Research
Regulation of cell cycle re-entry by ERK1/2 and ERK5
Extracellular signals (growth factors) instruct cells to proceed through the cell cycle and divide to form identical daughter cells. These signals are relayed in part by a three-tier, hierarchical array of protein kinases (Raf–>MEK1/–>ERK1/2), in which each kinase serves to phosphorylate and activate the next kinase in the pathway.
When activated, ERK1/2 (extracellular signal-regulated kinases) accumulate in the nucleus where they phosphorylate transcription factors and so promote or repress gene expression.
For example, the ERK1/2 pathway promotes the expression of cyclin D1 and the repression of the CDK inhibitor p27KIP1 (see Figure 1) to drive quiescent cells through the G1 phase of the cell cycle and into S phase in which DNA is replicated. We are studying both the classical ERK1/2 pathway and the novel ERK5 pathway to try to understand their role in regulating cell cycle progression.
Translational Research — Identifying determinants of tumour cell sensitivity to novel chemotherapeutic drugs that target the ERK1/2 pathway
Activation of the ERK1/2 pathway is a normal response following engagement of growth factor receptors. However, the ERK1/2 pathway is frequently subject to inappropriate activation in a variety of human tumours due to activating mutations in the genes encoding BRAF, RAS or growth factor receptors (such as EGFR and FGFR3). Furthermore, genetic inhibition of this pathway, using interfering mutants of MEK1/2, has been shown to antagonise cellular transformation by RAS and RAF. As a result there is a lot of interest in isolating small molecule inhibitors of the ERK1/2 pathway as it is hoped that such molecules will represent novel anti-cancer drugs.
Indeed, there are now a variety of options for pharmacological intervention in the ERK1/2 pathway (See Figure 2). Drugs such as PD184352 (Squires et al 2002), PD0325901 and AZD6244 have proved to be very selective inhibitors of MEK1/2, thereby preventing activation of ERK1/2 and are currently in clinical trials for a variety of solid tumours. Studies have shown that tumour cells differ greatly in their sensitivity to such ‘MEK inhibitors’. This even applies to cell lines of the same tumour type; for example, colorectal cancer cell lines with identical KRAS mutations can differ by up to 2 orders of magnitude in their sensitivity to such drugs. It is clear that certain other mutations influence how cells respond to MEK inhibition.
In projects sponsored by AstraZeneca we are identifying genes that influence sensitivity to MEK inhibition or sensitivity to FGFR inhibition. This information may be used to interpret results from clinical trials and may help in the future to direct the most effective inhibitors to different patients based on their mutation profile (personalized therapies).
Inhibition of cell cycle re-entry by the cAMP/PKA pathway
We are also interested in how the ERK1/2 pathway is regulated by anti-proliferative signalling pathways. For example, it is well known that increasing intracellular cAMP content can cause many cell types to arrest in G1 of the cell cycle. We previously showed that cAMP could prevent the activation of the RAF–>MEK1/2–>ERK1/2 pathway. Whilst this would seem a plausible mechanism to account for the cAMP mediated growth arrest we now know that cAMP inhibits cell proliferation at other points downstream of the ERK1/2 pathway. For example, we have now been able to engineer cells so that ERK1/2 activation is insensitive to cAMP but these cells still undergo a cAMP-induced cell cycle arrest (Balmanno et al 2003). We are continuing to study the ‘cross talk' between the cAMP/PKA pathway and the cell cycle machinery to understand how cAMP antagonises cell cycle progression
Inhibition of apoptosis by the ERK1/2 pathway
Cells grown in culture require growth factors to stay alive as well as to divide; withdrawal of these ‘survival factors' results in programmed cell death or ‘apoptosis'. This apoptosis requires new gene expression and one gene that is important in initiating apoptosis in a variety of cells is called Bim. An increase in Bim expression, which is sufficient to kill cells, occurs rapidly in response to withdrawal of survival factors (Weston et al 2003; see Figure 3) and we have found that this is due, in part, to a decrease in ERK1/2 activity.
Indeed, selective activation of the ERK1/2 pathway is sufficient to inhibit the accumulation of Bim mRNA, promote the phosphorylation and proteasomal degradation of the mature Bim protein (Ley et al 2003; Ley et al 2005b) and protect cells from cell death (see Figure 4). Thus the ERK1/2 pathway can promote cell division and cell survival. Since the ERK1/2 pathway is regulated by the RAS and BRAF oncoproteins this may explain the ability of many cancer cells to survive and proliferate even when growth factors are scarce.
Activation of the G1 and G2 checkpoints by the stress kinase pathways — taking the stress out of stress kinase signalling
When cells are exposed to stressful or toxic stimuli that cause DNA damage, they undergo cell cycle arrest at G1 or G2 to prevent the replication or segregation of mutated DNA. If the damage is too severe they will even undergo apoptosis. Mammalian cells possess at least two stress-activated protein kinases (SAPKs), called p38 and JNK, which are activated by hierarchical protein kinase cascades similar to the RAF–>MEK1/2–>ERK1/2 pathway (see Figure 5).
Understanding the role of the stress kinases in cell cycle control is hampered by the fact that the majority of stimuli that activate these pathways are inherently toxic and also activate an array of other signalling pathways. To simplify this we use conditional protein kinases that allow us to activate defined combinations of MAPK and SAPK pathways without any overt cellular stress or damage (see Figure 6)
We have used these to investigate the role of the SAPKs in regulating cell cycle progression. Activation of p38 can cooperate with ERK1/2 to cause cells to arrest at G1 by increasing the expression of the cell cycle inhibitor protein p21CIP1 (Todd et al 2004). p38 can also promote an arrest at G2 by inhibiting the expression of cyclin B1 (Garner et al 2002); this function of p38 may have been conserved from as far back as yeast. The JNK pathway evolved with multi-cellular organisms such as Drosophila where it regulates cell movement and cell viability during development, in part by phosphorylation of the transcription factor c-Jun.
Updated 23 August, 2011