Investigation of the causal relationship between inflammatory bowel disease and type 2 diabetes mellitus: a Mendelian randomization study.

Background: Type 2 diabetes mellitus (T2DM) and inflammatory bowel disease (IBD) have been associated, according to various epidemiological research. This study uses Mendelian randomization (MR) to investigate the causal link between T2DM and IBD. Methods: To investigate the causal relationship between IBD and T2DM risk using European population data from the genome-wide association study (GWAS) summary datasets, we constructed a two-sample MR study to evaluate the genetically predicted impacts of liability towards IBD outcomes on T2DM risk. As instrumental variables (IVs), we chose 26 single nucleotide polymorphisms (SNPs) associated with IBD exposure data. The European T2DM GWAS data was obtained from the IEU OpenGWAS Project database, which contains 298,957 cases as the outcome data. The causal relationship between T2DM and IBD using a reverse MR analysis was also performed. Results: The two-sample MR analysis, with the Bonferroni adjustment for multiple testing, revealed that T2DM risk in Europeans is unaffected by their IBD liability (odds ratio (OR): 0.950-1.066, 95% confidence interval (CI): 0.885-1.019, p = 0.152-0.926). The effects of liability to T2DM on IBD were not supported by the reverse MR analysis either (OR: 0.739-1.131, 95% confidence interval (CI): 0.651-1.100, p = 0.058-0.832). MR analysis of IBS on T2DM also have no significant causal relationship (OR: 0.003-1.007, 95% confidence interval (CI): 1.013-5.791, p = 0.069-0.790). FUMA precisely mapped 22 protein-coding genes utilizing significant SNPs of T2DM acquired from GWAS. Conclusion: The MR study showed that the existing evidence did not support the significant causal effect of IBD on T2DM, nor did it support the causal impact of T2DM on IBD.

The anti-inflammatory effects of exercise on autoimmune diseases: A 20-year systematic review.

BACKGROUND: The anti-inflammatory effect of exercise may be an underlying factor in improving several autoimmune diseases. The aim of this systematic review was to examine the evidence on the role of exercise training in mitigating inflammation in adolescents and adults with autoimmune disease. METHODS: PubMed, Web of Science, and Embase databases were systematically reviewed for related studies published between January 1, 2003, and August 31, 2023. All randomized and non-randomized controlled trials of exercise interventions with autoimmune disease study participants that evaluated inflammation-related biomarkers were included. The quality of evidence was assessed using the Tool for the assEssment of Study qualiTy and reporting in EXercise scale and Cochrane bias risk tool. RESULTS: A total of 14,565 records were identified. After screening the titles, abstracts, and full texts, 87 were eligible for the systematic review. These studies were conducted in 25 different countries and included a total of 2779 participants (patients with autoimmune disease, in exercise or control groups). Overall, the evidence suggests that inflammation-related markers such as C-reactive protein, interleukin 6, and tumor necrosis factor α were reduced by regular exercise interventions. Regular exercise interventions combined with multiple exercise modes were associated with greater benefits. CONCLUSION: Regular exercise training by patients with autoimmune disease exerts an anti-inflammatory influence. This systematic review provides support for the promotion and development of clinical exercise intervention programs for patients with autoimmune disease. Most patients with autoimmune disease can safely adopt moderate exercise training protocols, but changes in inflammation biomarkers will be modest at best. Acute exercise interventions are ineffective or even modestly but transiently pro-inflammatory.

Vitamin E stabilizes iron and mitochondrial metabolism in pulmonary fibrosis.

