ANXA1 Promotes Tumor Immune Evasion by Binding PARP1 and Upregulating Stat3-Induced Expression of PD-L1 in Multiple Cancers.

The deregulation of Annexin A1 (ANXA1), a regulator of inflammation and immunity, leads to cancer growth and metastasis. However, whether ANXA1 is involved in cancer immunosuppression is still unclear. Here, we report that ANXA1 knockdown (i) dramatically downregulates programmed cell death-ligand 1 (PD-L1) expression in breast cancer, lung cancer, and melanoma cells; (ii) promotes T cell-mediated killing of cancer cells in vitro; and (iii) inhibits cancer immune escape in immune-competent mice via downregulating PD-L1 expression and increasing the number and killing activity of CD8+ T cells. Mechanistically, ANXA1 functioned as a sponge molecule for interaction of PARP1 and Stat3. Specifically, binding of ANXA1 to PARP1 decreased PARP1's binding to Stat3, which reduced poly(ADP-ribosyl)ation and dephosphorylation of Stat3 and thus, increased Stat3's transcriptional activity, leading to transcriptionally upregulated expression of PD-L1 in multiple cancer cells. In clinical samples, expression of ANXA1 and PD-L1 was significantly higher in breast cancer, non-small cell lung cancer, and skin cutaneous melanoma compared with corresponding normal tissues and positively correlated in cancer tissues. Moreover, using both ANXA1 and PD-L1 proteins for predicting efficacy of anti-PD-1 immunotherapy and patient prognosis was superior to using individual proteins. Our data suggest that ANXA1 promotes cancer immune escape via binding PARP1 and upregulating Stat3-induced expression of PD-L1, that ANXA1 is a potential new target for cancer immunotherapy, and combination of ANXA1 and PD-L1 expression is a potential marker for predicting efficacy of anti-PD-1 immunotherapy in multiple cancers.

ANXA1-derived peptide for targeting PD-L1 degradation inhibits tumor immune evasion in multiple cancers.

BACKGROUND: Immune checkpoint inhibitors (ICIs) therapy targeting programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) shows promising clinical benefits. However, the relatively low response rate highlights the need to develop an alternative strategy to target PD-1/PD-L1 immune checkpoint. Our study focuses on the role and mechanism of annexin A1 (ANXA1)-derived peptide A11 degrading PD-L1 and the effect of A11 on tumor immune evasion in multiple cancers. METHODS: Binding of A11 to PD-L1 was identified by biotin pull-down coupled with mass spectrometry analysis. USP7 as PD-L1's deubiquitinase was found by screening a human deubiquitinase cDNA library. The role and mechanism of A11 competing with USP7 to degrade PD-L1 were analyzed. The capability to enhance the T cell-mediated tumor cell killing activity and antitumor effect of A11 via suppressing tumor immune evasion were investigated. The synergistic antitumor effect of A11 and PD-L1 mAb (monoclonal antibody) via suppressing tumor immune evasion were also studied in mice. The expression and clinical significance of USP7 and PD-L1 in cancer tissues were evaluated by immunohistochemistry. RESULTS: A11 decreases PD-L1 protein stability and levels by ubiquitin proteasome pathway in breast cancer, lung cancer and melanoma cells. Mechanistically, A11 competes with PD-L1's deubiquitinase USP7 for binding PD-L1, and then degrades PD-L1 by inhibiting USP7-mediated PD-L1 deubiquitination. Functionally, A11 promotes T cell ability of killing cancer cells in vitro, inhibits tumor immune evasion in mice via increasing the population and activation of CD8+ T cells in tumor microenvironment, and A11 and PD-1 mAb possess synergistic antitumor effect in mice. Moreover, expression levels of both USP7 and PD-L1 are significantly higher in breast cancer, non-small cell lung cancer and skin melanoma tissues than those in their corresponding normal tissues and are positively correlated in cancer tissues, and both proteins for predicting efficacy of PD-1 mAb immunotherapy and patient prognosis are superior to individual protein. CONCLUSION: Our results reveal that A11 competes with USP7 to bind and degrade PD-L1 in cancer cells, A11 exhibits obvious antitumor effects and synergistic antitumor activity with PD-1 mAb via inhibiting tumor immune evasion and A11 can serve as an alternative strategy for ICIs therapy in multiple cancers.

BCAT2 Shapes a Noninflamed Tumor Microenvironment and Induces Resistance to Anti-PD-1/PD-L1 Immunotherapy by Negatively Regulating Proinflammatory Chemokines and Anticancer Immunity.

