René Bernards ¹

¹ Division of Molecular Carcinogenesis, Center for Biomedical Genetics and Cancer Genomics Center, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
Email: r.bernards@nki.nl

A medley of molecules, and the interactions between them, mediate cancer. The latest news is that the enzyme CDK8 orchestrates cross-talk between two signalling pathways that are frequently deregulated in human cancers.

There is no simple answer to the question of which cellular proteins or signalling pathways are responsible for making a cell cancerous. For example, the Wnt signalling pathway, which normally plays a pivotal part in development, is often deregulated by mutation in cancer cells (1). Mutations in the gene for the retinoblastoma tumour-suppressor protein, which is part of another signalling pathway, are also frequently associated with cancer. When put together, the findings of Firestein et al. (2) and Morris et al. (3), presented in this issue, lead to the identification of a point at which these important pathways could communicate with each other in colorectal cancer.

When Wnt proteins bind to their cell-surface receptors, this leads to the stabilization of a protein called b-catenin in the cytoplasm. b-Catenin then moves to the nucleus, where it interacts with gene transcription factors called TCFs to activate a specific set of genes. Firestein and colleagues (2) (page 547) set out to search for genes whose suppression alters b-catenin-dependent gene transcription in colon cancer cells. Of some 1,000 genes (mostly encoding kinase enzymes) that they screened, 34 seemed to be required for b-catenin activity. They then tested the same 1,000 genes for a role in the proliferation of colon cancer cells, and came up with a second list of 166 genes. The two 'hit lists' share nine genes — required for both b-catenin activity and proliferation of colorectal cancer cells. But which of these, if any, are relevant to the biology of human colon cancer?

To address this question, Firestein et al. investigated whether any of the nine genes resides in a region of the human genome that is amplified (meaning that additional copies of the gene are present) in colon cancer. Only one — CDK8 — was located in a frequently amplified region, suggesting that its increased level of expression contributes to the development of colorectal tumours. Indeed, increased expression of this gene turned normal mouse fibroblast cells cancerous, and this ability depended on the presence of functional b-catenin–TCF. This observation supported the results of the authors' initial screen, which indicated that CDK8 contributes to oncogenesis by stimulating b-catenin/TCF activity. But how does it do this?

CDK8 is a member of a family of protein kinases that must associate with regulatory proteins called cyclins to become active. CDK8 and its regulatory subunit cyclin C are components of the multi-protein Mediator complex, which couples the actions of transcription factors with the molecular machinery that carries out transcription.

When Firestein et al. studied the promoter sequence for one of the best-characterized target genes of b-catenin–TCF — the c-MYC oncogene — they found that CDK8 also interacts with this promoter close to where b-catenin–TCF binds. Decreasing the level of CDK8 by the technique of RNA interference also reduced b-catenin binding to the promoter, indicating that CDK8 (presumably as part of the Mediator complex) stabilizes the interaction of b-catenin with the c-MYC promoter, thereby facilitating transcription of the gene (Fig. 1a). So, amplification and overexpression of CDK8 contributes to colon carcinogenesis, at least in part, by aiding b-catenin–TCF activity.

Figure 1: Cross-talk between signalling pathways in colorectal cancer (2, 3).

Figure 1: Cross-talk between signalling pathways in colorectal cancer (2, 3).

a, The product of the CDK8 gene, which is amplified in half of the cases of colorectal cancer, stimulates transcriptional activation of b-catenin–TCF target genes, possibly as part of the Mediator multi-protein complex. CycC, cyclin C.

b, E2F1, in contrast, inhibits the activity of the b-catenin–TCF complex.

c, In colorectal cancer, E2F1 is itself inhibited both through phosphorylation by the CycC–CDK8 complex and by direct binding of the retinoblastoma protein pRB. Increased expression of both RB1 and CDK8 therefore promotes colon cancer by inhibiting E2F1 and stimulating oncogenic transcriptional activity of b-catenin–TCF.

How does the retinoblastoma tumour-suppressor protein (pRB) fit into the picture? This protein exerts its tumour-suppressive effects mainly by restraining the activity of three closely related transcription factors of the E2F family, which regulate the expression of genes involved in both cellular proliferation and programmed cell death (apoptosis) (4). Working with the fruitfly Drosophila, Morris and colleagues (3) (page 552) find that E2F1 counteracts the activity of b-catenin.

