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Role of protein kinase N2 (PKN2) in cigarette smoke-mediated oncogenic transformation of oral cells

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Journal of Cell Communication and Signaling Aims and scope

Abstract

Smoking is the leading cause of preventable death worldwide. Though cigarette smoke is an established cause of head and neck cancer (including oral cancer), molecular alterations associated with chronic cigarette smoke exposure are poorly studied. To understand the signaling alterations induced by chronic exposure to cigarette smoke, we developed a cell line model by exposing normal oral keratinocytes to cigarette smoke for a period of 12 months. Chronic exposure to cigarette smoke resulted in increased cellular proliferation and invasive ability of oral keratinocytes. Proteomic and phosphoproteomic analyses showed dysregulation of several proteins involved in cellular movement and cytoskeletal reorganization in smoke exposed cells. We observed overexpression and hyperphosphorylation of protein kinase N2 (PKN2) in smoke exposed cells as well as in a panel of head and neck cancer cell lines established from smokers. Silencing of PKN2 resulted in decreased colony formation, invasion and migration in both smoke exposed cells and head and neck cancer cell lines. Our results indicate that PKN2 plays an important role in oncogenic transformation of oral keratinocytes in response to cigarette smoke. The current study provides evidence that PKN2 can act as a potential therapeutic target in head and neck squamous cell carcinoma, especially in patients with a history of smoking.

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Fig. 1: Chronic exposure to cigarette smoke induces phenotypic changes in oral keratinocytes.
Fig. 2
Fig. 3: PKN2 mediated signaling in smoke exposed cells
Fig. 4: Silencing of PKN2 decreases cellular proliferation.
Fig. 5: Inhibition of PKN2 decreases the invasive and migratory ability of smoke exposed cells and HNSCC cells exposed to cigarette smoke.

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Abbreviations

AGC:

Automatic Gain Control

BCA:

Bicinchoninic acid

EDTA:

Ethylenediaminetetraacetic acid

EMT:

Epithelial–mesenchymal transition

HCD:

Higher energy collisional dissociation

HPLC:

High Pressure Liquid Chromatography

SDS:

Sodium Dodecyl Sulfate

TEABC:

Triethyl ammonium bicarbonate

TMT:

Tandem Mass Tag

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Acknowledgements

We thank the Department of Biotechnology (DBT), Government of India for research support to the Institute of Bioinformatics (IOB), Bangalore. Pavithra Rajagopalan is a recipient of Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), New Delhi, India. Kiran K. Mangalaparthi is a recipient of Senior Research Fellowship from University Grants Commission (UGC), New Delhi, India. Harsha Gowda is a Wellcome Trust/DBT India Alliance Intermediate Career Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Institute of Bioinformatics, 7th floor, Discoverer Building, International Tech Park, Bangalore, 560 066, India

    Pavithra Rajagopalan, Vishalakshi Nanjappa, Krishna Patel, Ankit P. Jain, Kiran K. Mangalaparthi, Arun H. Patil, T. S. Keshava Prasad, Harsha Gowda & Aditi Chatterjee

  2. School of Biotechnology, Kalinga Institute of Industrial Technology, Bhubaneswar, 751024, India

    Pavithra Rajagopalan, Ankit P. Jain, Arun H. Patil & Premendu P. Mathur

  3. School of Biotechnology, Amrita Vishwa Vidyapeetham, Kollam, 690 525, India

    Krishna Patel, Kiran K. Mangalaparthi & Bipin Nair

  4. Department of Biochemistry & Molecular Biology, Pondicherry University, Pondicherry, 605014, India

    Premendu P. Mathur

  5. NIMHANS-IOB Bioinformatics and Proteomics Laboratory, Neurobiology Research Centre, National Institute of Mental Health and Neurosciences, Bangalore, 560 029, India

    T. S. Keshava Prasad

  6. Center for Systems Biology and Molecular Medicine, Yenepoya, Mangalore, 575020, India

    T. S. Keshava Prasad

  7. Department of Surgery, UC San Diego, Moores Cancer Center, La Jolla, CA, 92093, USA

    Joseph A. Califano

  8. Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, 21231, USA

    David Sidransky

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  1. Pavithra Rajagopalan
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  2. Vishalakshi Nanjappa
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  3. Krishna Patel
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  4. Ankit P. Jain
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  12. Harsha Gowda
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  13. Aditi Chatterjee
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Contributions

AC and HG participated in study conception and study design. PR and VN were involved in cell culture and performed all assays and experiments. APJ and KKM carried out fractionation and mass spectrometric analysis of samples. PR and KP prepared the manuscript and manuscript figures. PR, KP and AHP were involved in data analyses and interpretation. AC, HG, DS, JAC, PPM, TSKP and BN edited, critically read and revised the manuscript. All the authors have read and approved the final manuscript.

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Correspondence to Harsha Gowda or Aditi Chatterjee.

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Supplementary Figure 1

Schematic workflow employed to study the proteomic and phosphoproteomic alterations in normal oral keratinocytes chronically exposed to cigarette smoke (TIFF 1988 kb)

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Rajagopalan, P., Nanjappa, V., Patel, K. et al. Role of protein kinase N2 (PKN2) in cigarette smoke-mediated oncogenic transformation of oral cells. J. Cell Commun. Signal. 12, 709–721 (2018). https://doi.org/10.1007/s12079-017-0442-2

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