Energy status dictates PD-L1 protein abundance and anti-tumor immunity to enable checkpoint blockade.

Aberrant energy status contributes to multiple metabolic diseases, including obesity, diabetes, and cancer, but the underlying mechanism remains elusive. Here, we report that ketogenic-diet-induced changes in energy status enhance the efficacy of anti-CTLA-4 immunotherapy by decreasing PD-L1 protein levels and increasing expression of type-I interferon (IFN) and antigen presentation genes. Mechanistically, energy deprivation activates AMP-activated protein kinase (AMPK), which in turn, phosphorylates PD-L1 on Ser283, thereby disrupting its interaction with CMTM4 and subsequently triggering PD-L1 degradation. In addition, AMPK phosphorylates EZH2, which disrupts PRC2 function, leading to enhanced IFNs and antigen presentation gene expression. Through these mechanisms, AMPK agonists or ketogenic diets enhance the efficacy of anti-CTLA-4 immunotherapy and improve the overall survival rate in syngeneic mouse tumor models. Our findings reveal a pivotal role for AMPK in regulating the immune response to immune-checkpoint blockade and advocate for combining ketogenic diets or AMPK agonists with anti-CTLA4 immunotherapy to combat cancer.

Prognostic significance and immune characteristics of CMTM4 in hepatocellular carcinoma.

BACKGROUND: Previous study has shown that chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing family member 4 (CMTM4) can bind and maintain programmed cell death ligand 1 (PD-L1) expression to promote tumor progression by alleviating the suppression of tumor-specific T cell activity, suggesting its potential role in tumor immunotherapy. However, the role of CMTM4 in tumor immunity has not been well clarified, especially in hepatocellular carcinoma (HCC). METHODS: The protein expression of CMTM4/PD-L1/CD4/CD8 was detected by immunohistochemistry (IHC) detection in 90 cases of HCC tissues. The mRNA expression profiles and related prognosis data were obtained from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC). Two immune therapy cohorts were from Imvigor210 and GSE176307. RESULTS: Though the single protein expression of CMTM4, PD-L1, CD4 or CD8 in HCC tissues by IHC detection didn't show a significant relationship with the prognosis of HCC patients, we found that high co-expression of CMTM4/PD-L1/CD4 showed a good prognosis of HCC patients. Further Timer 2.0 analysis identified that HCC patients with high expression of CMTM4/PD-L1 and high infiltration of CD4+ T cells had a better overall survival than those with low infiltration of CD4+ T cells. Moreover, a series of bioinformatics analyses revealed that CMTM4-related genes posed important effects on prognosis and immunity in HCC patients, and CMTM4 had a positive correlation with infiltration of CD4+ and CD8+ T cells in HCC. At last, we used two immunotherapy cohorts to verify that the combination of CMTM4 with PD-L1 could improve the prognosis of tumor patients underwent immunotherapy. CONCLUSIONS: CMTM4 and PD-L1 co-expression with T cell infiltration shows prognostic significance in HCC, suggesting combined effect from multiple proteins should be considered in HCC treatment.

CMTM4 regulates epithelial-mesenchymal transition and PD-L1 expression in head and neck squamous cell carcinoma.

The epithelial-mesenchymal transition (EMT) is a pivotal step involved in cancer recurrence and metastasis. In addition, the activation of the EMT program can induce a cancer stem cell (CSC)-like phenotype and programmed death-ligand 1 (PD-L1) expression in head and neck squamous cell carcinoma (HNSCC). The CMTM family has reported as an important regulator in this process. Here, we investigated the role of CMTM4 in HNSCC. We indicated that CMTM4 was overexpressed in human and mouse HNSCC samples and in HNSCC cell lines by immunohistochemistry and Western blot. A high expression level of CMTM4 was correlated with advanced lymph node metastasis and a negative prognosis. CMTM4-knockdown by small interfering RNA downregulated the EMT process and inhibited the migration and invasion abilities of tumor cells. Moreover, knockdown of CMTM4 decreased CSC-associated markers via the protein kinase B pathway. Notably, CMTM4-knockdown inhibited the expression of interferon-γ induced PD-L1 in HNSCC cells. A positive correlation was found between CMTM4 expression and CD8+ and PD-1+ cell density in the stroma. Our findings indicated that CMTM4 may play an important role in regulating EMT/CSC phenotypes and PD-L1 expression. This study may reinforce the interest in CMTM4 as a potential target for the prognosis and treatment of HNSCC.

Integrated Analyses of Phenotype and Quantitative Proteome of CMTM4 Deficient Mice Reveal Its Association with Male Fertility.

The chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing family (CMTM) is a gene family that has been implicated in male reproduction. CMTM4 is an evolutionarily conserved member that is highly expressed in the testis. However, its function in male fertility remains unknown. Here, we demonstrate that CMTM4 is associated with spermatogenesis and sperm quality. Using Western blotting and immunohistochemical analyses, we found CMTM4 expression to be decreased in poor-quality human spermatozoa, old human testes, and testicular biopsies with nonobstructive azoospermia. Using CRISPR-Cas9 technology, we knocked out the Cmtm4 gene in mice. These Cmtm4 knockout (KO) mice showed reduced testicular daily sperm production, lower epididymal sperm motility and increased proportion of abnormally backward-curved sperm heads and bent sperm midpieces. These mice also had an evident sub-fertile phenotype, characterized by low pregnancy rates on prolonged breeding with wild type female mice, reduced in vitro fertilization efficiency and a reduced percentage of acrosome reactions. We then performed quantitative proteomic analysis of the testes, where we identified 139 proteins to be downregulated in Cmtm4-KO mice, 100 (71.9%) of which were related to sperm motility and acrosome reaction. The same proteomic analysis was performed on sperm, where we identified 3588 proteins with 409 being differentially regulated in Cmtm4-KO mice. Our enrichment analysis showed that upregulated proteins were enriched with nucleosomal DNA binding functions and the downregulated proteins were enriched with actin binding functions. These findings elucidate the roles of CMTM4 in male fertility and demonstrates its potential as a promising molecular candidate for sperm quality assessment and the diagnosis or treatment of male infertility.

CMTM4 regulates angiogenesis by promoting cell surface recycling of VE-cadherin to endothelial adherens junctions.

Vascular endothelial (VE) cadherin is a key component of endothelial adherens junctions (AJs) and plays an important role in maintaining vascular integrity. Endocytosis of VE-cadherin regulates junctional strength and a decrease of surface VE-cadherin reduces vascular stability. However, disruption of AJs is also a requirement for vascular sprouting. Identifying novel regulators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we evaluated the angiogenic potential of (CKLF-like MARVEL transmembrane domain 4) CMTM4 and assessed in which molecular pathway CMTM4 is involved during angiogenesis. Using a 3D vascular assay composed of GFP-labeled HUVECs and dsRED-labeled pericytes, we demonstrated in vitro that siRNA-mediated CMTM4 silencing impairs vascular sprouting. In vivo, CMTM4 silencing by morpholino injection in zebrafish larvae inhibits intersomitic vessel growth. Intracellular staining revealed that CMTM4 colocalizes with Rab4+ and Rab7+ vesicles, both markers of the endocytic trafficking pathway. CMTM4 colocalizes with both membrane-bound and internalized VE-cadherin. Adenovirus-mediated CMTM4 overexpression enhances the endothelial endocytic pathway, in particular the rapid recycling pathway, shown by an increase in early endosomal antigen-1 positive (EEA1+), Rab4+, Rab11+ , and Rab7+ vesicles. CMTM4 overexpression enhances membrane-bound VE-cadherin internalization, whereas CMTM4 knockdown decreases internalization of VE-cadherin. CMTM4 overexpression promotes endothelial barrier function, shown by an increase in recovery of transendothelial electrical resistance (TEER) after thrombin stimulation. We have identified in this study a novel regulatory function for CMTM4 in angiogenesis. CMTM4 plays an important role in the turnover of membrane-bound VE-cadherin at AJs, mediating endothelial barrier function and controlling vascular sprouting.

CMTM4 inhibits cell proliferation and migration via AKT, ERK1/2, and STAT3 pathway in colorectal cancer.

CMTM4 (CKLF-like MARVEL transmembrane domain containing 4), a potential tumor suppressor gene, is involved in several types of malignancies. It has been reported to be downregulated and exhibit anti-tumorigenic activities by regulating cell growth and cell cycle in clear cell renal cell carcinoma. It has also been identified as a tumor suppressor in hepatocellular carcinoma (HCC), and its negative expression is a risk factor for poor prognosis of HCC patients. In the present study, an integrated bioinformatics analysis based on The Cancer Genome Atlas (TCGA) database showed that CMTM4 was frequently reduced in colorectal cancer (CRC) and high expression of CMTM4 was associated with increased overall survival rates. Based on these findings, we adopted gain-of-function and lost-of-function strategies using SW480 and HT29 CRC cell lines which have relatively low and high endogenous CMTM4 levels, respectively. We observed impeded cell proliferation and migration upon overexpression of CMTM4 in SW480 cells, and the opposite effects were observed upon knockdown of CMTM4 in HT-29 cells. Cell signaling pathways essential for CRC progression were then examined, and the phosphorylation levels of AKT, ERK1/2, and STAT3 were found to be decreased by CMTM4 overexpression in SW480 cells and elevated by CMTM4 silencing in HT29 cells. Their inhibitors were used to validate that the three signaling pathways contributed to the inhibitory effects of CMTM4 on CRC cells. Taken together, our results suggest that CMTM4 plays a tumor suppressive role in CRC.

