title: Nuclear heterogeneous nuclear ribonucleoprotein D is associated with poor prognosis and interactome analysis reveals its novel binding partners in oral cancer.
abstract: BACKGROUND
  Post-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).
  
  
  METHODS
  Immunoprecipitation (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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  Our 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.
authors: Department of Biochemistry, All India Institute of Medical Sciences, Room No. 3009, New Delhi, 110029, India. manishbhu@gmail.com.
authors: Manish
authors: M
authors: Kumar
authors: Alex and Simona Shnaider Laboratory of Molecular Oncology, Mount Sinai Hospital, 6-500, Toronto, ON, M5G 1X5, Canada. matta.ajay@gmail.com.
authors: Ajay
authors: A
authors: Matta
authors: Department of Chemistry, Centre for Research in Mass Spectrometry, York University, Toronto, ON, Canada. olena.masui@york.ca.
authors: Olena
authors: O
authors: Masui
authors: Alex and Simona Shnaider Laboratory of Molecular Oncology, Mount Sinai Hospital, 6-500, Toronto, ON, M5G 1X5, Canada. gsrivastava123@gmail.com.
authors: Gunjan
authors: G
authors: Srivastava
authors: Department of Biochemistry, All India Institute of Medical Sciences, Room No. 3009, New Delhi, 110029, India. jatinkaur@gmail.com.
authors: Jatinder
authors: J
authors: Kaur
authors: Department of Otorhinolaryngology, All India Institute of Medical Sciences, New Delhi, India. drathakar@gmail.com.
authors: Alok
authors: A
authors: Thakar
authors: Department of Surgery, Dr. B. R. A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India. nootan.shukla@yahoo.co.in.
authors: Nootan Kumar
authors: NK
authors: Shukla
authors: Department of Dental Surgery, All India Institute of Medical Sciences, New Delhi, India. ajoyroy@hotmail.com.
authors: Ajoy
authors: A
authors: RoyChoudhury
authors: Department of Pathology, All India Institute of Medical Sciences, New Delhi, India. sharmamehar@yahoo.co.in.
authors: Meherchand
authors: M
authors: Sharma
authors: Alex and Simona Shnaider Laboratory of Molecular Oncology, Mount Sinai Hospital, 6-500, Toronto, ON, M5G 1X5, Canada. PWalfish@mtsinai.on.ca.|Department of Otolaryngology-Head and Neck Surgery, Joseph and Mildred Sonshine Family Centre for Head and Neck Diseases, Mount Sinai Hospital, Toronto, ON, Canada. PWalfish@mtsinai.on.ca.|Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Joseph & Wolf Lebovic Health Complex, Toronto, ON, M5G 1X5, Canada. PWalfish@mtsinai.on.ca.|Department of Otolaryngology-Head and Neck Surgery, University of Toronto, Toronto, ON, Canada. PWalfish@mtsinai.on.ca.|Department of Medicine, Endocrine Division, Mount Sinai Hospital and University of Toronto, Toronto, ON, Canada. PWalfish@mtsinai.on.ca.
authors: Paul G
authors: PG
authors: Walfish
authors: Department of Chemistry, Centre for Research in Mass Spectrometry, York University, Toronto, ON, Canada. kwmsiu@yorku.ca.|Department of Chemistry, University of Windsor, Windsor, ON, Canada. kwmsiu@yorku.ca.
authors: K W
authors: KW
authors: Michael Siu
authors: Department of Biochemistry, All India Institute of Medical Sciences, Room No. 3009, New Delhi, 110029, India. s_s_chauhan@hotmail.com.
authors: Shyam Singh
authors: SS
authors: Chauhan
authors: Alex and Simona Shnaider Laboratory of Molecular Oncology, Mount Sinai Hospital, 6-500, Toronto, ON, M5G 1X5, Canada. ralhanr@gmail.com.|Department of Otolaryngology-Head and Neck Surgery, Joseph and Mildred Sonshine Family Centre for Head and Neck Diseases, Mount Sinai Hospital, Toronto, ON, Canada. ralhanr@gmail.com.|Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Joseph & Wolf Lebovic Health Complex, Toronto, ON, M5G 1X5, Canada. ralhanr@gmail.com.|Department of Otolaryngology-Head and Neck Surgery, University of Toronto, Toronto, ON, Canada. ralhanr@gmail.com.
authors: Ranju
authors: R
authors: Ralhan
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002294:Carcinoma, Squamous Cell / Q000175:diagnosis / Q000378:metabolism* / Q000401:mortality; D045744:Cell Line, Tumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000378:metabolism*; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000378:metabolism; D006801:Humans; D053208:Kaplan-Meier Estimate; D008297:Male; D008875:Middle Aged; D009062:Mouth Neoplasms / Q000175:diagnosis / Q000378:metabolism* / Q000401:mortality; D015999:Multivariate Analysis; D011379:Prognosis; D016016:Proportional Hazards Models; D011485:Protein Binding; D040901:Proteomics; D055815:Young Adult
chemical_list: D048948:14-3-3 Proteins; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D034641:Heterogeneous-Nuclear Ribonucleoprotein K