Nuclear HIF-1α Research Articles

A transcription-independent role for HIF-1α in modulating microprocessor assembly.

Microprocessor is an essential nuclear complex responsible for the initial RNase-mediated cleavage of primary miRNA, which is a tightly controlled maturation process that requires the proper assembly of Drosha and DGCR8. Unlike previously identified mechanisms directly targeting the enzymatic subunit Drosha, current knowledge about the biological ways of controlling miRNA nuclear maturation through DGCR8 is less addressed. In this study, we unveiled that the microprocessor assembly is governed by a master gene regulator HIF-1α irrespective of its canonical transcriptional activity. First, a widespread protein binding of HIF-1α with DGCR8 instead of Drosha was observed in response to biological stimulations. Similar protein interactions between their corresponding orthologues in model organisms were also observed. After dissecting the essential protein domains, we noticed that HIF-1α suppresses microprocessor assembly via binding to DGCR8. Furthermore, our results showed that HIF-1α hijacks monomeric DGCR8 thus reducing its dimer formation prior to microprocessor assembly, and consequently, the suppressed microprocessor formation and nuclear processing of primary miRNA were demonstrated. In conclusion, here we unveiled the mechanism of how microprocessor assembly is regulated by HIF-1α, which not only demonstrates a non-transcriptional function of nuclear HIF-1α but also provides new molecular insights into the regulation of microprocessor assembly through DGCR8.

Hypoxia-inducible factor-1α mediates reflux-induced epithelial-mesenchymal plasticity in Barrett’s oesophagus patients

IntroductionEpithelial-mesenchymal plasticity (EMP), the process through which epithelial cells acquire mesenchymal features, is needed for wound repair but also might contribute to cancer initiation. Earlier, in vitro studies showed that...

Activation of the hypoxia-inducible factor pathway by roxadustat improves glucose metabolism in human primary myotubes from men

Aims/hypothesisHypoxia-inducible factor prolyl 4-hydroxylase (HIF-P4H) enzymes regulate adaptive cellular responses to low oxygen concentrations. Inhibition of HIF-P4Hs leads to stabilisation of hypoxia-inducible factors (HIFs) and activation of the HIF pathway affecting multiple biological processes to rescue cells from hypoxia. As evidence from animal models suggests that HIF-P4H inhibitors could be used to treat metabolic disorders associated with insulin resistance, we examined whether roxadustat, an HIF-P4H inhibitor approved for the treatment of renal anaemia, would have an effect on glucose metabolism in primary human myotubes.MethodsPrimary skeletal muscle cell cultures, established from biopsies of vastus lateralis muscle from men with normal glucose tolerance (NGT) (n=5) or type 2 diabetes (n=8), were treated with roxadustat. Induction of HIF target gene expression was detected with quantitative real-time PCR. Glucose uptake and glycogen synthesis were investigated with radioactive tracers. Glycolysis and mitochondrial respiration rates were measured with a Seahorse analyser.ResultsExposure to roxadustat stabilised nuclear HIF1α protein expression in human myotubes. Treatment with roxadustat led to induction of HIF target gene mRNAs for GLUT1 (also known as SLC2A1), HK2, MCT4 (also known as SLC16A4) and HIF-P4H-2 (also known as PHD2 or EGLN1) in myotubes from donors with NGT, with a blunted response in myotubes from donors with type 2 diabetes. mRNAs for LDHA, PDK1 and GBE1 were induced to a similar degree in myotubes from donors with NGT or type 2 diabetes. Exposure of myotubes to roxadustat led to a 1.4-fold increase in glycolytic rate in myotubes from men with NGT (p=0.0370) and a 1.7-fold increase in myotubes from donors with type 2 diabetes (p=0.0044), with no difference between the groups (p=0.1391). Exposure to roxadustat led to a reduction in basal mitochondrial respiration in both groups (p<0.01). Basal glucose uptake rates were similar in myotubes from donors with NGT (20.2 ± 2.7 pmol mg−1 min−1) and type 2 diabetes (25.3 ± 4.4 pmol mg−1 min−1, p=0.4205). Treatment with roxadustat enhanced insulin-stimulated glucose uptake in myotubes from donors with NGT (1.4-fold vs insulin-only condition, p=0.0023). The basal rate of glucose incorporation into glycogen was lower in myotubes from donors with NGT (233 ± 12.4 nmol g−1 h−1) than in myotubes from donors with type 2 diabetes (360 ± 40.3 nmol g−1 h−1, p=0.0344). Insulin increased glycogen synthesis by 1.9-fold (p=0.0025) in myotubes from donors with NGT, whereas roxadustat did not affect their basal or insulin-stimulated glycogen synthesis. Insulin increased glycogen synthesis by 1.7-fold (p=0.0031) in myotubes from donors with type 2 diabetes. While basal glycogen synthesis was unaffected by roxadustat, pretreatment with roxadustat enhanced insulin-stimulated glycogen synthesis in myotubes from donors with type 2 diabetes (p=0.0345).Conclusions/interpretationRoxadustat increases glycolysis and inhibits mitochondrial respiration in primary human myotubes regardless of diabetes status. Roxadustat may also improve insulin action on glycogen synthesis in myotubes from donors with type 2 diabetes.Graphical

