Aberrant Methylation of the Imprinted C19MC and MIR371-3 Clusters in Patients with Non-Small Cell Lung Cancer.

Epigenetic mechanisms have emerged as an important contributor to tumor development through the modulation of gene expression. Our objective was to identify the methylation profile of the imprinted C19MC and MIR371-3 clusters in patients with non-small cell lung cancer (NSCLC) and to find their potential target genes, as well as to study their prognostic role. DNA methylation status was analyzed in a NSCLC patient cohort (n = 47) and compared with a control cohort including COPD patients and non-COPD subjects (n = 23) using the Illumina Infinium Human Methylation 450 BeadChip. Hypomethylation of miRNAs located on chromosome 19q13.42 was found to be specific for tumor tissue. We then identified the target mRNA-miRNA regulatory network for the components of the C19MC and MIR371-3 clusters using the miRTargetLink 2.0 Human tool. The correlations of miRNA-target mRNA expression from primary lung tumors were analyzed using the CancerMIRNome tool. From those negative correlations identified, we found that a lower expression of 5 of the target genes (FOXF2, KLF13, MICA, TCEAL1 and TGFBR2) was significantly associated with poor overall survival. Taken together, this study demonstrates that the imprinted C19MC and MIR371-3 miRNA clusters undergo polycistronic epigenetic regulation leading to deregulation of important and common target genes with potential prognostic value in lung cancer.

Real-World Analysis of Nivolumab and Atezolizumab Efficacy in Previously Treated Patients with Advanced Non-Small Cell Lung Cancer.

Nivolumab (anti-PD-1 antibody) and atezolizumab (anti-PD-L1 antibody) have shown superior survival outcomes and improved adverse effects compared to standard chemotherapy in advanced non-small cell lung cancer (NSCLC) patients. However, the efficacy of both treatments has not been directly compared in clinical trials. This retrospective, single-centre study was performed from June 2015 to December 2020 and included a cohort of 158 previously treated patients with stage IV or recurrent NSCLC who received PD-1 (nivolumab) (n = 89) or PD-L1 (atezolizumab) (n = 69) inhibitors at the Virgen del Rocío Hospital in Seville. The objective response rate (ORR) was 22.5% in the nivolumab group and 14.5% in the atezolizumab group (p = 0.140). Multivariate analysis did not show significant differences between the two groups for PFS and OS (PFS hazard ratio (HR): 0.80, 95% confidence interval (CI): 0.55−1.17, p = 0.260; OS HR: 0.79, 95% CI: 0.52−1.21, p = 0.281). Adverse events of all grades occurred in 68 patients in the nivolumab group (76.4%) and in 34 patients in the atezolizumab group (49.3%) (p < 0.001). Atezolizumab and nivolumab did not show statistically significant differences in survival outcomes in patients with NSCLC, even when stratified by histological subtype (squamous versus nonsquamous). However, the safety analysis suggested a more favourable toxicity profile for atezolizumab.

PKP1 and MYC create a feedforward loop linking transcription and translation in squamous cell lung cancer.

PURPOSE: Plakophilin 1 (PKP1) is well-known as an important component of the desmosome, a cell structure specialized in spot-like cell-to-cell adhesion. Although desmosomes have generally been associated with tumor suppressor functions, we recently found that PKP1 is recurrently overexpressed in squamous cell lung cancer (SqCLC) to exert an oncogenic role by enhancing the translation of MYC (c-Myc), a major oncogene. In this study, we aim to further characterize the functional relationship between PKP1 and MYC. METHODS: To determine the functional relationship between PKP1 and MYC, we performed correlation analyses between PKP1 and MYC mRNA expression levels, gain/loss of function models, chromatin immunoprecipitation (ChIP) and promoter mutagenesis followed by luciferase assays. RESULTS: We found a significant correlation between the mRNA levels of MYC and PKP1 in SqCLC primary tumor samples. In addition, we found that MYC is a direct transcription factor of PKP1 and binds to specific sequences within its promoter. In agreement with this, we found that MYC knockdown reduced PKP1 protein expression in different SqCLC models, which may explain the PKP1-MYC correlation that we found. Conversely, we found that PKP1 knockdown reduced MYC protein expression, while PKP1 overexpression enhanced MYC expression in these models. CONCLUSIONS: Based on these results, we propose a feedforward functional relationship in which PKP1 enhances MYC translation in conjunction with the translation initiation complex by binding to the 5'-UTR of MYC mRNA, whereas MYC promotes PKP1 transcription by binding to its promoter. These results suggest that PKP1 may serve as a therapeutic target for SqCLC.