Introduction: Pulmonary fibrosis (PF) is a fatal chronic lung disease that causes structural damage and decreased lung function and has a poor prognosis. Currently, there is no medicine that can truly cure PF. Vitamin E (VE) is a group of natural antioxidants with anticancer and antimutagenic properties. There have been a few reports about the attenuation of PF by VE in experimental animals, but the molecular mechanisms are not fully understood. Methods: Bleomycin-induced PF (BLM-PF) mouse model, and cultured mouse primary lung fibroblasts and MLE 12 cells were utilized. Pathological examination of lung sections, immunoblotting, immunofluorescent staining, and real-time PCR were conducted in this study. Results: We confirmed that VE significantly delayed the progression of BLM-PF and increased the survival rates of experimental mice with PF. VE suppressed the pathological activation and fibrotic differentiation of lung fibroblasts and epithelial-mesenchymal transition and alleviated the inflammatory response in BLM-induced fibrotic lungs and pulmonary epithelial cells in vitro. Importantly, VE reduced BLM-induced ferritin expression in fibrotic lungs, whereas VE did not exhibit iron chelation properties in fibroblasts or epithelial cells in vitro. Furthermore, VE protected against mitochondrial dysmorphology and normalized mitochondrial protein expression in BLM-PF lungs. Consistently, VE suppressed apoptosis in BLM-PF lungs and pulmonary epithelial cells in vitro. Discussion: Collectively, VE markedly inhibited BLM-induced PF through a complex mechanism, including improving iron metabolism and mitochondrial structure and function, mitigating inflammation, and decreasing the fibrotic functions of fibroblasts and epithelial cells. Therefore, VE presents a highly potential therapeutic against PF due to its multiple protective effects with few side effects.

Association Between Visceral Fat Area and Glycated Hemoglobin in Type 2 Diabetics: A Retrospective Study.

Introduction: Obesity is a risk factor for the development of type 2 diabetes (T2DM) as well as its associated metabolic complications. Central obesity, characterized by an increased visceral fat area (VFA), is contributed to the development of T2DM. However, the relationship between VFA and HbA1c is not particularly clear. Methods: A total of 3173 patients with T2DM participated in the study at the Metabolic Management Center (MMC), with anthropometric and biochemical measurements recorded. To examine the association between HbA1c and VFA, fitting curves were plotted, facilitating a comprehensive observation of their relationship. Results: HbA1c was inversely associated with VFA (ß -1.79, 95% CI -2.34~-1.24, P < 0.001). The fitted curve shows that VFA increased with the increase of HbA1c when it was less than 8.62%. When it was greater than 8.62%, VFA decreased as HbA1c increased. Using linear inflection point analysis, we found that its inflection point interval falls within 8.36%~8.88%. Conclusion: VFA was positively associated with HbA1c in individuals with T2DM. Furthermore, the relationship between the two variables was an inverted U-shaped association.

Uncovering interactions of mitochondrial proteins BNIP3 and BNIP3L with necroptosis-associated kinase RIPK3: Insights into kinase activation.

Mitochondria, an important organelle implicated in programmed cell death, assumes a crucial role in necroptosis. However, the regulatory mechanisms through which mitochondria participates in necroptosis are largely unknown. To address this knowledge gap, our study aimed to identify mitochondrial proteins that engage in interactions with receptor-interacting protein kinase 3 (RIPK3), a significant upstream kinase involved in necroptosis. Among the candidates, BNIP3 and BNIP3L exhibited significant higher binding scores to RIPK3 compared to others. Computational modeling revealed specific interactions, as RIPK3 specifically binds to a conserved α-helix region within BNIP3 and BNIP3L. Validation experiments confirmed the significance of these helical peptides for RIPK3 binding. Conserved peptides were also identified in BNIP3 and BNIP3L proteins from various animal species, including humans. The binding between human RIPK3 and BNIP3/BNIP3L peptides demonstrated perfect shape and charge complementation, with highly conserved interface residues. Moreover, peptide binding stabilized an active conformation of RIPK3, potentially enhancing its kinase activity. These findings uncover the interactions between RIPK3 and BNIP3/BNIP3L, providing insights into RIPK3 regulation and its role in necroptosis.

