Elsevier

Lung Cancer

Volume 89, Issue 2, August 2015, Pages 189-196
Lung Cancer

Methyl(R217)HuR and MCM6 are inversely correlated and are prognostic markers in non small cell lung carcinoma

https://doi.org/10.1016/j.lungcan.2015.05.008Get rights and content

Highlights

  • Methyl(R217)HuR is a marker of good prognosis.

  • MCM6 and Ki-67 are markers of poor prognosis and are correlated with HuR expression.

  • MiR16 and miR519 are downregulated in lung cancer.

  • MiR519 correlates inversely to HuR expression.

Abstract

Objectives

In non small cell lung carcinoma (NSCLC), earlier studies supported a prognostic value of intra-cytoplasmic HuR expression. HuR is a RNA binding protein previously shown to stimulate proliferation, but the link between HuR and proliferation in NSCLC has not yet been evaluated. The first objective of this study was to analyze the expression of HuR in a series of NSCLC and to correlate this to two proliferation markers, Ki-67 and MCM6. As potential post-transcriptional regulatory mechanisms for HuR expression, two miRNAs, miR16 and miR519, were also analyzed. Finally, because HuR methylation could be involved in its nucleocytoplasmic shuttling, the expression of methyl(R217)HuR and its relation to cancer survival were determined.

Materials and methods

Immunohistochemistry was used to evaluate the expression of HuR, methy(R217)HuR, Ki-67 and MCM6 in a series of 190 NSCLCs. The level of miR16 and miR519 was determined by qRT-PCR.

Results

Higher cytoplasmic HuR staining was found in tumor vs. control paired normal lung (p < 0.0001), but without correlation with survival. The level of methyl(R217)HuR was correlated both significantly with intra-cytoplasmic HuR staining (p < 0.001), and overall survival (p = 0.01). MCM6 correlated to a poorer overall survival (p < 0.01). Both MCM6 and Ki-67 were positively correlated with HuR nuclear staining (p < 0.0001 and p < 0.001, respectively). On the contrary, MCM6 and Ki-67 correlated inversely to methyl(R217)HuR (p < 0.001 and p = 0.01, respectively). The levels of miR16 and miR519 were significantly lower in tumor tissue vs. paired normal lung (p < 0.0001), but only miR519 correlated inversely to HuR expression (p = 0.01).

Conclusion

While overall cytoplasmic HuR level was higher in tumor tissues, we found unexpectedly that methyl(R217)HuR was a marker of good prognosis. Furthermore, our data suggest that HuR level could be regulated by miR519. Finally, we demonstrated that Ki-67 and MCM6, both correlated with HuR, are valuable markers of poor prognosis in NSCLC.

Introduction

Lung cancer remains the top worldwide cause for cancer related death. For advanced-stage tumors, the prognosis rests rather poor, while for lower-grade tumors, it is often difficult to predict the outcome of the disease. Few prognostic markers have been described for lung cancers and to date and none has been validated for routine clinical practice. Proliferation markers, most notably Ki-67, have been evaluated in numerous studies, however, with contradictory results [1], [2], [3]. Interestingly, Martin et al., in a systematic review with meta-analysis, concluded Ki-67 to be a bad prognosis marker for survival in 15 out of 37 studies [1]. Among other proliferation markers, the mini-chromosome maintenance (MCM) proteins – key proteins in the initiation of DNA synthesis and DNA replication – were shown to be associated with histological grades in various neoplastic processes [4]. In lung cancer, few studies have evaluated the prognostic value of the MCMs proteins, including MCM2, MCM4, and MCM7 [2], [5], [6]. To our knowledge, MCM6 has not yet been evaluated in lung carcinoma.

HuR (ELAV-like 1) – a RNA-binding protein capable of binding and stabilizing AU-rich mRNAs coding for proteins with diverse functions ranging from cell proliferation, survival, angiogenesis, invasion, metastatic process – plays a central role in cancer proliferation [7]. HuR is mainly localized in the nucleus, and its role in this localization remains relatively obscure, even if few data indicate that it could have a role in alternative splicing [8], [9], [10]. On the contrary, several studies showed that when translocated into the cytoplasm, HuR is able to stabilize or modulate the translation of many mRNAs including cyclin D1, Bcl-2, Mdm2, VEGF, SIRT1 and HIF-1α mRNAs [7], [11], [12].

Previous in vitro studies have shown that HuR promotes cell proliferation and chemoresistance in various cancer cell lines, including lung cancer cell lines [13], [14], [15], [16], [17], [18], [19], [20], [21]. In lung cancer, several studies have reported the prognostic value of cytoplasmic HuR [22], [23], [24], [25], [26]. If most of the evidence associates cytoplasmic HuR to cancer proliferation, the unfavorable prognostic value of nuclear HuR has, on the other hand, been demonstrated in ovarian [27] and colo-rectal carcinomas [28].

