Intermittent Hypoxia Mediates Cancer Development and Progression Through HIF-1 and miRNA Regulation

Introduction: There is still a debate for the link between obstructive sleep apnoea (OSA) and cancer. The mechanisms underlying this causality are poorly understood. Several miRNAs are involved in cancer development and progression with expression being influenced by hypoxia. The aims of this work were (i) to compare miRNAs expression in controls versus patients affected by OSA without or with cancer (ONCO-OSA) and (ii) in colorectal cancer cells exposed to intermittent hypoxia (IH), to evaluate miRNAs impact on tumor progression in vitro. Methods: We detected miRNAs by qRT-PCR in patients’ sera and in CaCo2 cells exposed to 2–32h of IH with or without acriflavine (ACF), a HIF-1 inhibitor. Viability and transwell invasion test were applied to investigate the proliferation and migration of CaCo2 exposed to IH and treated with miRNA inhibitors or acriflavine. HIF-1α activity was evaluated in CaCo2 cells after IH. Results: The levels of miR-21, miR-26a and miR-210 increased in OSA and ONCO-OSA patients compared to controls. MiR-23b increased in ONCO-OSA patients, and miR-27b and miR-145 increased in OSA but not ONCO-OSA patients. MiR-21, miR-26a, miR-23b and miR-210 increased in cells after IH. IH stimulated cell proliferation and migration. This effect was reduced after either miRNA inhibition or acriflavine treatment. MiRNA inhibition reduces HIF-1α gene expression. Conversely, acriflavine reduced the expression of these miRNAs. Conclusions: We identified a signature of miRNAs, induced by the IH environment. They could be implicated in cancer development and progression through a regulatory loop involving HIF-1. (AU)

Intermittent Hypoxia Mediates Cancer Development and Progression Through HIF-1 and miRNA Regulation.

INTRODUCTION: There is still a debate for the link between obstructive sleep apnoea (OSA) and cancer. The mechanisms underlying this causality are poorly understood. Several miRNAs are involved in cancer development and progression with expression being influenced by hypoxia. The aims of this work were (i) to compare miRNAs expression in controls versus patients affected by OSA without or with cancer (ONCO-OSA) and (ii) in colorectal cancer cells exposed to intermittent hypoxia (IH), to evaluate miRNAs impact on tumor progression in vitro. METHODS: We detected miRNAs by qRT-PCR in patients' sera and in CaCo2 cells exposed to 2-32h of IH with or without acriflavine (ACF), a HIF-1 inhibitor. Viability and transwell invasion test were applied to investigate the proliferation and migration of CaCo2 exposed to IH and treated with miRNA inhibitors or acriflavine. HIF-1α activity was evaluated in CaCo2 cells after IH. RESULTS: The levels of miR-21, miR-26a and miR-210 increased in OSA and ONCO-OSA patients compared to controls. MiR-23b increased in ONCO-OSA patients, and miR-27b and miR-145 increased in OSA but not ONCO-OSA patients. MiR-21, miR-26a, miR-23b and miR-210 increased in cells after IH. IH stimulated cell proliferation and migration. This effect was reduced after either miRNA inhibition or acriflavine treatment. MiRNA inhibition reduces HIF-1α gene expression. Conversely, acriflavine reduced the expression of these miRNAs. CONCLUSIONS: We identified a signature of miRNAs, induced by the IH environment. They could be implicated in cancer development and progression through a regulatory loop involving HIF-1.

Evaluation of drug cost savings related to clinical trials from the perspective of a university hospital.

