Stat3 downstream genes serve as biomarkers in human lung carcinomas and chronic obstructive pulmonary disease

doi:10.1016/j.lungcan.2008.05.025

Lung Cancer

Volume 63, Issue 3, March 2009, Pages 341-347

https://doi.org/10.1016/j.lungcan.2008.05.025 Get rights and content

Summary

Smoking causes lung cancer and chronic obstructive pulmonary disease (COPD) that impose severe health problem to humans. Both diseases are related to each other and can be induced by chronic inflammation in the lung. To identify the molecular mechanism for lung cancer formation, a CCSP-rtTA/(teto)₇Stat3C bitransgenic model was generated recently. In this model, persistent activation of the Stat3 signaling pathway induced pulmonary inflammation and adenocarcinoma formation in the lung. A group of Stat3 downstream genes were identified by Affymetrix GeneChip microarray analysis that can be used as biomarkers for lung cancer diagnosis and prognosis. To determine which human lung cancers are related to the Stat3 pathway, multiple Stat3 downstream genes were screened in human lung cancers (adenocarcinomas and squamous cell carcinomas) and lung tissue with COPD. In both cancer and COPD, the Stat3 gene was up-regulated. A panel of Stat3-up-regulated downstream genes in mice was up-regulated in human adenocarcinomas, but not in human squamous cell carcinomas. This panel of genes was also modestly up-regulated in lung tissue with COPD from patients with a history of smoking and not up-regulated in those without histories of smoking. Several Stat3-down-regulated downstream genes also showed differential expression patterns in carcinoma and COPD. These studies support a concept that Stat3 is a potent oncogenic molecule that plays a role in formation of lung adenocarcinomas in both mice and humans. The carcinogenesis of adenocarcinoma and squamous cell carcinoma is mediated by different molecular mechanisms and pathways in vivo. Stat3 and its downstream genes can serve as biomarkers for lung adenocarcinoma and COPD diagnosis and prognosis in mice and humans.

Introduction

Lung cancer is one of the biggest public health challenges facing the United States and many other countries. It accounts for 28% of all cancer deaths (more than colonic, mammary, prostatic and pancreatic cancers combined). Although incidence rates have stabilized, more than 173,000 new cases of lung cancer are diagnosed each year, and it continues to be the most common cause of cancer death among men and women, with more than 163,000 people succumbing each year (http://www.cancer.gov/ncicancerbulletin/NCI_Cancer_Bulletin_052306/page3). Lung cancer is difficult disease to detect in its early stages. In most cases, the tumors are detected at advanced stages and the overall 5-year survival rate is ∼15%. Thus, it is essential to better understand the early events that initiate lung carcinogenesis and to find biomarkers for early lung cancer detection. Approximately 80% of human lung cancers are adenocarcinomas and squamous cell carcinomas. Human chronic obstructive pulmonary disease (COPD) (especially with smoking history) is a pre-cancer disease and is the fourth leading cause for human death in the United States. Importantly, both COPD and lung cancer can be induced by repeated exposure to smoking. It is well known that smokers with COPD are elevated at risk to develop lung cancer. Both lung diseases are associated with inflammation in the lung. Nevertheless, the molecular mechanisms connecting inflammation and COPD and lung cancer are poorly understood.

Inflammation is a protective process to facilitate pathogen clearance and to repair tissue injury in the lung. The magnitudes of inflammatory responses are precisely regulated by pro- and anti-inflammatory molecules. Exuberant inflammation can cause severe consequences and lead to carcinogenesis. One commonly induced pro-inflammatory gene group during pulmonary inflammation is the interleukin 6 (IL-6) family of cytokines. Pro-inflammatory IL-6 cytokines share the common gp130 receptor subunit [1], [2]. Upon binding to their cell-surface receptors, IL-6 family members trigger Stat3 phosphorylation by Janus-activated kinases (JAKs). As we demonstrated previously, IL-6, JAKs and Stat3 are co-expressed in alveolar type II (AT II) epithelial cells in the lung [3], [4], [5]. Over-expression of dominant negative Stat3 to subvert endogenous Stat3 activity in respiratory epithelial cells causes alveolar destruction, suggesting its role in maintaining alveolar structure and function [4]. On the other hand, persistent activation of Stat3 signaling has the potential to induce lung cancer [6]. Carcinogenesis is a complex process that is influenced by both genetics and the tissue microenvironment. Based on our studies in the CCSP-rtTA/(teto)₇Stat3C bitransgenic mouse model, Stat3 induces lung adenocarcinoma formation in two steps. The first step involves aberrant expression of cytokines/chemokines and abnormal inflammatory cell infiltration, which hijack the immune system to inhibit immune surveillance and promote neoplasia. The second step involves reactivation of developmental genes that stimulate epithelial cell over-growth. These observations are in agreement with previous findings that Stat3 is an oncogene [7].