The authors show that high levels of fly b-catenin negate the apoptotic effects of increased Drosophila E2F1 expression in the developing wing. Moreover, when Drosophila E2F1 was expressed in a mutant fly that expresses high b-catenin levels in the eye, the abnormal effects of the high b-catenin were greatly suppressed. What's more, E2F1 also suppressed activation of transcription by b-catenin–TCF in human cells (3). At least in part, E2F1 exerts this effect by activating three genes (AXIN1, AXIN2 and SIAH1), which are involved in the pathway leading to b-catenin degradation. Together, these results establish E2F1 as a negative regulator of b-catenin–TCF activity.

Given the potent effects of E2F1 on b-catenin, Morris et al. performed a genetic screen in Drosophila to identify other genes that control E2F1 activity. They generated flies that have decreased expression of Drosophila E2F1 — and so have an aberrant eye morphology — and looked for genes that could overcome the effect of this mutation. They identified one mutant that had a partial loss of CDK8 function. This finding suggests that, in the fly, CDK8 normally acts to suppress the activity of E2F1. Indeed, expression of E2F1-regulated genes is increased in Drosophila larvae with mutations in the genes for either CDK8 or cyclin C (ref. 3). The observations that, in both Drosophila and human cells, CDK8 forms a complex with E2F1 and phosphorylates it, points to a potential mechanism for the effect of CDK8 on E2F1.

The combined results from the two studies(2, 3) point to a dual effect of CDK8 on b-catenin activation. As well as the direct stimulatory effect of CDK8 on b-catenin's transcriptional activity that Firestein and colleagues (2) describe (Fig. 1a), CDK8 seems to have a second (less direct) stimulatory effect on b-catenin, by protecting it from inhibition by E2F1 (Fig. 1b). This interplay between the Wnt/b-catenin and the pRB/E2F1 signalling pathways might be even more complex, as a previous study (5) showed that cyclin D1, which when complexed with the kinase CDK4 can inactivate pRB, is a transcriptional target of b-catenin–TCF.

Together, these results(2, 3) make a strong case that, at least in colon cancer, E2F1 activity must be suppressed in order to fully unleash the oncogenic activity of b-catenin–TCF. As pRB, like CDK8, is a potent inhibitor of E2F1, this means that, in the context of colorectal cancer, pRB is more likely to be acting as an oncoprotein than as a tumour suppressor (Fig. 1b). Indeed, in support of this counterintuitive notion, although the gene encoding pRB (RB1) is mutated and inactivated in some 30% of all human cancers, its mutation in colorectal cancer has not been identified (6,7). On the contrary, RB1 is often overexpressed and even amplified in colorectal cancer (2,8). Moreover, reduced expression of pRB inhibits proliferation of human colon cancer cells, consistent with the growth-promoting activity of this protein (3, 9).

Dual oncogenic and tumour-suppressor activity has also been previously reported (10) for E2F1, as mice lacking this gene show tissue-specific proliferation defects as well as oncogenesis. Given the complexity of the networks mediating cancer, with new entanglements continually being revealed, there is a compelling case for the generation of a comprehensive map of genetic interactions in the human cancer genome. Undoubtedly, much more can be gleaned from studying the cross-talk between signalling pathways.

References:

1. Clevers, H. Cell 127, 469–480 (2006).

2. Firestein, R. et al. Nature 455, 547–551 (2008).

3. Morris, E. J. et al. Nature 455, 552–556 (2008).

4. Rowland, B. D. & Bernards, R. Cell 127, 871–874 (2006).

5. Tetsu, O. & McCormick, F. Nature 398, 422–426 (1999).

6. Horowitz, J. M. et al. Proc. Natl Acad. Sci. USA 87, 2775–2779 (1990).

7. Hildebrandt, B. et al. Oncology 59, 344–346 (2000).

8. Gope, R. et al. J. Natl Cancer Inst. 82, 310–314 (1990).

9. Williams, J. P. et al. Mol. Cell Biol. 26, 1170–1182 (2006).

10. Yamasaki, L. et al. Cell 85, 537–548 (1996).

1. Morris EJ, Ji J-Y, Yang F, Di Stefano L, Herr A, Moon N-S, Kwon E-J, Haigis KM, Näär AM, and Dyson NJ, "E2F1 represses b-catenin transcription and is antagonized by both pRB and CDK8".
http://www.nature.com/nature/journal/v455/n7212/full/nature07310.html

2. Firestein R  and Hahn WC, "Revving the Throttle on an Oncogene: CDK8 Takes the Driver Seat", Cancer Research, 69: (20), 7899-7901 (October 15, 2009).

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