OBJECTIVES: Clinical trials are an opportunity for patients to access innovative therapy, but patient inclusion in clinical trials can also result in cost savings for hospitals. Our objective was to evaluate the economic impact of clinical trials drug cost savings in a French academic institution from the perspectives of both the French Health Insurance (FHI) and hospitals. METHODS: A retrospective, observational, cost saving analysis was performed on all the clinical trials initiated in our university hospital between 2015 and 2020. Only trials involving an investigational medicinal product were considered. Drug cost savings were defined as the best standard of care, defined in the protocol, whose cost was covered by a sponsor. RESULTS: Of the 646 trials undertaken during the 6 years analysed, 21% (212/646) led to cost savings, mostly driven by the industrial sponsor (92%, €6 984 283/€7 591 612) for a total of €7 591 612 (91% from the FHI's perspective (€6 959 115/€7 591 612)). Oncology trials generated 79.1% (€6 004 966/€7 591 612) of global cost savings, mostly driven by onco-haematology (33.1%, €1 983 146/€6 004 966), onco-pneumology (29.2%, €1 754 333/€6 004 966) and onco-dermatology (23.5%, €1 409 553/€6 004 966) followed by hepatogastroenterology trials (6.9%, €413 113/€6 004 966). Of the 162 drugs, the top 15 generated 75.3% (€5 715 479/€7 591 612) of savings and were grouped together: 12 antineoplastic agents (six per os and six intravenous) and three per os antiviral for hepatitis C. CONCLUSIONS: With ever-changing prices and new innovative treatments, such cost avoidance must be regularly evaluated. We provided objective evidence that clinical trials could achieve potential cost savings for the FHI and hospitals, in addition to the potential benefit to patients of having access to innovative investigational medicinal products.

Differential Impact of Intermittent vs. Sustained Hypoxia on HIF-1, VEGF and Proliferation of HepG2 Cells.

Obstructive sleep apnea (OSA) is an emerging risk factor for cancer occurrence and progression, mainly mediated by intermittent hypoxia (IH). Systemic IH, a main landmark of OSA, and local sustained hypoxia (SH), a classical feature at the core of tumors, may act separately or synergistically on tumor cells. Our aim was to compare the respective consequences of intermittent and sustained hypoxia on HIF-1, endothelin-1 and VEGF expression and on cell proliferation and migration in HepG2 liver tumor cells. Wound healing, spheroid expansion, proliferation and migration were evaluated in HepG2 cells following IH or SH exposure. The HIF-1α, endothelin-1 and VEGF protein levels and/or mRNA expression were assessed, as were the effects of HIF-1 (acriflavine), endothelin-1 (macitentan) and VEGF (pazopanib) inhibition. Both SH and IH stimulated wound healing, spheroid expansion and proliferation of HepG2 cells. HIF-1 and VEGF, but not endothelin-1, expression increased with IH exposure but not with SH exposure. Acriflavine prevented the effects of both IH and SH, and pazopanib blocked those of IH but not those of SH. Macitentan had no impact. Thus, IH and SH stimulate hepatic cancer cell proliferation via distinct signaling pathways that may act synergistically in OSA patients with cancer, leading to enhanced tumor progression.

Chronic intermittent hypoxia, a hallmark of obstructive sleep apnea, promotes 4T1 breast cancer development through endothelin-1 receptors.

The association between obstructive sleep apnea (OSA) and cancer is still debated and data are scarce regarding the link between OSA and breast cancer progression. Since conclusive epidemiological studies require large sample sizes and sufficient duration of exposure before incident cancer occurrence, basic science studies represent the most promising approach to appropriately address the topic. Here we assessed the impact of intermittent hypoxia (IH), the major hallmark of OSA, on the development of breast cancer and explored the specific involvement of the endothelin signaling pathway. Original in vitro and in vivo models were used where 3D-spheroids or cultures of murine 4T1 breast cancer cells were submitted to IH cycles, and nude NMRI mice, orthotopically implanted with 4T1 cells, were submitted to chronic IH exposure before and after implantation. The role of the endothelin-1 in promoting cancer cell development was investigated using the dual endothelin receptor antagonist, macitentan. In vitro exposure to IH significantly increased 4T1 cell proliferation and migration. Meta-analysis of 4 independent in vivo experiments showed that chronic IH exposure promoted tumor growth, assessed by caliper measurement (overall standardized mean difference: 1.00 [0.45-1.55], p < 0.001), bioluminescence imaging (1.65 [0.59-2.71]; p < 0.01) and tumor weight (0.86 [0.31-1.41], p < 0.01), and enhanced metastatic pulmonary expansion (0.77 [0.12-1.42]; p = 0.01). Both in vitro and in vivo tumor-promoting effects of IH were reversed by macitentan. Overall, these findings demonstrate that chronic intermittent hypoxia exposure promotes breast cancer growth and malignancy and that dual endothelin receptor blockade prevents intermittent hypoxia-induced tumor development.

