Downregulation of HOXA1 gene affects small cell lung cancer cell survival and chemoresistance under the regulation of miR-100

https://doi.org/10.1016/j.ejca.2014.01.024Get rights and content

Abstract

Chemoresistance is often developed in small cell lung cancer (SCLC) patients and leads to poor prognosis. Hox genes, a highly conserved family, play a crucial role in apoptosis, receptor signalling and differentiation. MicroRNAs (miRNAs) have also been shown to play a crucial role in these biological processes by regulating the target genes. Several studies reported that both Hox genes and miRNAs are involved in chemoresistance. The aim of our study is to characterise the clinical significance and functional roles of HOXA1 in SCLC. Expression of HOXA1 was examined in 63 cases of SCLC tissues and 29 cases of blood by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methods. Multivariate analysis confirmed the prognostic significance of HOXA1 in SCLC patients. Restoration of HOXA1 expression was carried out in SCLC multidrug resistant cell line H69AR and its parental cell line H69 to assess its influence on chemoresistance. Luciferase reporter assay was used to assess HOXA1 as a target of miR-100. The results showed that HOXA1 was expressed in 46% (29/63) of SCLC. Low HOXA1 expression was associated with the poor prognosis of SCLC (P < 0.05 by the Fisher’s Exact Test) and the shorter survival rate (P < 0.001 by the Kaplan–Meier method). HOXA1 expression on both mRNA and protein levels significantly correlated with chemotherapy response. Enforced expression of HOXA1 in resistant H69AR cells led to increased chemosensitivity through increasing cell apoptosis and cell-cycle arrest. Inhibition of HOXA1 expression using HOXA1 siRNA in H69 cells resulted in cell resistance to therapeutic drugs through reducing drug-induced cell apoptosis accompanied with cell cycle arrest. Expression of endogenous miR-100 was significantly elevated in resistant H69AR cells and negatively related with HOXA1 expression. The expression of HOXA1 in SCLC tissues correlated inversely with the expression levels of miR-100. Reporter assays confirmed that miR-100 targeted predicted sites in 3′-untranslated region (3′-UTR) of HOXA1 gene. Our data suggested that HOXA1-mediated SCLC chemoresistance is under the regulation of miR-100. HOXA1 may be a prognostic predictor and potential therapeutic target in human SCLC.

Introduction

Small cell lung cancer (SCLC) which accounts for approximately 15% of all human lung cancers is a very aggressive disease because of its high propensity for relapse [1], [2]. According to the Veterans Administration Lung Group system, SCLC is traditionally defined as two-stage classification system including limited disease (LD) and extensive disease (ED). The combination of platinum and etoposide is the most widely used regimen for treatment of SCLC. Although 80–90% of SCLC patients are responsive to chemotherapy, they are seldom curable by it because of rapidly developed drug resistance. The 2-year survival rate of SCLC patients is less than 5% [3], [4]. Therefore, chemoresistance has become one of the major problems in the treatment of SCLC and is clinically a very important issue for improving poor prognosis of SCLC.

