Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

Abstract

We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer.

Keywords: Cervical cancer; HPV16-mediated transformation; Human keratinocytes; Human papillomavirus; Ski; TGF-beta.

Fig. 1

TGF-β decreases Ski protein levels in normal HKc, HKc/HPV16 and HKc/DR. (A) Western blot of ski protein in normal HKc, HKc/HPV16 and HKc/DR. Cells were cultured in the absence (−) or the presence (+) of TGF-β (40 pM for 24 h) and cell extracts prepared for Western blot analysis of Ski. Western analysis for TFIID was used as a loading control. (B) Detection of Ski protein by immunofluorescence in normal HKc, HKc/HPV16 and HKc/DR with or without TGF-β treatment. Cells were cultured in the absence (−) or the presence (+) of TGF-β (40 pM for 24 h) and Ski was detected by indirect immunofluorescence (green). Nuclei were stained with far red. Shown are the images for Ski alone (green) and the merged images of Ski and nuclei (green and red).

Fig. 2

Delayed loss of nuclear Ski and Ski degradation in response to TGF-β in HKc/DR, compared to normal HKc. Cells were immunostained for Ski (green) at various times (0 to 60 min) after treatment with TGF-β (40 pM) and nuclei visualized with far red. (a–f) Ski staining only (green). (a′–f′) merged Ski (green) and nuclei (red) staining.

Fig. 3

Ski protein levels in HKc/DR increase throughout the cell cycle and Ski is a phosphoprotein. (A) HKc/DR were synchronized by feeding with growth factor-free medium (no serum, EGF, insulin, or BPE) for 48 h. Cells were then refed with complete medium and total cell protein collected at different times (0 to 27 h) following refeeding for detection of Ski by Western Blot. TFIID was used as a loading control. (B) Nuclear extracts from HKc/DR were incubated in the absence (−PP) or the presence (+PP) of λ-phosphatase for 1 h at 30° C or in the presence of λ-phosphatase and the phosphatase inhibitors NaF and Na₃VO₄ (+PP+PPI). Ski protein was detected by Western blot and TFIID was used as a loading control.

Fig. 4

Ski protein is localized to the centrosomes and spindles in HKc/DR. HKc/DR were treated with nocodazole for 16 h, to block the cells at prophase. Ski was then detected by indirect immunofluorescence (green) and nuclei were stained with DAPI (blue). (a) Ski (green) staining only; (b) merged Ski (green) and nuclei (blue) staining.

Fig. 5

Ski Protein levels and cell proliferation are decreased by a Ski shRNA in HKc/DR. (A) HKc/DR were stably transfected with either the pSUPER.retro vector (Control) or a pSUPER.retro SkiRNAi plasmid, encoding a Ski shRNA (Ski shRNA). Ski protein levels in cell extracts were determined by Western blot. TFIID was used as a loading control. (B) HKc/DR were plated in 6-well plates in equal numbers, transfected with either the control pSUPER.retro vector (closed circles) or the pSUPER.retro SkiRNAi plasmid (squares), and selected with puromycin for three days. Cell numbers were determined by manually counting the cells beginning 3 days after the end of puromycin selection.

Fig. 6

Ski protein levels increase in cervical squamous cell carcinoma. Ski protein (green) in adjacent normal cervical tissue and cervical squamous cell carcinoma tissue was detected by indirect immunofluorescence and confocal microscopy. Nuclei were stained with DAPI 405 (blue). (a, b) Ski staining only (green); (a′, b′) nuclei staining only (blue); (a″, b″) merged Ski and nuclei staining.