Fasting Plasma Glucose and Glycated Hemoglobin Levels as Risk Factors for the Development of Hypertension: A Retrospective Cohort Study.

Introduction: Hypertension (HTN) is a significant risk factor for cardiovascular disease. Identifying new risk factors for hypertension is crucial. This study aims to determine the predictive value of fasting plasma glucose (FPG) and glycated hemoglobin (HbA1c) in the development of hypertension. Methods: In this study, we examined 16,026 individuals without diabetes and other cardiovascular risk factors who were underwent annual screening at the People's Hospital of Yuxi, Yunnan, China from 2013 to 2016. The participants were divided into two groups: normoglycemic and prediabetic. Normoglycemia was defined as having an HbA1c level of less than 5.7% and an FPG level of less than 5.6 mmol/ L. Prediabetes was defined according to the ADA criteria, which includes having an HbA1c level between 5.7% and 6.5%, or an impaired fasting glucose level between 5.6 mmol/L and 7.0 mmol/L. The participants were further divided into four subgroups based on their FPG and HbA1c levels: normoglycemia, impaired HbA1c only, FPG only, and both parameters impaired. Results: The cohort study was conducted on 16,026 participants from Yunnan, China, consisting of 60.6% males and 39.4% females, with a mean age of 44.6 ± 12.5 years. The study revealed that prediabetes was independently associated with an increased risk for HTN (OR 1.53, 95% CI 1.41~1.67, P < 0.001). The analysis of different subgroups of HbA1c and FPG showed that FPG was a better predictor of HTN than HbA1c, regardless of the group. Conclusion: FPG and HbA1c were significantly associated with the future development of HTN in individuals with prediabetes.

Glycemic dispersion: a new index for screening high glycemic variability.

OBJECTIVE: For patients with diabetes, high-frequency and -amplitude glycemic variability may be more harmful than continuous hyperglycemia; however, there is still a lack of screening indicators that can quickly and easily assess the level of glycemic variability. The aim of this study was to investigate whether the glycemic dispersion index is effective for screening high glycemic variability. METHODS: A total of 170 diabetes patients hospitalized in the Sixth Affiliated Hospital of Kunming Medical University were included in this study. After admission, the fasting plasma glucose, 2-hour postprandial plasma glucose, and glycosylated hemoglobin A1c were measured. The peripheral capillary blood glucose was measured seven times in 24 h, before and after each of three meals and before bedtime. The standard deviation of the seven peripheral blood glucose values was calculated, and a standard deviation of > 2.0 was used as the threshold of high glycemic variability. The glycemic dispersion index was calculated and its diagnostic efficacy for high glycemic variability was determined by the Mann-Whitney U test, receiver operating characteristic (ROC) curve and, Pearson correlation analysis. RESULTS: The glycemic dispersion index of patients with high glycemic variability was significantly higher than that of those with low glycemic variability (p < 0.01). The best cutoff value of the glycemic dispersion index for screening high glycemic variability was 4.21. The area under the curve (AUC) was 0.901 (95% CI: 0.856-0.945) and had a sensitivity of 0.781 and specificity of 0.905. It was correlated with the standard deviation of blood glucose values (r = 0.813, p < 0.01). CONCLUSIONS: The glycemic dispersion index had good sensitivity and specificity for screening high glycemic variability. It was significantly associated with the standard deviation of blood glucose concentration and is simple and easy to calculate. It was an effective screening indicator of high glycemic variability.

Comparative study of type 2 diabetes mellitus-associated gut microbiota between the Dai and Han populations.