To improve response rate of monotherapy of immune checkpoint blockade (ICB), it is necessary to find an emerging target in combination therapy. Through analyzing tumor microenvironment (TME)-related indicators, it is validated that BCAT2 shapes a noninflamed TME in bladder cancer. The outcomes of multiomics indicate that BCAT2 has an inhibitory effect on cytotoxic lymphocyte recruitment by restraining activities of proinflammatory cytokine/chemokine-related pathways and T-cell-chemotaxis pathway. Immunoassays reveal that secretion of CD8+ T-cell-related chemokines keeps a robust negative correlation with BCAT2, generating a decreasing tendency of CD8+ T cells around BCAT2+ tumor cells from far to near. Cotreatment of BCAT2 deficiency and anti-PD-1 antibody has a synergistic effect in vivo, implying the potential of BCAT2 in combination therapy. Moreover, the value of BCAT2 in predicting efficacy of immunotherapy is validated in multiple immunotherapy cohorts. Together, as a key molecule in TME, BCAT2 is an emerging target in combination with ICB and a biomarker of guiding precision therapy.

Cuproptosis-related modification patterns depict the tumor microenvironment, precision immunotherapy, and prognosis of kidney renal clear cell carcinoma.

Background: Due to the different infiltration abundance of immune cells in tumor, the efficacy of immunotherapy varies widely among individuals. Recently, growing evidence suggested that cuproptosis has impact on cancer immunity profoundly. However, the comprehensive roles of cuproptosis-related genes in tumor microenvironment (TME) and in response to immunotherapy are still unclear. Methods: Based on 43 cuproptosis-related genes, we employed unsupervised clustering to identify cuproptosis-related patterns and single-sample gene set enrichment analysis algorithm to build a cuproptosis signature for individual patient's immune cell infiltration and efficacy of immune checkpoint blockade (ICB) evaluation. Then, the cuproptosis-related genes were narrowed down using univariate Cox regression model and least absolute shrinkage and selection operator algorithm. Finally, a cuproptosis risk score was built by random survival forest based on these narrowed-down genes. Results: Two distinct cuproptosis-related patterns were developed, with cuproptosis cluster 1 showing better prognosis and higher enrichment of immune-related pathways and infiltration of immune cells. For individual evaluation, the cuproptosis signature that we built could be used not only for predicting immune cell infiltration in TME but also for evaluating an individual's sensitivity to ICBs. Patients with higher cuproptosis signature scores exhibited more activated cancer immune processes, higher immune cell infiltration, and better curative efficacy of ICBs. Furthermore, a robust cuproptosis risk score indicated that patients with higher risk scores showed worse survival outcomes, which could be validated in internal and external validation cohorts. Ultimately, a nomogram which combined the risk score with the prognostic clinical factors was developed, and it showed excellent prediction accuracy for survival outcomes. Conclusion: Distinct cuproptosis-related patterns have significant differences on prognosis and immune cell infiltration in kidney renal clear cell carcinoma (KIRC). Cuproptosis signature and risk score are able to provide guidance for precision therapy and accurate prognosis prediction for patients with KIRC.

ANXA1 Binds and Stabilizes EphA2 to Promote Nasopharyngeal Carcinoma Growth and Metastasis.

Overexpression of ANXA1 and EphA2 has been linked to various cancers and both proteins have attracted considerable attention for the development of new anticancer drugs. Here we report that ANXA1 competes with Cbl for binding EphA2 and increases its stability by inhibiting Cbl-mediated EphA2 ubiquitination and degradation in nasopharyngeal carcinoma (NPC). Binding of ANXA1 to EphA2 promoted NPC cell growth and metastasis in vitro and in vivo by elevating EphA2 levels and increasing activity of EphA2 oncogenic signaling (pS897-EphA2). Expression of ANXA1 and EphA2 was positively correlated and both were significantly higher in NPC tissues than in the normal nasopharyngeal epithelial tissues. Patients with high expression of both proteins presented poorer disease-free survival and overall survival relative to patients with high expression of one protein alone. Furthermore, amino acid residues 20-30aa and 28-30aa of the ANXA1 N-terminus bound EphA2. An 11 amino acid-long ANXA1-derived peptide (EYVQTVKSSKG) was developed on the basis of this N-terminal region, which disrupted the connection of ANXA1 with EphA2, successfully downregulating EphA2 expression and dramatically suppressing NPC cell oncogenicity in vitro and in mice. These findings suggest that ANXA1 promotes NPC growth and metastasis via binding and stabilization of EphA2 and present a strategy for targeting EphA2 degradation and treating NPC with a peptide. This therapeutic strategy may also be extended to other cancers with high expression of both proteins. SIGNIFICANCE: These findings show that EphA2 is a potential target for NPC therapeutics and an ANXA1-derived peptide suppresses NPC growth and metastasis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/20/4386/F1.large.jpg.