Differential effects of coverslip-induced hypoxia and cobalt chloride mimetic hypoxia on cellular stress, metabolism, and nuclear structure

Hypoxia has profound effects on cell physiology, both in normal or pathological settings like cancer. In this study, we asked whether a variant of coverslip-induced hypoxia that recapitulates the conditions found in the tumor microenvironment would elicit similar cellular responses compared to the well established model of cobalt chloride-induced hypoxia. Comparable levels of nuclear HIF-1α were observed after 24 h of coverslip-induced hypoxia or cobalt chloride treatment in CAL-27 oral squamous carcinoma cells. However, cellular stress levels assessed by reactive oxygen species production and lipid droplet accumulation were markedly increased in coverslip-induced hypoxia compared to cobalt chloride treatment. Conversely, mitochondrial ATP production sharply decreased after coverslip-induced hypoxia but was preserved in the presence of cobalt chloride. Coverslip-induced hypoxia also had profound effects in nuclear organization, assessed by changes in nuclear dry mass distribution, whereas these effects were much less marked after cobalt chloride treatment. Taken together, our results show that coverslip-induced hypoxia effects on cell physiology and structure are more pronounced than mimetic hypoxia induced by cobalt chloride treatment. Considering also the simplicity of coverslip-induced hypoxia, our results therefore underscore the usefulness of this method to recapitulate in vitro the effects of hypoxic microenvironments encountered by cells in vivo.

Expression of HIF‑α and their association with clinicopathological parameters in clinical renal cell carcinoma.

This study aimed to assess the cellular localization and expression levels of hypoxia-inducible factor (HIF) -α proteins (specifically HIF-1α, HIF-2α, and HIF-3α) that play a role in the hypoxia pathway and to determine their correlation with clinicopathological parameters and patient survival in renal cell carcinoma (RCC). Tissue microarray (TMA) with cores from 150 clear cell RCCs and 31 non-ccRCC samples. HIF-1α, HIF-2α, and HIF-3α antibodies were used for immunohistochemistry (IHC) of TMA to evaluate the cellular localization and expression levels of HIF-α proteins, specifically in relation to the hypoxia pathway. The expression levels of the HIF-α proteins were higher in the nucleus than in the cytoplasm. Furthermore, the nuclear expression levels of all HIF-α proteins were significantly higher in clear cell RCC (ccRCC) than in non-ccRCC. Cytoplasmic HIF-3α expression was also higher in ccRCC than in non-ccRCC, whereas cytoplasmic HIF-1α and HIF-2α expression levels were similar between the different RCC types. In ccRCC, nuclear HIF-1α expression levels correlated with both nuclear HIF-2α and HIF-3α levels, whereas cytoplasmic HIF-3α expression levels were associated with HIF-1α only.In non-ccRCC, there was a positive correlation observed between nuclear HIF-1α and HIF-3α expression, but no correlation was found with HIF-2α. In patients with ccRCC, the nuclear expressions of HIF-1α and HIF-3α was significantly associated with cancer-specific survival (CSS) in univariate analysis. This association was no longer evident in multivariate analysis. Notably, there was no correlation observed between nuclear HIF-2α expression and CSS in these patients. In contrast, cytoplasmic expression levels showed no association with CSS. The expression levels of the three primary HIF-α proteins were found to be higher in the nucleus than in the cytoplasm. Furthermore, the results indicated that HIF-3α and HIF-1α expression levels were significant univariate factors associated with CSS in patients with clear cell RCC. These results highlight the critical role that HIF-3α and HIF-1α play in the hypoxia pathway.

TRIM2 Selectively Regulates Inflammation-Driven Pathological Angiogenesis without Affecting Physiological Hypoxia-Mediated Angiogenesis.

