Cancer Letters

Cancer Letters

Volume 434, 10 October 2018, Pages 132-143
Cancer Letters

Original Articles
Genome-wide identification of transcription factors that are critical to non-small cell lung cancer

https://doi.org/10.1016/j.canlet.2018.07.020Get rights and content

Highlights

  • To identify transcription factors (TFs) that are critical to lung carcinogenesis, 1530 TFs were silenced by siRNAs.

  • Eleven potential tumor suppressing TFs and 10 potential oncogenic TFs were unveiled.

  • IRX5 was required for lung cancer cell proliferation in vitro and in vivo.

  • IRX5 was upregulated by tobacco carcinogen benzo(a)pyrene, and Cyclin D1 was a downstream target of this TF.

Abstract

To systematically unveil transcription factors (TFs) that are critical to lung carcinogenesis, here we conducted a genome-wide lethality screening in non-small cell lung cancer (NSCLC) cells and reported that among the 1530 TFs tested, 21 genes were required for NSCLC cell proliferation and were negatively or positively associated with overall survival (OS) of patients with NSCLC. These included 11 potential tumor suppressing genes (AFF3, AhR, AR, CBFA2T3, CHD4, KANK2, NR3C2, PTEN, PRDM16, RB1, and STK11) and 10 potential oncogenic TFs (BARX1, DLX6, ELF3, EN1, ETV1, FOXE1, HOXB7, IRX4, IRX5, and SALL1). The expression levels of IRX5 were positively associated with OS of smoker and inversely associated with OS of non-smoker patients with lung adenocarcinoma. We showed that tobacco carcinogen benzo(a)pyrene (BaP) induced upregulation of IRX5 in lung epithelial cells, and Cyclin D1 was a downstream target of IRX5. Furthermore, silencing of IRX5 by lentivirus mediated transfection of short hairpin RNA significantly inhibited tumor growth in nude mice. These results indicate that tobacco smoke can modulate TFs to facilitate lung carcinogenesis, and inhibition of IRX5 may have therapeutic potentials in NSCLCs.

Introduction

Lung cancer is the most common cause of cancer related mortality, killing 1.59 million people each year worldwide [1]. Lung cancer comprises of small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), whereas NSCLC consists of lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and large cell carcinoma [2]. Tremendous efforts have been made to dissect lung carcinogenesis, and a large amount of somatic mutations and abnormally activated or inactivated oncogenes/tumor suppressor genes have been unveiled [3]. Tyrosine kinase represents one of the most common type of driver molecules. For example, epidermal growth factor receptor (EGFR) [4], the anaplastic lymphoma kinase (ALK) [5], ROS1 [6], fibroblast growth factor receptor 1 (FGFR1) [7], and phosphoinositide 3-kinase (PI3K) [8], are frequently mutated or activated in NSCLCs and serve as therapeutic targets. EGFR [9] and ALK fusion proteins [10] also induce programmed cell death 1 ligand (PD-L1) to confer cancer cells the capacity of immune evasion. Kirsten rat sarcoma viral oncogene homolog (KRAS), a GTPase that is mutated in about 30% of LUADs [11], promotes lung cancer in mice [12]. However, druggable driver molecules are found in only a minority of patients, hence efforts should be made to uncover the driver genes in a majority of lung cancers [13].

Abnormalities in transcription factors (TFs) have been frequently detected in lung cancer. TP53, the most frequently mutated gene in both NSCLCs and SCLCs [11,14,15], is usually inactivated by loss-of-function mutations, copy number loss, and epigenetic changes [16,17]. PTEN and Rb1 are frequently inactivated by loss-of-function mutations or suppression of expression [11,14,15]. Overexpression or copy-number gain is seen in MYC [18], SOX2 [19], and KLF5 [20], and persistently activated STAT3 is detected in about 50% of NSCLCs [[21], [22], [23]]. Moreover, activation of STAT3 [24] and deficiency in PTEN [25] or p53 [26] result in PD-L1 overexpression. However, the roles of other TFs in lung carcinogenesis remain to be determined.

To systematically identify TFs that are critical to lung tumorigenesis, we performed a genome-wide silencing of TFs in NSCLC cells and investigated their clinical significance using the Online Survival Analysis Software [27] and The Cancer Genome Atlas (TCGA) datasets. We reported that among the 1530 TFs investigated, 21 TFs were critical to NSCLC cell proliferation and associated with clinical outcome of the patients. We further dissected the role of a candidate, Iroquois Homeobox 5 (IRX5) [28], in lung carcinogenesis.

Section snippets

Patients

This study was approved by the local research ethics committees of all participating sites; all lung cancer samples were collected with informed consent. The diagnosis of lung cancer was confirmed by at least two pathologists. Tissue samples were taken at the time of surgery and quickly frozen in liquid nitrogen. The tumor samples contained a tumor cellularity of greater than 60% and the matched control samples had no tumor content.

Reagents

Anti-FLAG, anti-Actin, anti-IRX5 and anti-LC-3 antibodies were

A genome-wide silencing of TFs in lung cancer cells

The small interfering RNAs (siRNAs) of the Dharmacon human siGENOME SMARTpool library that contains siRNAs for 1530 TFs (Table S1), were transfected into A549 NSCLC cells using the DharmFECT transfection reagent. The cell viability was analyzed 48 h after transfection and the Z-scores [29] from duplicate experiments for each SMARTpool were determined. A total of 293 TFs with Z scores ≥2 (cell proliferation was increased when the genes were silenced; n = 160) or ≤ −2 (cell proliferation was

Discussion

TFs which recognize specific DNA sequences to control chromatin and transcription and guide the expression of the genome, are key cellular components that determine how cells function and respond to the environment [59]. Normal lung development requires orchestration of TFs [60], and abnormalities in TFs may lead to lung diseases. In this study, we conducted a genome-wide siRNA assay to systematically identify TFs that are critical to NSCLC, and reported that 21 TFs were required for cell

Conflicts of interest

The authors have declared that no competing interests exist.

Author contributions

The project was conceived and designed by G.B.Z. The experiments were conducted by D.L.Z, L.W.Q., C.Z., L.M., G.Z.W., X.C.Z., Y.F.Z., and X.C. Biospecimens were harvested/provided by Y.C.Z., Y.C.H. Data were analyzed by G.B.Z. The manuscript was written by G.B.Z.

Acknowledgements

This work was supported by the National Natural Science Funds for Distinguished Young Scholar (81425025), the National Key Research and Development Program of China (2016YFC0905500), the “Personalized Medicines——Molecular Signature-based Drug Discovery and Development", Strategic Priority Research Program of the Chinese Academy of Sciences (XDA12010307), the National Natural Science Foundation of China (81672765), and grants from the State Key Laboratory of Membrane Biology. The study sponsor

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