HMGA1 sensitizes esophageal squamous cell carcinoma to mTOR inhibitors through the ETS1-FKBP12 axis.

Esophageal carcinoma is amongst the prevalent malignancies worldwide, characterized by unclear molecular classifications and varying clinical outcomes. The PI3K/AKT/mTOR signaling, one of the frequently perturbed dysregulated pathways in human malignancies, has instigated the development of various inhibitory agents targeting this pathway, but many ESCC patients exhibit intrinsic or adaptive resistance to these inhibitors. Here, we aim to explore the reasons for the insensitivity of ESCC patients to mTOR inhibitors. We assessed the sensitivity to rapamycin in various ESCC cell lines by determining their respective IC50 values and found that cells with a low level of HMGA1 were more tolerant to rapamycin. Subsequent experiments have supported this finding. Through a transcriptome sequencing, we identified a crucial downstream effector of HMGA1, FKBP12, and found that FKBP12 was necessary for HMGA1-induced cell sensitivity to rapamycin. HMGA1 interacted with ETS1, and facilitated the transcription of FKBP12. Finally, we validated this regulatory axis in in vivo experiments, where HMGA1 deficiency in transplanted tumors rendered them resistance to rapamycin. Therefore, we speculate that mTOR inhibitor therapy for individuals exhibiting a reduced level of HMGA1 or FKBP12 may not work. Conversely, individuals exhibiting an elevated level of HMGA1 or FKBP12 are more suitable candidates for mTOR inhibitor treatment.

O-GlcNAc of STING mediates antiviral innate immunity.

BACKGROUND: O-GlcNAcylation modification affects multiple physiological and pathophysiolocal functions of cells. Altered O-GlcNAcylation was reported to participate in antivirus response. Stimulator of interferon genes (STING) is an adaptor mediating DNA virus-induced innate immune response. Whether STING is able to be modified by O-GlcNAcylation and how O-GlcNAcylation affects STING-mediated anti-DNA virus response remain unknown. METHODS: Metabolomics analysis was used for detecting metabolic alterations in HSV-1 infection cells. Succinylated wheat germ agglutinin (sWGA), co-immunoprecipitation, and pull-down assay were employed for determining O-GlcNAcylation. Mutagenesis PCR was applied for the generation of STING mutants. WT and Sting1-/- C57BL/6 mice (KOCMP-72512-Sting1-B6NVA) were infected with HSV-1 and treated with O-GlcNAcylation inhibitor for validating the role of STING O-GlcNAcylation in antiviral response. RESULTS: STING was functionally activated by O-GlcNAcylation in host cells challenged with HSV-1. We demonstrated that this signaling event was initiated by virus infection-enhanced hexosamine biosynthesis pathway (HBP). HSV-1 (or viral DNA mimics) promotes glucose metabolism of host cells with a marked increase in HBP, which provides donor glucosamine for O-GlcNAcylation. STING was O-GlcNAcylated on threonine 229, which led to lysine 63-linked ubiquitination of STING and activation of antiviral immune responses. Mutation of STING T229 to alanine abrogated STING activation and reduced HSV-1 stimulated production of interferon (IFN). Application of 6-diazo-5-oxonorleucine (DON), an agent that blocks the production of UDP-GlcNAc and inhibits O-GlcNAcylation, markedly attenuated the removal of HSV-1 in wild type C57BL/6 mice, leading to an increased viral retention, elevated infiltration of inflammatory cells, and worsened tissue damages to those displayed in STING gene knockout mice. Together, our data suggest that STING is O-GlcNAcylated in HSV-1, which is crucial for an effective antiviral innate immune response. CONCLUSION: HSV-1 infection activates the generation of UDP-Glc-NAc by upregulating the HBP metabolism. Elevated UDP-Glc-NAc promotes the O-GlcNAcylation of STING, which mediates the anti-viral function of STING. Targeting O-GlcNAcylation of STING could be a useful strategy for antiviral innate immunity.

HMGA1 drives chemoresistance in esophageal squamous cell carcinoma by suppressing ferroptosis.

