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. 2018 Dec;12(4):709-721.
doi: 10.1007/s12079-017-0442-2. Epub 2018 Feb 26.

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

Affiliations

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

Pavithra Rajagopalan et al. J Cell Commun Signal. 2018 Dec.

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.

Keywords: Carcinogenesis; Cell adhesion; High-throughput; Orbitrap fusion; Smoking.

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Conflict of interest statement

The authors declare that no competing financial interests exist.

Figures

Fig. 1
Fig. 1. Chronic exposure to cigarette smoke induces phenotypic changes in oral keratinocytes.
a Cellular morphology of OKF6/TERT1-Parental cells and OKF6/TERT1-Smoke cells (magnification 20X) (b) Growth curve depicting cellular proliferation rates of OKF6/TERT1-Parental and OKF6/TERT1-Smoke cells. c Transwell-based invasion assays were performed using Matrigel-coated chambers where number of cells that invade into the lower chamber were visualized (10× magnification) with methylene blue staining in OKF6/TERT1-Parental and OKF6/TERT1-Smoke cells. d Invaded cells were counted and relative changes in invasive ability of OKF6/TERT1-Parental and OKF6/TERT1-Smoke cells were calculated and represented graphically (***p < 0.0001). e Colony formation assay of smoke exposed and parental cells visualized (3× magnification) after staining with methylene blue. f Wound migration assays were carried out using OKF6/TERT1-Smoke and parental cells between 0 and 10 h. Cells were imaged at 10× magnification
Fig. 2
Fig. 2
a Quadrant plot depicting log fold change of proteins at proteome and phosphoproteome level in OKF6/TERT1-Smoke cells compared to OKF6/TERT1-Parental cells. b Ingenuity pathway analysis of dysregulated proteins (≥2 fold) and differentially phosphorylated proteins (≥2 fold) in smoke exposed cells compared to parental cells. Top ten significant (p ≤ 0.05) molecular and cellular functions as observed in OKF6/TERT1-Smoke cells. c Top twelve significant (p ≤ 0.05) functions annotated under ‘Cellular Movement’
Fig. 3
Fig. 3. PKN2 mediated signaling in smoke exposed cells
a Western blot analysis shows endogenous expression of PKN2 in OKF6/TERT1-Smoke cells and in a panel of HNSCC cell lines compared to OKF6/TERT1-Parental cells (b) OKF6/TERT1 Smoke and HNSCC cell lines JHU-O11, FaDu, CAL 27, JHU-O22 and JHU-O29 were transfected with PKN2 siRNA. Immunoblot analysis of total PKN2, p-PKN2 (Thr816), CD44, p-CD44 (Ser706) and DSC3 was performed. β-actin was used as loading control
Fig. 4
Fig. 4. Silencing of PKN2 decreases cellular proliferation.
Proliferation curve of (a) OKF6/TERT1-Smoke (b) JHU-O11 (c) JHU-O22 (d) JHU-O29 (e) FaDu (f) CAL 27 cells upon siRNA-mediated silencing of PKN2 (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)
Fig. 5
Fig. 5. Inhibition of PKN2 decreases the invasive and migratory ability of smoke exposed cells and HNSCC cells exposed to cigarette smoke.
a–b Colony formation assays were carried out using OKF6/TERT1-Smoke, JHU-O11, FaDu, CAL 27, JHU-O22 and JHU-O29 cells following siRNA-mediated silencing of PKN2 or control siRNA (scrambled siRNA), A graphical representation of the colony forming ability of the indicated cells upon PKN2 silencing (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001). Colonies were visualized at 3× magnification. c–d Invasion assays were carried out using OKF6/TERT1-Smoke, JHU-O11, FaDu and CAL 27 cells. Cells were transfected with either control (Scrambled) or PKN2 siRNA and invaded cells were photographed at 10× magnification. A graphical representation of the invasive ability of the cells upon PKN2 silencing (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001) (e) Wound migration assays were carried out using OKF6/TERT1-Smoke and HNSCC cells with or without PKN2 SiRNA. Distance between migrated cells were calculated between 0 and 10 h and represented as bar graph. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)

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