Inhibition of yes‐associated protein down‐regulates PD‐L1 (CD274) expression in human malignant pleural mesothelioma

Although tumour PD‐L1 (CD274) expression had been used as a predictive biomarker in checkpoint immunotherapy targeting the PD1/PD‐L1 axis in various cancers, the regulation of PD‐L1 (CD274) expression is unclear. Yes‐associated protein (YAP), an important oncogenic protein in Hippo signalling pathway, reportedly promotes cancer development. We investigated whether inhibition of YAP down‐regulates PD‐L1 (CD274) in human malignant pleural mesothelioma (MPM). Western blotting showed that 2 human MPM cell lines (H2052 and 211H) had increased PD‐L1 protein expression compared to H290, MS‐1 and H28 cells. In H2052 and 211H cells, PD‐L1 mRNA expression was significantly increased compared to other MPM cell lines; YAP knockdown by small interfering RNA decreased PD‐L1 protein and mRNA expression. Forced overexpression of the YAP gene increased PD‐L1 protein expression in H2452 cells. Chromatin immunoprecipitation (ChIP) assay showed the precipitation of PD‐L1 enhancer region encompassing 2 putative YAP‐TEAD‐binding sites in H2052 cells. We found that, in human MPM tissue microarray samples, YAP and PD‐L1 concurrently expressed in immunohistochemistry stain (n = 70, P < .05, chi‐square). We conclude that PD‐L1 is correlated with YAP expression, and inhibition of YAP down‐regulates PD‐L1 expression in human MPM. Further study of how YAP regulates PD‐L1 in MPM is warranted.     

PINCH‐2 expression in cancers involving serosal effusions using quantitative PCR

Y. Yuan, H. P. Dong, D. A. Nymoen, J. M. Nesland, C. Wu and B. Davidson
PINCH‐2 expression in cancers involving serosal effusions using quantitative PCR Objective: The PINCH‐2 gene was previously shown to be overexpressed in malignant mesothelioma compared with ovarian/peritoneal serous carcinoma in Affymetrix array analysis. The objective of the present study was to validate this finding at the mRNA and protein level. Methods: Effusions (n = 91; 71 ovarian and 10 breast carcinomas, 10 malignant mesotheliomas) were assayed for PINCH‐2 mRNA expression using quantitative PCR. PINCH‐2 protein expression was analysed in 37 effusions using flow cytometry. Results: Quantitative PCR analysis showed significantly higher PINCH‐2 mRNA levels in mesotheliomas compared with carcinomas (P = 0.004). Values of <10 copies were found exclusively in carcinoma effusions (25.4% of ovarian and 50% of breast carcinomas). However, PINCH‐2 protein expression by flow cytometry did not differ significantly between the three cancer types. No association was observed between PINCH‐2 levels and patient survival or expression of previously‐studied molecules related to adhesion, metastasis and apoptosis inhibition in ovarian carcinoma. Conclusions: Our data suggest that PINCH‐2 mRNA is overexpressed in malignant mesothelioma compared with carcinomas involving serosal cavities, and that low levels of this gene argue against the diagnosis of mesothelioma. The frequent PINCH‐2 protein expression in all three studied cancers suggests a role for this molecule in cancer cell biology in effusions and merits further research.     

Aquaporin‐1 inhibition reduces metastatic formation in a mouse model of melanoma

Aquaporin‐1 (AQP1) is a proangiogenic water channel protein promoting endothelial cell migration. We previously reported that AQP1 silencing by RNA interference reduces angiogenesis‐dependent primary tumour growth in a mouse model of melanoma. In this study, we tested the hypothesis that AQP1 inhibition also affects animal survival and lung nodule formation. Melanoma was induced by injecting B16F10 cells into the back of C57BL6J mice. Intratumoural injection of AQP1 siRNA and CTRL siRNA was performed 10 days after tumour cell implantation. Lung nodule formation was analysed after the death of the mice. Western blot was used to quantify HIF‐1α, caspase‐3 (CASP3) and metalloproteinase‐2 (MMP2) protein levels. We found that AQP1 knock‐down (KD) strongly inhibited metastatic lung nodule formation. Moreover, AQP1 siRNA‐treated mice showed a twofold survival advantage compared to mice receiving CTRL siRNAs. The reduced AQP1‐dependent tumour angiogenesis caused a hypoxic condition, evaluated by HIF‐1α significant increase, in turn causing an increased level of apoptosis in AQP1 KD tumours, assessed by CASP3 quantification and DNA fragmentation. Importantly, a decreased level of MMP2 after AQP1 KD indicated a decreased activity against extracellular matrix associated with reduced vascularization and metastatic formation. In conclusion, these findings highlight an additional role for AQP1 as an important determinant of tumour dissemination by facilitating tumour cell extravasation and metastatic formation. This study adds knowledge on the role played by AQP1 in tumour biology and supports the view of AQP1 as a potential drug target for cancer therapy.     

