## 4 Discussion
Indeed, a positive feedback loop has been described between these two proteins in hepatocellular carcinoma cells, whereby TGF\(\beta\) induces EMMPRIN expression through its effects on _Slug_, and EMMPRIN can enhance TGF\(\beta\) bioavailability through its impact on MMPs that cleave pro-TGF\(\beta\) and also increase TGF\(\beta\) transcription through \(\beta\)-catenin [53; 54]. We suggest that such a loop also exists in CT26 colon carcinoma cells. The process of EMT and its reversal by MET are both necessary for successful metastatic outgrowth. In fact, the expression of the EMT-TFs (e.g., Snail, Slug, Zeb), stem-like markers (e.g., NANOG, SOX2), and dormancy markers (e.g., NR2F1, p21, p27) was shown to be linked, as dormant cells need to arrest their cell cycle and maintain the stem-like properties that allow them to survive for long periods, and then self-renew upon receiving the appropriate signals from the microenvironment [47]. We show here that the CT26-WT cells do not show any significant change in proliferation and in the expression of vimentin, EMT-TFs and dormancy markers when co-cultured with macrophages, although the angiogenic potential was increased in these co-cultured cells. In contrast, the addition of recombinant TGF\(\beta\) or low levels of recombinant EMMPRIN caused an increase in proliferation and in the angiogenic potential, but not in the EMT-TFs and those dormancy markers that we examined. We suggest that these results indicate that while TGF\(\beta\) and EMMPRIN are sufficient in determining the EMT and dormancy status of the cell, other pathways, perhaps the PI3K/Akt pathway [55], need to cooperate to determine the state of proliferation and angiogenesis. The lack of change in most of the EMT and dormancy markers upon the addition of TGF\(\beta\), or low levels of EMMPRIN in the CT26-WT cells, may suggest that additional factors (e.g., FGFs, IGF, ROS, S100A8/9 [56]) that are lacking when only recombinant proteins are added may be needed to enhance the production of the EMT and -dormancy markers in the CT26-WT tumor cells. We did not look for such factors in our analyses, and this should be elaborated on in a following study. Alternatively, this lack of change in the co-culture vs. single culture of CT26-WT can be explained by the known aggressiveness of the CT26 cells [57; 58], such that the untreated cells already express high, almost maximal basal levels of these markers. Therefore, any increase in their expression in the co-cultured cells is only marginal and not significant. However, the increase in proliferation and angiogenic potential indicate that the addition of TGF\(\theta\) or low levels of EMMPRIN is sufficient to induce these pathways. We propose that EMMPRIN serves as a gatekeeper to prevent entry into a dormant state, and that when EMMPRIN levels are reduced in the CT26-KD cells, not only are the dormancy markers elevated, but also the EMT-TFs levels are increased. This is in contradiction to previous reports that demonstrated EMMPRIN as a protein that promotes EMT and the expression of the EMT-TFs Snail and Slug [34; 35; 59; 60]. For example, in the LNCaPP prostate cancer cell line, knockdown of EMMPRIN resulted in an increased expression of E-cadherin and decreased expression of \(\beta\)-catenin and Snail [35]. In the Huh7 and HepG2 HCC cells, overexpression of EMMPRIN resulted in increased expression of MMPs that cleaved and activated TGF\(\beta\), leading to the induced expression of Snail and Slug, the latter being a regulator of EMMPRIN expression, again demonstrating the positive feedback loop between TGF\(\beta\) and EMMPRIN [34; 53]. To explain this discrepancy, we suggest that the state of ERK/p38 activation in the CT26 cells is crucial to the final outcome. ERK1/2 activation has been shown to cooperate with the TGF\(\beta\) signaling to induce the expression of EMT-TFs [45]. Mutations in the RAS gene, which is upstream of ERK1/2, and specifically the RAS\({}^{G12D}\) mutation, markedly enhance Snail expression [61].