Although extensive surveys of primary tumors and cell lines derived from tumors indicate that inappropriate activation of Stat3 occurs with surprisingly high frequency (50–90%) in a wide variety of human cancers, including lung cancers [8], [9], [10], [11], [12], [13], no Stat3 downstream gene has been used to link the Stat3 pathway to lung cancer formation. During characterization of the CCSP-rtTA/(teto)₇Stat3C bitransgenic mouse model, multiple Stat3 up or down-regulated downstream genes were identified by Affymetrix GeneChip Microarray analysis, which have been confirmed by Real-Time PCR and immunohistochemistry analyses [6]. These genes can potentially serve as molecular biomarkers for cancer patient diagnosis or prognosis.

In this report, a panel of Stat3-most-up-regulated and down-regulated downstream genes in the CCSP-rtTA/(teto)₇Stat3C bitransgenic mouse model was studied in human lung carcinomas and COPD tissues to assess if they can be used as biomarkers for diagnostic or prognostic purposes. Although expression of the Stat3 gene was up-regulated in human carcinomas and tissues with COPD, its downstream genes were differentially expressed in adenocarcinomas and squamous cell carcinomas and in tissues with COPD from smokers and non-smokers and therefore can be used to distinguish them. These studies also suggest that Stat3 and its downstream genes more actively participate in the carcinogenesis of adenocarcinomas than in squamous cell carcinomas in the lungs. This is in agreement with observations made in the CCSP-rtTA/(teto)₇Stat3C bitransgenic mouse model in which only adenocarcinomas were observed. In COPD, smokers seem more skewed towards the COPD–adenocarcinoma transition based on Stat3-up-regulated gene analysis.

Section snippets

Human tissue

Human lung tissues were obtained from the Indiana University Simon Cancer Center Tissue Procurement Lab and the IU/Lilly Clinically Annotated Tissue Databank. The protocol for an exempt study using these human tissues has been approved by the IUPUI/Clarian Institutional Review Committee. All samples are surgically collected from human tissues. Normal samples represent lung tissues without cancer and COPD.

Isolation of total RNAs and Real-Time PCR

Total RNA from whole lung tissues was purified using the Qiagen total RNA purification kit

Pre-screening of human samples

Samples from 20 normal human lung tissues, 30 human pulmonary adenocarcinomas, 29 human pulmonary squamous cell carcinomas, 29 human lung tissues with COPD from non-smokers and 33 human lung tissues with COPD from smokers were obtained. The quality of all human samples was pre-screened by two standards. First, the total RNA was purified. Only samples with high yields of RNA were selected for the Real-Time PCR study. Second, the selected samples were subject to initial Real-Time PCR analyses for

Discussion

In this study, Stat3 gene up-regulation was observed in human pulmonary adenocarcinomas and squamous cell carcinomas (Fig. 1), suggesting that Stat3 plays a role in initiation and progression of these cancers. This is in agreement with our previous findings in animals. Up-regulation of Stat3 in the CCSP-rtTA/(teto)₇Stat3C bitransgenic mouse model induced lung adenocarcinomas [6]. The Stat3 process of carcinogenesis is initiated by increased expression of a subset of pro-inflammatory cytokines,

Conflict of interest

None declared.

Acknowledgements

This study was supported by National Institute of Health Grants HL-061803 and HL-067862 (C. Yan and H. Du), and Indiana University Cancer Center ITRAC Award (C. Yan).

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Stat3 downstream genes serve as biomarkers in human lung carcinomas and chronic obstructive pulmonary disease

Summary

Introduction

Section snippets

Human tissue

Isolation of total RNAs and Real-Time PCR

Pre-screening of human samples

Discussion

Conflict of interest

Acknowledgements

J Biol Chem

Cell

Am J Pathol

Biochem Biophys Res Commun

Biochem Biophys Res Commun

J Biol Chem

J Biol Chem

Jaks and STATs: biological implications

Annu Rev Immunol

Gp130 and the interleukin-6 family of cytokines

Annu Rev Immunol

Synergy between signal transducer and activator of transcription 3 and retinoic acid receptor-alpha in regulation of the surfactant protein B gene in the lung

Mol Endocrinol

Overexpression of Stat3C in Pulmonary Epithelium Protects against Hyperoxic Lung Injury

J Immunol