BACKGROUND: The global prevalence of type 2 diabetes mellitus (T2DM) is increasing. T2DM is associated with alterations of the gut microbiota, which can be affected by age, illness, and genetics. Previous studies revealed that there are discriminating microbiota compositions between the Dai and the Han populations. However, the specific gut microbiota differences between the two populations have not been elucidated. AIM: To compare the gut microbiota differences in subjects with and without T2DM in the Dai and Han populations. METHODS: A total of 35 subjects of the Han population (including 15 healthy children, 8 adult healthy controls, and 12 adult T2DM patients) and 32 subjects of the Dai population (including 10 healthy children, 10 adult healthy controls, and 12 adult T2DM patients) were enrolled in this study. Fasting venous blood samples were collected from all the subjects for biochemical analysis. Fecal samples were collected from all the subjects for DNA extraction and 16S rRNA sequencing, which was followed by analyses of the gut microbiota composition. RESULTS: No significant difference in alpha diversity was observed between healthy children and adults. The diversity of gut microbiota was decreased in T2DM patients compared to the healthy adults in both the Dai and Han populations. There was a significant difference in gut microbiota between healthy children and healthy adults in the Han population with an increased abundance of Bacteroidetes and decreased Firmicutes in children. However, this difference was less in the Dai population. Significant increases in Bacteroidetes in the Han population and Proteobacteria in the Dai population and decreases in Firmicutes in both the Han and Dai population were observed in T2DM patients compared to healthy adults. Linear discriminant analysis Effect Size analysis also showed that the gut microbiota was different between the Han and Dai populations in heathy children, adults, and T2DM patients. Four bacteria were consistently increased and two consistently decreased in the Han population compared to the Dai population. CONCLUSION: Differences in gut microbiota were found between the Han and Dai populations. A significant increase in Bacteroidetes was related to the occurrence of T2DM in the Han population, while a significant increase in Proteobacteria was related to the occurrence of T2DM in the Dai population.

Circadian clock-A promising scientific target in oral science.

The oral and maxillofacial organs play vital roles in chewing, maintaining facial beauty, and speaking. Almost all physiological processes display circadian rhythms that are driven by the circadian clock, allowing organisms to adapt to the changing environment. In recent years, increasing evidence has shown that the circadian clock system participates in oral and maxillofacial physiological and pathological processes, such as jaw and tooth development, salivary gland function, craniofacial malformations, oral carcinoma and other diseases. However, the roles of the circadian clock in oral science have not yet been comprehensively reviewed. Therefore, This paper provides a systematic and integrated perspective on the function of the circadian clock in the fields of oral science, reviews recent advances in terms of the circadian clock in oral and maxillofacial development and disease, dialectically analyzes the importance of the circadian clock system and circadian rhythm to the activities of oral and maxillofacial tissues, and focuses on analyzing the mechanism of the circadian clock in the maintenance of oral health, affecting the common diseases of the oral and maxillofacial region and the process of oral-related systemic diseases, sums up the chronotherapy and preventive measures for oral-related diseases based on changes in tissue activity circadian rhythms, meanwhile, comes up with a new viewpoint to promote oral health and human health.

Inflammatory Microenvironment Accelerates Bone Marrow Mesenchymal Stem Cell Aging.

MSC senescence is considered a contributing factor in aging-related diseases. We investigated the influence of the inflammatory microenvironment on bone marrow mesenchymal stem cells (BMSCs) under aging conditions and the underlying mechanism to provide new ideas for stem cell therapy for age-related osteoporosis. The BMSCs were cultured until passage 3 (P3) (young group) and passage 10 (P10) (aging group) in vitro. The supernatant was collected as the conditioned medium (CM). The young BMSCs were cultured in the CM of P3 or P10 cells. The effects of CM from different groups on the aging and stemness of the young BMSCs were examined. A Quantibody® mouse inflammation array on serum extracts from young (aged 8 weeks) and old (aged 78 weeks) mice was performed, and differentially expressed factors were screened out. We discovered that the CM from senescent MSCs changed the physiology of young BMSCs. Systemic inflammatory microenvironments changed with age in the mice. In particular, the pro-inflammatory cytokine IL-6 increased, and the anti-inflammatory cytokine IL-10 decreased. The underlying mechanism was investigated by GO and KEGG analyses, and there was a change in the JAK-STAT signaling pathway, which is closely related to IL-6 and IL-10. Collectively, our results demonstrated that the age-related inflammatory microenvironment has a significant effect on the biological functions of BMSCs. Targeted reversal of this inflammatory environment may provide a new strategy for stem cell therapy to treat aging-related skeletal diseases.

