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The serine protease inhibitor serpinE2 is a novel target of ERK signaling involved in human colorectal tumorigenesis.

Bergeron S, Lemieux E, Durand V, Cagnol S, Carrier JC, Lussier JG, Boucher MJ, Rivard N - Mol. Cancer (2010)

Bottom Line: Among the most harmful of all genetic abnormalities that appear in colorectal cancer (CRC) development are mutations of KRAS and its downstream effector BRAF as they result in abnormal extracellular signal-related kinase (ERK) signaling.Our data indicate that serpinE2 is up-regulated by oncogenic activation of Ras, BRAF and MEK1 and contributes to pro-neoplastic actions of ERK signaling in intestinal epithelial cells.Hence, serpinE2 may be a potential therapeutic target for colorectal cancer treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cellular Biology, CIHR Team on Digestive Epithelium, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada.

ABSTRACT

Background: Among the most harmful of all genetic abnormalities that appear in colorectal cancer (CRC) development are mutations of KRAS and its downstream effector BRAF as they result in abnormal extracellular signal-related kinase (ERK) signaling. In a previous report, we had shown that expression of a constitutive active mutant of MEK1 (caMEK) in normal rat intestinal epithelial cells (IECs) induced morphological transformation associated with epithelial to mesenchymal transition, growth in soft agar, invasion and metastases in nude mice. Results from microarrays comparing control to caMEK-expressing IECs identified the gene encoding for serpinE2, a serine protease inhibitor, as a potential target of activated MEK1.

Results: 1- RT-PCR and western blot analyses confirmed the strong up-regulation of serpinE2 expression and secretion by IECs expressing oncogenic MEK, Ras or BRAF. 2- Interestingly, serpinE2 mRNA and protein were also markedly enhanced in human CRC cells exhibiting mutation in KRAS and BRAF. 3- RNAi directed against serpinE2 in caMEK-transformed rat IECs or in human CRC cell lines HCT116 and LoVo markedly decreased foci formation, anchorage-independent growth in soft agarose, cell migration and tumor formation in nude mice. 4- Treatment of CRC cell lines with U0126 markedly reduced serpinE2 mRNA levels, indicating that expression of serpinE2 is likely dependent of ERK activity. 5- Finally, Q-PCR analyses demonstrated that mRNA levels of serpinE2 were markedly increased in human adenomas in comparison to healthy adjacent tissues and in colorectal tumors, regardless of tumor stage and grade.

Conclusions: Our data indicate that serpinE2 is up-regulated by oncogenic activation of Ras, BRAF and MEK1 and contributes to pro-neoplastic actions of ERK signaling in intestinal epithelial cells. Hence, serpinE2 may be a potential therapeutic target for colorectal cancer treatment.

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SerpinE2 silencing in caMEK-expressing IECs decreases foci formation, growth in soft agar and migration. A- caMEK-expressing IECs were stably infected with lentiviruses encoding for a control shRNA (scrambled sequence) or encoding a serpinE2-specific shRNA. These stable cell populations were thereafter lysed at confluence and equal amounts of concentrated culture medium were analyzed by Western blotting with specific antibodies against serpinE2. The graph illustrates densitometric analysis performed with the western blot data shown on the left to determine the % of serpinE2 downregulation. The level of serpinE2 observed in shScrambled cells was set at 100%. A representative experiment of three independent experiments is shown. B- wtMEK/IEC-6 or caMEK/IEC-6 cells expressing either shScrambled or shSerpinE2 were seeded in a 6-well plate at 100 000 cells per well. Cells were harvested and counted. Values are means of 4 experiments ± SE. C and D- caMEK-transformed cells expressing or not shSerpinE2 were seeded on parental IEC-6 cell monolayer during 15 days. Thereafter, the cells were stained with crystal violet and images of colonies were acquired under light microscopy. The size of the foci was calculated using Image J software and expressed as % of shScrambled cells (control) which was set at 100%. E- Cells were cultured in soft agarose for 3 weeks before MTT. The number of colonies was determined using Image J software and expressed as % of shSrcambled cells (control) set at 100%. F- Cell migration to the undersurface of the polycarbonate membrane of Boyden chambers coated with fibronectin (FN) or vitronectin (VN) was evaluated 24 h after seeding, in presence of 20 mM hydroxyurea. Values were expressed as a % of shScrambled cells migrating on fibronectin. *, significantly different from shScrambled cells at p < 0.05 (Student's t test).
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Figure 2: SerpinE2 silencing in caMEK-expressing IECs decreases foci formation, growth in soft agar and migration. A- caMEK-expressing IECs were stably infected with lentiviruses encoding for a control shRNA (scrambled sequence) or encoding a serpinE2-specific shRNA. These stable cell populations were thereafter lysed at confluence and equal amounts of concentrated culture medium were analyzed by Western blotting with specific antibodies against serpinE2. The graph illustrates densitometric analysis performed with the western blot data shown on the left to determine the % of serpinE2 downregulation. The level of serpinE2 observed in shScrambled cells was set at 100%. A representative experiment of three independent experiments is shown. B- wtMEK/IEC-6 or caMEK/IEC-6 cells expressing either shScrambled or shSerpinE2 were seeded in a 6-well plate at 100 000 cells per well. Cells were harvested and counted. Values are means of 4 experiments ± SE. C and D- caMEK-transformed cells expressing or not shSerpinE2 were seeded on parental IEC-6 cell monolayer during 15 days. Thereafter, the cells were stained with crystal violet and images of colonies were acquired under light microscopy. The size of the foci was calculated using Image J software and expressed as % of shScrambled cells (control) which was set at 100%. E- Cells were cultured in soft agarose for 3 weeks before MTT. The number of colonies was determined using Image J software and expressed as % of shSrcambled cells (control) set at 100%. F- Cell migration to the undersurface of the polycarbonate membrane of Boyden chambers coated with fibronectin (FN) or vitronectin (VN) was evaluated 24 h after seeding, in presence of 20 mM hydroxyurea. Values were expressed as a % of shScrambled cells migrating on fibronectin. *, significantly different from shScrambled cells at p < 0.05 (Student's t test).