Chemotherapy is a primary treatment for esophageal squamous cell carcinoma (ESCC). Resistance to chemotherapeutic drugs is an important hurdle to effective treatment. Understanding the mechanisms underlying chemotherapy resistance in ESCC is an unmet medical need to improve the survival of ESCC. Herein, we demonstrate that ferroptosis triggered by inhibiting high mobility group AT-hook 1 (HMGA1) may provide a novel opportunity to gain an effective therapeutic strategy against chemoresistance in ESCC. HMGA1 is upregulated in ESCC and works as a key driver for cisplatin (DDP) resistance in ESCC by repressing ferroptosis. Inhibition of HMGA1 enhances the sensitivity of ESCC to ferroptosis. With a transcriptome analysis and following-up assays, we demonstrated that HMGA1 upregulates the expression of solute carrier family 7 member 11 (SLC7A11), a key transporter maintaining intracellular glutathione homeostasis and inhibiting the accumulation of malondialdehyde (MDA), thereby suppressing cell ferroptosis. HMGA1 acts as a chromatin remodeling factor promoting the binding of activating transcription factor 4 (ATF4) to the promoter of SLC7A11, and hence enhancing the transcription of SLC7A11 and maintaining the redox balance. We characterized that the enhanced chemosensitivity of ESCC is primarily attributed to the increased susceptibility of ferroptosis resulting from the depletion of HMGA1. Moreover, we utilized syngeneic allograft tumor models and genetically engineered mice of HMGA1 to induce ESCC and validated that depletion of HMGA1 promotes ferroptosis and restores the sensitivity of ESCC to DDP, and hence enhances the therapeutic efficacy. Our finding uncovers a critical role of HMGA1 in the repression of ferroptosis and thus in the establishment of DDP resistance in ESCC, highlighting HMGA1-based rewiring strategies as potential approaches to overcome ESCC chemotherapy resistance. Schematic depicting that HMGA1 maintains intracellular redox homeostasis against ferroptosis by assisting ATF4 to activate SLC7A11 transcription, resulting in ESCC resistance to chemotherapy.

Akkermansia muciniphila protects the intestine from irradiation-induced injury by secretion of propionic acid.

Intestinal dysbiosis frequently occurs in abdominal radiotherapy and contributes to irradiation (IR)-induced intestinal damage and inflammation. Akkermansia muciniphila (A. muciniphila) is a recently characterized probiotic, which is critical for maintaining the dynamics of the intestinal mucus layer and preserving intestinal microbiota homeostasis. However, the role of A. muciniphila in the alleviation of radiation enteritis remains unknown. In this study, we reported that the abundance of A. muciniphila was markedly reduced in the intestines of mice exposed to abdominal IR and in the feces of patients who received abdominal radiotherapy. Abundance of A. muciniphila in feces of radiotherapy patients was negatively correlated with the duration of diarrhea in patients. Administration of A. muciniphila substantially mitigated IR-induced intestinal damage and prevented mouse death. Analyzing the metabolic products of A. muciniphila revealed that propionic acid, a short-chain fatty acid secreted by the microbe, mediated the radioprotective effect. We further demonstrated that propionic acid bound to G-protein coupled receptor 43 (GRP43) on the surface of intestinal epithelia and increased histone acetylation and hence enhanced the expression of tight junction proteins occludin and ZO-1 and elevated the level of mucins, leading to enhanced integrity of intestinal epithelial barrier and reduced radiation-induced intestinal damage. Metformin, a first-line agent for the treatment of type II diabetes, promoted intestinal epithelial barrier integrity and reduced radiation intestinal damage through increasing the abundance of A. muciniphila. Together, our results demonstrated that A. muciniphila plays a critical role in the reduction of abdominal IR-induced intestinal damage. Application of probiotics or their regulators, such as metformin, could be an effective treatment for the protection of radiation exposure-damaged intestine.

Development and management of gastrointestinal symptoms in long-term COVID-19.

Background: Emerging evidence reveals that SARS-CoV-2 possesses the capability to disrupt the gastrointestinal (GI) homeostasis, resulting in the long-term symptoms such as loss of appetite, diarrhea, gastroesophageal reflux, and nausea. In the current review, we summarized recent reports regarding the long-term effects of COVID-19 (long COVID) on the gastrointestine. Objective: To provide a narrative review of abundant clinical evidence regarding the development and management of long-term GI symptoms in COVID-19 patients. Results: Long-term persistent digestive symptoms are exhibited in a majority of long-COVID patients. SARS-CoV-2 infection of intestinal epithelial cells, cytokine storm, gut dysbiosis, therapeutic drugs, psychological factors and exacerbation of primary underlying diseases lead to long-term GI symptoms in COVID-19 patients. Interventions like probiotics, prebiotics, fecal microbiota transplantation, and antibiotics are proved to be beneficial in preserving intestinal microecological homeostasis and alleviating GI symptoms. Conclusion: Timely diagnosis and treatment of GI symptoms in long-COVID patients hold great significance as they may contribute to the mitigation of severe conditions and ultimately lead to the improvement of outcomes of the patients.

Retinoic acid signaling during priming licenses intestinal CD103+ CD8 TRM cell differentiation.

