Cell-cell contacts induce STAT3 activity in colon carcinoma cells through an autocrine stimulation loop.
OBJECTIVE
Signal transducer and activator of transcription 3 (STAT3) is persistently activated in colorectal carcinoma (CRC) cells in the tumor context, but inactive in cultivated CRC cell lines. We approached mechanisms leading to STAT3 activation in CRC by mimicking "tumor-like" growth conditions and forcing cell-cell contacts of HT-29 CRC cells in culture. Further aims were to analyze in how far HT-29 cells growing in a tumorous manner spread STAT3 activity by secretion of soluble factors.


METHODS
Non-adhesive growth and aggregation of HT-29 cells were achieved by cultivation on non-coated plastic surfaces. STAT3 activity was assessed by Western blot employing a phospho-STAT3-specific antibody as well as by electrophoretic mobility shift assay (EMSA). Expression changes of STAT3 target gene mmp-1 were quantified by real-time RT-PCR, Cytokine/chemokine patterns in conditioned media were characterized by cytokine arrays.


RESULTS
Forced aggregation and non-adhesive growth of HT-29 CRC cells resulted in enhanced tyrosine phosphorylation of STAT3 and elevated expression of matrix metalloproteinase MMP-1. Furthermore, an activity was secreted into the medium that evoked STAT3 phosphorylation in adhesively growing HT-29 cells. The degree of activity in the conditioned medium was enhanced when wild-type STAT3 was overexpressed and reduced when a dominant variant of STAT3 was expressed in HT-29 cells. A characteristic panel of chemokines appeared in STAT3-activating conditioned medium.


CONCLUSIONS
Changing cultivation conditions of the CRC cell line HT-29 toward detachment and aggregation, thus toward the situation in tumors, induces STAT3 activity and evokes an autocrine STAT3 activation loop.
Tsareva
Svetlana A
SA
Institute of Biochemistry II, Jena University Hospital, Nonnenplan 2, 07743, Jena, Germany.
Wagner
Stefan
S
Müller
Annekatrin
A
Corvinus
Florian
F
Friedrich
Karlheinz
K
D019898:Autocrine Communication; D002448:Cell Adhesion; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000473:pathology*; D024202:Electrophoretic Mobility Shift Assay; D019073:HT29 Cells; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000235:genetics; D050796:STAT3 Transcription Factor / Q000502:physiology*
D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D020781:Matrix Metalloproteinase 1