Biological Behaviors And Cx43 Expression Of Fibroblasts During CRC Cell-fibroblast Interactions

Colorectal cancer (CRC) is a common malignant digestive system tumor. Report by the American Cancer Society in 2014 shows that the morbidity and mortality of colorectal cancer still ranks the third in the world. Although great progression has been achieved, problems such as metastasis, recurrence and drug resistance of CRC remain unsolved and the underlying mechanism is still unclear. Therefore, researches on how colorectal cancer regulated are necessary and meaningful for clinical therapy.Tumor stroma plays an important role in tumor development. Studies show that fibroblasts in tumor stroma can regulate proliferation, migration, invasion and angiogenesis of tumor cells which are also involved in tumor metastasis and recurrence. The activation from fibroblasts to cancer associated fibroblasts has been regarded as a key process for the effects of fibroblasts on tumor. Connexin 43 (Cx43) which is one of the gap junction proteins, plays critical roles on regulating cell-cell communication. Our previous study by immunohistochemistry found that the expression of Cx43 was abnormally increased on the infiltration front of colorectal cancer tissues. Direct co-culture assay by two-layer cell spheroid model and monolayer co-culture model showed that Cx43 was up-regulated on the contact interface of CRC cells and fibroblasts along with the activation of fibroblasts. Further more, fibroblasts were activated when cultured within CRC cell culture supernatants, as TGF-β and Wnt signaling in these fibroblasts were also activated. Indirect co-culture assay by double layer co-culture model showed that when co-cultured with CRC cells, fibroblasts could be activated. And activated fibroblasts could activate Wnt signaling and up-regulate Cx43 expression in CRC cells. However, Cx43 expression and behaviors of fibroblasts as well as the underlying mechanism during CRC cell-fibroblast interactions are unclarified, which need more investigations now.To confirm our suppose, HELFs were co-cultured with CRC cells in vitro to detect the effects of CRC cell-fibroblast interactions on fibroblasts’ behaviors. Our results showed that CRC cell-fibroblast interactions induced fibroblast activation and promoted its proliferation and migration. The expression of Cx43 and Wnt signaling related proteins Dvl3 and TCF4 in fibroblasts were up-regulated, and nuclear P-catenin was increased. The up-regulation of Cx43 and α-SMA were partially cancelled by Wnt signaling inhibitor DKK1. TGF β1 promoted the activation of Wnt signaling, up-regulation of Cx43 and activation of fibroblasts. However, the up-regulation of Cx43 in activated fibroblasts does not effect the GJIC level of fibroblasts. As Cx43 is reported to be a target gene of Wnt signaling, we speculate that CRC cell-fibroblast interactions induce activation, proliferation and migration of fibroblasts, which may be contributed to the up-regulation of Cx43 expression and Wnt signaling pathway.From the above findings, we can get the following conclusions:1. CRC cell-fibroblast interactions promote the activation, proliferation and migration of fibroblasts.2. CRC cell-fibroblast interactions promoting the up-regulation of Cx43 and activation of Wnt signaling in fibroblasts.3. Wnt signaling induce the up-regulation of Cx43 and activation of fibroblasts.4. TGFβ1 promoting the activation of Wnt signaling, up-regulation of Cx43 and activation of fibroblasts.5. The up-regulation of Cx43 in activated fibroblasts does not effect GJIC level of fibroblasts, which suggests that Cx43 may work in a gap junction independent way.