MicroRNAs (miRs) are short non-coding RNAs that bind complementary sequences in mRNA resulting in translation repression and/or mRNA degradation. We investigated expression of the reported metastasis-associated miRs-335, 206, 135a, 146a, 146b, 10b, 21, let7a and let7b in normal mucosa, non-metastatic and metastatic colorectal cancer (CRC). Expression of target miRs in micro-dissected paraffin embedded tissues was evaluated in 15 primary tumours with adjacent normal tissue from patients that were disease-free at 4 years (cohort A) and 19 paired primary tumours with corresponding liver metastases (cohort B) by quantitative real-time PCR. Increased expression of miR-21, mir-135a and miR-335 was associated with clinical progression of CRC, while miR-206 demonstrated an opposite trend. The levels of mir-21 did not associate with the expression of PTEN, an important tumour suppressor in CRC and one of many putative targets of miR-21, but interestingly was associated with stage of disease in the PTEN expressing tumours. Surprisingly, let7a, a KRAS-targeting miR, showed elevated expression in metastatic disease compared to normal mucosa or non-metastatic disease, and only in KRAS mutation positive tumors. Finally, a prognostic signature of miR 21,135a, 335, 206 and let-7a for detecting the presence of metastases had a specificity of 87% and sensitivity of 76% for the presence of metastases. In summary, we have shown stage-associated differential expression of five out of nine tested metastasis-associated miRs. We have further found that an analysis of these five miRs expression levels in primary tumors significantly correlates with the presence of metastatic disease, making this a potential clinically useful prognostic tool.

Extracellular signal-regulated kinases (ERKs) are signaling molecules that regulate many cellular processes. We have previously identified an alternatively spliced 46-kDa form of ERK1 that is expressed in rats and mice and named ERK1b. Here we report that the same splicing event in humans and monkeys causes, due to sequence differences in the inserted introns, the production of an ERK isoform that migrates together with the 42-kDa ERK2. Because of the differences of this isoform from ERK1b, we named it ERK1c. We found that its expression levels are about 10% of ERK1. ERK1c seems to be expressed in a wide variety of tissues and cells. Its activation by MEKs and inactivation by phosphatases are slower than those of ERK1, which is probably the reason for its differential regulation in response to extracellular stimuli. Unlike ERK1, ERK1c undergoes monoubiquitination, which is increased with elevated cell density concomitantly with accumulation of ERK1c in the Golgi apparatus. Elevated cell density also causes enhanced Golgi fragmentation, which is facilitated by overexpression of native ERK1c and is prevented by dominant-negative ERK1c, indicating that ERK1c mediates cell density-induced Golgi fragmentation. The differential regulation of ERK1c extends the signaling specificity of MEKs after stimulation by various extracellular stimuli.Extracellular signal-regulated kinases (ERKs) 1 and 2 (ERK1 and ERK2, which are 44 and 42 kDa, respectively) are key signaling enzymes that are activated by a large number of extracellular stimuli and play an important role in physiological processes, such as proliferation, differentiation, and development (28,32,40). ERKs are ubiquitously expressed and share a high degree of similarity (85% [5]). Under most conditions, ERK1 and ERK2 demonstrate identical patterns of regulation, which is mainly manifested in their activation by MEK1 and MEK2 (MEKs) through the phosphorylation of threonine and tyrosine in their characteristic Thr-Glu-Tyr motif. These two isoforms also share substrate recognition and subcellular localization, suggesting a redundant functionality of these ERKs (34). Consistent with these observations, ERK1 knockout mice are viable, without any apparent defects, which might be due to increased levels of ERK2 expression that compensate for the lack of ERK1 (24). On the other hand, ERK2 knockout mice die as embryos, due to failure in mesoderm induction, which is not compensated for by ERK1 that is not expressed at this stage of embryonic development (45). The ability of the ERKs to transmit different and even opposing signals despite the pronounced similarities between the two ERK isoforms raises the question of how is the specificity of the different signals regulated.It has previously been demonstrated that signaling specificity is determined by duration and strength of the signals (23) as well as compartmentalization (29,39). Another mechanism that contributes to specificity is the extensive cross talk and interplay between the ERK cascade and other intracellular signal...

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