Correlation of peripheral blood biomarkers with clinical outcomes in NSCLC patients with high PD-L1 expression treated with pembrolizumab.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are currently the standard therapy in advanced non-small cell lung cancer (NSCLC); however, there is no well-established prognostic biomarker. We investigated the relationship between survival outcomes and three peripheral blood biomarkers, including the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and monocyte-to-lymphocyte ratio (MLR), as well as a new score termed the risk blood biomarker (RBB), calculated from the combination of the neutrophil-monocyte-to-lymphocyte ratio (NMLR) and white blood cell count (WBC). METHODS: This study included patients with stage IV or recurrent NSCLC confirmed with programmed death ligand 1 (PD-L1) expression ≥50% who received pembrolizumab monotherapy as first-line treatment at the Virgen del Rocío University Hospital in Seville, Spain. To establish the relationship between baseline peripheral blood biomarkers and survival outcomes, progression free survival (PFS) and overall survival (OS), we used the Kaplan-Meier method and multivariable Cox regression models. RESULTS: A total of 51 patients were included in this study. In multivariate analysis, baseline NLR and PLR showed a strong association with PFS [NLR hazard ratio (HR): 0.19, 95% confidence interval (CI): 0.09-0.44, P<0.001; PLR HR: 0.46, 95% CI: 0.23-0.92, P=0.03] and OS (NLR HR: 0.07, 95% CI: 0.02-0.19, P<0.001; PLR HR: 0.29, 95% CI: 0.13-0.67, P=0.004), and the MLR was associated with OS (MLR HR: 0.34, 95% CI: 0.15-0.76, P=0.01). According to the RBB score, groups with lower scores were associated with superior PFS (group 0: HR: 0.16, 95% CI: 0.06-0.41, P<0.001 and group 1: HR: 0.29, 95% CI: 0.12-0.73, P=0.01) and OS (group 0: HR: 0.04, 95% CI: 0.01-0.17, P<0.001 and group 1: HR: 0.15, 95% CI: 0.05-0.42, P<0.001). CONCLUSIONS: Low baseline NLR, MLR and PLR are significantly associated with better PFS, and low baseline NLR and PLR are associated with better OS. Additionally, we identified three subgroups of patients using the RBB score, and low scores were associated with improved survival outcomes and response to therapy.

Primary and Acquired Resistance to Immunotherapy in Lung Cancer: Unveiling the Mechanisms Underlying of Immune Checkpoint Blockade Therapy.

After several decades without maintained responses or long-term survival of patients with lung cancer, novel therapies have emerged as a hopeful milestone in this research field. The appearance of immunotherapy, especially immune checkpoint inhibitors, has improved both the overall survival and quality of life of patients, many of whom are diagnosed late when classical treatments are ineffective. Despite these unprecedented results, a high percentage of patients do not respond initially to treatment or relapse after a period of response. This is due to resistance mechanisms, which require understanding in order to prevent them and develop strategies to overcome them and increase the number of patients who can benefit from immunotherapy. This review highlights the current knowledge of the mechanisms and their involvement in resistance to immunotherapy in lung cancer, such as aberrations in tumor neoantigen burden, effector T-cell infiltration in the tumor microenvironment (TME), epigenetic modulation, the transcriptional signature, signaling pathways, T-cell exhaustion, and the microbiome. Further research dissecting intratumor and host heterogeneity is necessary to provide answers regarding the immunotherapy response and develop more effective treatments for lung cancer.

The Roles of Imprinted SLC22A18 and SLC22A18AS Gene Overexpression Caused by Promoter CpG Island Hypomethylation as Diagnostic and Prognostic Biomarkers for Non-Small Cell Lung Cancer Patients.

Genomic imprinting is a process that involves one gene copy turned-off in a parent-of-origin-dependent manner. The regulation of imprinted genes is broadly dependent on promoter methylation marks, which are frequently associated with both oncogenes and tumor suppressors. The purpose of this study was to assess the DNA methylation patterns of the imprinted solute-carrier family 22 member 18 (SLC22A18) and SLC22A18 antisense (SLC22A18AS) genes in non-small cell lung cancer (NSCLC) patients to study their relevance to the disease. We found that both genes were hypomethylated in adenocarcinoma and squamous cell carcinoma patients. Due to this imprinting loss, SLC22A18 and SLC22A18AS were found to be overexpressed in NSCLC tissues, which is significantly more evident in lung adenocarcinoma patients. These results were validated through analyses of public databases of NSCLC patients. The reversed gene profile of both genes was achieved in vitro by treatment with ademetionine. We then showed that high SLC22A18 and SLC22A18AS expression levels were significantly associated with worsening disease progression. In addition, low levels of SLC22A18AS were also correlated with better overall survival for lung adenocarcinoma patients. We found that SLC22A18 and SLC22A18AS knockdown inhibits cell proliferation in vitro. All these results suggest that both genes may be useful as diagnostic and prognostic biomarkers in NSCLC, revealing novel therapeutic opportunities.

Correction: Plakophilin 1 enhances MYC translation, promoting squamous cell lung cancer.

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

Plakophilin 1 enhances MYC translation, promoting squamous cell lung cancer.

