Damage-induced G1 checkpoint in mammalian cells involves upregulation of p53, which activates transcription of p21Waf1 (CDKN1A). Inhibition of cyclin-dependent kinase (CDK)2 and CDK4/6 by p21 leads to dephosphorylation and activation of Rb. We now show that ectopic p21 expression in human HT1080 fibrosarcoma cells causes not only dephosphorylation but also depletion of Rb; this effect was p53-independent and susceptible to a proteasome inhibitor. CDK inhibitor p27 (CDKN1B) also caused Rb dephosphorylation and depletion, but another CDK inhibitor p16 (CDKN2A) induced only dephosphorylation but not depletion of Rb. Rb depletion was observed in both HT1080 and HCT116 colon carcinoma cells, where p21 was induced by DNA-damaging agents. Rb depletion after DNA damage did not occur in the absence of p21, and it was reduced when p21 induction was inhibited by p21-targeting short hairpin RNA or by a transdominant inhibitor of p53. These results indicate that p21 both activates Rb through dephosphorylation and inactivates it through degradation, suggesting negative feedback regulation of damage-induced cell-cycle checkpoint arrest.Oncogene ( Keywords: p21; Rb; p27; damage response p53-inducible cyclin-dependent kinase (CDK) inhibitor p21 (CDKN1A) is the key mediator of damage-induced cell-cycle arrest. p21 interacts with different cyclin/CDK complexes and other regulators of transcription and signal transduction, exerting broad effects on cell survival, gene expression and morphology (Roninson, 2002). p21 effects are partially mediated by Rb, which is inactivated in proliferating cells through phosphorylation by CDK2 and CDK4/6, both of which are inhibited by p21. As a result, p21 induction leads to Rb dephosphorylation and activation, with ensuing G1 arrest.Whereas p21 activates Rb by dephosphorylation, several oncoproteins inactivate Rb by degradation via the proteasome. Proteasome-mediated Rb degradation is promoted by Mdm2 (Sdek et al., 2005) and gankyrin (Higashitsuji et al., 2000), E7 of papilloma virus (Boyer et al., 1996) and Tax of HTLV1 (Kehn et al., 2005). Oncoprotein-induced proteasomal degradation of Rb is one of the mechanisms for Rb inactivation in carcinogenesis (Ying and Xiao, 2006), but Rb degradation has not been described in DNA damage response.Changes in Rb phosphorylation are most commonly detected by immunoblotting through changes in the protein's electrophoretic mobility. Examination of numerous Rb immunoblots published by different groups showed that in many (but not all) cases Rb dephosphorylation, which results from drug treatment, cell senescence or ectopic p21 expression, is associated with a reduction in the Rb protein signal. In the present study, we have asked (i) whether a decrease in the Rb signal in response to p21 reflects protein degradation or merely altered immunoreactivity of dephosphorylated Rb, (ii) if p53 plays a p21-independent role in the decrease in Rb, (iii) whether such decrease can be induced by other CDK inhibitors that induce Rb dephosphorylation and (iv) if the decrease...