Angiogenesis is a critical physiological response to ischemia but becomes pathological when dysregulated and driven excessively by inflammation. We recently identified a novel angiogenic role for tripartite-motif-containing protein 2 (TRIM2) whereby lentiviral shRNA-mediated TRIM2 knockdown impaired endothelial angiogenic functions in vitro. This study sought to determine whether these effects could be translated in vivo and to determine the molecular mechanisms involved. CRISPR/Cas9-generated Trim2-/- mice that underwent a periarterial collar model of inflammation-induced angiogenesis exhibited significantly less adventitial macrophage infiltration relative to wildtype (WT) littermates, concomitant with decreased mRNA expression of macrophage marker Cd68 and reduced adventitial proliferating neovessels. Mechanistically, TRIM2 knockdown in endothelial cells in vitro attenuated inflammation-driven induction of critical angiogenic mediators, including nuclear HIF-1α, and curbed the phosphorylation of downstream effector eNOS. Conversely, in a hindlimb ischemia model of hypoxia-mediated angiogenesis, there were no differences in blood flow reperfusion to the ischemic hindlimbs of Trim2-/- and WT mice despite a decrease in proliferating neovessels and arterioles. TRIM2 knockdown in vitro attenuated hypoxia-driven induction of nuclear HIF-1α but had no further downstream effects on other angiogenic proteins. Our study has implications for understanding the role of TRIM2 in the regulation of angiogenesis in both pathophysiological contexts.

Association between the apoptotic effect of Cabazitaxel and its pro-oxidant efficacy on the redox adaptation mechanisms in prostate cancer cells with different resistance phenotypes

ABSTRACT Redox adaptation causes poor prognosis by adapting cancer cells to excessive oxidative stress. Previously, we introduced an oxidative stress-resistant metastatic prostate cancer (mPC) model (LNCaP-HPR) that redox adaptation reduced the effect of Cabazitaxel (Cab), the last taxane-derivative for metastatic castration-resistant PC (mCRPC). Whereas, we investigated for the first time whether there is an association between the altered apoptotic effect and pro-oxidant efficacy of Cab on the redox adaptation in PC cells with different phenotypes, including LNCaP mPC, LNCaP-HPR, C4–2 mCRPC, and RWPE-1 cells. Cab was shown pro-oxidant efficacy proportionally with the apoptotic effect, more prominent in the less aggressive LNCaP cells, by increasing the endogenous ROS, mitochondrial damage, and inhibiting nuclear ROS scavengers, p-Nrf2 and HIF-1α. However, the pro-oxidant and apoptotic effect was lower in the LNCaP-HPR and C4–2 cells, indicating that the drug sensitivity of the cells adapted to survive with more ROS was reduced via altered regulation of redox adaptation. Additionally, unlike LNCaP, Cab caused an increase in the p-NF-κB activation, suggesting that the p-NF-κB might accompany maintaining survival with the increased ROS in the aggressive PC cells. Moreover, the cytotoxic and apoptotic effects of Cab were less on RWPE-1 cells compared to LNCaP but were closer to those on the more aggressive LNCaP-HPR and C4–2 cells, except for the changing pro-oxidant effect of Cab. Consequently, this study indicates the variable pro-oxidant effects of Cab on redox-sensitive proteins, which could be a target for improving Cab’s apoptotic effect more in aggressive PC cells.

MITF regulates the subcellular location of HIF1α through SUMOylation to promote the invasion and metastasis of daughter cells derived from polyploid giant cancer cells.

High concentrations of cobalt chloride (CoCl2) can induce the formation of polyploid giant cancer cells (PGCCs) in various tumors, which can produce daughter cells with strong proliferative, migratory and invasive abilities via asymmetric division. To study the role of hypoxia‑inducible factor (HIF) 1α in the formation of PGCCs, colon cancer cell lines Hct116 and LoVo were used as experimental subjects. Western blotting, nuclear and cytoplasmic protein extraction and immunocytochemical experiments were used to compare the changes in the expression and subcellular localization of HIF1α, microphthalmia‑associated transcription factor (MITF), protein inhibitor of activated STAT protein4 (PIAS4) and von Hippel‑Lindau disease tumor suppressor (VHL) after treatment with CoCl2. The SUMOylation of HIFα was verified by co‑immunoprecipitation assay. After inhibiting HIF1α SUMOylation, the changes in proliferation, migration and invasion abilities of Hct116 and LoVo were compared by plate colony formation, wound healing and Transwell migration and invasion. In addition, lysine sites that led to SUMOylation of HIF1α were identified through site mutation experiments. The results showed that CoCl2 can induce the formation of PGCCs with the expression level of HIF1α higher in treated cells than in control cells. HIF1α was primarily located in the cytoplasm of control cell. Following CoCl2 treatment, the subcellular localization of HIF1α was primarily in the nuclei of PGCCs with daughter cells (PDCs). After treatment with SUMOylation inhibitors, the nuclear HIF1α expression in PDCs decreased. Furthermore, their proliferation, migration and invasion abilities also decreased. After inhibiting the expression of MITF, the expression of HIF1α decreased. MITF can regulate HIF1α SUMOylation. Expression and subcellular localization of VHL and HIF1α did not change following PIAS4 knockdown. SUMOylation of HIF1α occurs at the amino acid sites K391 and K477 in PDCs. After mutation of the two sites, nuclear expression of HIF1α in PDCs was reduced, along with a significant reduction in the proliferation, migration and invasion abilities. In conclusion, the post‑translation modification regulated the subcellular location of HIF1α and the nuclear expression of HIF1α promoted the proliferation, migration and invasion abilities of PDCs. MITF could regulate the transcription and protein levels of HIF1α and participate in the regulation of HIF1α SUMOylation.