Mentions: In order to determine the contribution of serpinE2 in intestinal transformation induced by activated MEK, foci from post-confluent caMEK-expressing IECs were retrieved by aspiration with a pipette and pooled as one caMEK-expressing cell population. All further experiments were performed with this previously characterized caMEK-expressing IEC population [14] and compared with wtMEK-expressing cell populations. Recombinant lentiviruses encoding anti-serpinE2 short hairpin RNA (shRNA) were therefore developed to stably suppress serpinE2 levels in these cells. Several lentiviral constructs were generated and tested for their ability to knock down serpinE2 protein. One of these viral shRNAs was selected and designated as shSerpinE2. caMEK-expressing cells were henceforth infected with shSerpinE2 lentiviruses or with lentiviruses expressing a control shRNA (shScrambled). Secretion of serpinE2 protein was analyzed 14 days after selection with blasticidin S in these populations. As shown in Figure 2A, secreted serpinE2 levels were markedly reduced (> 60%) in cells-expressing shSerpinE2; in contrast, shScrambled had no effect on the secretion of serpinE2 (data not shown).


The serine protease inhibitor serpinE2 is a novel target of ERK signaling involved in human colorectal tumorigenesis.

Bergeron S, Lemieux E, Durand V, Cagnol S, Carrier JC, Lussier JG, Boucher MJ, Rivard N - Mol. Cancer (2010)