Cmtm4 deficiency exacerbates colitis by inducing gut dysbiosis and S100a8/9 expression.

The dysfunction of innate immunity components is one of the major drivers for ulcerative colitis (UC), and increasing reports indicate that the gut microbiome serves as an intermediate between genetic mutations and UC development. Here, we find that the IL-17 receptor subunit, CMTM4, is reduced in UC patients and dextran sulfate sodium (DSS)-induced colitis. The deletion of CMTM4 (Cmtm4-/-) in mice leads to a higher susceptibility to DSS-induced colitis than in wild-type, and the gut microbiome significantly changes in composition. The causal role of the gut microbiome is confirmed with a cohousing experiment. We further identify that S100a8/9 is significantly up-regulated in Cmtm4-/- colitis, with the block of its receptor RAGE that reverses the phenotype associated with the CMTM4 deficiency. CMTM4 deficiency rather suppresses S100a8/9 expression in vitro via the IL17 pathway, further supporting that the elevation of S100a8/9 in vivo is most likely a result of microbial dysbiosis. Taken together, the results suggest that CMTM4 is involved in the maintenance of intestinal homeostasis, suppression of S100a8/9, and prevention of colitis development. Our study further shows CMTM4 as a crucial innate immunity component, confirming its important role in UC development and providing insights into potential targets for the development of future therapies.

CMTM6 and CMTM4 as two novel regulators of PD-L1 modulate the tumor microenvironment.

The tumor microenvironment (TME) plays crucial roles in regulating tumor occurrence, progress, metastasis and drug resistance. However, it remains largely elusive how the components of TME are regulated to govern its functions in tumor biology. Here, we discussed how the two novel functional proteins, chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing 6 (CMTM6) and CMTM4, which involved in the post-translational regulation of PD-L1, modulate the TME functions. The roles of CMTM6 and CMTM4 in regulating TME components, including immune cells and tumor cells themselves were discussed in this review. The potential clinical applications of CMTM6 and CMTM4 as biomarkers to predict therapy efficacy and as new or combined immunotherapy targets are also highlighted. Finally, the current hot topics for the biological function of CMTM6/4 and several significant research directions for CMTM6/4 are also briefly summarized in the review.

Co-Expression with Membrane CMTM6/4 on Tumor Epithelium Enhances the Prediction Value of PD-L1 on Anti-PD-1/L1 Therapeutic Efficacy in Gastric Adenocarcinoma.

Anti-PD-1/L1 immunotherapy has been intensively used in heavily treated population with advanced gastric adenocarcinoma. However, the immunotherapeutic efficacy is low even in PD-L1 positive patients. We aimed to establish a new strategy based on the co-expression of CMTM6/4 and PD-L1 for patient stratification before immunotherapy. By analyzing the data obtained from TCGA and single-cell RNA sequencing at the mRNA level, and 6-color multiplex immunofluorescence staining of tumor tissues in tissue array and 48-case pre-immunotherapy patients at the protein level, we found that CMTM6/4 and PD-L1 co-expressed in both epithelial and mesenchymal regions of gastric adenocarcinoma. The tumor tissues had higher levels of CMTM6/4 expression than their adjacent ones. A positive correlation was found between the expression of CMTM6/4 and the expression of PD-L1 in tumor epithelium. Epithelial co-expression of CMTM6/4 and PD-L1 in gastric tumor region was associated with shorter overall survival but better short-term response to anti-PD-1/L1 immunotherapy. Thus, we developed a predictive model and three pathological patterns based on the membrane co-expression of CMTM6/4 and PD-L1 in tumor epithelial cells for pre-immunotherapy patient screening in gastric adenocarcinoma.

Expression of CMTM4 shows clinical significance in lung cancer.

BACKGROUND: Human chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing family (CMTM) is implicated in the pathogenesis of immune, reproductive systems and various cancers. However, the expression and clinical significance of CMTM4 in lung cancer have not been identified. METHODS: We performed immunohistochemistry to detect the expression of CMTM4 in 75 paired lung adenocarcinoma and non-tumor lung tissues. The correlation between CMTM4 expression and clinical significance of lung cancer patients was analyzed by Chi-square test. Kaplan-Meier method and Log-Rank test were used to calculate the survival time of lung cancer patients. RESULTS: We found that CMTM4 was positively expressed in 34/75 (45.3%) cases of lung adenocarcinoma tissues, while positively expressed in 59/75 (78.6%) cases of non-tumor lung tissues, suggesting a lower expression of CMTM4 in lung adenocarcinoma tissues than non-tumor lung tissues (P<0.05). In addition, the negative expression of CMTM4 was associated with gender, smoking, and metastasis in lung cancer patients. Moreover, lung cancer patients with negative expression of CMTM4 had a shorter survival time than the patients with positive expression of CMTM4. COX regression analysis showed that CMTM4 was an independent prognostic factor for the overall survival of lung cancer patients. CONCLUSIONS: Our study supports that CMTM4 can be used as a new marker for the treatment and prognosis of lung cancer.