Chronic Intermittent Hypoxia Increases Cell Proliferation in Hepatocellular Carcinoma.

Obstructive sleep apnea (OSA) syndrome is characterized by chronic intermittent hypoxia and is associated with an increased risk of all-cause mortality, including cancer mortality. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, characterized by increasing incidence and high mortality. However, the link between HCC and OSA-related chronic intermittent hypoxia remains unclear. Herein, we used a diethylnitrosamine (DEN)-induced HCC model to investigate whether OSA-related chronic intermittent hypoxia has an impact on HCC progression. To elucidate the associated mechanisms, we first evaluated the hypoxia status in the DEN-induced HCC model. Next, to simulate OSA-related intermittent hypoxia, we exposed cirrhotic rats with HCC to intermittent hypoxia during six weeks. We performed histopathological, immunohistochemical, RT-qPCR, and RNA-seq analysis. Chronic DEN injections strongly promoted cell proliferation, fibrosis, disorganized vasculature, and hypoxia in liver tissue, which mimics the usual events observed during human HCC development. Intermittent hypoxia further increased cell proliferation in DEN-induced HCC, which may contribute to an increased risk of HCC progression. In conclusion, our observations suggest that chronic intermittent hypoxia may be a factor worsening the prognosis of HCC.

Alcohol-related hepatocellular carcinoma is a heterogenous condition: Lessons from a latent class analysis.

BACKGROUND: Alcohol-associated hepatocellular carcinoma (AL-HCC) poor prognosis has been attributed to diagnosis at a later stage. However, host factors and specific health trajectories have been associated with severe outcomes in alcohol-related liver disease. We hypothesize AL-HCC is not a homogeneous condition but encompasses subgroups yielding different outcomes. AIMS: Our aim was to provide a first attempt at a clinical phenotyping of AL-HCC. METHODS: We analysed data for the calendar years 2007-2013 from the French nationwide administrative hospital database. We selected patients with AL-HCC only. Clustering of AL-HCC phenotypes was performed by latent class analysis (LCA). RESULTS: The study included 11 363 patients with AL-HCC, mainly male (89.6%), median age 67 years [IQR: 61; 74] of which 71.2% had at least one metabolic comorbidity. Five phenotypes were identified. Phenotype 1 (41.4%) displayed high rates of unrecognized cirrhosis prior to HCC diagnosis (81%), low rates of metabolic comorbidities (diabetes 13%), and mostly compensated liver disease at HCC diagnosis while the four other phenotypes displayed high rates of metabolic comorbidities (diabetes up to 100%), various patterns of liver disease trajectories and overall 42% unrecognized cirrhosis. In adjusted survival analysis, compared to phenotype 1, risk of death after HCC diagnosis was significantly different for all phenotypes. CONCLUSION: LCA uncovers AL-HCC is a heterogeneous condition with distinct phenotypes yielding specific survival outcomes. Frequent unrecognized cirrhosis prior to HCC underlines the urgent need for implementing strategies to identify the underlying liver disease prior to HCC onset in patients with documented alcohol use disorders and metabolic comorbidities.

An innovative intermittent hypoxia model for cell cultures allowing fast Po2 oscillations with minimal gas consumption.

