Tumor suppressor p16INK4A is necessary for survival of cervical carcinoma cell lines

Abstract

The tumor suppressor p16(INK4A) inhibits formation of enzymatically active complexes of cyclin-dependent kinases 4 and 6 (CDK4/6) with D-type cyclins. Oncogenic stress induces p16(INK4A) expression, which in turn triggers cellular senescence through activation of the retinoblastoma tumor suppressor. Subversion of oncogene-induced senescence is a key step during cancer development, and many tumors have lost p16(INK4A) activity by mutation or epigenetic silencing. Human papillomavirus (HPV)-associated tumors express high levels of p16(INK4A) in response to E7 oncoprotein expression. Induction of p16(INK4A) expression is not a consequence of retinoblastoma tumor suppressor inactivation but is triggered by a cellular senescence response and is mediated by epigenetic derepression through the H3K27-specific demethylase (KDM)6B. HPV E7 expression causes an acute dependence on KDM6B expression for cell survival. The p16(INK4A) tumor suppressor is a critical KDM6B downstream transcriptional target and its expression is critical for cell survival. This oncogenic p16(INK4A) activity depends on inhibition of CDK4/CDK6, suggesting that in cervical cancer cells where retinoblastoma tumor suppressor is inactivated, CDK4/CDK6 activity needs to be inhibited in order for cells to survive. Finally, we note that HPV E7 expression creates a unique cellular vulnerability to small-molecule KDM6A/B inhibitors.

Keywords: apoptosis; biomarker; cancer therapy; synthetic lethality.

Fig. 1.

KDM6B addiction of cervical cancer lines. KDM6B was depleted in the HPV16⁺ SiHa and CaSKi cervical carcinoma cell lines, the HPV39⁺ cervical cancer cell line Me-180, and the HPV18⁺ cervical cancer cell line HeLa. Cell viability was measured by AlamarBlue assay. Averages and SDs for three independent experiments are shown. Statistically significant changes are indicated, **P < 0.01.

Fig. 2.

KDM6B addiction is caused by the HPV E7 protein. KDM6B was depleted in: (A) HFKs expressing control vector, HPV16 E7, E6, or E6 and E7; (B) HFKs expressing HPV18 E7, E6, or E6 and E7; and (C) U2OS-tet on cells with doxycycline-inducible expression of HPV16 E7. Cell viability was measured by AlamarBlue assay. Averages and SDs for three independent experiments are shown. Statistically significant changes are indicated, **P < 0.01.

Fig. 3.

Cervical cancer cell addiction to p16^INK4A. p16^INK4A was depleted in the HPV16⁺ SiHa and CaSki cervical carcinoma cell lines, and the HPV18⁺ HeLa cervical cancer line. Three independent p16 shRNA constructs (shp16AB, shp16CD, shp16EF) were used. Cell viability was measured by AlamarBlue assay. Averages and SDs for three independent experiments are shown. Statistically significant changes are indicated, **P < 0.01.

Fig. 4.

SiHa cervical cancer cells show evidence of cell death and caspase 3 cleavage upon KDM6B and p16^INK4A depletion. p16^INK4A and KDM6B were depleted in the HPV16⁺ cervical carcinoma cell line SiHa. (A) SiHa cells with control shRNA (magnification, 10×); (B) SiHa cells with KDM6B shRNA (magnification, 10×); (C) SiHa cells with p16^INK4A shRNA (magnification, 10×). (D) Western blot analysis of p16^INK4A, procaspase 3, and cleaved caspase 3 levels. Lysates were separated by SDS/PAGE, transferred, and probed for p16^INK4A and procaspase 3. An actin blot is included as a loading control.

Fig. 5.

HPV16 E7 induces p16^INK4A addiction. p16^INK4A was depleted in (A) HFKs expressing control vector, HPV16 E7, E6, or E6 and E7; and (B) U2OS-tet on cells with doxycycline-inducible expression of HPV16 E7. Three independent p16 shRNA constructs (shp16AB, shp16CD, shp16EF) were used in A and two in B. (C) p16^INK4A was depleted in U2OS-tet on cells with doxycycline-inducible expression of HPV16 E7. Two independent p16 shRNA constructs (shp16AB, shp16CD) and their respective rescue constructs (p16, 16CD) were used. (D) KDM6B was depleted in U2OS-tet on cells with doxycycline-inducible expression of HPV16 E7 and rescued with p16^INK4A. Cell viability was measured by AlamarBlue assay. Averages and SDs for three independent experiments are shown. Statistically significant changes are indicated: *P < 0.05, **P < 0.01.

Fig. 6.

Cell death caused by p16^INK4A depletion in p16^INK4A-addicted cells is dependent on CDK4 and CDK6. (A) p16^INK4A or p16^INK4A, together with CDK4 and CDK6, were depleted in U2OS-tet on cells with doxycycline-inducible expression of HPV16 E7 by transfection with p16^INK4A, CDK4-, or CDK6-specific siRNA duplexes. Transfection of the nontargeting siRNA pool was used as a control (siCtrl). (B) KDM6B or KDM6B in combination with CDK4 and CDK6 were depleted in U2OS-tet on cells with doxycycline-inducible expression of HPV16 E7 by transfection with KDM6B, CDK4-, or CDK6-specific siRNA duplexes. Transfection of a nontargeting siRNA pool was used as a control (siCtrl). (C) U2OS-tet on cells with doxycycline-induced HPV16 E7 expression were transfected with CDK4, CDK6, and oncogenic CDK4 or CDK6 mutants that cannot be inhibited by p16^INK4A (R24C and R31C, respectively). Cell viability was measured by AlamarBlue assay. Averages and SDs for three independent experiments are shown. (D) Kinase-defective CDK4 or CDK6 mutants were transfected into U2OS-tet on cells with doxycycline-induced HPV16 E7 expression and did do not affect viability. Averages and SDs for three independent experiments are shown. Statistically significant changes are indicated: *P < 0.05.

Fig. 7.

Effect of GSK-J4 on cervical cancer cells. Cervical cancer cell lines SiHa, CaSki, and HeLa were treated with 0–30 μM GSK-J4 (CaSki) or 25–100 μM GSK-J4 (SiHa and HeLa) for 72 h. Cell viability was measured by AlamarBlue assay. Averages and SDs for three independent experiments are shown.

Fig. 8.

Induction and abrogation of the OIS tumor suppressor response by HPV16 E7. (A) Oncogenic stimuli such as RAS/RAF signaling or HPV16 E7 expression causes KDM6B expression and derepression of p16^INK4A expression, which inhibits CDK4/6 activity and pRB phosphorylation, causing G1 cell cycle arrest and senescence (OIS). (B) HPV16 E7 has evolved to evade OIS by targeting pRB for ubiquitin-mediated proteasomal degradation.