ZINC RESTRICTION BY INTESTINAL E. COLI - PRODUCED YERSINIABACTIN ACTIVATES HIF-1α IN MACROPHAGES TO PROMOTE CD-ASSOCIATED FIBROSIS

Abstract Intestinal fibrosis is a significant complication in Crohn’s disease (CD) patients, causing severe intestinal strictures and obstructions that are ultimately relieved by surgical bowel resection. Mechanisms driving CD-associated fibrosis are poorly understood but implicate the microbiome, especially adherent-invasive E. coli (AIEC). Previously, our work demonstrated that AIEC production of the metallophore yersiniabactin (Ybt) promotes fibrosis in the well-established gnotobiotic Il10-/- CD mouse model. These pro-fibrotic effects do not require bacterial uptake/utilization of Ybt, suggesting Ybt directly targets the host. Here we show that CD11b+ F4/80+ MHCIIlow macrophages are abundant in the submucosa and muscularis of fibrotic lesions in our mouse model. Similarly, CD68+ macrophages are abundant in strictures, not adjacent margins, of ileocolonic CD patients. We hypothesized that Ybt targets host macrophages to drive fibrosis. A critical metal-dependent pathway for immune cell function during inflammation is HIF-1α. We demonstrate that AIEC infection activates HIF-1α in macrophages, which is dependent upon Ybt production. Importantly, in fibrotic lesions of CD mouse models and resected strictures of ileocolonic CD patients, HIF-1α is activated and localized in nuclei of F4/80+ or CD68+ macrophages. We identified that zinc sequestration by Ybt is responsible for HIF-1α activation, a novel mechanism of HIF-1α stabilization. Furthermore, Ybt-producing AIEC induce potent pro-fibrotic gene expression in macrophage:fibroblast co-cultures, a process dependent on macrophage HIF-1α. This implies that Ybt induces pro-fibrotic macrophages, activating adjacent fibroblasts and fostering intestinal fibrosis. In summary, our findings suggest that as intestinal macrophages encounter AIEC, Ybt sequesters zinc, causes an accumulation of nuclear HIF-1α, and this imbalance of metal homeostasis drives pro-fibrotic macrophage and fibroblast activation. Ultimately, effective fibrotic remodeling fails and leads to intestinal fibrosis in CD patients.

HIF-1α/MMP-9 Axis Is Required in the Early Phases of Skeletal Myoblast Differentiation under Normoxia Condition In Vitro.

Hypoxia-inducible factor (HIF)-1α represents an oxygen-sensitive subunit of HIF transcriptional factor, which is usually degraded in normoxia and stabilized in hypoxia to regulate several target gene expressions. Nevertheless, in the skeletal muscle satellite stem cells (SCs), an oxygen level-independent regulation of HIF-1α has been observed. Although HIF-1α has been highlighted as a SC function regulator, its spatio-temporal expression and role during myogenic progression remain controversial. Herein, using biomolecular, biochemical, morphological and electrophysiological analyses, we analyzed HIF-1α expression, localization and role in differentiating murine C2C12 myoblasts and SCs under normoxia. In addition, we evaluated the role of matrix metalloproteinase (MMP)-9 as an HIF-1α effector, considering that MMP-9 is involved in myogenesis and is an HIF-1α target in different cell types. HIF-1α expression increased after 24/48 h of differentiating culture and tended to decline after 72 h/5 days. Committed and proliferating mononuclear myoblasts exhibited nuclear HIF-1α expression. Differently, the more differentiated elongated and parallel-aligned cells, which are likely ready to fuse with each other, show a mainly cytoplasmic localization of the factor. Multinucleated myotubes displayed both nuclear and cytoplasmic HIF-1α expression. The MMP-9 and MyoD (myogenic activation marker) expression synchronized with that of HIF-1α, increasing after 24 h of differentiation. By means of silencing HIF-1α and MMP-9 by short-interfering RNA and MMP-9 pharmacological inhibition, this study unraveled MMP-9's role as an HIF-1α downstream effector and the fact that the HIF-1α/MMP-9 axis is essential in morpho-functional cell myogenic commitment.