CD8 tissue-resident memory T (TRM) cells provide frontline protection at barrier tissues; however, mechanisms regulating TRM cell development are not completely understood. Priming dictates the migration of effector T cells to the tissue, while factors in the tissue induce in situ TRM cell differentiation. Whether priming also regulates in situ TRM cell differentiation uncoupled from migration is unclear. Here, we demonstrate that T cell priming in the mesenteric lymph nodes (MLN) regulates CD103+ TRM cell differentiation in the intestine. In contrast, T cells primed in the spleen were impaired in the ability to differentiate into CD103+ TRM cells after entry into the intestine. MLN priming initiated a CD103+ TRM cell gene signature and licensed rapid CD103+ TRM cell differentiation in response to factors in the intestine. Licensing was regulated by retinoic acid signaling and primarily driven by factors other than CCR9 expression and CCR9-mediated gut homing. Thus, the MLN is specialized to promote intestinal CD103+ CD8 TRM cell development by licensing in situ differentiation.

Mitochondrial fission induces immunoescape in solid tumors through decreasing MHC-I surface expression.

Mitochondrial dynamics can regulate Major Histocompatibility Complex (MHC)-I antigen expression by cancer cells and their immunogenicity in mice and in patients with malignancies. A crucial role in the mitochondrial fragmentation connection with immunogenicity is played by the IRE1α-XBP-1s axis. XBP-1s is a transcription factor for aminopeptidase TPP2, which inhibits MHC-I complex cell surface expression likely by degrading tumor antigen peptides. Mitochondrial fission inhibition with Mdivi-1 upregulates MHC-I expression on cancer cells and enhances the efficacy of adoptive T cell therapy in patient-derived tumor models. Therefore mitochondrial fission inhibition might provide an approach to enhance the efficacy of T cell-based immunotherapy.

A Pan-Histone Deacetylase Inhibitor Enhances the Antitumor Activity of B7-H3-Specific CAR T Cells in Solid Tumors.

PURPOSE: The limited efficacy of chimeric antigen receptor (CAR) T-cell therapies with solid malignancies prompted us to test whether epigenetic therapy could enhance the antitumor activity of B7-H3.CAR T cells with several solid cancer types. EXPERIMENTAL DESIGN: We evaluated B7-H3 expression in many human solid cancer and normal tissue samples. The efficacy of the combinatorial therapy with B7-H3.CAR T cells and the deacetylase inhibitor SAHA with several solid cancer types and the potential underlying mechanisms were characterized with in vitro and ex vivo experiments. RESULTS: B7-H3 is expressed in most of the human solid tumor samples tested, but exhibits a restricted expression in normal tissues. B7-H3.CAR T cells selectively killed B7-H3 expressing human cancer cell lines in vitro. A low dose of SAHA upregulated B7-H3 expression in several types of solid cancer cells at the transcriptional level and B7-H3.CAR expression on human transgenic T-cell membrane. In contrast, the expression of immunosuppressive molecules, such as CTLA-4 and TET2, by T cells was downregulated upon SAHA treatment. A low dose of SAHA significantly enhanced the antitumor activity of B7-H3.CAR T cells with solid cancers in vitro and ex vivo, including orthotopic patient-derived xenograft and metastatic models treated with autologous CAR T-cell infusions. CONCLUSIONS: Our results show that our novel strategy which combines SAHA and B7-H3.CAR T cells enhances their therapeutic efficacy with solid cancers and justify its translation to a clinical setting.

LncRNA LTSCCAT promotes tongue squamous cell carcinoma metastasis via targeting the miR-103a-2-5p/SMYD3/TWIST1 axis.

