Elsevier

Lung Cancer

Volume 66, Issue 3, December 2009, Pages 292-297
Lung Cancer

Targeted reduction of KLF6-SV1 restores chemotherapy sensitivity in resistant lung adenocarcinoma

https://doi.org/10.1016/j.lungcan.2009.02.014Get rights and content

Abstract

Kruppel-like factor 6 splice variant 1 (KLF6-SV1) is an oncogenic splice variant of the KLF6 tumor suppressor gene that is specifically overexpressed in a number of human cancers. Previously, we have demonstrated that increased expression of KLF6-SV1 is associated with decreased survival in lung adenocarcinoma patient samples and that targeted reduction of KLF6-SV1 using siRNA induced apoptosis both alone and in combination with the chemotherapeutic drug cisplatin. Here, we demonstrate that chemoresistant lung cancer cells express increased levels of KLF6-SV1. Furthermore, targeted reduction of KLF6-SV1 using RNA interference restores chemotherapy sensitivity to lung cancer cells both in culture and in vivo through induction of apoptosis. Conversely, overexpression of KLF6-SV1 resulted in a marked reduction in chemotherapy sensitivity in a tumor xenograft model. Combined, these findings highlight a functional role for the KLF6-SV1 splice variant in the regulation of chemotherapy response in lung cancer and could provide novel insight into lung cancer therapy.

Introduction

Lung cancer is a leading cause of caner death in the United States, with over 162,460 deaths from lung cancer in the US exceeding cancer mortality from colorectal, breast, prostate, and pancreatic cancer combined [1]. The aggregate survival benefit from use of chemotherapy in the treatment of lung cancer is supported by evidence from dozens of randomized controlled trials [2]. Though the data supports a benefit in large groups of patients, variability in individual response to chemotherapy and the development of resistance after either discontinuation of treatment or during treatment itself is a major cause of patient morbidity and mortality [2], [3]. Treatment failure results from the development of resistance to chemotherapy and is characterized by the selection for cancer cells with either defects in the apoptotic cascade, increased expression of nucleotide excision repair pathway members, and/or increased activity of drug efflux transporters [4], [5]. Targeted therapy to the specific molecular alterations underlying the development of chemotherapy resistance represents an appealing therapeutic strategy.

KLF6 (Zf9/CPBP) (GeneBank accession number AF001461) is a member of the Krüppel-like factor (KLF) family, that was originally shown to be functionally inactivated by loss of heterozygosity (LOH) and somatic mutation in sporadic prostate adenocarcinomas [6]. More recent reports have extended the range of human tumors and the mechanisms by which KLF6 can be inactivated to include deletion of the KLF6 locus and mutation in colorectal cancers [7], hepatocellular and gastric carcinomas [8], [9], LOH in ovarian carcinoma and gliobastoma [10], [11], decreased KLF6 expression in non-small cell lung cancer [12], [13], hypermethylation of the promoter region in esophageal SCC cell lines and hepatocellular carcinoma patient samples [14], [15]. In addition, three alternatively spliced KLF6 isoforms have been identified [16] and at least one of them, KLF6-SV1, has been shown to be biologically active, antagonizing the tumor suppressor function of KLF6 and promoting tumor growth and dissemination [10], [16], [17].

A role of KLF6 in non-small cell lung cancer (NSCLC) was first suggested by microarray studies comparing gene expression between normal and lung cancer specimens [18]. COPEB/KLF6 was decreased in malignant compared to benign lung tissue but high KLF6 expression levels in tumor specimens were associated with advanced disease stages and contributed to a prognostic gene signature of poor survival [18]. Furthermore, recent reports suggest that decreased KLF6 expression is a common event in lung adenocarcinoma and overexpression of KLF6 in lung cancer cell lines induced spontaneous apoptosis [13]. Previously, we have demonstrated that increased expression of KLF6-SV1 is associated with decreased survival in lung adenocarcinoma patient samples and that targeted reduction of KLF6-SV1 using siRNA induced apoptosis both alone and in combination with the chemotherapeutic drug cisplatin [19]. Given the evidence for a role for the KLF6 tumor suppressor gene in lung cancer [12], [13], [19], we sought to define the role of the oncogenic splice variant, KLF6-SV1 in the development of chemotherapy resistance and whether targeted reduction of KLF6-SV1 could restore chemotherapy sensitivity in chemoresistant lung cancer cell lines.

Section snippets

Cell culture and cell line generation

All cell lines were obtained from the American Tissue Culture Collection (ATCC). Retroviral infection with KLF6-SV1 was performed as previously described [10], [20], [21]. The A549 resistant cell lines were generated by serial selection in cisplatin (final concentration 20 μM) for 2 passages for the A549-IR cell line and 8 passages for the A549-CR cell line. Transient transfection of a non-targeting control and SV1 siRNA was performed with Lipofectamine 2000 (Invitrogen) in the A549 lung cancer

KLF6-SV1 expression is increased in a chemotherapy resistant lung cancer cell line

The A549 lung adenocarcinoma cell line was serially selected in media containing chemotherapy for between 2 and 8 passages. Two derivative cell lines were generated after selection that were then tested for their sensitivity to cisplatin. While the addition of cisplatin to the parental A549 cell line (A549-NR) resulted in significant induction of apoptosis, a marked reduction in apoptosis was seen in both the A549 cisplatin resistant cell line (A549-CR) and the A549 Intermediate Resistant cell

Discussion

Lung cancer is the leading cause of cancer-related death in the United States. While early stage disease is treatable with a 5 yr survival exceeding 70%, the prognosis of patients with metastatic lung cancer remains dismal [4]. Treatment failure and the development of chemotherapy resistance are common and contribute significantly to the poor prognosis of patients with advanced disease. A better understanding of the molecular mechanisms underlying the development of resistance will allow for the

Acknowledgements

We would like to thank the Howard Hughes Medical Institute for providing funding for the work. Goutham Narla is a recipient of the HHMI Physician-Scientist Early Career Award.

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