High Erk activity suppresses expression of the cell cycle inhibitor p27Kip1 in colorectal cancer cells
© Kress et al; licensee BioMed Central Ltd. 2010
Received: 31 December 2009
Accepted: 2 February 2010
Published: 2 February 2010
The molecular heterogeneity of human cancer cells at the level of signaling protein activities remains poorly understood. Using a panel of 64 colorectal (CRC) cancer cell lines the activity status of the MAP kinases Erk1 and Erk2 was investigated. Erk1/2 activity varied greatly within the CRC cell line panel and was not detectably associated with the speed of cell growth in 10 CRC lines analyzed. As expected, mutations in K-Ras or B-Raf were often, albeit not always, linked to high Erk1/2 activity. The phosphorylation of several known Erk1/2 targets investigated did not generally reflect Erk1/2 activity in the 10 CRC lines analyzed. However, the reduction of Erk1/2 activity with MEK inhibitors generally abolished cell growth but only led to an increase of cellular p27Kip1 levels in CRC cells with high Erk1/2 activity levels. The results indicate that high Erk1/2 activation is utilized by some CRC lines to override the cell cycle brake p27Kip1, while others presumably rely on different mechanisms in order to inactivate this important cell cycle brake. Such detailed knowledge of the molecular diversity of cancer cell signaling mechanisms may eventually help to develop molecularly targeted, patient-specific therapeutic strategies and treatments.
The limited knowledge about the heterogeneity of cancers on the signaling protein activity level is a major obstacle for better, individualized cancer therapies with signal transduction-modulating drugs. It is now well feasible to comprehensively analyze mutations and mRNA expression changes in tumor biopsies and isolated tumor cells with high-throughput techniques. By contrast, in-depth biochemical analyses of signaling protein activities are currently all but impossible with patient biopsy material. However, important insight into the individual diversity of cancers can be gained by analyzing large panels of cancer cells from a specific tumor type [1–3].
Erk1 and 2 are multifunctional kinases which are employed in a very wide range of normal and pathological cell types, in many cases in order to regulate cell proliferation or differentiation [4–6]. However, these Erks also play, for example, a role in the trans-endothelial migration of some CRC cells  and can promote angiogenesis and invasion [8, 9]. The most studied signaling cascade engaging Erk1/2 is the Ras - Raf - MEK - Erk pathway that is transmitting the signals of numerous cell surface receptors. In many tumors, including CRC, Erk activation is linked to mutations of Ras GTPases or the S/T kinase B-Raf [10, 11]. By contrast, cancer-related mutations in MEK1/2 and Erk1/2 appear to be very rare, although different germline mutations in MEKs have been recently reported in human cardio-facio-cutaneous disorders .
The total Erk1/2 levels are similar in all 10 cell lines. Unexpectedly, the apparent activity of MEK1/2, analyzed by western blotting with a pMEK1/2 (pS217/pS221) antibody, did not correlate well with Erk1/2 activity (data not shown). We are at present unable to provide a molecular explanation for this, but a potential reason could again be the impact of Erk phosphatases, such as those of the MKP family.
In order to address the latter possibility, selected Erk substrates and targets, including Elk1, Msk1, Myc and p90Rsk, were analyzed with phosphoepitope specific antibodies in the 10 CRC lines. It is not possible to perform a comprehensive analysis at this point, since more than 160 substrates and targets of Erk have been reported so far  and phospho-specific antibodies are not yet available for many of these targets. In all cases initially tested we failed to detect a perfect correlation between Erk activation and phospho-levels of potential target proteins (data not shown), suggesting to us that probably none of these proteins is an essential substrate for all CRC lines with high Erk1/2 activity. Clearly, this does not preclude a functionally important role of the phosphorylation of these proteins by Erk1/2 in individual cases.
p27Kip1 is an important cell cycle regulatory protein previously discussed as a direct or indirect target of Erk1/2, with phosphorylation leading to its proteasomal destruction [14–16]. Therefore, elevations of p27Kip1 phospho-levels may be difficult to monitor in steady state, but the inhibition of Erk1/2 may lead to a change in p27Kip1 abundance which would be expected to be easily detectable.
