SP3-induced Timeless Transcription Contributes to Cell Growth of Lung Adenocarcinoma Cells

Abstract

Background

Timeless is well-known for its key role in replication checkpoints. Recent studies reveal the involvement of Timeless and specificity protein (SP) 1 in human malignancies. However, no evidence proved the interaction between SP3 and Timeless in lung adenocarcinoma (LUAD).

Methods

The expression and clinical significance of Timeless were analyzed using the LUAD dataset downloaded from the Cancer Genome Atlas (TCGA). Lentivirus-mediated Timeless knockdown in A549 cells was used to examine the role of Timeless in cell proliferation and pemetrexed (PEM) resistance. Transcription factors (TFs) bound to the Timeless promoter were identified by DNA pull-down technology with HPLC-MS/MS analysis and analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Dual-luciferase reporter assay was used to determine the activity of SP3 in Timeless transcription.

Introduction

As a common pathological type of non-small cell lung cancer (NSCLC), lung adenocarcinoma (LUAD) is mostly located in the bronchial mucosal epithelium [1]. Typical symptoms of the disease are hemoptysis, cough, and shortness of breath. LUAD is mainly caused by various factors, such as long-term smoking, environmental pollution, and occupational exposure [2]. Because of nonspecific clinical symptoms, most patients with LUAD have progressed to an intermediate or advanced stage at the time of initial diagnosis [3]. Despite improvements in surgery, radiotherapy, chemotherapy, and targeted therapy, the prognosis for lung cancer patients remains suboptimal, with a five-year survival rate of about 10%-20% [4]. Pemetrexed (PEM) is an anti-tumor drug that blocks the release process of enzymes required for purine and pyrimidine synthesis, thereby sustaining cell division activities in the S phase and significantly inhibiting the growth of cancer cells [5]. PEM has been approved as a first-line chemotherapy agent for non-squamous NSCLC [6]. PEM is used in patients with LUAD who cannot receive targeted therapy due to its low toxicity and low adverse effects. Moreover, it is more suitable for older patients with poor general conditions and has shown advantages in second-line therapy after failure of EGFR-tyrosine kinase inhibitor treatment [7, 8]. 

Materials and methods

Data collection and processing

The RNA-seq transcriptome data and clinical data were downloaded from the Cancer Genome Atlas (TCGA) database (http://www.cancergenome.nih.gov/). The differences in Timeless transcripts between LUAD samples and paired non-tumor samples (n = 58) were analyzed using stats [4.2.1] and car packages, and the data were visualized using the ggplot2 package [3.3.6]. For the receiver operating characteristic (ROC) analysis, the clinical data of LUAD samples (n = 539) and normal samples (n = 59) were downloaded. The pROC package [1.18.0] was used for data analysis, and the results were visualized using ggplot2 [3.3.6]. For OS analysis, the clinical data of LUAD patients (n = 500) were downloaded. The survival package [3.3.1] was used for proportional risk hypothesis testing and fitted survival regression, and the results were visualized using the survivor package and ggplot2 package [3.3.6]. 

Results

Timeless has a potential diagnostic and prognostic value for LUAD

To study the alteration in the transcription level of Timeless, we downloaded the RNA-seq data related to LUAD from the TCGA database. Our analysis showed a marked elevation in Timeless expression in LUAD samples in comparison with their non-tumor counterparts (Fig 1A). The ROC curves were utilized to make an evaluation of the diagnostic accuracy for Timeless. Fig 1B showed that the AUC of Timeless achieved 0.941 in LUAD, indicating that Timeless had a high diagnostic accuracy. Survival curves with log-rank tests showed that LUAD patients with high Timeless expression had shorter survival times (Fig 1C).

Discussion

Timeless is an important component of the biological rhythm system. As a replication fork-related TF, Timeless can combine with expected interacting proteins to form stable complexes, thus affecting replication checkpoints and normal DNA replication [36]. Timeless has been reported to be upregulated in a variety of cancers, and it is associated with cancer occurrence and prognosis. For example, overexpression of Timeless is associated with low clinical survival and lymph node metastasis in early-stage cervical cancer [37] and cisplatin sensitivity in cervical cancer [20, 38] and nasopharyngeal carcinoma [19]. Analysis of the expression profile of NSCLC cell lines using microarray analysis showed that Timeless expression in NSCLC cell lines is increased by 3.7 times compared to normal lung cells [22], and reduction of Timeless in lung cancer cell lines H157 and H460 inhibits cell proliferation. Also, Timeless is significantly higher in NSCLC samples [39]. Timeless has a significant correlation with OS for NSCLC patients and is correlated with immune checkpoint and immune infiltration levels in NSCLC [21]. These data suggest that Timeless has an important role in NSCLC.

Acknowledgments

I would like to express my gratitude to all those who have helped me during the writing of this thesis. Also, I would like to thank Dr. Ping Tian, Dajun Du, Li Yang, Nan Zhou, who contributed to the research work.

Citation: Tian P, Du D, Yang L, Zhou N, Tao L (2024) SP3-induced Timeless transcription contributes to cell growth of lung adenocarcinoma cells. PLoS ONE 19(2): e0298295. https://doi.org/10.1371/journal.pone.0298295

Editor: Divijendra Natha Reddy Sirigiri, BMSCE: BMS College of Engineering, INDIA

Received: August 30, 2023; Accepted: January 23, 2024; Published: February 14, 2024

Copyright: © 2024 Tian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: We have uploaded the mass spectrometry proteomics data to the iProX partner repository with the dataset identifier PXD046186 (https://www.iprox.cn//page/project.html?id=IPX0007322000). Some of the relevant data, including original western blot imgaes, of this article are also shown in Supporting Information.

Funding: • L T is Initials of the authors who received each award, • There is no grant number awarded to each author, • the full name of each funder is Science and technology research projects in Henan Province: Effect and mechanism of cryptotanshinone enhancing chemotherapeutic Pemetrexed on non-small cell lung cancer (No. 222102310520). • There is no URL of each funder website, • NO- The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviation: SP, Specificity protein; LUAD, lung adenocarcinoma; PEM, pemetrexed; TFs, transcription factors; KEGG, Kyoto Encyclopedia of Genes and Genomes; NSCLC, non-small cell lung cancer; OS, overall survival; ROC, receiver operating characteristic; FBS, fetal bovine serum; shRNA, short hairpin RNA; RT-qPCR, real time-quantitative PCR; EdU, 5-Ethynyl-2’deoxyuridine; CCK-8, Cell counting kit-8; OD, optical density; DAPI, 4’,6-diamidino-2-phenylindole; NOA, non-oncogene addiction

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0298295#abstract0