Elsevier

Lung Cancer

Volume 70, Issue 2, November 2010, Pages 136-145
Lung Cancer

Involvement of LKB1 in epithelial–mesenchymal transition (EMT) of human lung cancer cells

https://doi.org/10.1016/j.lungcan.2010.02.004Get rights and content

Abstract

Epithelial–mesenchymal transition (EMT) is a critical phenotypic alteration of cancer cells that triggers invasion and metastasis. Lung cancer cells often show mesenchymal phenotypes; however, a causative genetic alteration for the induction of EMT in lung cancer cells remains unknown. Recent studies have shown that the LKB1 gene is mutated in up to one-third of lung adenocarcinomas. Therefore, to pursue the possible involvement of LKB1 inactivation in the induction of EMT in lung carcinogenesis, we generated immortalized lung epithelial cells and lung adenocarcinoma cells with stable or transient LKB1 knockdown. LKB1 knockdown increased cell motility and invasiveness, and induced the expression of several mesenchymal marker proteins accompanied by the expression of ZEB1, a transcriptional repressor for E-cadherin and an EMT inducer. In agreement with the recent findings, expression of miR-200a/c was inversely correlated with that of ZEB1 in LKB1 knockdown clones with mesenchymal phenotype. Furthermore, transient knockdown of LKB1 induced ZEB1 mRNA and increased cell motility, and this motility was suppressed by ZEB1 repression. These results strongly indicate that LKB1 inactivation triggers EMT in lung cancer cells through the induction of ZEB1.

Introduction

Lung cancer is the leading cause of cancer death worldwide [1]. In particular, adenocarcinoma (AdC) is the most common histological type, and its highly invasive and metastatic phenotypes are the major reasons for poor prognosis of patients with lung AdC. The cellular and molecular mechanisms of lung AdC metastasis remain to be elucidated. Recently, epithelial–mesenchymal transition (EMT) has gained attention as a critical phenotypic alteration of cancer cells to acquire invasive and metastatic ability [2]. EMT is mediated through several transcription repressors, such as Snail, Slug, Twist and ZEB1, and these EMT inducers typically suppress the transcription of the E-cadherin gene, an epithelial cell marker and a potent suppressor of tumor cell invasion and metastasis [3], [4]. Indeed, a considerable fraction of lung cancer cells shows mesenchymal phenotypes with loss of E-cadherin expression [5], and ZEB1 expression was shown to correlate with E-cadherin loss in lung cancer cell lines [6]. Expression of Slug was also shown to be significantly associated with shortened survival of patients with lung AdC [7].

Multiple genetic alterations are accumulated in lung AdC cells during their progression. Inactivation of the p16 and p53 genes is common and frequent, and activating mutations of the EGFR and KRAS genes occur in a mutually exclusive manner [8], [9]. However, invasiveness and metastatic phenotypes of lung AdC cells cannot be explained by these alterations. Therefore, causative genetic alterations for the acquisition of invasive and metastatic phenotypes in lung AdC cells are still unclear. Recently, we showed that the LKB1 gene is inactivated preferentially in poorly differentiated AdCs in male smokers, and LKB1 inactivation often co-exists with KRAS mutation [10], [11]. Involvement of the LKB1 gene in the control of initiation, differentiation and metastasis in pulmonary tumorigenesis was also shown recently [12]. LKB1 is known to play important roles in multiple biological processes, including apoptosis, cell cycle arrest, cell polarity, chromatin remodeling, and energy metabolism [13], [14], [15], [16], [17], [18], [19], [20], [21]. Therefore, it is possible that inactivation of the LKB1 gene is involved in the acquisition of invasiveness and metastatic ability as well as the induction of EMT in lung AdC cells. Here, we investigated the pathogenic and biological significance of LKB1 inactivation in lung AdC progression by manipulating immortalized lung epithelial cells as well as a lung AdC cell line with epithelial phenotypes. Knockdown of LKB1 in these cells induced EMT through the enhanced ZEB1 expression. Thus, it was strongly indicated that LKB1 inactivation is a genetic alteration for the EMT induction in lung AdC cells.

Section snippets

Expression vectors and antibodies

Two shRNA mediated silencing vectors for LKB1, pTER-LKB1-sh#1 and pTER-LKB1-sh#4, were used in this study [18]. Targeting sequences for LKB1 of these vectors are pTER-LKB1-sh#1; 5′-GATCCTCAAGAAGAAGAAG-3′ and pTER-LKB1-sh#4; 5′-CGAAGAGAAGCAGAAAATG-3′. A human telomerase reverse transcriptase (hTERT) retroviral expression vector, pMSCVpuro-hTERT, was constructed as described previously [22], [23]. Primary antibodies, LKB1 (sc-32245), ZEB1 (sc-25388) and Twist (sc-15393), were purchased from Santa

Establishment of immortalized hSAECs by hTERT

It has been shown that both hTERT expression and inactivation of either RB or p16 are required for the immortalization of primary human keratinocytes and mammary epithelial cells [29], [30]. Introduction of hTERT into hSAECs led to their immortalization with a retarded growth until PD = 35. Then, fast growing cells emerged and continued to grow up to PD = 120 (Fig. 1A). Therefore, immortalized cells were cloned and karyotyped. Among 5 clones analyzed, one clone designated as hSAEC-T1 showed minimal

Discussion

Inactivation of the p53 and p16 genes is common in lung AdCs, and the majority of AdC cells carry mutations of the p53 gene and either methylation or homozygous deletion of the p16 gene [8]. The LKB1 gene is inactivated in lung cancer generally by either homozygous deletion or hemizygous deletion plus point mutation that results in the absence/truncation of LKB1 protein products, and in most cases, LKB1 inactivation is accompanied by inactivation of both the p53 and p16 genes [10], [11]. In

Conflict of interest

None declared.

Acknowledgments

B.C. Roy is a recipient of an invitation program for foreign researchers from the Foundation for Promotion of Cancer Research. We thank Drs. J.D. Minna, E. Shimizu, Y. Hayata and M. Perucho for providing us with cell lines, and Dr. H.C. Clevers for providing us with LKB1 shRNA vectors. This work was supported by Grants-in-Aid from the Ministry of Health, Labor and Welfare for the 3rd-term Comprehensive 10-year Strategy for Cancer Control and for Cancer Research (16-1), and a Grant-in-Aid from

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