Circadian rhythms affect bone reconstruction by regulating bone energy metabolism.

Metabolism is one of the most complex cellular biochemical reactions, providing energy and substances for basic activities such as cell growth and proliferation. Early studies have shown that glucose is an important nutrient in osteoblasts. In addition, amino acid metabolism and fat metabolism also play important roles in bone reconstruction. Mammalian circadian clocks regulate the circadian cycles of various physiological functions. In vertebrates, circadian rhythms are mediated by a set of central clock genes: muscle and brain ARNT like-1 (Bmal1), muscle and brain ARNT like-2 (Bmal2), circadian rhythmic motion output cycle stagnates (Clock), cryptochrome 1 (Cry1), cryptochrome2 (Cry2), period 1 (Per1), period 2 (Per2), period 3 (Per3) and neuronal PAS domain protein 2 (Npas2). Negative feedback loops, controlled at both the transcriptional and posttranslational levels, adjust these clock genes in a diurnal manner. According to the results of studies on circadian transcriptomic studies in several tissues, most rhythmic genes are expressed in a tissue-specific manner and are affected by tissue-specific circadian rhythms. The circadian rhythm regulates several activities, including energy metabolism, feeding time, sleeping, and endocrine and immune functions. It has been reported that the circadian rhythms of mammals are closely related to bone metabolism. In this review, we discuss the regulation of the circadian rhythm/circadian clock gene in osteoblasts/osteoclasts and the energy metabolism of bone, and the relationship between circadian rhythm, bone remodeling, and energy metabolism. We also discuss the therapeutic potential of regulating circadian rhythms or changing energy metabolism on bone development/bone regeneration.

[Organoids derived from the digestive system and their perspective applications in exercise physiology].

Organoid, formed from organ-specific cells, is a group of self-renewal and self-organizing cells growing in a 3-dimensional structure. With the recent progress on microenvironment regulation, stem cell differentiation and organ development, organoids have been constructed and used as promising tools for a wide range of multidisciplinary biomedical applications. Exercise disrupts the internal environment homeostasis, which brings a series of physiological alterations to the digestive system. The current animal or human models are necessary, but not sufficient to monitor the fluctuating microenvironment of gastrointestinal epithelial cells or hepatocytes during exercise. This review described the construction and application of digestive system organoids, as well as the effect of exercise on the microenvironment of intestinal epithelial cells and hepatocytes. The perspective applications of digestive system organoids in exercise physiology were also stated. Using organoid technologies, the possible mechanisms of the exercise-induced dynamic physiological changes would be explored in a new dimension.

[Corrigendum] Connexin 43 reduces susceptibility to sympathetic atrial fibrillation.

Following the publication of the above article, the authors have realized that the data shown in Fig. 3B were published previously in Fig 1A of following publication, on which several of were co­authors [Shu C, Huang W, Zeng Z, He Y, Luo B, Liu H, Li J and Xu J: Connexin 43 is involved in the sympathetic atrial fibrillation in canine and canine atrial myocytes. Anatol J Cardiol 18: 3­9, 2017]. This error arose inadvertently; the corrected version of Fig. 3, also containing the correct data for Fig. 3B, is shown opposite. The authors are grateful to the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this Corrigendum, and stress that this error did not significantly influence either the results or the conclusions of the paper. Furthermore, the authors apologize to the readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 42: 1125-1133, 2018; DOI: 10.3892/ijmm.2018.3648].

Chemerin/CMKLR1 Axis Promotes Inflammation and Pyroptosis by Activating NLRP3 Inflammasome in Diabetic Cardiomyopathy Rat.