Abnormal expression of long-noncoding RNA is involved in the tumorigenesis and progression of various cancers, but the potential molecular regulatory mechanisms are unclear. Microbial flora and chronic inflammation, such as periodontitis, which is associated with oral cancer, affect the occurrence and progression of tumors. Accordingly, we stimulated the tongue squamous cell carcinoma (TSCC) cell lines CAL27 and SCC15 with a low concentration of lipopolysaccharide (LPS) from Porphyromonas gingivalis (P.g) for 6 days and then performed LncRNA sequencing on P.g-LPS-treated CAL27 cells and untreated CAL27 cells. LTSCCAT was upregulated in P.g-LPS-treated CAL27 cells compared with untreated CAL27 cells. LTSCCAT induced epithelial-mesenchymal transition and promoted the invasion and metastasis of TSCC in vitro and in vivo. LncRNA LTSCCAT was upregulated in TSCC patients with periodontitis and was correlated with metastasis and poor prognosis. We predicted through an online database and confirmed by dual-luciferase reporter assays that LTSCCAT is a competitive endogenous RNA for the regulation of miR-103a-2-5p. Another dual-luciferase reporter assay confirmed that miR-103a-2-5p has a binding site at the 3'-UTR of the histone methylation transferase SMYD3 and inhibits its translation. Chromatin immunoprecipitation experiments demonstrated that SMYD3 binds directly to the promoter region of TWIST1 and promotes its transcription, which is related to H3K4 trimethylation. The effect of pcDNA/LTSCCAT on expression was attenuated by miR-103a-2-5p mimics. The RF and SVM classifier predicts that LTSCCAT can bind to SMYD3, whereas the RNA immunoprecipitation (RIP) assay confirms that it cannot. In addition, we predicted the combination of LTSCCAT and SMYD3 through software, but the RIP assay confirmed that LTSCCAT could not be combined with SMYD3. For the first time, we showed that periodontitis promotes the invasion and metastasis of TSCC and clarified the molecular mechanism of LTSCCAT to promote invasion and metastasis of TSCC, providing a potential therapeutic target for clinical treatment of TSCC.

lncRNA CISAL Inhibits BRCA1 Transcription by Forming a Tertiary Structure at Its Promoter.

Cisplatin-based neoadjuvant chemotherapy has been shown to improve survival in patients with squamous cell carcinoma (SCC), but clinical biomarkers to predict chemosensitivity remain elusive. Here, we show the long noncoding RNA (lncRNA) LINC01011, which we termed cisplatin-sensitivity-associated lncRNA (CISAL), controls mitochondrial fission and cisplatin sensitivity by inhibiting BRCA1 transcription in tongue SCC (TSCC) models. Mechanistically, we found CISAL directly binds the BRCA1 promoter and forms an RNA-DNA triplex structure, sequestering BRCA1 transcription factor-GABPA away from the downstream regulatory binding region. Importantly, the clinical relevance of these findings is suggested by the significant association of CISAL and BRCA1 expression levels in TSCC tumors with neoadjuvant chemosensitivity and overall survival. We propose a new model where lncRNAs are tethered at gene promoter by RNA-DNA triplex formation, spatially sequestering transcription factors away from DNA-binding sites. Our study uncovers the potential of CISAL-BRCA1 signaling as a potential target to predict or improve chemosensitivity.

Decreased expression of mitochondrial miR-5787 contributes to chemoresistance by reprogramming glucose metabolism and inhibiting MT-CO3 translation.

MicroRNAs (miRNAs) have been recently found in the mitochondria, and were named "mitomiRs", but their function has remained elusive. Here, we aimed to assess the presence and function(s) of mitomiRs in tongue squamous cell carcinoma (TSCC). Methods: miRNA microarray was performed in paired TSCC cell lines, Cal27 and its chemoresistant counterpart, Cal27-re. Decreased expression of mitomiRs in chemoresistant cells was characterized. The functions of mitomiRs were investigated by a series of in vitro and in vivo experiments. Results: Differential microarray analysis identified downregulation of mitomiR-5787 in Cal27-re cells. We knocked down mitomiR-5787 in parental cells and upregulated its expression in cisplatin-resistant cells. The sensitivity of TSCC cells to cisplatin was regulated by miR-5787. The glucose metabolism assay suggested that reduced expression of miR-5787 changed the balance of glucose metabolism by shifting it from oxidative phosphorylation to aerobic glycolysis. Xenograft experiments in BALB/c-nu mice further verified the in vitro results. Reduced expression of miR-5787 contributes to chemoresistance in TSCC cells by inhibiting the translation of mitochondrial cytochrome c oxidase subunit 3 (MT-CO3). The prognostic analysis of 126 TSCC patients showed that the patients with low expression of miR-5787 and/or MT-CO3 had poor cisplatin sensitivity and prognosis. Conclusions: Mitochondrial miR-5787 could regulate cisplatin resistance of TSCC cells and affect oxidative phosphorylation and aerobic glycolysis. Downregulation of miR-5787 inhibited the translation of MT-CO3 to regulate cisplatin resistance of TSCC. Mitochondrial miR-5787 and MT-CO3 can be used as predictive biomarkers or therapeutic targets for cisplatin chemotherapy resistance.

Long Noncoding RNA MPRL Promotes Mitochondrial Fission and Cisplatin Chemosensitivity via Disruption of Pre-miRNA Processing.