Mutational landscape and genetic signatures of cell‐free DNA in tumour‐induced osteomalacia

Tumour‐induced osteomalacia (TIO) is a very rare paraneoplastic syndrome with bone pain, fractures and muscle weakness, which is mostly caused by phosphaturic mesenchymal tumours (PMTs). Cell‐free DNA (cfDNA) has been regarded as a non‐invasive liquid biopsy for many malignant tumours. However, it has not been studied in benign tumours, which prompted us to adopt the targeted next‐generation sequencing approach to compare cfDNAs of 4 TIO patients, four patients with bone metastasis (BM) and 10 healthy controls. The mutational landscapes of cfDNA in TIO and BM groups were similar in the spectrum of allele frequencies and mutation types. Markedly, deleterious missense mutations in FGFR1 and loss‐of‐function mutations in MED12 were found in 3/4 TIO patients but none of BM patients. The gene ontology analysis strongly supported that these mutated genes found in TIOs would play a potential role in PMTs' process. The genetic signatures and corresponding change in expression of FGFR1 and FGF23 were further validated in PMT tissues from a test cohort of another three TIO patients. In summary, we reported the first study of the mutational landscape and genetic signatures of cfDNA in TIO/PMTs.     

Aquaporin‐4 deficiency reduces TGF‐β1 in mouse midbrains and exacerbates pathology in experimental Parkinson's disease

Aquaporin‐4 (AQP4), the main water‐selective membrane transport protein in the brain, is localized to the astrocyte plasma membrane. Following the establishment of a 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease (PD) model, AQP4‐deficient (AQP4^?/?) mice displayed significantly stronger microglial inflammatory responses and remarkably greater losses of tyrosine hydroxylase (TH⁺)‐positive neurons than did wild‐type AQP4 (AQP4^+/+) controls. Microglia are the most important immune cells that mediate immune inflammation in PD. However, recently, few studies have reported why AQP4 deficiency results in more severe hypermicrogliosis and neuronal damage after MPTP treatment. In this study, transforming growth factor‐β1 (TGF‐β1), a key suppressive cytokine in PD onset and development, failed to increase in the midbrain and peripheral blood of AQP4^?/? mice after MPTP treatment. Furthermore, the lower level of TGF‐β1 in AQP4^?/? mice partially resulted from impairment of its generation by astrocytes; reduced TGF‐β1 may partially contribute to the uncontrolled microglial inflammatory responses and subsequent severe loss of TH⁺ neurons in AQP4^?/? mice after MPTP treatment. Our study provides not only a better understanding of both aetiological and pathogenical factors implicated in the neurodegenerative mechanism of PD but also a possible approach to developing new treatments for PD via intervention in AQP4‐mediated immune regulation.     

Proteomic differential display analysis identified upregulated astrocytic phosphoprotein PEA‐15 in human malignant pleural mesothelioma cell lines

We performed proteomic differential display analysis of human malignant pleural mesothelioma (MPM) cell lines and a human pleural mesothelial cell line by using 2‐DE and LC‐MS/MS. The human MPM cell lines were NCI‐H28, NCI‐H2052 and NCI‐H2452, and the human pleural mesothelial cell line was MeT‐5A. Between MeT‐5A and NCI‐H2052, we found 38 protein spots whose expression levels were different, from the results of 2‐DE; 28 protein spots appeared higher, and 10 other protein spots lower in NCI‐H2052 than in MeT‐5A. These spots were analyzed by LC‐MS/MS analysis and identified by a peptide sequence tag. However, from the results of 2‐DE of the other cell lines, there was only one consistently upregulated protein, astrocytic phosphoprotein PEA‐15, in all three MPM cell lines. Western blotting using specific antibodies against PEA‐15 confirmed the elevated expression level of PEA‐15 in all three MPM cell lines compared with MeT‐5A cells and normal pleura tissues from patients. PEA‐15 was knocked down in NCI‐H2052 cells, and the proliferation of PEA‐15‐silenced NCI‐H2052 cells was suppressed 7–15% compared with negative control cells. These results suggest that PEA‐15 expression is likely to be associated with the tumorigenesis of MPM.     