Chemerin and its receptor CMKLR1 (a G-protein-coupled receptor) are inducers of inflammation, and play an important role in diabetic cardiomyopathy (DCM). In this study, we investigated the role of the chemerin/CMKLR1 axis in mediating inflammation and cell death in DCM. Sprague-Dawley rats, treated with a high-fat diet and low-dose of streptozotocin, were used as a DCM model. CMKLR1 expression was knocked down by siRNA (CMKLR1-siRNA) to evaluate the role of CMKLR1 in DCM. Chemerin-treated H9c2 cells were used to investigate the factors acting downstream of the chemerin/CMKLR1 axis. LDH release and EthD-III staining were used to measure the ratio of cell death in vitro. CMKLR1-siRNA and siRNA against nucleotide-binding oligomerization domain-like receptors 3 (NLRP3-siRNA) were used to explore the mechanism underlying chemerin-induced inflammation and cell death. The results showed that the expression of chemerin, CMKLR1, NLRP3, pro-caspase-1, activated caspase-1, and mature IL-1ß was increased in the DCM model rat. Myocardium of DCM model rats exhibited fibrosis, hypertrophy, a disorganized ultrastructure, and impaired function. Pyroptosis was observed in vivo and in vitro. Silencing of CMKLR1 in vivo attenuated the expression of NLRP3 and activated caspase-1 and IL-1ß. CMKLR1-siRNA treatment attenuated cardiac inflammation, fibrosis, hypertrophy, and pyroptosis, and improved cardiac function in vivo. Silencing of either CMKLR1 or NLRP3 suppressed the levels of activated caspase-1, IL-1ß, and pyroptosis; however, silencing of both CMKLR1 and NLRP3 further decreased the levels of mature IL-1ß and pyroptosis. Overall, the results showed that the chemerin/CMKLR1 axis contributed to the development of DCM and that the NLRP3 inflammasome mediated the chemerin/CMLR1-induced inflammation and pyroptosis. These data indicate that silencing of the CMKLR1 gene might exert a protective effect against DCM.

Effect of Aerobic and Resistance Training on Endothelial Progenitor Cells in Mice with Type 2 Diabetes.

Since no study has explored whether exercise could improve impaired proliferation, migration, and angiogenesis of endothelial progenitor cells (EPCs) in animal models or humans with type 2 diabetes, we aimed to explore the effect of different models of exercise on EPC function and expression of caveolin-1, PI3K, and AKT in mice with type 2 diabetes. Male db/db mice (age: 8 weeks) with type 2 diabetes were subjected to aerobic training (AT), resistance training (RT), or combined aerobic and resistance training (AT+RT) 3 or 4 days/week. Mice in the control group remained sedentary with no specific training requirement. Bone marrow-derived EPCs were isolated, and the protein concentrations of caveolin-1, Pi3k, and AKT, and EPC function, were identified in the 1st, 4th, 8th, and 12th weeks of the intervention. Greater increases in proliferation, migration, and angiogenesis were observed in the AT, RT, and AT+RT groups than in the control group. AT+RT was more effective than AT or RT in improving the migratory and angiogenesis function of EPCs in mice with type 2 diabetes and achieved maximum improvement after 8 weeks of intervention. Western blot analysis showed that caveolin-1, p-PI3k, and p-Akt levels were obviously increased in the AT, RT, and AT+RT groups compared with the control group. The expression level of these proteins in the AT+RT group was higher than that in the AT and RT groups. AT+RT may be a helpful reference when choosing exercise methods for the prevention of diabetes-related cardiovascular diseases.

Retraction Note: Resveratrol rescues hyperglycemia-induced endothelial dysfunction via activation of Akt.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Exercise induces tissue hypoxia and HIF-1α redistribution in the small intestine.