Gene Signatures and Prognostic Values of m6A Genes in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignant tumor with a high rate of local invasion and early distant metastasis. Accumulating studies suggest that N6-methyladenosine methylation (m6A) is closely related to tumorigenesis. However, the relationship between m6A-related genes and prognosis of NPC is poorly understood. Our research aims to discover the prognostic value of m6A RNA methylation genes in NPC. In this study, we analyzed the differentially expressed m6A-related genes between NPC samples and normal control samples and found that two upregulated genes (YTHDF3 and IGF2BP2) and one downregulated gene (METTL3) were overlapped in GSE68799 and GSE53819. Next, we found that high expression of IGF2BP1 and low expression of METTL3 and YTHDF3 in NPC patients showed poor progression-free survival (PFS). Subsequently, the four m6A genes were selected for consensus cluster analysis, and risk models were established. The risk signature, using three genes (GF2BP1 + IGF2BP2 + METTL3), was an independent prognostic factor and predicts the clinicopathological features of NPC. Additionally, the GO, KEGG analysis, and CIBERSORT algorithm revealed that the risk signature was closely associated to immune infiltration in NPC. Finally, the expression and clinical significance of METTL3 were successfully validated in NPC tissues using immunohistochemical techniques. In conclusion, our finding revealed the potential role of m6A modification in NPC, providing novel insight into NPC prognosis and therapeutic strategies.

HDAC7 promotes the oncogenicity of nasopharyngeal carcinoma cells by miR-4465-EphA2 signaling axis.

HDAC7 plays a crucial role in cancers, and is the main drug target of several HDAC inhibitors. However, the role and mechanism of HDAC7 in nasopharyngeal carcinoma (NPC) are still unclear. In this study, we observed that HDAC7 was significantly upregulated in the NPC tissues relative to normal nasopharyngeal mucosa (NNM) tissues, HDAC7 expression levels were positively correlated with NPC progression and negatively correlated with patient prognosis, and HDAC7 knockdown dramatically inhibited the in vitro proliferation, migration, and invasion of NPC cells, and the growth of NPC xenografts in mice, indicating the HDAC7 promotes the oncogenicity of NPC. Mechanistically, HDAC7 promoted the in vitro proliferation, migration, and invasion of NPC cells by upregulating EphA2, in which miR-4465 mediated HDAC7-regulating EphA2, a direct target gene of miR-4465. We further showed that miR-4465 was significantly downregulated in the NPC tissues relative to NNM tissues, and inhibited the in vitro proliferation, migration, and invasion of NPC cells by targeting EphA2 expression. Moreover, we observed that the expressions of HDAC7, miR-4465, and EphA2 in NPC tissues were correlated. The results suggest that HDAC7 promotes the oncogenicity of NPC by downregulating miR-4465 and subsequently upregulating EphA2, highlighting HDAC7 as a potential therapeutic target for NPC.

Facial nerve schwannoma mimicking chronic suppurative otitis media: A case report.

RATIONALE: Facial nerve schwannoma (FNS) is a rare slow-growing nerve sheath tumor derived from Schwann cells. FNS with normal facial nerve function may sometimes be misdiagnosed as otitis media because of similar ontological symptoms such as purulence, tympanic membrane damage, and hearing loss. PATIENT CONCERNS: A 68-year-old woman was referred to our department because of otorrhea and hearing loss in the right ear for 20 years. Otoscopy revealed abundant purulent secretions deep in the right external auditory canal, and granulation proliferation in the posterior part of membranae tensa. Audiogram showed a right mixed hearing loss with an 85-dB pure-tone average and 35-dB air-bone gap. DIAGNOSIS: This patient was misdiagnosed as chronic suppurative otitis media before surgery. During surgery, a mass was found, and intraoperative frozen section histopathology confirmed an FNS. INTERVENTIONS: This patient was subjected to mastoidectomy for curing chronic suppurative otitis media initially. During surgery, a mass was found attached and widely extended into the tympanic and mastoid segments. We removed most part of the mass, however found the mass deriving from the vertical part of the facial nerve. Intraoperative frozen section histopathology confirmed an FNS. So we removed the incurs and malleus, and searched for the edge of the mass. The mass involved multisegments of facial nerve including the tympanic, vertical and pyramidal segments. The tumor was removed completely, and nerves were repaired using greater auricular nerves. OUTCOMES: After surgery, the patient had facial nerve paralysis of House-Brackmann (HB) Grade VI. Facial function recovered to HB Grade III at 30 months after surgery. The patient was followed up for 5 years. She had a facial function of HB grade III at the most recent follow-up. LESSONS: FNS is rare and tend to be misdiagnosed. It is important to combine the imaging modalities of computed tomography and magnetic resonance imaging to evaluate FNS before surgery. The primary goal of managing FNS is to maintain normal facial function as long as possible; therefore, tailored strategy should be taken for managing FNS.

