IFITM3 knockdown reduces the expression of CCND1 and CDK4 and suppresses the growth of oral squamous cell carcinoma cells

Abstract

Purpose: Oral squamous cell carcinoma (OSCC) is a challenging disease to treat. Up to 50% of OSCC patients with advanced disease develop recurrences. Elucidation of key molecular mechanisms underlying OSCC development may provide opportunities to target specific genes and, thus, to improve patient survival. In this study, we examined the expression and functional role of interferon transmembrane protein 3 (IFITM3) in OSCC development.

Methods: The expression of IFITM3 in OSCC and normal oral mucosal tissues was assessed by qRT-PCR and immunohistochemistry. The role of IFITM3 in driving OSCC cell proliferation and survival was examined using siRNA-mediated gene knockdown, and the role of IFITM3 in driving cell cycle regulators was examined using Western blotting.

Results: We found that IFITM3 is overexpressed in more than 79% of primary OSCCs. We also found that IFITM3 knockdown led to impaired OSCC cell growth through inhibition of cell proliferation, induction of cell cycle arrest, senescence and apoptosis. In addition, we found that IFITM3 knockdown led to reduced expressions of CCND1 and CDK4 and reduced RB phosphorylation, leading to inhibition of OSCC cell growth. This information may be instrumental for the design of novel targeted therapeutic strategies.

Conclusions: From our data we conclude that IFITM3 is overexpressed in OSCC and may regulate the CCND1-CDK4/6-pRB axis to mediate OSCC cell growth.

Keywords: CCND1; CDK4; IFITM3; Oral squamous cell carcinoma; Proliferation.

Fig. 1. IFITM3 is overexpressed in OSCC but not in normal oral mucosa (NOM).

a Quantitative RT-PCR (qRT-PCR) analysis showing that IFITM3 mRNA levels are significantly up-regulated in OSCC tissues. As indicated by the box and whiskers plot, OSCC tissues exhibited a mean 2.9 ± 0.46-fold increase in IFITM3 expression, with a maximum level of 18-fold overexpression compared to NOM tissues. b IHC staining analysis showing that IFITM3 is significantly overexpressed in primary tumours compared to NOM tissues. c Representative IHC images showing that IFITM3 is expressed at high levels in primary OSCC tissues (i & ii), while the NOM tissues do not exhibit IFITM3 expression (iii & iv). Original magnification: 200x

Fig. 2. siRNA knockdown of IFITM3 inhibits OSCC cell growth.

a Western blot showing reduction in IFITM3 expression after siRNA transfection in ORL-150 and ORL-204 cells. b siRNA knockdown of IFITM3 in OSCC cells significantly inhibits cell proliferation in comparison to cells transfected with non-targeting (NT) siRNA (p value: *** ≤ 0.001, ** ≤ 0.001, * ≤0.05). c Colony formation ability of cells transfected with siRNA targeting IFITM3 or NT siRNA. Cells were stained with crystal violet after 12 days of incubation, after which colonies of more than 50 cells were counted. d Significantly reduced colony formation of IFITM3 knockdown cells compared to cells transfected with NT siRNA. e siRNA-mediated knockdown of IFITM3 inhibits OSCC colony formation in semi-solid media

Fig. 3. OSCC growth inhibition by IFITM3 knockdown is mediated by induction of apoptosis, cell senescence and cell cycle arrest.

a Apoptosis was assessed by staining OSCC cells with Annexin V and PI and analysis by flow cytometry. Apoptotic cells are positive for Annexin V as indicated by the cells in the lower right quadrant of the scatter plot. b Increased populations of apoptotic cells after IFITM3 knockdown. c Senescent cells stained in blue (arrows) indicating activation of senescent-associated β-galactosidase activity (magnification: 200x). d IFITM3 knockdown significantly induces cellular senescence. e Cell cycle analysis using propidium iodide staining. Histograms indicate the distribution of cells in different stages of the cell cycle. f IFITM3 knockdown significantly arrests OSCC cells at the G1-S phase of the cell cycle

Fig. 4. IFITM3 knockdown modulates levels of cell cycle proteins.

Western blots showing the expression of cell cycle markers in OSCC cells post-transfection with siRNA for IFITM3. a TP53 is not detected in ORL-150 cells due to presence of an inactivating mutation, while ORL-204 cells show up-regulation of TP53 upon IFITM3 knockdown. ORL-166 cells were used as positive control for p16 expression. b Reduced CCND1, CDK4 and pRB expression in IFITM3 knockdown cells

Fig. 5. Bioinformatics analysis of IFITM3 associations with cell cycle pathways.

a Data mining from the STRING database indicate that IFITM3 is associated with genes involved in KEGG cell cycle and cancer pathways. b Gene co-expression network depicting genes positively co-expressed with IFITM3 in ORL cell lines enriched in cell cycle signalling pathways