Plakophilin 1 (PKP1) is a member of the arm-repeat (armadillo) and plakophilin gene families and it is an essential component of the desmosomes. Although desmosomes have generally been associated with tumor suppressor functions, we have consistently observed that PKP1 is among the top overexpressed proteins in squamous cell lung cancer. To explore this paradox, we developed in vivo and in vitro functional models of PKP1 gain/loss in squamous cell lung cancer. CRISPR-Cas9 PKP1 knockout severely impaired cell proliferation, but it increased cell dissemination. In addition, PKP1 overexpression increased cell proliferation, cell survival, and in vivo xenograft engraftment. We further investigated the molecular mechanism of the mainly oncogenic function of PKP1 by combining transcriptomics, proteomics, and protein-nucleic acid interaction assays. Interestingly, we found that PKP1 enhances MYC translation in collaboration with the translation initiation complex by binding to the 5'-UTR of MYC mRNA. We propose PKP1 as an oncogene in SqCLC and a novel posttranscriptional regulator of MYC. PKP1 may be a valuable diagnostic biomarker and potential therapeutic target for SqCLC. Importantly, PKP1 inhibition may indirectly target MYC, a primary anticancer target.

Survival, classifications, and desmosomal plaque genes in non-small cell lung cancer.

Novel biomarkers are required to improve prognostic predictions obtained with lung cancer staging systems. This study of 62 surgically-treated Non-Small Cell Lung Cancer (NSCLC) patients had two objectives: i) to compare the predictive value of T-stage classifications between the 6(th) and 7(th) editions of the Tumor, Node, and Metastasis staging system (TNM); and ii) to examine the association of Pkp1 and/or Krt15 gene expression with survival and outcomes. Multivariate and Kaplan-Meier survival analyses were performed, examining the relationship of survival with T-stage, recurrence, and TNM-stage (by each TNM edition) and with the single/combined expression of Pkp1 and/or Krt15 genes. Five-year survival rates only significantly differed as a function of T-stage in patients without recurrence when estimated using the 6(th) edition of the TNM classification and only in patients in pathologic TNM-stage IA using the 7(th). Overall survival for patients with elevated expression of both genes was 13.5 months in those with adenocarcinoma and 34.6 months in those with squamous cell carcinoma. Overall survival was 30.4 months in patients with Pkp1 gene upregulation and 30.9 months in those with Krt15 gene upregulation. In conclusion, survival estimations as a function of T-staging differed between the 6(th) and 7(th) editions of TNM. Overall survival differed according to the expression of Pkp1 and/or Krt15 genes, although this relationship did not reach statistical significance.

Differential immunohistochemical localization of desmosomal plaque-related proteins in non-small-cell lung cancer.

AIMS: Immunohistochemistry is a highly valuable and widely used tool in the subtyping of lung carcinomas. The aim of this study was to identify markers for the differential diagnosis of non-small-cell carcinomas. METHODS AND RESULTS: We report on the immunohistochemical localization of plakophilin-1 (PKP1), keratin-15 (KRT15) and desmoglein-3 (DSG3) intercellular adhesion proteins in samples from 75 primary non-small-cell lung cancers in non-treated patients. The staining pattern of these proteins differed between squamous cell carcinomas and adenocarcinomas, with no membrane staining in the latter. Membrane staining for all three proteins was characteristic of squamous cell carcinomas. We observed a relationship between the presence/absence of these proteins in the membranes of squamous cell carcinomas and the differentiation grade, with more intense staining in better differentiated areas. CONCLUSIONS: Staining for these proteins marked intercellular junctions that are characteristic of stratified squamous epithelium and of neoplasias with this type of differentiation, and can be useful in the diagnosis of patients with squamous cell carcinoma of the lung. The high specificity of membrane staining for PKP1 and DSG3 and high sensitivity of cytoplasmic and membrane staining for KRT15 for the diagnosis of squamous cell carcinoma may be useful for the differential diagnosis of non-small-cell carcinomas.

Gene expression profiling reveals novel biomarkers in nonsmall cell lung cancer.

The development of reliable gene expression profiling technology is having an increasing impact on our understanding of lung cancer biology. Our study aimed to determine any correlation between the phenotypic heterogeneity and genetic diversity of lung cancer. Microarray analysis was performed on a set of 46 tumor samples and 45 paired nontumor samples of nonsmall cell lung cancer (NSCLC) samples to establish gene signatures in primary adenocarcinomas and squamous-cell carcinomas, determine differentially expressed gene sequences at different stages of the disease and identify sequences with biological significance for tumor progression. After the microarray analysis, the expression level of 92 selected genes was validated by qPCR and the robust Bonferroni test in an independent set of 70 samples composed of 48 tumor samples and 22 nontumor samples. Gene sequences were differentially expressed as a function of tumor type, stage and differentiation grade. High upregulation was observed for KRT15 and PKP1, which may be good markers to distinguish squamous-cell carcinoma samples. High downregulation was observed for DSG3 in stage IA adenocarcinomas.