Proteomic and phosphoproteomic profiling of shammah induced signaling in oral keratinocytes

Shankargouda Patil,Jayshree Advani,Mohd Younis Bhat,Nezar Al-Hebshi,Sonali V Mohan,Firdous A Bhat,Tejaswini Subbannayya,Aditi Chatterjee,Niraj Babu,Keshava K Datta,Pavithra Rajagopalan,Harsha Gowda,David Sidransky

doi:10.1038/s41598-021-88345-x

Abstract

Shammah is a smokeless tobacco product often mixed with lime, ash, black pepper and flavorings. Exposure to shammah has been linked with dental diseases and oral squamous cell carcinoma. There is limited literature on the prevalence of shammah and its role in pathobiology of oral cancer. In this study, we developed a cellular model to understand the effect of chronic shammah exposure on oral keratinocytes. Chronic exposure to shammah resulted in increased proliferation and invasiveness of non-transformed oral keratinocytes. Quantitative proteomics of shammah treated cells compared to untreated cells led to quantification of 4712 proteins of which 402 were found to be significantly altered. In addition, phosphoproteomics analysis of shammah treated cells compared to untreated revealed hyperphosphorylation of 36 proteins and hypophosphorylation of 83 proteins (twofold, p-value ≤ 0.05). Bioinformatics analysis of significantly altered proteins showed enrichment of proteins involved in extracellular matrix interactions, necroptosis and peroxisome mediated fatty acid oxidation. Kinase-Substrate Enrichment Analysis showed significant increase in activity of kinases such as ROCK1, RAF1, PRKCE and HIPK2 in shammah treated cells. These results provide better understanding of how shammah transforms non-neoplastic cells and warrants additional studies that may assist in improved early diagnosis and treatment of shammah induced oral cancer.

Highlights

Shammah is a smokeless tobacco product often mixed with lime, ash, black pepper and flavorings
We found reduced expression of several genes mapped to necroptic pathway such as Fas associated via death domain (FADD) (0.49-fold; p-value ≤ 0.05), peptidylprolyl isomerase A (PPIA) (0.43-fold; p-value ≤ 0.05), caspase 8 (CASP8) (0.48-fold; p-value ≤ 0.05), caspase 1 (CASP1) (0.37-fold; p-value ≤ 0.05), glycogen phosphorylase (PYGL) (0.39-fold; p-value ≤ 0.05), calpain 2 (CAPN2) (0.51-fold; p-value ≤ 0.05), PYD and CARD domain containing (PYCARD) (0.43-fold; p-value ≤ 0.05), eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2) (0.44-fold; p-value ≤ 0.05) and high mobility group box 1 (HMGB1) (0.41-fold; p-value ≤ 0.05) in OKF6/TERT1-Shammah cells
We have developed an in vitro cellular model to study the effect of chronic shammah exposure on oral keratinocytes

Summary

Introduction

Shammah is a smokeless tobacco product often mixed with lime, ash, black pepper and flavorings. We generated an in vitro cellular model using normal, non-transformed oral keratinocytes (OKF6/TERT1)[19], which were chronically treated with shammah for a period of 6 months. Upon establishment of the cellular model, we employed tandem mass tag (TMT) based quantitative proteomic and TiO2-based phosphoenrichment approach to study global proteomic and phosphoproteomic alterations in OKF6/TERT1 cells upon chronic exposure to shammah.

Results

Conclusion