Colorectal Carcinogenesis: Connecting K-RAS-Induced Transformation and CREB Activity In Vitro and In Vivo.
Oncogenic transformation is often associated with an increased expression of the cAMP response element binding (CREB) transcription factor controlling the expression of genes involved in cell proliferation, cell cycle, apoptosis, and tumor development, but a link between K-RAS(V12)-induced transformation and CREB has not yet been determined. Therefore, the constitutive and/or inhibitor-regulated mRNA and protein expression of CREB and signal transduction components and growth properties of parental fibroblasts, K-RAS(V12)-transformed counterparts, shCREB K-RAS(V12) transfectants and human colon carcinoma cells were determined. Increased CREB transcript and protein levels accompanied by an enhanced CREB activity was detected in K-RAS(V12)-transformed murine fibroblasts and K-RAS(V12)-mutated human tumor cells, which is dependent on the MAPK/MEK, PI3K, and/or PKC signal transduction. Immunohistochemical (IHC) staining of colorectal carcinoma lesions and murine tumors, with known KRAS gene mutation status, using antibodies specific for CREB and phospho-CREB, revealed a mechanistic link between CREB expression and K-RAS(V12)-mutated colorectal carcinoma lesions when compared with control tissues. Silencing of CREB by shRNA and/or treatment with a CREB inhibitor (KG-501) reverted the neoplastic phenotype of K-RAS(V12) transformants as demonstrated by a more fibroblast-like morphology, enhanced apoptosis sensitivity, increased doubling time, decreased migration, invasion and anchorage-independent growth, reduced tumorigenesis, and enhanced immunogenicity in vivo. The impaired shCREB-mediated invasion of K-RAS(V12) transformants was accompanied by a transcriptional downregulation of different matrix metalloproteinases (MMP) coupled with their reduced enzymatic activity.


CONCLUSIONS
CREB plays a key role in the K-RAS(V12)-mediated neoplastic phenotype and represents a suitable therapeutic target for murine and human K-RAS(V12)-induced tumors.
Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
André
A
Steven
Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Max
M
Heiduk
Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Christian V
CV
Recktenwald
Center for Medical Research, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Bernhard
B
Hiebl
Institute of Pathology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Claudia
C
Wickenhauser
Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Chiara
C
Massa
Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany. barbara.seliger@uk-halle.de.
Barbara
B
Seliger
D000818:Animals; D017209:Apoptosis; D002277:Carcinoma / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D011905:Genes, ras; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D009361:Neoplasm Invasiveness; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D018631:ras Proteins / Q000378:metabolism*
C115163:CREB1 protein, human; C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018631:ras Proteins