Expression of HIF-1α in pediatric asthmatic patients.

Several studies have suggested that HIF-1α regulates eosinophil activity and induces epithelial inflammation via NF-κB activation in the pathophysiology of asthma. The purpose of this study was to examine the expression of the transcription factors HIF-1α and nuclear HIF in mononuclear cells obtained from peripheral blood samples of healthy pediatric patients, asthmatic patients, and asthmatic exacerbations, regardless of disease severity. HIF-1 levels were measured using immunocytochemistry in 133 patients aged 6 to 17 years in this crosssectional and comparative study. A microscope was used to examine glass slides, and positive cells were counted in four fields per slide using an image analyzer. HIF-1α and nuclear HIF levels were significantly higher in asthma patients and even higher in patients experiencing asthma attacks (p<0.0001, 95% CI). There was no significant difference in the percentage of HIF-1α expression between groups with intermittent asthma and those with mild persistent asthma, nor between patients with asthma and those experiencing asthma exacerbations. When compared to healthy individuals, the expression of nuclear HIF and HIF-1α is increased in peripheral mononuclear cells in asthma patients and even more so in asthma exacerbations. This suggests that HIF-1α is important in the pathogenesis of this disease.

Expression of hypoxia-inducible genes is suppressed in altered gravity due to impaired nuclear HIF1α accumulation

Extravehicular activities, the backbone of manned space exploration programs, set astronauts into mild hypoxia. Unfortunately, microgravity aggravates threatening symptoms of hypoxia such as vision impairment and brain edema. Hypoxia-inducible factors (HIFs) sense cellular hypoxia and, subsequently, change the cells’ expression profile instantaneously by rapidly translocating—most likely cytoskeleton-dependently—into the nucleus and subsequently forming transcription complexes with other proteins. We tested the hypothesis that this fundamental process could be altered by sudden changes in gravitational forces in parabolic flights using a newly developed pocket-size cell culture lab that deoxygenizes cells within 15 min. Sudden gravity changes (SGCs 1g–1.8g–0g–1.8g–1g) during hypoxic exposure suppressed expression of the HIF1α-dependent genes investigated as compared with hypoxia at constant 1g. Normoxic cells subjected to SGCs showed reduced nuclear but not cytoplasmatic HIF1α signal and appeared to have disturbed cytoskeleton architecture. Inhibition of the actin-dependent intracellular transport using a combination of myosin V and VI inhibitors during hypoxia mimicked the suppression of the HIF1α-dependent genes observed during hypoxic exposure during SGCs. Thus, SGCs seem to disrupt the cellular response to hypoxia by impairing the actin-dependent translocation of HIF1α into the nucleus.

Cav3.2 T-Type calcium channels downregulation attenuates bone cancer pain induced by inhibiting IGF-1/HIF-1α signaling pathway in the rat spinal cord

BackgroundBone cancer pain (BCP) is one of the most ubiquitous and refractory symptoms of cancer patients that needs to be urgently addressed. Substantial studies have revealed the pivotal role of Cav3.2 T-type calcium channels in chronic pain, however, its involvement in BCP and the specific molecular mechanism have not been fully elucidated. MethodsThe expression levels of Cav3.2, insulin-like growth factor 1(IGF-1), IGF-1 receptor (IGF-1R) and hypoxia-inducible factor-1α (HIF-1α) were detected by Western blot in tissues and cells. X-ray and Micro CT used to detect bone destruction in rats. Immunofluorescence was used to detect protein expression and spatial location in the spinal dorsal horn. Electrophoretic mobility shift assay used to verify the interaction between HIF-1α and Cav3.2. ResultsThe results showed that the expression of Cav3.2 channel was upregulated and blockade of this channel alleviated mechanical allodynia and thermal hyperalgesia in BCP rats. Additionally, inhibition of IGF-1/IGF-1R signaling not only reversed the BCP-induced upregulation of Cav3.2 and HIF-1α, but also decreased nociceptive hypersensitivity in BCP rats. Inhibition of IGF-1 increased Cav3.2 expression levels, which were abolished by pretreatment with HIF-1α siRNA in PC12 cells. Furthermore, nuclear HIF-1α bound to the promoter of Cav3.2 to regulate the Cav3.2 transcription level, and knockdown of HIF-1α suppresses the IGF-1-induced upregulation of Cav3.2 and pain behaviors in rats with BCP. ConclusionThese findings suggest that spinal Cav3.2 T-type calcium channels play a central role during the development of bone cancer pain in rats via regulation of the IGF-1/IGF-1R/HIF-1α pathway.