SerpinE2 silencing in caMEK-expressing IECs decreases foci formation, growth in soft agar and migration. A- caMEK-expressing IECs were stably infected with lentiviruses encoding for a control shRNA (scrambled sequence) or encoding a serpinE2-specific shRNA. These stable cell populations were thereafter lysed at confluence and equal amounts of concentrated culture medium were analyzed by Western blotting with specific antibodies against serpinE2. The graph illustrates densitometric analysis performed with the western blot data shown on the left to determine the % of serpinE2 downregulation. The level of serpinE2 observed in shScrambled cells was set at 100%. A representative experiment of three independent experiments is shown. B- wtMEK/IEC-6 or caMEK/IEC-6 cells expressing either shScrambled or shSerpinE2 were seeded in a 6-well plate at 100 000 cells per well. Cells were harvested and counted. Values are means of 4 experiments ± SE. C and D- caMEK-transformed cells expressing or not shSerpinE2 were seeded on parental IEC-6 cell monolayer during 15 days. Thereafter, the cells were stained with crystal violet and images of colonies were acquired under light microscopy. The size of the foci was calculated using Image J software and expressed as % of shScrambled cells (control) which was set at 100%. E- Cells were cultured in soft agarose for 3 weeks before MTT. The number of colonies was determined using Image J software and expressed as % of shSrcambled cells (control) set at 100%. F- Cell migration to the undersurface of the polycarbonate membrane of Boyden chambers coated with fibronectin (FN) or vitronectin (VN) was evaluated 24 h after seeding, in presence of 20 mM hydroxyurea. Values were expressed as a % of shScrambled cells migrating on fibronectin. *, significantly different from shScrambled cells at p < 0.05 (Student's t test).
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Figure 2: SerpinE2 silencing in caMEK-expressing IECs decreases foci formation, growth in soft agar and migration. A- caMEK-expressing IECs were stably infected with lentiviruses encoding for a control shRNA (scrambled sequence) or encoding a serpinE2-specific shRNA. These stable cell populations were thereafter lysed at confluence and equal amounts of concentrated culture medium were analyzed by Western blotting with specific antibodies against serpinE2. The graph illustrates densitometric analysis performed with the western blot data shown on the left to determine the % of serpinE2 downregulation. The level of serpinE2 observed in shScrambled cells was set at 100%. A representative experiment of three independent experiments is shown. B- wtMEK/IEC-6 or caMEK/IEC-6 cells expressing either shScrambled or shSerpinE2 were seeded in a 6-well plate at 100 000 cells per well. Cells were harvested and counted. Values are means of 4 experiments ± SE. C and D- caMEK-transformed cells expressing or not shSerpinE2 were seeded on parental IEC-6 cell monolayer during 15 days. Thereafter, the cells were stained with crystal violet and images of colonies were acquired under light microscopy. The size of the foci was calculated using Image J software and expressed as % of shScrambled cells (control) which was set at 100%. E- Cells were cultured in soft agarose for 3 weeks before MTT. The number of colonies was determined using Image J software and expressed as % of shSrcambled cells (control) set at 100%. F- Cell migration to the undersurface of the polycarbonate membrane of Boyden chambers coated with fibronectin (FN) or vitronectin (VN) was evaluated 24 h after seeding, in presence of 20 mM hydroxyurea. Values were expressed as a % of shScrambled cells migrating on fibronectin. *, significantly different from shScrambled cells at p < 0.05 (Student's t test).
Mentions: In order to determine the contribution of serpinE2 in intestinal transformation induced by activated MEK, foci from post-confluent caMEK-expressing IECs were retrieved by aspiration with a pipette and pooled as one caMEK-expressing cell population. All further experiments were performed with this previously characterized caMEK-expressing IEC population [14] and compared with wtMEK-expressing cell populations. Recombinant lentiviruses encoding anti-serpinE2 short hairpin RNA (shRNA) were therefore developed to stably suppress serpinE2 levels in these cells. Several lentiviral constructs were generated and tested for their ability to knock down serpinE2 protein. One of these viral shRNAs was selected and designated as shSerpinE2. caMEK-expressing cells were henceforth infected with shSerpinE2 lentiviruses or with lentiviruses expressing a control shRNA (shScrambled). Secretion of serpinE2 protein was analyzed 14 days after selection with blasticidin S in these populations. As shown in Figure 2A, secreted serpinE2 levels were markedly reduced (> 60%) in cells-expressing shSerpinE2; in contrast, shScrambled had no effect on the secretion of serpinE2 (data not shown).

Bottom Line: Among the most harmful of all genetic abnormalities that appear in colorectal cancer (CRC) development are mutations of KRAS and its downstream effector BRAF as they result in abnormal extracellular signal-related kinase (ERK) signaling.Our data indicate that serpinE2 is up-regulated by oncogenic activation of Ras, BRAF and MEK1 and contributes to pro-neoplastic actions of ERK signaling in intestinal epithelial cells.Hence, serpinE2 may be a potential therapeutic target for colorectal cancer treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cellular Biology, CIHR Team on Digestive Epithelium, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada.

ABSTRACT

Background: Among the most harmful of all genetic abnormalities that appear in colorectal cancer (CRC) development are mutations of KRAS and its downstream effector BRAF as they result in abnormal extracellular signal-related kinase (ERK) signaling. In a previous report, we had shown that expression of a constitutive active mutant of MEK1 (caMEK) in normal rat intestinal epithelial cells (IECs) induced morphological transformation associated with epithelial to mesenchymal transition, growth in soft agar, invasion and metastases in nude mice. Results from microarrays comparing control to caMEK-expressing IECs identified the gene encoding for serpinE2, a serine protease inhibitor, as a potential target of activated MEK1.

Results: 1- RT-PCR and western blot analyses confirmed the strong up-regulation of serpinE2 expression and secretion by IECs expressing oncogenic MEK, Ras or BRAF. 2- Interestingly, serpinE2 mRNA and protein were also markedly enhanced in human CRC cells exhibiting mutation in KRAS and BRAF. 3- RNAi directed against serpinE2 in caMEK-transformed rat IECs or in human CRC cell lines HCT116 and LoVo markedly decreased foci formation, anchorage-independent growth in soft agarose, cell migration and tumor formation in nude mice. 4- Treatment of CRC cell lines with U0126 markedly reduced serpinE2 mRNA levels, indicating that expression of serpinE2 is likely dependent of ERK activity. 5- Finally, Q-PCR analyses demonstrated that mRNA levels of serpinE2 were markedly increased in human adenomas in comparison to healthy adjacent tissues and in colorectal tumors, regardless of tumor stage and grade.

Conclusions: Our data indicate that serpinE2 is up-regulated by oncogenic activation of Ras, BRAF and MEK1 and contributes to pro-neoplastic actions of ERK signaling in intestinal epithelial cells. Hence, serpinE2 may be a potential therapeutic target for colorectal cancer treatment.

Show MeSH
Related in: MedlinePlus