Cyclin-dependent kinase 8 (CDK8) and its paralog CDK19 are transcriptional regulators that, in complex with CCNC, MED12 and MED13, mediate several carcinogenic signalling pathways such as NFκβ, TGFβ/BMP, WNT/β-catenin, HIF1A and serum growth factor network. Using immunohistochemical analysis, we found that CDK8/19 protein is overexpressed in invasive ductal carcinomas of the breast relative to non-malignant mammary tissues. TCGA database analysis showed that gene amplification is the most frequent type of genetic alterations of CDK8, CDK19, CCNC and MED13 in breast cancers, with MED13 appearing as one of the most frequently amplified genes in breast cancer (amplified in 9.7% of samples), whereas point mutations are more common in MED12. CDK8, CDK19 and CCNC expression was strongly increased but MED12 expression was decreased in tumors with mutant p53. Meta-analysis of transcriptome databases revealed that higher expression of CDK8, CDK19, CCNC and MED13 (but not MED12) is associated with shorter relapse-free survival (RFS) in the four molecular subtypes of breast cancer. The RFS correlations were much stronger in patients who underwent systemic adjuvant therapy than in untreated patients, suggesting that CDK8 and its interactive genes impact the failure of systemic therapy. This result is in agreement with the role of CDK8 as a mediator of the chemotherapy-induced paracrine network that promotes drug resistance and metastasis (Porter et al., PNAS, 109, 13799, 2012) and with our finding that a small-molecule CDK8/19 inhibitor augmented the efficacy of doxorubicin in a triple-negative breast cancer xenograft model. The expression levels of CDK8, CDK19, CCNC and MED13 in breast cancer samples were directly correlated with each other and with the expression of MYC but inversely correlated with estrogen receptor (ER)α expression. Since MYC is known to be a positive downstream mediator of the ER activity, we hypothesized that CDK8 may play a similar role, with an increase in CDK8 augmenting estrogen mitogenic signalling in tumors with decreased ER. Confirming this hypothesis, we have found that CDK8 inhibition by selective small-molecule CDK8/19 inhibitors or by shRNA knockdown suppresses estrogen-induced transcription in ER-positive breast cancer cell lines. CDK8/19 inhibition abrogates the mitogenic effect of estrogen on ER-positive cells and synergizes with the ER antagonist fulvestrant. Treatment of estrogen-deprived ER-positive cells with a CDK8/19 inhibitor significantly impeded the outgrowth of estrogen-independent cells, to a greater extent than did mTOR or HER2 targeted drugs. These results indicate that the expression of CDK8 and its interactive genes has a profound impact on the response to treatment in breast cancer and may provide novel biomarkers for relapse-free survival after adjuvant therapy. CDK8/19 inhibition may be useful to augment chemotherapy and hormone therapy of breast cancer and to prevent the development of tumors resistant to estrogen deprivation. Citation Format: McDermott MSJ, Györffy B, Chumanevich AA, Kaza V, Porter DC, Catroppo JF, Chen M, Oliver D, Shtutman M, Roninson IB, Broude EV. CDK8 protein complex as a potential biomarker and therapeutic target in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-07.
Over 70% of breast cancer patients are estrogen receptor (ER) positive and 25% of patients over-express HER2 making these patients susceptible to therapeutic intervention with ER- and HER2-targeted therapies, respectively. However, intrinsic and acquired resistance to targeted therapies is a significant clinical issue and new therapeutic approaches aimed to preventing and overcoming resistance are urgently needed. We have previously shown that high expression of CDK8, a transcription regulating kinase, is associated with shorter relapse free survival in both ER and HER2 positive breast cancer. We have found that CDK8 inhibition by a selective small molecule inhibitor (Senexin B), by shRNA knockdown or by CRISPR/CAS9 knockout, strongly inhibits estrogen signaling in ER-positive breast cancer cells. Senexin B produces a synergistic growth inhibitory effect with an antiestrogen fulvestrant in all the tested ER-positive breast cancer cell lines in vitro and in MCF7 xenograft model in vivo. Senexin B treatment also inhibited invasive growth of MCF7 xenografts. CDK8 inhibition suppressed the emergence of estrogen independence upon long-term estrogen deprivation. A highly synergistic growth inhibitory effect occurred when Senexin B was combined with an anti-HER2 monoclonal antibody (a biosimilar of trastuzumab) or with the HER2/EGFR small molecule inhibitor lapatinib. These synergistic effects were observed in all HER2 positive breast cancer cell lines tested including those that exhibit innate and acquired resistance to HER2 targeting therapy. Furthermore, combining lapatinib with Senexin B completely abrogated the emergence of acquired lapatinib resistance. Taken together these results suggest that CDK8 inhibition, when combined with either ER- or HER2-targeted therapies, offers a rational approach to improving the efficacy of targeted drugs in breast cancer. Citation Format: McDermott MS, Chumanevich A, Liang J, Chen M, Altilia S, Hennes C, Roninson IB, Broude EV. CDK8 inhibition improves the efficacy of ER- and HER2-targeted drugs in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-05.
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