CRISPR-mediated MECOM depletion retards tumor growth by reducing cancer stem cell properties in lung squamous cell carcinoma.

Targeted therapy for lung squamous cell carcinoma (LUSC) remains a challenge due to the lack of robust targets. Here, we identified MECOM as a candidate of therapeutic target for LUSC by screening 38 genes that were commonly amplified in three pairs of primary tumors and patient-derived xenografts (PDXs) using a clustered regularly interspaced short palindromic repeats (CRISPR)-mediated approach. High MECOM expression levels were associated with poor prognosis. Forced expression of MECOM in LUSC cell lines promoted cancer stem cell (CSC) properties, and its knockout inhibited CSC phenotypes. Furthermore, systemic delivery of CRISPR-mediated MECOM depletion cassette using adenovirus with an adaptor, which is composed of a single-chain fragment variable (scFv) against epithelial cell adhesion molecules (EpCAM) fused to the ectodomain of coxsackievirus and adenovirus receptor, and a protector, which consists of the scFv connected to the hexon symmetry of the adenovirus, could specifically target subcutaneous and orthotopic LUSC and retard tumor growth. This study could provide a novel therapeutic strategy for LUSC with high efficacy and specificity.

Molecular Subtyping and Prognostic Assessment Based on Tumor Mutation Burden in Patients with Lung Adenocarcinomas.

The distinct molecular subtypes of lung cancer are defined by monogenic biomarkers, such as EGFR, KRAS, and ALK rearrangement. Tumor mutation burden (TMB) is a potential biomarker for response to immunotherapy, which is one of the measures for genomic instability. The molecular subtyping based on TMB has not been well characterized in lung adenocarcinomas in the Chinese population. Here we performed molecular subtyping based on TMB with the published whole exome sequencing data of 101 lung adenocarcinomas and compared the different features of the classified subtypes, including clinical features, somatic driver genes, and mutational signatures. We found that patients with lower TMB have a longer disease-free survival, and higher TMB is associated with smoking and aging. Analysis of somatic driver genes and mutational signatures demonstrates a significant association between somatic RYR2 mutations and the subtype with higher TMB. Molecular subtyping based on TMB is a potential prognostic marker for lung adenocarcinoma. Signature 4 and the mutation of RYR2 are highlighted in the TMB-High group. The mutation of RYR2 is a significant biomarker associated with high TMB in lung adenocarcinoma.

Genomic sequencing and editing revealed the GRM8 signaling pathway as potential therapeutic targets of squamous cell lung cancer.

The study sought to explore novel genetic aberration driving squamous cell lung carcinoma (LUSC). The whole exome (WES), whole genome (WGS) and target region (TS) sequencings and CRISPR-Cas9 genome editing techniques were integrated to explore and validate novel targeting candidates from LUSC primary tumors and corresponding patient-derived xenografts (PDXs). Seven genes (FGFR2, GRM1,PIK3CG, PIK3CA,ZFHX4, CSMD3, GRM8) with high frequencies of both single nucleotide variants (SNVs) and copy number variants (CNVs), and two genes (CLDN1 and RIT1) only with CNVs were identified by bioinformatics analysis. The functions of these candidates were validated through CRISPR-Cas9 system in primary PDX cells. Furthermore, we focused on the genetic and functional analysis of Metabotropic glutamate receptor 8 (GRM8), whose transcriptional activation was elucidated to promote the survival of LUSC tumor cell through inhibiting cAMP pathway and activating MAPK pathway. The SNV identified in GRM8, A112G, activated downstream signaling pathway and induced cell proliferation, which could be reversed by cAMP stimulator and MEK inhibitor. In conclusion, the components of GRM8 signaling pathway could serve as potential targets of squamous cell lung cancer carrying GRM8 activating variants.

Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells.

BACKGROUND: Viral infection causes multiple forms of human cancer, and HPV infection is the primary factor in cervical carcinomas. Recent single-cell RNA-seq studies highlight the tumor heterogeneity present in most cancers, but virally induced tumors have not been studied. HeLa is a well characterized HPV+ cervical cancer cell line. RESULT: We developed a new high throughput platform to prepare single-cell RNA on a nanoliter scale based on a customized microwell chip. Using this method, we successfully amplified full-length transcripts of 669 single HeLa S3 cells and 40 of them were randomly selected to perform single-cell RNA sequencing. Based on these data, we obtained a comprehensive understanding of the heterogeneity of HeLa S3 cells in gene expression, alternative splicing and fusions. Furthermore, we identified a high diversity of HPV-18 expression and splicing at the single-cell level. By co-expression analysis we identified 283 E6, E7 co-regulated genes, including CDC25, PCNA, PLK4, BUB1B and IRF1 known to interact with HPV viral proteins. CONCLUSION: Our results reveal the heterogeneity of a virus-infected cell line. It not only provides a transcriptome characterization of HeLa S3 cells at the single cell level, but is a demonstration of the power of single cell RNA-seq analysis of virally infected cells and cancers.

Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing.

BACKGROUND: Single-cell resequencing (SCRS) provides many biomedical advances in variations detection at the single-cell level, but it currently relies on whole genome amplification (WGA). Three methods are commonly used for WGA: multiple displacement amplification (MDA), degenerate-oligonucleotide-primed PCR (DOP-PCR) and multiple annealing and looping-based amplification cycles (MALBAC). However, a comprehensive comparison of variations detection performance between these WGA methods has not yet been performed. RESULTS: We systematically compared the advantages and disadvantages of different WGA methods, focusing particularly on variations detection. Low-coverage whole-genome sequencing revealed that DOP-PCR had the highest duplication ratio, but an even read distribution and the best reproducibility and accuracy for detection of copy-number variations (CNVs). However, MDA had significantly higher genome recovery sensitivity (~84 %) than DOP-PCR (~6 %) and MALBAC (~52 %) at high sequencing depth. MALBAC and MDA had comparable single-nucleotide variations detection efficiency, false-positive ratio, and allele drop-out ratio. We further demonstrated that SCRS data amplified by either MDA or MALBAC from a gastric cancer cell line could accurately detect gastric cancer CNVs with comparable sensitivity and specificity, including amplifications of 12p11.22 (KRAS) and 9p24.1 (JAK2, CD274, and PDCD1LG2). CONCLUSIONS: Our findings provide a comprehensive comparison of variations detection performance using SCRS amplified by different WGA methods. It will guide researchers to determine which WGA method is best suited to individual experimental needs at single-cell level.

Frequent alterations in cytoskeleton remodelling genes in primary and metastatic lung adenocarcinomas.

The landscape of genetic alterations in lung adenocarcinoma derived from Asian patients is largely uncharacterized. Here we present an integrated genomic and transcriptomic analysis of 335 primary lung adenocarcinomas and 35 corresponding lymph node metastases from Chinese patients. Altogether 13 significantly mutated genes are identified, including the most commonly mutated gene TP53 and novel mutation targets such as RHPN2, GLI3 and MRC2. TP53 mutations are furthermore significantly enriched in tumours from patients harbouring metastases. Genes regulating cytoskeleton remodelling processes are also frequently altered, especially in metastatic samples, of which the high expression level of IQGAP3 is identified as a marker for poor prognosis. Our study represents the first large-scale sequencing effort on lung adenocarcinoma in Asian patients and provides a comprehensive mutational landscape for both primary and metastatic tumours. This may thus form a basis for personalized medical care and shed light on the molecular pathogenesis of metastatic lung adenocarcinoma.