HOX genes, a highly conserved subgroup of the homeobox superfamily which characteristically encode a 60-amino acid long DNA-binding motif, have been reported to play important roles during embryonic development, regulating cell apoptosis, differentiation and receptor signalling [5], [6], [7]. In vertebrates, there are 39 Hox transcription factor genes organised into four separated chromosome clusters, each containing from 9 to 11 genes [8], [9]. During mammalian development, Hox gene expression controls the identity of various regions along the body axis according to the rules of spatiotemporal colinearity [9]. Numerous examples of aberrant Hox expression have been found in many cancers such as prostate cancer, gastric cancer and lung cancer [10], [11], [12], [13], [14], [15], [16], [17], [18]. Deregulation of Hox genes influences tumourigenesis and cancer cells biology through differentiation, apoptosis, receptor signalling and other unknown mechanisms. Some studies concerned about the relationship between Hox genes and apoptosis or drug resistance [19], [20], [21]. Blocking Hox activity by targeting the interaction between Hox and its cofactor Pbx caused apoptotic and necrotic cell death in the renal cancer cell lines CaKi-2 and 769-P [19]. Knockdown of HOXA5 expression partially abrogates retinoid-induced apoptosis and promotes cell survival upon RA treatment [20]. The Abl kinase inhibitors and PI3K inhibitor LY294002 induced the expression of HOXA10, and enhanced apoptosis in CML cells. Moreover, the reduction of HOXA10 expression by siRNA in chronic myeloid leukaemia (CML) cells inhibited apoptosis by treatment with the Abl kinase inhibitors and LY294002 [20].microRNAs (miRNAs) are a class of endogenous phylogenetically conserved small (∼22 nucleotides) non-coding regulatory RNA molecules. They are responsible for the posttranscriptional regulation of their target mRNA translation and stability mainly by binding to the 3′-untranslated region (3′-UTR) of their target mRNA. The levels of individual miRNA are dramatically changed in different cell types and different developmental stages, suggesting that miRNA plays a role in cell growth, differentiation and programmed cell death [22]. In the last few years, an increasing amount of experimental evidence has strongly supported the involvement of miRNAs in these processes [23], [24], [25], [26]. Recently, some studies have also been presented that miRNAs are closely associated with the drug sensitivity or resistance [27], [28], [29], [30], [31]. Valeri et al. declared that miR-21 targets and down-regulates the core mismatch repair (MMR) recognition protein complex, human mutS homologue 2 (hMSH2) and 6 (hMSH6) in colorectal tumour. Cells that overproduce miR-21 exhibit significantly reduced 5-fluorouracil (5-FU)-induced G2/M damage arrest and apoptosis that is characteristic of defects in the core MMR component. Moreover, xenograft studies demonstrate that miR-21 overexpression dramatically reduces the therapeutic efficacy of 5-FU [27]. Rao et al reported that fulvestrant treatment of estradiol (E2)- and fulvestrant-sensitive MCF7 cells resulted in increased expression of endogenous miR-221/222. Ectopic upregulation of miR-221/222 in oestrogen receptor-α (ER-α)-positive cell lines counteracted the effects of E2 depletion or fulvestrant-induced cell death. MiR-221/222 overexpression resulted in deregulation of multiple oncogenic signalling pathways previously associated with drug resistance [28]. Upregulation of miR-214 promotes survival of ovarian cancer cells and induces cancer cells resistance to cisplatin [29]. MiR-140 is involved in the resistance to 5-FU in osteosarcoma and colon cancer by reduced cell proliferation through G1 and G2 phase arrest mediated in part through the suppression of HDAC4 [30]. A restored miR-122 expression can trigger doxorubicin-induced apoptosis and miR-122, increases sensitivity to doxorubicin challenge in human hepatocarcinoma cells [31]. Our previous study showed that a number of miRNAs are involved in the drug resistance of SCLC cells using miRNA microarray [32].

Some reports suggested there was a correlation between miRNAs and Hox genes. Analysis of the genomic location of miRNA genes indicates a strong correlation between the location of specific miRNAs and Hox genes by bioinformatic analysis [33], [34], [35], [36]. The miR-10a and miR-196-1 are both located within the HOX B cluster on 17q21, whereas miR-196-2 is within the HOX C cluster at 12q13, and miR-10b maps to the HOX D cluster at 2q31 [33]. miR-196 has extensive, evolutionarily conserved complementarity to messages of HOXB8, HOXC8 and HOXD8. MiR-196 induces cleavage of HOXB8 directly both in mouse embryos and cultured cells [34]. Moreover, miR-196 inhibits HOXB8 expression in myeloid differentiation of HL60 cells [35]. MiR-130a downregulated HoxA5 expression and antagonises the antiangiogenic activity of HoxA5 [36].