Few data are available concerning the molecular events leading to HuR overexpression and its nuclear-to-cytoplasm shuttling. Global HuR expression could be influenced by microRNAs, such as miR16 and miR519 – both capable of decreasing the HuR level through inhibiting its mRNA translation [29], [30], [31], [32]. Interestingly, it was reported that miR16 was down regulated in NSCLC [33], but in vivo correlation between miR16 and HuR has not yet been studied in these tumors. Regarding HuR subcellular localization, CARM 1-dependant HuR methylation on arginine 217 was reported to be involved in its nucleocytoplasmic translocation [34], but the impact of HuR methylation on its function remains relatively obscure.

The first objective of this study was to analyze the expression of HuR in a series of NSCLC, and, as HuR could stimulate cell proliferation, to study the correlation between HuR and two proliferation markers (Ki-67 and MCM6). In addition, to explore possible post-transcriptional regulations by miRNAs on HuR expression, the expression of miR16 and miR519 in these tumor tissues relative to those in the paired non-tumor tissues were determined. Finally, as previous experimental data suggested that HuR methylation could be involved in its nucleocytoplasmic shuttling, we evaluated the expression of methyl(R217)HuR and determined how it correlated with overall HuR expression and with tumor prognosis.

Section snippets

Population and clinical data

One hundred and ninety surgical specimens were retrieved from the clinical and biological cohort of the Centre de Ressources Biologiques (BB-0033-00035, CHU Nancy, France), from 2005 to 2007, including 109 cases of adenocarcinoma and 81 squamous cell carcinomas (SCC). Clinical data were prospectively recorded, including age, sex, stage (TNM 7th edition and IASLC stage [35]), tobacco consumption, treatment and global survival. All cases were examined and reviewed by two experienced pathologists

Clinical data

Mean age was 62 years (min.: 41; max.: 83), with a male-to-female ratio of 3.3 (Table 1). Ten percent of the patients were nonsmokers. No significant difference was found between adenocarcinoma and SCC in terms of progression free and overall survival (p > 0.05). Clinical stage was significantly correlated with progression free and overall survival (p < 0.0001), as well as T status (p = 0.02) or N status (p < 0.0001). In adenocarcinoma, histological subtype was not correlated with overall survival or

HuR is overexpressed in lung cancer

HuR was suggested to play a central role in cancer initiation and progression. In lung cancer, several teams reported significant cytoplasmic HuR overexpressions in the cancer tissues, with an impact on the prognosis of the disease. In a series of 132 NSCLC, Wang et al. found that cytoplasmic expression of HuR was an independent prognostic factor for survival in multivariate analysis [24], [25]. Similarly, Lauriola et al. showed, in 54 lung adenocarcinomas of stage I and II, an unfavorable

Conflicts of interest

The authors have no conflicts of interest or funding to disclose.

Acknowledgements

The authors thank: GIRCI Est and La Région Lorraine for financial support; all the team of the Pathology Department of Nancy (CHU) for technical help; Mrs. Christine RONTONDA and Mrs. Kossar HOSSEINI (Clinical Epidemiology and Evaluation, CHU Nancy, France) for methodological support.

References (44)

  • B. Martin et al.

    Ki-67 expression and patients survival in lung cancer: systematic review of the literature with meta-analysis

    Br J Cancer

    (2004)
  • B. Werynska et al.

    Correlation between expression of metallothionein and expression of Ki-67 and MCM-2 proliferation markers in non-small cell lung cancer

    Anticancer Res

    (2011)
  • J. Yang et al.

    Prognostic significance of MCM2, Ki-67 and gelsolin in non-small cell lung cancer

    BMC Cancer

    (2006)
  • G. Gauchotte et al.

    Expression of minichromosome maintenance MCM6 protein in meningiomas is strongly correlated with histologic grade and clinical outcome

    Am J Surg Pathol

    (2012)
  • K. Abdelmohsen et al.

    Posttranscriptional regulation of cancer traits by HuR

    Wiley Interdiscip Rev RNA

    (2010)
  • M.N. Hinman et al.

    Diverse molecular functions of Hu proteins

    Cell Mol Life Sci

    (2008)
  • I. Lopez de Silanes et al.

    HuR: post-transcriptional paths to malignancy

    RNA Biol

    (2005)
  • H. Hasegawa et al.

    HuR is exported to the cytoplasm in oral cancer cells in a different manner from that of normal cells

    Br J Cancer

    (2009)
  • T.K. Williams et al.

    pp32 (ANP32A) expression inhibits pancreatic cancer cell growth and induces gemcitabine resistance by disrupting HuR binding to mRNAs

    PLoS ONE

    (2010)
  • N. Filippova et al.

    The RNA-binding protein HuR promotes glioma growth and treatment resistance

    Mol Cancer Res

    (2011)
  • W. Kakuguchi et al.

    HuR knockdown changes the oncogenic potential of oral cancer cells

    Mol Cancer Res

    (2010)
  • S. Danilin et al.

    Role of the RNA-binding protein HuR in human renal cell carcinoma

    Carcinogenesis

    (2010)
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