Cul4A overexpression associated with Gli1 expression in malignant pleural mesothelioma

Malignant pleural mesothelioma (mesothelioma) is a highly aggressive cancer without an effective treatment. Cul4A, a scaffold protein that recruits substrates for degradation, is amplified in several human cancers, including mesothelioma. We have recently shown that Cul4A plays an oncogenic role in vitro and in a mouse model. In this study, we analysed clinical mesothelioma tumours and found moderate to strong expression of Cul4A in 70.9% (51/72) of these tumours, as shown by immunohistochemistry. In 72.2% mesothelioma tumours with increased Cul4A copy number identified by fluorescence in situ hybridization analysis, Cul4A protein expression was moderate to strong. Similarly, Cul4A was overexpressed and Cul4A copy number was increased in human mesothelioma cell lines. Because Gli1 is highly expressed in human mesothelioma cells, we compared Cul4A and Gli1 expression in mesothelioma tumours and found their expression associated (P < 0.05, chi‐square). In mesothelioma cell lines, inhibiting Cul4A by siRNA decreased Gli1 expression, suggesting that Gli1 expression is, at least in part, regulated by Cul4A in mesothelioma cells. Our results suggest a linkage between Cul4A and Gli1 expression in human mesothelioma.     

Diagnostic value of GLUT-1 in distinguishing malignant mesothelioma from reactive mesothelial cells: a meta-analysis

Abstract

Background: Previous studies have demonstrated the diagnostic value of glucose transporter 1 (GLUT-1) to distinguish malignant mesothelioma (MM) from reactive mesothelial cells (RMC), but the results are inconsistent. The purpose of this meta-analysis is to investigate the diagnostic accuracy of GLUT-1 in distinguishing MM from RMC.

Methods: A systematical search was conducted until May 2019 in PubMed, Medline, Embase and the Cochrane Library. The revised tool for the quality assessment of diagnostic accuracy studies (QUADAS-2) was used to assess the quality of the eligible studies. The Stata15 and Review Manager5.3 software programmes were used to perform the meta-analysis.

Results: A total of 24 studies, including 969?MM patients and 1080 RMC individuals were explored in the meta-analysis. The summary assessments revealed that the pooled sensitivity was 0.73 (95% CI, 0.62–0.81) and the pooled specificity was 0.95 (95% CI, 0.91–0.98). The area under the summary ROC curve (AUC) was 0.93 (95% CI: 0.91–0.95).

Conclusions: GLUT-1 is highly accurate to distinguish MM from RMC.     

Tumour cell‐intrinsic CTLA4 regulates PD‐L1 expression in non‐small cell lung cancer

Cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death protein 1 (PD‐1) are immune checkpoint proteins expressed in T cells. Although CTLA4 expression was found in multiple tumours including non‐small cell lung cancer (NSCLC) tissues and cells, its function in tumour cells is unknown. Recently, PD‐1 was found to be expressed in melanoma cells and to promote tumorigenesis. We found that CTLA4 was expressed in a subset of NSCLC cell lines and in a subgroup of cancer cells within the lung cancer tissues. We further found that in NSCLC cells, anti‐CTLA4 antibody can induce PD‐L1 expression, which is mediated by CTLA4 and the EGFR pathway involving phosphorylation of MEK and ERK. In CTLA4 knockout cells, EGFR knockout cells or in the presence of an EGFR tyrosine kinase inhibitor, anti‐CTLA4 antibody was not able to induce PD‐L1 expression in NSCLC cells. Moreover, anti‐CTLA4 antibody promoted NSCLC cell proliferation in vitro and tumour growth in vivo in the absence of adaptive immunity. These results suggest that tumour cell‐intrinsic CTLA4 can regulate PD‐L1 expression and cell proliferation, and that anti‐CTLA4 antibody, by binding to the tumour cell‐intrinsic CTLA4, may result in the activation of the EGFR pathway in cancer cells.