Previous study from our laboratory has shown HOXA1 downexpression and miR-100 upexpression based on genome-wide gene expression analyses in SCLC multidrug resistance cells (H69AR) as compared to parental H69 cells (unpublished data). Bioinformatic analysis indicated HOXA1 might be directly regulated by miR-100. In the present study, we further investigate the roles of HOXA1 upregulation in drug resistance using cellular model of human SCLC resistant cell line (H9AR). The relevance of HOXA1 expression in terms of the clinical progression was dissected in SCLC patients. In addition, HOXA1 is proved to be targeted and regulated by miR-100 using luciferase assays. We propose that HOXA1 regulated by miR-100 has a significant impact on progression and chemoresistance in SCLC.

Section snippets

Cell culture

Human SCLC cell line NCI-H69 and the drug-resistant subline NCI-H69AR were purchased from the American Type Culture Collection (ATCC, United States of America (USA)) and maintained in RPMI 1640 medium contain in l-glutamine with 10% and 20% foetal calf serum respectively in an incubator at 37 °C with 5% CO2. NCI-H69AR was alternately fed with drug-free medium and medium containing 0.8 μM of adriamycin (ADM). The resistant cell line was tested regularly for maintained resistance to the selected

HOXA1 expression is correlated with clinical stage, chemotherapy response and survival in SCLC patients

To investigate the clinical features of HOXA1 expression in SCLC, immunohistochemistry staining was performed in 63 samples from SCLC patients. HOXA1 expression was localised on the membrane of the cancer cells (Fig. 1A). No positive staining for HOXA1 was presented in normal lung alveolar epithelium (Fig. 1B). The positive rate of HOXA1 expression was 46% (29/63) in SCLC. Table 1 summarises the relationship between HOXA1 expression and clinical characteristics in SCLC patients. By Fisher’s

Discussion

HOXA1, a member of the HOXA group which was first identified in Drosophila, has been previously report to play a pivotal role during the normal growth and differentiation of mammalian tissues. Forced expression of HOXA1 in immortalised human mammary epithelial cells results in oncogenic transformation and tumour formation in vivo [37]. HOXA1 mutant lost its ability to stimulate mammary cells proliferation, anchorage-independent cell growth and loss of contact inhibition [38]. HOXA1 expression

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81172241).

References (45)

  • X. Zhang et al.

    Human growth hormone-regulated HOXA1 is a human mammary epithelial oncogene

    J Biol Chem

    (2003)
  • X. Zhang et al.

    HOXA1 is required for E-cadherin-dependent anchorage-independent survival of human mammary carcinoma cells

    J Biol Chem

    (2006)
  • R. Govindan et al.

    Changing epidemiology of small-cell lung cancer in the United States over the last 30 years: analysis of the surveillance, epidemiologic, and end results database

    J Clin Oncol

    (2006)
  • A. Jemal et al.

    Cancer Stat

    (2009)
  • J.P. Chute et al.

    Tweenty years of phase I trials for patients with extensive-stage small cell lung cancer: perceptible progress

    J Clin Oncol

    (1999)
  • C. Cillo et al.

    Homeobox genes in normal and malignant cells

    J Cell Physiol

    (2001)
  • D.G. Grier et al.

    The pathophysiology of HOX genes and their role in cancer

    J Pathol

    (2005)
  • N. Shah et al.

    The Hox genes and their roles in oncogenesis

    Nat Rev Cancer

    (2010)
  • W.J. Gehring et al.

    Homeotic genes and the homeobox

    Annu Rev Genet

    (1986)
  • D. Waltregny et al.

    Overexpression of the homeobox gene HOXC8 in human prostate cancer correlates with loss of tumor differentiation

    Prostate

    (2002)
  • T. Ota et al.

    HOXA7 in epithelial ovarian cancer: interrelationships between differentiation and clinical features

    Reprod Sci

    (2007)
  • K. Makiyama et al.

    Aberrant expression of HOX genes in human invasive breast carcinoma

    Oncol Rep

    (2005)
  • Cited by (0)

    1

    The first two authors contributed equally to this work.

    View full text