Nuclear heterogeneous nuclear ribonucleoprotein D is associated with poor prognosis and interactome analysis reveals its novel binding partners in oral cancer.

Manish Kumar,K W Michael Siu,Nootan Kumar Shukla,Meherchand Sharma,Ajoy Roychoudhury,Jatinder Kaur,Paul G Walfish,Ajay Matta,Shyam Singh Chauhan,Ranju Ralhan,Olena Masui,Alok Thakar,Gunjan Srivastava

doi:10.1186/s12967-015-0637-3

Abstract

BackgroundPost-transcriptional regulation by heterogeneous ribonucleoproteins (hnRNPs) is an important regulatory paradigm in cancer development. Our proteomic analysis revealed hnRNPD overexpression in oral dysplasia as compared with normal mucosa; its role in oral carcinogenesis remains unknown. Here in we determined the hnRNPD associated protein networks and its clinical significance in oral squamous cell carcinoma (OSCC).MethodsImmunoprecipitation (IP) followed by tandem mass spectrometry was used to identify the binding partners of hnRNPD in oral cancer cell lines. Ingenuity pathway analysis (IPA) was carried out to unravel the protein interaction networks associated with hnRNPD and key interactions were confirmed by co-IP-western blotting. hnRNPD expression was analyzed in 183 OSCCs, 44 oral dysplasia and 106 normal tissues using immunohistochemistry (IHC) and correlated with clinico-pathological parameters and follow up data over a period of 91 months. Kaplan–Meier survival and Cox-multivariate-regression analyses were used to evaluate the prognostic significance of hnRNPD in OSCC.ResultsWe identified 345 binding partners of hnRNPD in oral cancer cells. IPA unraveled novel protein–protein interaction networks associated with hnRNPD and suggested its involvement in multiple cellular processes: DNA repair, replication, chromatin remodeling, cellular proliferation, RNA splicing and stability, thereby directing the fate of oral cancer cells. Protein–protein interactions of hnRNPD with 14-3-3ζ, hnRNPK and S100A9 were confirmed using co-IP-western blotting. IHC analysis showed significant overexpression of nuclear hnRNPD in oral dysplasia [p = 0.001, Odds ratio (OR) = 5.1, 95 % CI = 2.1–11.1) and OSCCs (p = 0.001, OR = 8.1, 95 % CI = 4.5–14.4) in comparison with normal mucosa. OSCC patients showing nuclear hnRNPD overexpression had significantly reduced recurrence free survival [p = 0.026, Hazard ratio = 1.95, 95 % CI = 1.0–3.5] by Kaplan–Meier survival and Cox-multivariate-regression analyses and has potential to define a high-risk subgroup among OSCC patients with nodal negative disease.ConclusionsOur findings suggest novel functions of hnRNPD in cellular proliferation and survival, besides RNA splicing and stability in oral cancer. Association of nuclear hnRNPD with poor prognosis in OSCC patients taken together with its associated protein networks in oral cancer warrant future studies designed to explore its potential as a plausible novel target for molecular therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0637-3) contains supplementary material, which is available to authorized users.

Highlights

Post-transcriptional regulation by heterogeneous ribonucleoproteins is an important regulatory paradigm in cancer development
Expression of heterogeneous nuclear ribonucleoprotein D (hnRNPD) in oral cancer cells and tissues Real time PCR and Western blotting were performed to determine the expression of hnRNPD transcripts and protein expression levels respectively in oral cancer cells (SCC4, HSC2, Tu167 and MDA1986), oral squamous cell carcinoma (OSCC) and normal oral mucosa tissues
Identification of binding partners of hnRNPD in OSCC cells To gain an insight into the role of hnRNPD in OSCCs, we identified its binding partners in oral cancer cell lines (SCC4 and MDA1986)

Summary

Introduction

Post-transcriptional regulation by heterogeneous ribonucleoproteins (hnRNPs) is an important regulatory paradigm in cancer development. Post-transcriptional regulation of mRNA stability and translation by RNA binding proteins (RBPs) is a key determinant of gene expression [1,2,3] These RNA–protein interactions dictate the ultimate fate of the transcripts and are emerging as an important regulatory paradigm in cancer development [3, 4]. AUrich RNA-binding factor (AUF1)/heterogeneous nuclear ribonucleoprotein D (hnRNPD) is an ARE-binding protein which regulates the mRNA stability of many genes involved in cell cycle, proliferation, survival, senescence and stress response [1, 2, 3, 4, 5, 7, 8, 9, 10, 11]. Nuclear hnRNPD has been shown to activate the transcription promoter for telomerase catalytic subunit Tert, and links maintenance of telomere length and normal aging to attenuation of inflammatory cytokine expression and inhibition of cellular senescence [20]

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