MicroRNA­16 inhibits interleukin­13­induced inflammatory cytokine secretion and mucus production in nasal epithelial cells by suppressing the IκB kinase ß/nuclear factor­κB pathway.

Chronic inflammation of the nasal mucosal tissue plays important roles in the pathogenesis of allergic rhinitis (AR). Aberrantly expressed microRNAs (miRNAs) have been found to have strong associations with inflammatory reactions in allergic diseases; however, its functional significance and molecular mechanism underlying in AR remains unclear. The aim of the present study was to investigate the biological functions of miRNA and reveal its underlying molecular mechanisms in AR. miRNA microarray was performed to analyze miRNAs expression levels in 3 paired nasal mucosal samples from patients with AR and a control group. Subsequently, human nasal epithelial cells (JME/CF15) were used as an in vitro model to further explore the functions of miRNAs. Microarray data revealed that miR­16 was one of the miRNAs being most significantly downregulated. Interleukin (IL)­13 stimulation gradually decreased the levels of miR­16 in JME/CF15 cells. Moreover, upregulation of miR­16 inhibited inflammatory cytokines, including granulocyte­macrophage colony­stimulating factor (GM­CSF), eotaxin, IL­1ß, IL­6 and IL­10 in IL­13­treated JME/CF15 cells. Furthermore, overexpression of miR­16 significantly decreased the mRNA and protein expression levels of mucin 5AC (MUC5AC). IκB kinase ß (IKKß) was identified as a direct target of miR­16 and its expression was negatively regulated by miR­16 at mRNA and protein levels. Notably, forced expression of miR­16 blocked NF­κB signaling by decreasing the expression of nuclear p­p65 and p­IκB­α, as well as increasing the expression of IκB­α in IL­13­treated nasal epithelial cells. Moreover, enhanced IKKß reactivated the NF­κB pathway that was blocked by miR­16 mimics and then effectively suppressed the miR­16­mediated inhibitory effects on inflammatory response. These findings suggested that miR­16 suppressed the inflammatory response by inhibiting the activation of IKKß/NF­κB signaling pathways.

Effects of micronutrients on the reproduction of infertility rat model induced by adenine.

Male infertility is a serious global medical and social issue demanding more specific and effective treatments. In this study, we generated a male infertility rat model using adenine induction to study the effects of certain micronutrients on reproduction. Fifty male SD rats were used in the study, and thirty of them received daily intra-gastric administration of 300 mg/kg adenine for four weeks. The thirty adenine treated mice were evenly divided into 3 groups to receive intra-gastric administration of micronutrient mixture of vitamin A, vitamin C, vitamin E, Zinc, and selenium (micronutrient group), normal saline (model control group), and methyl testosterone solution (androgen group). The other twenty rats used were normal male SD rats that were evenly divided into two groups to receive intra-gastric administration of normal saline (normal control group) and micronutrient mixture first then adenine 40 min later (micronutrient prevention group). After four weeks of micronutrient and other treatments, all rats were sacrificed for analyses. Compared with those in the model control group, the rats in the micronutrient group showed significantly improved physical signs, significantly increased body weights, significantly increased testis index, significantly increased sperm counts and motility, significantly decreased sperm malformation, and significantly repaired testis tissue. Compared with those in the model control group, the rats in the micronutrient group showed significantly decreased FSH levels and recovered LH and testosterone levels. The rats in the micronutrient prevention group did not show significant differences in sperm counts, sperm motility, sperm malformation, and hormonal levels from those in the normal control group. The findings from this study provide evidence for the potential application of micronutrients in male infertility treatments.