Background: Exercise induces blood flow redistribution among tissues, leading to splanchnic hypoperfusion. Intestinal epithelial cells are positioned between the anaerobic lumen and the highly metabolic lamina propria with an oxygen gradient. Hypoxia-inducible factor (HIF)-1α is pivotal in the transcriptional response to the oxygen flux. Methods: In this study, the pimonidazole hydrochloride staining was applied to observe the tissue hypoxia in different organs, which might be affected by the blood flow redistribution. The HIF-1α luciferase reporter ROSA26 oxygen-dependent degradation domain (ODD)-Luc/+ mouse model (ODD domain-Luc; female, n = 3-6/group) was used to detect the HIF-1α expression in the intestine. We used 3 swimming models: moderate exercise for 30 min, heavy-intensity exercise bearing 5% bodyweight for 1.5 h, and long-time exercise for 3 h. Results: We found that 1 session of swimming at different intensities could induce tissue hypoxia redistribution in the small intestine, colon, liver and kidney, but not in the spleen, heart, and skeletal muscle. Our data showed that exercise exacerbated the extent of physiological hypoxia in the small intestine. Next, using ODD-Luc mice, we found that moderate exercise increased the in vivo HIF-1α level in the small intestine. The post-exercise HIF-1α level was gradually decreased in a time-dependent manner. Interestingly, the redistribution of tissue hypoxia and the increase of HIF-1α expression were not related to the exercise intensity and duration. Conclusion: This study provided evidence that the small intestine is the primary target organ for exercise-induced tissue hypoxia and HIF-1α redistribution, suggesting that HIF-1α may be a potential target for the regulation of gastrointestinal functions after exercise.

Improved Stability of Metal Nanowires via Electron Beam Irradiation Induced Surface Passivation.

Suppressing the corrosion of nanoscaled metal materials is a critical issue for various devices. Herein, we demonstrate the electron beam irradiation can be a simple and efficient method to realize silver/copper nanowires protection by transforming the original organic capping agents into dense carbonaceous shells. Single nanowire tests prove the significant stability improvement from 4 days to 20 days for silver nanowire and from 20 h to at least 1 week for copper nanowire. The comprehensive advantages such as solution/pollution-free and continuous process with high precision offer this method substantial potential applications in bottom-up assembled electronic and optoelectronic devices.

The FGFR1 V561M Gatekeeper Mutation Drives AZD4547 Resistance through STAT3 Activation and EMT.

FGFR1 has been implicated in numerous cancer types including squamous cell lung cancer, a subset of non-small cell lung cancer with a dismal 5-year survival rate. Small-molecule inhibitors targeting FGFR1 are currently in clinical trials, with AZD4547 being one of the furthest along; however, the development of drug resistance is a major challenge for targeted therapies. A prevalent mechanism of drug resistance in kinases occurs through mutation of the gatekeeper residue, V561M in FGFR1; however, mechanisms underlying V561M resistance to AZD4547 are not fully understood. Here, the cellular consequences of the V561M gatekeeper mutation were characterized, and it was found that although AZD4547 maintains nanomolar affinity for V561M FGFR1, based on in vitro binding assays, cells expressing V561M demonstrate dramatic resistance to AZD4547 driven by increased STAT3 activation downstream of V561M FGFR1. The data reveal that the V561M mutation biases cells toward a more mesenchymal phenotype, including increased levels of proliferation, migration, invasion, and anchorage-independent growth, which was confirmed using CyTOF, a novel single-cell analysis tool. Using shRNA knockdown, loss of STAT3 restored sensitivity of cancer cells expressing V561M FGFR1 to AZD4547. Thus, the data demonstrate that combination therapies including FGFR and STAT3 may overcome V561M FGFR1-driven drug resistance in the clinic. IMPLICATIONS: The V561M FGFR1 gatekeeper mutation leads to devastating drug resistance through activation of STAT3 and the epithelial-mesenchymal transition; this study demonstrates that FGFR1 inhibitor sensitivity can be restored upon STAT3 knockdown.