Performing hypoxia-reoxygenation cycles in cell culture with a cycle duration accurately reflecting what occurs in obstructive sleep apnea (OSA) patients is a difficult but crucial technical challenge. Our goal was to develop a novel device to expose multiple cell culture dishes to intermittent hypoxia (IH) cycles relevant to OSA with limited gas consumption. With gas flows as low as 200 ml/min, our combination of plate holders with gas-permeable cultureware generates rapid normoxia-hypoxia cycles. Cycles alternating 1 min at 20% O2 followed by 1 min at 2% O2 resulted in Po2 values ranging from 124 to 44 mmHg. Extending hypoxic and normoxic phases to 10 min allowed Po2 variations from 120 to 25 mmHg. The volume of culture medium or the presence of cells only modestly affected the Po2 variations. In contrast, the nadir of the hypoxia phase increased when measured at different heights above the membrane. We validated the physiological relevance of this model by showing that hypoxia inducible factor-1α expression was significantly increased by IH exposure in human aortic endothelial cells, murine breast carcinoma (4T1) cells as well as in a blood-brain barrier model (2.5-, 1.5-, and 6-fold increases, respectively). In conclusion, we have established a new device to perform rapid intermittent hypoxia cycles in cell cultures, with minimal gas consumption and the possibility to expose several culture dishes simultaneously. This device will allow functional studies of the consequences of IH and deciphering of the molecular biology of IH at the cellular level using oxygen cycles that are clinically relevant to OSA.

Nonalcoholic fatty liver disease in chronic obstructive pulmonary disease.

Nonalcoholic fatty liver disease (NAFLD) is independently linked to cardiometabolic morbidity and mortality. Low-grade inflammation, oxidative stress and ectopic fat, common features of chronic obstructive pulmonary disease (COPD), might contribute to the development of NAFLD.We aimed to investigate the prevalence of NAFLD and to evaluate the relationship between various types of liver damage and COPD severity, comorbidities and circulating inflammatory cytokines. Validated noninvasive tests (FibroMax: SteatoTest, NashTest and FibroTest) were used to assess steatosis, nonalcoholic steatohepatitis (NASH) and liver fibrosis. Patients underwent an objective assessment of COPD comorbidities, including sleep studies. Biological parameters included a complete lipid profile and inflammatory markers.In COPD patients the prevalence of steatosis, NASH and fibrosis were 41.4%, 36.9% and 61.3%, respectively. In multivariate analysis, SteatoTest and FibroTest were significantly associated with sex, body mass index (BMI), untreated sleep apnoea and insulin resistance, and, in addition, COPD Global Initiative for Chronic Obstructive Lung Disease stage for SteatoTest. Patients with steatosis had higher tumour necrosis factor-α levels and those with NASH or a combination of liver damage types had raised leptin levels after adjustment for age, sex and BMI.We concluded that NAFLD is highly prevalent in COPD and might contribute to cardiometabolic comorbidities.

Comparison between two generic questionnaires to assess satisfaction with medication in chronic diseases.

OBJECTIVE: The objective of this work was to compare two generic questionnaires assessing patients' satisfaction with medication. In addition we tested whether satisfaction can predict adherence to medication regimens in patients with chronic diseases, and which dimensions of satisfaction are most involved. METHODS: This prospective, observational study was conducted over one year in a heterogeneous population of patients with various chronic diseases. Satisfaction with medication was assessed by using the TSQM® vII and the SatMed-Q® questionnaires, and adherence to treatment was assessed with the Morisky-Green questionnaire. Clinical pharmacists interviewed patients to collect clinical, demographic and therapeutic data. RESULTS: 190 patients were enrolled. Both questionnaires showed excellent reliability and correlation was high (R=0.70; p<0.001). Adherence was correlated with satisfaction with medication whether assessed with the SatMed-Q® (R=0.23; p=0.002) or the TSQM® (R=0.17; p=0.02). Among different dimensions of satisfaction, convenience of use and side effects are prominent predictors of adherence. CONCLUSION: Adherence is related to the patient's satisfaction with medication whether assessed with the TSQM® vII or the SatMed-Q®. Therefore, these simple questionnaires could be used as predictive tools to identify patients whos' adherence needs to be improved.