B4GALT1 Is a New Candidate to Maintain the Stemness of Lung Cancer Stem Cells.

Claudia De Vitis,Christian Napoli,Francesca De Nicola,Francesca Ascenzi,Valentina Salvati,Maurizio Fanciulli,Simona Di Martino,Gennaro Ciliberto,Giacomo Corleone,Massimiliano Bassi,Sara Bruschini,Federico Venuta,Alberto Ricci,Rita Mancini,Matteo Pallocca,Erino Angelo Rendina

doi:10.3390/jcm8111928

Abstract

Background: According to the cancer stem cells (CSCs) hypothesis, a population of cancer cells with stem cell properties is responsible for tumor propagation, drug resistance, and disease recurrence. Study of the mechanisms responsible for lung CSCs propagation is expected to provide better understanding of cancer biology and new opportunities for therapy. Methods: The Lung Adenocarcinoma (LUAD) NCI-H460 cell line was grown either as 2D or as 3D cultures. Transcriptomic and genome-wide chromatin accessibility studies of 2D vs. 3D cultures were carried out using RNA-sequencing and Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq), respectively. Reverse transcription polymerase chain reaction (RT-PCR) was also carried out on RNA extracted from primary cultures derived from malignant pleural effusions to validate RNA-seq results. Results: RNA-seq and ATAC-seq data disentangled transcriptional and genome accessibility variability of 3D vs. 2D cultures in NCI-H460 cells. The examination of genomic landscape of genes upregulated in 3D vs. 2D cultures led to the identification of 2D cultures led to the identification of Beta-1,4-galactosyltranferase 1 (B4GALT1) as the top candidate. B4GALT1 as the top candidate. B4GALT1 was validated as a stemness factor, since its silencing caused strong inhibition of 3D spheroid formation. Conclusion: Combined transcriptomic and chromatin accessibility study of 3D vs. 2D LUAD cultures led to the identification of B4GALT1 as a new factor involved in the propagation and maintenance of LUAD CSCs.

Highlights

Lung cancer is the leading cause of cancer mortality worldwide [1]
Lung cancer can be divided into two major types, non-small cell lung cancer (NSCLC), which accounts for 80% of cases, and small cell lung cancer (SCLC)
We used the 3D spheroid assay on the malignant pleural effusions (MPEs) derived Lung Adenocarcinoma (LUAD) cell line NCI-H460 and intersected transcriptomic with epigenetic signatures of cancer stem cells (CSCs)-enriched (3D cultures) vs. differentiated cells (2D cultures) by RNA-seq and ATAC-seq, respectively

Summary

Introduction

Lung cancer is the leading cause of cancer mortality worldwide [1]. Lung cancer can be divided into two major types, non-small cell lung cancer (NSCLC), which accounts for 80% of cases, and small cell lung cancer (SCLC). SCD1 inhibition induced the inactivation of β-catenin and YAP/TAZ signalling pathways [18] Based on these observations and on those of several other laboratories, we consider the 3D spheroid assay a valuable approach to identify factors responsible for the maintenance and propagation of lung CSCs [19]. We have applied this approach to identify regulatory principles responsible for CSCs maintenance For this purpose, we used the 3D spheroid assay on the MPEs derived LUAD cell line NCI-H460 and intersected transcriptomic with epigenetic signatures of CSCs-enriched (3D cultures) vs differentiated cells (2D cultures) by RNA-seq and ATAC-seq, respectively. We singled out and validated the role of the B4GALT1 as an emerging gene in CSCs propagation and as a target for their inhibition

Cell Cultures

RNA-Sequencing and Bioinformatic Analysis

Transcriptome Analyses of Public Datasets

Comparative Analysis of DNA Accessibility

Results

Conclusions