Combined expression of HIF1α, VEGF and HER2 predicts metastasis, relapse and response to combination chemotherapy in colorectal cancer patients

Addressing a single target is the frequent development of drug resistance followed by cancer relapse and treatment failure. Therefore, assessment of simultaneous expression of target molecules is essential to choose the optimal combination therapy for each colorectal cancer patient.This study aims to evaluate the immunohistochemical expression of HIF1α, HER2 and VEGF and to clarify their clinical significance as prognostic factors and predictive markers of FOLFOX (combination chemotherapy inclusive of Leucovorin calcium, Fluorouracil and Oxaliplatin response).Marker expression was retrospectively evaluated by immunohistochemistry in 111 patients with colorectal adenocarcinomas from south Tunisia, followed by statistical analysis.The immunohistochemical staining revealed that 45 %, 80.2 %, 86.5 % and 25.5 % of specimen were positive for nuclear, cytoplasmic HIF1α expression, VEGF and HER2 respectively. Nuclear HIF1α and VEGF were associated with worst prognosis while cytoplasmic HIF1α and HER2 were correlated with favourable prognosis. Multivariate analysis confirms the association between nuclear HIF1α, distant metastasis, relapse, FOLFOX response and 5 years overall survival. HIF1α positivity and HER2 negativity were significantly associated to short survival. Combined immunoprofiles HIF1α+/VEGF+, HIF1α+/HER2-, HIF1α+/VEGF+/HER2- were associated to distant metastasis, cancer relapse and short survival. Interestingly, our findings confirmed that patients bearing a HIF1α positive tumor were significantly more resistant to FOLFOX compared to negative ones (p = 0.002, p ≤ 0.001). Positive expression of HIF1α and VEGF, or decreased expression of HER2 was each associated with poor prognosis and short overall survival. In summary, we found that expression of nuclear HIF1α, alone or combined with VEGF and HER2 serves as a predictive marker of poor prognosis and FOLFOX response in colorectal cancer from south Tunisia.

Abstract P2-20-14: Relationship of HIF-1α and tumor infiltrating lymphocytes in patients with HER2-negative early-stage breast cancer treated with neoadjuvant chemotherapy

Abstract Title: Relationship of HIF-1α and tumor infiltrating lymphocytes in patients with HER2-negative early-stage breast cancer treated with neoadjuvant chemotherapy Authors: Grabinski VF, Oza HH, Gilkes DM, Shah MY, Cimino-Mathews A, Gabrielson E, Zhi I, Pipa Z, Lehman J, Ibanez HE, Ke S, Tsai HL, Stearns V, Santa-Maria CA Introduction: Hypoxia inducible factor 1α (HIF-1α) is an oxygen-dependent transcription factor expressed in areas of hypoxia associated with regulation of immune escape mechanisms, metabolism (CAIX), and results in cancer treatment resistance. High stromal tumor infiltrating lymphocytes (sTILs) are associated with pathologic complete response (pCR) to neoadjuvant chemotherapy (NACT) in breast cancer. We investigated associations between HIF-1α, sTILs and their relationship with pCR after NACT in patients with HER2-negative early-stage breast cancer. Methods: We identified patients with early-stage HER2-negative breast cancer at the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center from 2000-2009 who received NACT, and had accessible breast tissue from biopsy and/or surgery. A pathologist scored nuclear HIF-1α (0, 1, 2, or 3), membranous CAIX (0, 1, 2, or 3), sTILs (0-100) from baseline (ie pre-NACT) and surgical tissue (if non-pCR). Statistical analysis considered the following variables: age, histology, race, menopausal status, tumor size, and node status. Chemotherapy regimens were divided into either anthracycline AND taxane versus anthracycline OR taxane. Wilcoxon rank-sum, Fisher exact, and Jonckheere trends tests were utilized to assess differences between groups. Results: 43 patients met the predefined criteria and were included in the analysis, 16% (n=7) obtained a pCR. Patient race, ethnicity, clinical tumor size, histology, clinical subtype (estrogen receptor positive (ER+) vs. triple negative (TN)), and NACT regimen were not associated with pCR. Older age (p=0.019), postmenopausal status (p=0.009), smaller surgical tumor size (p &amp;lt; 0.001), and surgical node-negative status (p &amp;lt; 0.001) were associated with pCR. Mean TILs score was 32.5% in patients achieving pCR versus 20% in those with non-pCR (p=0.46); mean HIF-1α was 2.42 in pCR group versus 2.57 in non-pCR (p=0.84). There was a trend of lower baseline sTILs with increasing nuclear HIF-1α score (p=0.085). Membranous CAIX was not associated with nuclear HIF-1α or baseline TILs, however, in the small proportion of patients (n=11) with CAIX scoring, higher membranous CAIX score at baseline (1 vs. 0) was associated with pCR (p=0.024). Conclusions: We observed that patients with pCR had a numerically higher sTILs level at baseline consistent with other studies, but did not find a correlation of HIF-1α and pCR. Patients with higher HIF-1α scores tended to have lower sTILS, suggesting that hypoxia may be leading to an immunosuppressive tumor microenvironment. Further characterizing the HIF-1α pathway and its effects on the immune microenvironment may help identify resistance mechanisms to chemotherapy and immunotherapy. Citation Format: Harsh Oza, Victoria Grabinski, Hector Ibanez, Ines Godet, Daniele Gilkes, Ashley Cimino-Mathews, Edward Gabrielson, Mirat Shah, W. Iris Zhi, Zoe Pipa, Jennifer Lehman, Suqi Ke, Hua-Ling Tsai, Vered Stearns, Cesar Augusto Santa-Maria. Relationship of HIF-1α and tumor infiltrating lymphocytes in patients with HER2-negative early-stage breast cancer treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-14.