The simplest explanation for these findings is that a subset of CRC cells utilizes the strong activation of Erk1/2 to down-regulate p27Kip1 expression. This may be mediated by targeting the Thr187 residue on p27Kip1, which is linked to its ubiquitinylation and degradation [14–16].
Despite these interesting results, we can, of course, by no means exclude the possibility that high Erk1/2 activity is also required to phosphorylate other key substrates in this subset of 'high Erk' CRC cells.
Erk1 and Erk2 boast a plethora of known substrates  and very likely more remain to be detected. In many cases, the subcellular distribution and expression levels of both, Erk kinases and their potential substrates, could determine which substrates are actually becoming phosphorylated in a specific cell or disease context. What is a critical Erk substrate in each individual cancer may thus depend greatly on the specific genetic composition of that particular tumor.
Nevertheless, from our study it would appear that down-regulation of p27Kip1 expression is at least a common, if not ubiquitous occurrence in CRC cells with high Erk1/2 activity. Similar findings have been reported in other tumor types . In some genetic contexts, however, p27Kip1 may not need to be inactivated for tumors to develop, and could possibly even take on oncogenic functions according to recent results [18, 19].
Interestingly, the absolute expression levels of p27Kip1 do not correlate with Erk activity levels (Additional file 3). Several reasons could explain this finding. Firstly, p27Kip1 may be differentially localized in different CRC cells, allowing in some cases only a portion of p27Kip1 to act as a cell cycle break that requires counter-action by high Erk activity. Secondly, the individual variability of the overall genetic composition of each tumor cells could lead to distinct levels of p27Kip1 being tolerated before a prominent effect on the cell cycle machinery is elicited.
It is presently unclear whether p27Kip1 is commonly a direct Erk target in CRC cells with high Erk activity, although phosphorylation of p27Kip1 by Erk1/2 in CRC has been suggested and complex formation of endogenous p27Kip1 and Erk1/2 was detected in LoVo cells by co-immunoprecipitation . Indirect mechanisms of p27Kip1 regulation by Erk have also been reported .
p27Kip1 downregulation through high Erk activity levels is only one molecular route to eliminate the normal function of this important cell cycle regulator [22, 23]. Many CRC cells of the analyzed panel with low Erk1/2 activity have presumably found alternative means to accomplish this task. This does, of course, not imply that those cells do not require Erk activity; in fact, as discussed above, inhibiting Erks close allies, MEK1/2, clearly reduces Erk activity and inhibits proliferation in these cells (data not shown).
We also observed that U0126 treatment of CRC cells led to marked morphology changes in some of the cell lines (see Additional file 4) further supporting multiple functional roles of MEK1/2 and, presumably, Erk1/2.
The results reported here imply that different Erk signal intensities are used in CRC cells to accomplish distinct molecular tasks, an observation that was also made in a previous study analyzing the activity of Src family kinases in this cell line panel . Understanding the importance of different signal strengths in individual cancers is not only of interest to learn more about these still poorly grasped diseases, it may eventually also impact on the therapeutic use of signal transduction-modulating drugs. In the case of p27Kip1, which is not usually mutated in cancers, the inhibition of its proteasomal degradation could become a useful therapeutic option for some CRC patients, including those with constitutively high Erk activity.
List of Abbreviations
Extracellular signal-regulated kinase
MAP kinase (MAPK) or Erk kinase
a MEK inhibitor compound.
We are grateful to Walter Bodmer and many other colleagues for providing cell lines, and to Cancer Research UK, Heads Up and Oxford University for financial support. TK was kindly supported by the Bayerische Forschungsstiftung (Munich, Germany).
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