PURPOSE: The overall biological roles and clinical significance of most long noncoding RNAs (lncRNA) in chemosensitivity are not fully understood. We investigated the biological function, mechanism, and clinical significance of lncRNA NR_034085, which we termed miRNA processing-related lncRNA (MPRL), in tongue squamous cell carcinoma (TSCC). EXPERIMENTAL DESIGN: LncRNA expression in TSCC cell lines with cisplatin treatment was measured by lncRNA microarray and confirmed in TSCC tissues. The functional roles of MPRL were demonstrated by a series of in vitro and in vivo experiments. The miRNA profiles, RNA pull-down, RNA immunoprecipitation, serial deletion analysis, and luciferase analyses were used to investigate the potential mechanisms of MPRL. RESULTS: We found that MPRL expression was significantly upregulated in TSCC cell lines treated with cisplatin and transactivated by E2F1. MPRL controlled mitochondrial fission and cisplatin sensitivity through miR-483-5p. In exploring the underlying interaction between MPRL and miR-483-5p, we identified that cytoplasmic MPRL directly binds to pre-miR-483 within the loop region and blocks pre-miR-483 recognition and cleavage by TRBP-DICER-complex, thereby inhibiting miR-483-5p generation and upregulating miR-483-5p downstream target-FIS1 expression. Furthermore, overexpression or knockdown MPRL altered tumor apoptosis and growth in mouse xenografts. Importantly, we found that high expression of MPRL and pre-miR-483, and low expression of miR-483-5p were significantly associated with neoadjuvant chemosensitivity and better TSCC patients' prognosis. CONCLUSIONS: We propose a model in which lncRNAs impair microprocessor recognition and are efficient of pre-miRNA cropping. In addition, our study reveals a novel regulatory network for mitochondrial fission and chemosensitivity and new biomarkers for prediction of neoadjuvant chemosensitivity in TSCC.These findings uncover a novel mechanism by which lncRNA determines mitochondrial fission and cisplatin chemosensitivity by inhibition of pre-miRNA processing and provide for the first time the rationale for lncRNA and miRNA biogenesis for predicting chemosensitivity and patient clinical prognosis.

Downregulation of RGMA by HIF-1A/miR-210-3p axis promotes cell proliferation in oral squamous cell carcinoma.

Repulsive guidance molecules comprise a group of proteins that play an important role in carcinogenesis through interactions with their receptors, but their function in oral squamous cell carcinoma (OSCC) is unclear. Here, we investigated the potential role of the RGM family members in oral cancer pathogenesis. Our study showed that only RGMA was significantly downregulated in the OSCC tissues analyzed by TCGA and validated this finding in OSCC cells. The decreased expression of RGMA was strongly associated with the T stage and with poor prognosis. The ectopic expression of RGMA significantly inhibited the proliferation of OSCC cells both in vitro and in vivo. Moreover, we confirmed that RGMA was a target of miR-210-3p in OSCC and miR-210-3p overexpression contributed to the acceleration of OSCC growth. Further experiments revealed that HIF1A specifically interacted with the promoter of miR-210-3p and enhanced its expression. In summary, our research indicates that RGMA is regulated by the HIF1A/miR-210-3p axis and inhibits OSCC cell proliferation; thus, in the future, the development of therapies that target the HIF1A/miR-210-3p/RGMA axis may aid in the treatment of aggressive cancers.

Mitochondrial miRNA Determines Chemoresistance by Reprogramming Metabolism and Regulating Mitochondrial Transcription.

miRNAs that translocate from the nucleus to mitochondria are referred to as mitochondrial microRNAs (mitomiR). mitomiRs have been shown to modulate the translational activity of the mitochondrial genome, yet their role in mitochondrial DNA (mtDNA) transcription remains to be determined. Here we report that the mitomiR-2392 regulates chemoresistance in tongue squamous cell carcinoma (TSCC) cells by reprogramming metabolism via downregulation of oxidative phosphorylation and upregulation of glycolysis. These effects were mediated through partial inhibition of mtDNA transcription by mitomiR-2392 rather than through translational regulation. This repression required specific miRNA-mtDNA base pairing and Argonaute 2. mitomiR-2392 recognized target sequences in the H-strand and partially inhibited polycistronic mtDNA transcription in a cell-specific manner. A retrospective analysis of TSCC patient tumors revealed a significant association of miR-2392 and regulated mitochondrial gene expression with chemosensitivity and overall survival. The clinical relevance of targeted mitochondrial genes was consistently validated by The Cancer Genome Atlas RNA sequencing in multiple types of cancer. Our study revealed for the first time the role of mitomiR in mtDNA transcription and its contribution to the molecular basis of tumor cell metabolism and chemoresistance.Significance: These findings uncover a novel mechanism by which mitomiRNA regulates mitochondrial transcription and provide rationale for use of mitomiRNA and mtDNA-encoded genes to predict chemosensitivity and patient clinical prognosis.