Air–liquid interface cultures trigger a metabolic shift in intestinal epithelial cells (IPEC-1)

An improved oxygen availability in air–liquid interface (ALI) cultures of enterocytes of the small intestine seems to be primarily responsible for morphological, metabolic, and functional changes. Intestinal porcine epithelial cells 1 (IPEC-1) are less investigated and are rarely used as model for intestinal barrier but showed a profound change of cell shape during ALI cultivation. We aim to answer the following question: Are the observed morphological effects accompanied by changes in metabolic function? A microarray analysis of submerged culture (SMC) and ALI cultures identified 830 significantly regulated genes. Subsequent functional clustering revealed alterations in 31 pathways, with the highest number of regulated genes in metabolic pathways, carbon metabolism, glycolysis, and hypoxia-inducible factor (HIF) signaling. Furthermore, HIF-1α as a mediator of a metabolic switch between glycolysis and oxidative phosphorylation showed a trend of increased mRNA levels in ALI in contrast to a reduced nuclear HIF-1α content in the nucleus. Candidate genes of oxidative phosphorylation such as a mitochondrial marker exhibited enhanced mRNA levels, which was confirmed by western blot analysis. Cytochrome C oxidase (COX) subunit 5B protein was decreased in ALI, although mRNA level was increased. The oxidation of ferrocytochrome C to ferricytochrome C was used for detection of cytochrome C oxidase activity of isolated mitochondria and resulted in a trend of higher activity in ALI. Furthermore, quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. To evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenized cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In contrast, blocking with 2-desoxy-d-glucose (2DG) significantly reduced ATP content in ALI and SMC. These results indicate a metabolic shift in IPEC-1 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis.

BAP1 is a novel regulator of HIF-1α

BAP1 is a powerful tumor suppressor gene characterized by haplo insufficiency. Individuals carrying germline BAP1 mutations often develop mesothelioma, an aggressive malignancy of the serosal layers covering the lungs, pericardium, and abdominal cavity. Intriguingly, mesotheliomas developing in carriers of germline BAP1 mutations are less aggressive, and these patients have significantly improved survival. We investigated the apparent paradox of a tumor suppressor gene that, when mutated, causes less aggressive mesotheliomas. We discovered that mesothelioma biopsies with biallelic BAP1 mutations showed loss of nuclear HIF-1α staining. We demonstrated that during hypoxia, BAP1 binds, deubiquitylates, and stabilizes HIF-1α, the master regulator of the hypoxia response and tumor cell invasion. Moreover, primary cells from individuals carrying germline BAP1 mutations and primary cells in which BAP1 was silenced using siRNA had reduced HIF-1α protein levels in hypoxia. Computational modeling and co-immunoprecipitation experiments revealed that mutations of BAP1 residues I675, F678, I679, and L691 -encompassing the C-terminal domain-nuclear localization signal- to A, abolished the interaction with HIF-1α. We found that BAP1 binds to the N-terminal region of HIF-1α, where HIF-1α binds DNA and dimerizes with HIF-1β forming the heterodimeric transactivating complex HIF. Our data identify BAP1 as a key positive regulator of HIF-1α in hypoxia. We propose that the significant reduction of HIF-1α activity in mesothelioma cells carrying biallelic BAP1 mutations, accompanied by the significant reduction of HIF-1α activity in hypoxic tissues containing germline BAP1 mutations, contributes to the reduced aggressiveness and improved survival of mesotheliomas developing in carriers of germline BAP1 mutations.

GPER-mediated stabilization of HIF-1α contributes to upregulated aerobic glycolysis in tamoxifen-resistant cells.