Pancreatic stellate cells derived exosomal miR-5703 promotes pancreatic cancer by downregulating CMTM4 and activating PI3K/Akt pathway.

Exosomes play important role in tumor microenvironment, and mediate the crosstalk between pancreatic cancer (PC) cells and matrix components including pancreatic stellate cells (PSCs) to regulate pancreatic cancer progression. Here we isolated primary PSCs from PC patients, and demonstrated that PSC-derived exosomes could be internalized by PC cells to promote cell proliferation. Furthermore, we identified that miR-5703 in the exosomes acted as a driver of cell proliferation and its inhibitor suppressed the function of exosomes to promote PC cell proliferation. miR-5703 directly bound to the 3'UTR of CMTM4 and downregulated its expression. CMTM4 knockdown promoted PC cell proliferation, while overexpression of CMTM4 suppressed PC cell proliferation both in vivo and in vitro. Mechanistically, we revealed that CMTM4 suppressed PI3K/Akt pathway via downregulating PAK4. In conclusion, our results suggest that PSC-derived exosomal miR-5703 could target CMTM4 in PC cells and promote cell proliferation due to PAK4 activated PI3K/Akt pathway.

Expression Analysis of Canine CMTM6 and CMTM4 as Potential Regulators of the PD-L1 Protein in Canine Cancers.

Cancer is one of the most significant causes of death in dogs. Antibody drugs targeting the PD-1/PD-L1 axis represent a promising immunotherapy for both human and canine cancers. However, the regulation mechanisms of PD-L1 expression in canine cancers require further investigation to better understand the resistance mechanisms to anti-PD-L1 therapy. Recent reports have shown that CMTM6 and CMTM4 are critical regulators of PD-L1 protein expression in human cancer cells. By preventing PD-L1 from lysosome-mediated degradation, CMTM6 maintains PD-L1 expression on the cell surface. However, the literature has not reported on CMTM6 and CMTM4 in dogs, and their functions are completely unknown. To reveal a regulation mechanism of PD-L1 in canine cancers, this study firstly identified the gene sequences of CMTM6 and CMTM4. Then, the expression analysis of these proteins was performed by immunohistochemistry. Furthermore, the functions of CMTM6 and CMTM4 in regulating PD-L1 expression were examined by gene knockdown of CMTM6 and CMTM4. Canine CMTM6 and CMTM4 displayed high amino acid sequence identities compared with those of humans and mice. An immunohistochemical analysis using cross-reactive antibodies revealed that canine malignant melanoma and osteosarcoma express CMTM6, CMTM4, and PD-L1 simultaneously. Gene knockdown of CMTM6 and CMTM4 with RNA interference significantly reduced the cell surface expression of PD-L1 in a canine cell line. These results suggest that CMTM6 and CMTM4 are regulators of PD-L1 expression in canine cancers and could serve as potential therapeutic targets to enhance antitumor immunity.

MicroRNA-205 inhibits renal cells apoptosis via targeting CMTM4.

OBJECTIVES: MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression. They have important roles in kidney development, homeostasis and disease, and participate in the onset and progression of tubulointerstitial sclerosis and end-stage glomerular lesions that occur in various forms of chronic kidney disease (CKD). In the present study, we elucidated the role of microRNA 205 (miR-205) in cisplatin-induced renal cell apoptosis and explored the molecular mechanisms. MATERIALS AND METHODS: The chronic interstitial nephropathy rat model was induced, and the miRNA expression profile in the kidney cells from rats with CKD was screened. Cisplatin-induced apoptosis in normal renal HK-2 cells was evaluated using flow cytometry, and regulation of miR-205 on target gene was validated using luciferase assay, western blot and real time PCR assays. RESULTS: We found that miR-205 expression was significantly decreased in the cells from kidney of CKD rat (P<0.01). Our data showed that when miR-205 was overexpressed or silenced using the mimic or inhibitor, the percentages of apoptotic cells were suppressed or increased significantly (P<0.05), respectively. Moreover, we have identified CMTM4 gene, which is involved in cell proliferation and apoptosis, as a novel target for miR-205. In addition, miR-205 could inhibit apoptosis by binding to the 3'UTR of CMTM4 mRNA and inhibiting its transcriptional activity. CONCLUSION: This study elucidated that miR-205 plays an important role in the regulation of apoptosis in renal cells, suggesting a potential therapeutic target to hinder CKD development.