erythrocyte Slc7a5-deficient mice do not show differences in the number of circulating red blood cells in baseline or in anemic conditions when compared with control mice. However, circulating red blood cells in these mice are smaller with a reduced mean corpuscular volume leading to reduced hematocrit. Moreover, erythroid LAT1 (SLC7A5) deficiency reduces circulating hemoglobin as well as mean corpuscular hemoglobin. As a consequence, erythrocyte Slc7a5-deficient mice have elevated levels of circulating erythropoietin (EPO), which can be considered as a chronic compensation to the hypoxemia derived from the dysfunctional (smaller) circulating erythrocytes. Overall, our studies reveal a new physiopathological role of LAT1, an aspect that is relevant taking into consideration that the vast majority of studies about LAT1 were restricted to its role in cancer biology. Finally, we consider that this study might generate new clinical perspectives for anemia treatment and, in particular, those that limit the size of erythrocytes such as microcytic anemi

HIF1α Suppresses Tumor Cell Proliferation through Inhibition of Aspartate Biosynthesis

Cellular aspartate drives cancer cell proliferation, but signaling pathways that rewire aspartate biosynthesis to control cell growth remain largely unknown. Hypoxia-inducible factor-1α (HIF1α) can suppress tumor cell proliferation. Here, we discovered that HIF1α acts as a direct repressor of aspartate biosynthesis involving the suppression of several key aspartate-producing proteins, including cytosolic glutamic-oxaloacetic transaminase-1 (GOT1) and mitochondrial GOT2. Accordingly, HIF1α suppresses aspartate production from both glutamine oxidation as well as the glutamine reductive pathway. Strikingly, the addition of aspartate to the culture medium is sufficient to relieve HIF1α-dependent repression of tumor cell proliferation. Furthermore, these key aspartate-producing players are specifically repressed in VHL-deficient human renal carcinomas, a paradigmatic tumor type in which HIF1α acts as a tumor suppressor, highlighting the in vivo relevance of these findings. In conclusion, we show that HIF1α inhibits cytosolic and mitochondrial aspartate biosynthesis and that this mechanism is the molecular basis for HIF1α tumor suppressor activity.ISSN:2666-3864ISSN:2211-124

HIF1 alpha Suppresses Tumor Cell Proliferation through Inhibition of Aspartate Biosynthesis

Cellular aspartate drives cancer cell proliferation, but signaling pathways that rewire aspartate biosynthesis to control cell growth remain largely unknown. Hypoxia-inducible factor-1α (HIF1α) can suppress tumor cell proliferation. Here, we discovered that HIF1α acts as a direct repressor of aspartate biosynthesis involving the suppression of several key aspartate-producing proteins, including cytosolic glutamic-oxaloacetic transaminase-1 (GOT1) and mitochondrial GOT2. Accordingly, HIF1α suppresses aspartate production from both glutamine oxidation as well as the glutamine reductive pathway. Strikingly, the addition of aspartate to the culture medium is sufficient to relieve HIF1α-dependent repression of tumor cell proliferation. Furthermore, these key aspartate-producing players are specifically repressed in VHL-deficient human renal carcinomas, a paradigmatic tumor type in which HIF1α acts as a tumor suppressor, highlighting the in vivo relevance of these findings. In conclusion, we show that HIF1α inhibits cytosolic and mitochondrial aspartate biosynthesis and that this mechanism is the molecular basis for HIF1α tumor suppressor activity.status: publishe

Erythroid SLC7A5/SLC3A2 amino acid carrier controls red blood cell size and maturation

Summary: Inhibition of the heterodimeric amino acid carrier SLC7A5/SLC3A2 (LAT1/CD98) has been widely studied in tumor biology but its role in physiological conditions remains largely unknown. Here we show that the SLC7A5/SLC3A2 heterodimer is constitutively present at different stages of erythroid differentiation but absent in mature erythrocytes. Administration of erythropoietin (EPO) further induces SLC7A5/SLC3A2 expression in circulating reticulocytes, as it also occurs in anemic conditions. Although Slc7a5 gene inactivation in the erythrocyte lineage does not compromise the total number of circulating red blood cells (RBCs), their size and hemoglobin content are significantly reduced accompanied by a diminished erythroblast mTORC1 activity. Furthermore circulating Slc7a5-deficient reticulocytes are characterized by lower transferrin receptor (CD71) expression as well as mitochondrial activity, suggesting a premature transition to mature RBCs. These data reveal that SLC7A5/SLC3A2 ensures adequate maturation of reticulocytes as well as the proper size and hemoglobin content of circulating RBCs

Head and neck cancer surgery during the COVID-19 pandemic: An international, multicenter, observational cohort study

Background: The aims of this study were to provide data on the safety of head and neck cancer surgery currently being undertaken during the coronavirus disease 2019 (COVID-19) pandemic. Methods: This international, observational cohort study comprised 1137 consecutive patients with head and neck cancer undergoing primary surgery with curative intent in 26 countries. Factors associated with severe pulmonary complications in COVID-19–positive patients and infections in the surgical team were determined by univariate analysis. Results: Among the 1137 patients, the commonest sites were the oral cavity (38%) and the thyroid (21%). For oropharynx and larynx tumors, nonsurgical therapy was favored in most cases. There was evidence of surgical de-escalation of neck management and reconstruction. Overall 30-day mortality was 1.2%. Twenty-nine patients (3%) tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within 30 days of surgery; 13 of these patients (44.8%) developed severe respiratory complications, and 3.51 (10.3%) died. There were significant correlations with an advanced tumor stage and admission to critical care. Members of the surgical team tested positive within 30 days of surgery in 40 cases (3%). There were significant associations with operations in which the patients also tested positive for SARS-CoV-2 within 30 days, with a high community incidence of SARS-CoV-2, with screened patients, with oral tumor sites, and with tracheostomy. Conclusions: Head and neck cancer surgery in the COVID-19 era appears safe even when surgery is prolonged and complex. The overlap in COVID-19 between patients and members of the surgical team raises the suspicion of failures in cross-infection measures or the use of personal protective equipment. Lay Summary: Head and neck surgery is safe for patients during the coronavirus disease 2019 pandemic even when it is lengthy and complex. This is significant because concerns over patient safety raised in many guidelines appear not to be reflected by outcomes, even for those who have other serious illnesses or require complex reconstructions. Patients subjected to suboptimal or nonstandard treatments should be carefully followed up to optimize their cancer outcomes. The overlap between patients and surgeons testing positive for severe acute respiratory syndrome coronavirus 2 is notable and emphasizes the need for fastidious cross-infection controls and effective personal protective equipment