Tamoxifen is a first-line therapeutic drug for oestrogen-receptor positive breast cancer; however, like other therapeutics, its clinical use is limited by acquired resistance. Tamoxifen-resistant cells have demonstrated enhanced aerobic glycolysis; however, the mechanisms underlying this upregulation remain unclear. Here, we demonstrated that G-protein coupled oestrogen receptor (GPER) was involved in the upregulation of aerobic glycolysis via induction of hypoxia-inducible factor-1α (HIF-1α) expression and transcriptional activity in tamoxifen-resistant cells. Additionally, GPER stabilized HIF-1α through inhibiting its hydroxylation and ubiquitin-mediated degradation, which were associated with upregulation of C-terminal hydrolase-L1 (UCH-L1), downregulation of prolyl hydroxylase 2 (PHD2) and von Hippel-Lindau tumour suppressor protein (pVHL), induction of HIF-1α/UCH-L1 interaction, and suppression of HIF-1α/PHD2-pVHL association. The GPER/HIF-1α axis was functionally responsible for regulating tamoxifen sensitivity both in vitro and in vivo. Moreover, there was a positive correlation between GPER and HIF-1α expression in clinical breast cancer tissues, and high levels of GPER combined with nuclear HIF-1α indicated poor overall survival. High levels of the GPER/HIF-1α axis were also correlated with shorter relapse-free survival in patients receiving tamoxifen. Hence, our findings support a critical role of GPER/HIF-1α axis in the regulation of aerobic glycolysis in tamoxifen-resistant cells, offering a potential therapeutic target for tamoxifen-resistant breast cancer.

CoCl2 -simulated hypoxia potentiates the osteogenic differentiation of fibroblasts derived fromtympanosclerosis by upregulating the expression of BMP-2.

Tympanosclerosis (TS) is a result of long-standing middle ear inflammation characterized by fibroblasts ossification. Fibrosis is the last revertible stage in the progress of middle ear inflammation to TS. It was hypothesized that chronic hypoxia could be modulating fibrosis, which in turn additionally further aggravated hypoxia via decreasing oxygen diffusion. However, the effects of hypoxia on osteoinductive activity of fibroblasts have not been explored. Herein, we purposed to explore the role of hypoxia in osteogenic differentiation of fibroblasts derived from TS. The expression of bone morphogenetic protein-2 (BMP-2), hypoxia-inducible factor-1α (HIF-1α), and Vimentin in the human surgical specimens of tympansclerosis was investigated by immunofluorescent staining. Furthermore, cultured fibroblasts were stratified into the following study groups: control, 25, 50, and 100 μM cobaltous chloride (CoCl2 ) group. BMP-2, as well as HIF-1α levels of expression were detected via western blotting and immunofluorescence analysis. We found that the expression of BMP-2 and HIF-1α was significantly upregulated in TS tissues and these fibroblasts, which was vimentin positive surrounding sclerotic plaques, were also expressing HIF-1α positive. The results also demonstrated that CoCl2 treatment increased nuclear HIF-1α protein level in the fibroblast. Furthermore, treatment with CoCl2 significantly increased BMP-2 expression and remarkably elevated alkaline phosphatse activity and the mineralized nodules area. These data illustrate that hypoxia may play an osteogenic role in TS fibroblasts via the elevated expression of a possible osteogenic factor, BMP-2.

The Relationship Between the Tumor Cell Expression of Hypoxic Markers and Survival in Patients With ER-positive Invasive Ductal Breast Cancer.

The prognostic significance of hypoxia markers, hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), and carbonic anhydrase IX (CAIX), was investigated in estrogen receptor (ER)-positive breast cancer patients. Immunohistochemistry determined the expression of makers in two independent ductal ER-positive cohorts (Training set, n=373 and Validation set, n=285) and was related to clinicopathological parameters and disease-free survival (DFS). In the training cohort, nuclear HIF-1α (1) was independently associated with poorer DFS in luminal A tumors [hazard ratio (HR) = 0.53 95% confidence interval (CI): 0.30–0.94, p=0.030]. In the validation cohort, both HIF-1α (1) and CAIX were independently associated with decreased DFS in the entire cohort (HR = 1.85 95% CI: 1.10–3.11, p=0.019; HR = 1.74 95% CI: 1.08–2.82, p=0.023), in luminal A disease (HR = 1.98 95% CI: 1.02–3.83, p=0.042), and in luminal B disease (HR = 2.75 95% CI: 1.66–4.55, p<0.001), respectively. Taken together, elevated cytoplasmic HIF-1α (1) expression was an independent prognostic factor in luminal A disease, whereas CAIX was an independent prognostic factor in luminal B disease. Further work in large tissue cohorts is required.