Epigenomic study identifies a novel mesenchyme homeobox2-GLI1 transcription axis involved in cancer drug resistance, overall survival and therapy prognosis in lung cancer patients.

Leonel Armas-López,Patricio Santillán-Doherty,Enrique Guzman De Alba,David C Christiani,Blanca Ortiz-Quintero,Patricia Piña-Sánchez,Oscar Arrieta,Joaquín Zúñiga,Federico Ávila-Moreno

doi:10.18632/oncotarget.17715

Abstract

Several homeobox-related gene (HOX) transcription factors such as mesenchyme HOX-2 (MEOX2) have previously been associated with cancer drug resistance, malignant progression and/or clinical prognostic responses in lung cancer patients; however, the mechanisms involved in these responses have yet to be elucidated. Here, an epigenomic strategy was implemented to identify novel MEOX2 gene promoter transcription targets and propose a new molecular mechanism underlying lung cancer drug resistance and poor clinical prognosis. Chromatin immunoprecipitation (ChIP) assays derived from non-small cell lung carcinomas (NSCLC) hybridized on gene promoter tiling arrays and bioinformatics analyses were performed, and quantitative, functional and clinical validation were also carried out. We statistically identified a common profile consisting of 78 gene promoter targets, including Hedgehog-GLI1 gene promoter sequences (FDR≤0.1 and FDR≤0.2). The GLI-1 gene promoter region from −2,192 to −109 was occupied by MEOX2, accompanied by transcriptionally active RNA Pol II and was epigenetically linked to the active histones H3K27Ac and H3K4me3; these associations were quantitatively validated. Moreover, siRNA genetic silencing assays identified a MEOX2-GLI1 axis involved in cellular cytotoxic resistance to cisplatinum in a dose-dependent manner, as well as cellular migration and proliferation. Finally, Kaplan-Maier survival analyses identified significant MEOX2-dependent GLI-1 protein expression associated with clinical progression and poorer overall survival using an independent cohort of NSCLC patients undergoing platinum-based oncological therapy with both epidermal growth factor receptor (EGFR)-non-mutated and EGFR-mutated status. In conclusion, this is the first study to investigate epigenome-wide MEOX2-transcription factor occupation identifying a novel overexpressed MEOX2-GLI1 axis and its clinical association with platinum-based cancer drug resistance and EGFR-tyrosine kinase inhibitor (TKI)-based therapy responses in NSCLC patients.

Highlights

Lung cancer remains the leading cause of death due to malignant disease in both the U.S.A. and worldwide, with 1.6 million cases annually [1, 2, 3, 4]
We implemented an epigenomics approach involving immunoprecipitation assays targeting mesenchyme HOX-2 (MEOX2) and the RNA Pol II active enzyme using fragmented chromatin derived from 13 solid lung adenocarcinomas obtained from non-small cell lung carcinomas (NSCLC) patients ranging in age from 62 to 74 years, with clinical outcomes identified as lower (1 to 16 months) or higher overall survival (Table I)
The majority of cases demonstrated partial responses to first/ second-line oncological treatment regimens based on cisplatinum/paclitaxel-navelbine and were selected to isolate immunoprecipitated DNA (IP-DNA) in MEOX2 and RNA Pol II immunoprecipitation assays that was used for subsequent competitive hybridization on NimbleGen promoter tiling sequence arrays, following a pipeline strategy descripted in Supplementary Figure 1A

Summary

Introduction

Lung cancer remains the leading cause of death due to malignant disease in both the U.S.A. and worldwide, with 1.6 million cases annually [1, 2, 3, 4]. Non-small-cell lung carcinomas (NSCLCs) have been associated with exposure to carcinogenesis-promoting environmental risk factors, including tobacco smoke [5], as well several genetic and epigenetic aberrations in the lung [6]; notably, the number of lung malignancy-related deaths that are not tobacco-associated is continuously increasing [2, 6]. Several molecular mechanisms and cell-signaling pathways that may or may not be induced by exposure to environmental risk factors contribute to lung tumor biology, as reflected in the “Hallmarks of Cancer,” including genetic [8], transcriptional [9], and epigenetic aberrations in the cancer epigenome that are involved in [10,11,12,13] progression, survival and/or therapeutic responses in lung cancer patients [14,15,16]. The mechanisms associated with HOX-related genes such as MEOX2 in the context of lung cancer drug resistance, overall survival, and clinical prognosis has yet to be fully elucidated

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