We and others have recently reported that expression of a constitutively active mutant of MEK1 in normal intestinal epithelial cells is sufficient to induce growth factor relaxation for DNA synthesis, morphological transformation, growth in soft agar, epithelial to mesenchymal transition and to promote tumor invasion and metastasis [3, 4, 14, 15]. Thus, these data argue that a key role of sustained MEK activity resulting from the constitutive activation of KRAS or BRAF in colorectal carcinoma cells may be to provide signals inducing not only proliferation, but also transformation and tumorigenesis. However, in spite of the obvious role of MEK/ERK kinases in the induction and regulation of intestinal epithelial cell tumorigenesis, little is known as to the molecular mechanisms by which this signaling achieves such functions. In the present study, we show that serpinE2 gene is a MEK1 target in intestinal epithelial cells and that serpinE2 expression and secretion correlate with both MEK1 activity and intestinal epithelial cell transformation. Moreover, targeting of serpinE2 by mRNAi in human colorectal cancer cell lines decreased anchorage independent growth, migration, invasion as well as tumor formation in nude mice. Accordingly, we found an upregulation of serpinE2 mRNA levels in human adenomas and colorectal cancer tissues as compared to corresponding normal tissues.
Oncogenic mutations in KRAS or BRAF occur frequently in colorectal cancer and aberrant signaling through the ERK pathway has been correlated with both initiation  and progression  of CRC. Interestingly, KRAS and BRAF mutations seem to be mutually exclusive [39, 40], suggesting that they may have similar functions. These oncogenes primarily signal through the MEK/ERK pathway [41, 42]. Upon phosphorylation by MEK1/2, ERK1/2 translocate to the nucleus and phosphorylate various transcription factors regulating gene expression . Therefore, in order to define the genetic changes induced by persistent MEK activation, we and others [4, 15] have utilized oligonucleotide microarrays to determine which genes are regulated following the constitutive activation of MEK in normal intestinal epithelial cells. Our results revealed that serpinE2 gene was the gene mostly induced by activated MEK in intestinal epithelial cells. This observed altered level of expression of serpinE2 transcript was also noted in microarray analyses performed by Voisin and colleagues . In the present study, we were able to confirm that RAS-, BRAF- and caMEK-transformed intestinal epithelial cells express and secrete serpinE2. Furthermore, serpinE2 expression was rapidly enhanced (in 1 h) upon induction of oncogenic BRAF in normal intestinal epithelial cells, suggesting an early involvement of this protein in cell transformation. Of note, expression of serpinE2 in human colorectal cancer cell lines was shown to be dependent, at least in part, of endogenous activities of MEK/ERK. Other oncogenic pathways have been previously associated with induction of serpinE2 expression. Indeed, the very oncogenic receptor tyrosine kinase MET was also shown to promote serpinE2 gene expression in a xenograft colon tumor model . Additionally, PTEN deletion has been reported to up-regulate serpinE2 expression in MEF cells  and serpinE2 was shown to be overexpressed in cells transformed by adenovirus type 12 . Taken together, these results indicate that serpinE2 gene expression could be induced by different oncogenic pathways, emphasizing that this protein may be important in tumorigenesis.
Our results also led to the demonstration that serpinE2 contributes to transformation induced by activated MEK1 and to human colorectal carcinoma cell growth and migration. In agreement with the present study, data on serpinE2 expression in human cancer indicate that serpinE2 levels are elevated in pancreatic tumors , breast tumors , liposarcomas  and oral squamous carcinomas . Accordingly, we found a significantly higher level of serpinE2 mRNA when comparing affected tissues from advanced adenomas and carcinomas to adjacent healthy tissues. These results are in agreement with the study of Selzer-Plon et al. who recently reported that serpinE2 mRNA levels increase both at the transition between normal tissue and adenomas with mild/moderate dysplasia and again at the transition between severe dysplasia and colorectal cancer . In addition, no significant difference was observed when comparing serpinE2 mRNA levels in primary cancers classified into different TNM stages. Taken together, the above results suggest that enhanced serpinE2 expression may be implicated in tumor progression in colorectal tissue.
Although there is some evidence in the literature suggesting that serpinE2 may play a role in carcinogenesis, the precise function of this serpin in cancer still remains elusive. Through its ability to reduce proteolysis, this serine protease inhibitor is predicted to impair extracellular matrix degradation and consequently cancer cell invasion and metastasis. However, overexpression of serpinE2 appears to enhance the invasive potential of pancreatic tumors in xenograft models . Recently, using mammary tumor models, it has been reported that serpinE2 stimulates metastatic spread of mammary tumors . In addition, an analysis of 126 breast cancer patients revealed that patients with breast tumors showing elevated serpinE2 levels also had a significantly higher probability of developing lung metastasis . Finally, serpinE2 has recently been shown to promote lymph node metastasis in a testicular cancer model . Thus, increased function of serpinE2 appears to be associated with enhanced migration and metastasis. However, the biological roles of serpinE2 in colorectal carcinoma have never been studied. Herein, the present results show that endogenous expression of serpinE2 in rodent transformed intestinal epithelial cells and human CRC cells is correlated with enhanced cell migration and invasion abilities. The molecular mechanism by which serpinE2 modulates motility remains unknown. It is possible that serpinE2 may enhance signaling cascades mediating motility. In this regard, serpinE2 has recently been reported to stimulate ERK signaling by binding LRP-1  or syndecan-1 . However, preliminary results (data not shown) indicate that the phosphorylated levels of Akt and ERK1/2 were not affected following serpinE2 depletion in colon carcinoma cells. Alternatively, shSerpinE2-expressing cells may have a reduced migratory capacity which could result from a defect in cell adhesion. Indeed, typical cell movement across a two-dimensional substrate can be divided into three concerted steps: membrane protrusion, cell traction, deadhesion and tail retraction. Adhesion at the leading edge and deadhesion at the rear portion of cells are required for protrusion and tail retraction, respectively . As cellular migration and cellular adhesion are intimately related, changes in one could be expected to result in changes in the other. Binding of type-1 plasminogen activator inhibitor (PAI-1), the phylogenetically closest relative of serpinE2, to cell surface uPA promotes inactivation and internalization of adhesion receptors (e.g. urokinase receptor and integrins) and leads to cell detachment from a variety of extracellular matrixes . Recently, serpinE2 has been shown to also induce cell detachment from a variety of extracellular matrix proteins such as vitronectin, fibronectin and type-1 collagen in an uPA/uPAR-dependent manner . Interestingly, serpinE2 has been reported to co-localize with fibronectin  and to interact with vitronectin . Accordingly, we observed herein that the downregulation of serpinE2 significantly delayed colorectal carcinoma cell detachment after trypsinization, suggesting that serpinE2 expression does decrease adhesion and promote detachment of colorectal carcinoma cells. Moreover, we have recently demonstrated that uPA expression levels are enhanced in MEK1-transformed intestinal epithelial cells . Further experiments are hence necessary to clearly identify the molecular mechanisms involved in the deadhesive effects of serpinE2.