High RAC3 expression levels are required for induction and maintaining of cancer cell stemness

Abstract

RAC3 is a transcription coactivator, usually overexpressed in several tumors and required to maintain the pluripotency in normal stem cells.In this work we studied the association between RAC3 overexpression on cancer cell stemness and the capacity of this protein to induce cancer stem properties in non tumoral cells.We performed in vitro and in vivo experiments using two strategies: by overexpressing RAC3 in the non tumoral cell line HEK293 and by silencing RAC3 in the human colorectal epithelial cell line HCT116 by transfection. Furthermore, we analysed public repository microarrays data from human colorectal tumors in different developmental stages. We found that RAC3 overexpression was mainly associated to CD133+ side-population of colon cancer cells and also to early and advanced stages of colon cancer, involving increased expression of mesenchymal and stem markers. In turn, RAC3 silencing induced diminished tumoral properties and cancer stem cells as determined by Hoechst efflux, tumorspheres and clonogenic growth, which correlated with decreased Nanog and OCT4 expression. In non tumoral cells, RAC3 overexpression induced tumoral transformation; mesenchymal phenotype and stem markers expression. Moreover, these transformed cells generated tumors in vivo.Our results demonstrate that RAC3 is required for maintaining and induction of cancer cell stemness.

The inflammatory cytokine TNF contributes with RAC3-induced malignant transformation

“…As previously demonstrated (Panelo et al, 2018[ 25 ]), although HEK293 cells express moderated levels of the mesenchymal marker Vimentin, they were significantly increased when RAC3 was overexpressed, while E-Cadherin was significantly inhibited (Figure 1A and B (Fig. 1) ).…”

“…We have previously found that RAC3 has a transforming role in HEK293 cells when is overexpressed by stable transfection, acquiring not only the ability to grow in an anchorage-independent manner (Rubio et al, 2012[ 30 ]) but also to initiate tumors when are inoculated to mice (Panelo et al, 2018[ 25 ]).…”

“…Cancer propagation requires the migration and invasion of CSC and this is promoted by RAC3 overexpression (Panelo et al, 2018[ 25 ]). Therefore, as a biological correlation of the transformation induced by RAC3 overexpression and TNF stimulation, we analyzed the migratory potential of both cell clones stimulated or not by this cytokine.…”

“…HEK293 cells were transfected with a RAC3 expression vector pCMV-Tag 2B-RAC3 (RAC3) or with empty vector (EV) and selected for stable expression with Neomycin. The tumoral cell lines were transfected with an expression vector for shRNA-RAC3 (pRV-GFP-puromycin) or the scramble control, as previously described in our laboratory and selected for stable expression with puromycin (Panelo et al, 2018[ 25 ]).…”

“…Most of the studies that allowed to define RAC3 as an oncogene were performed in models where it is naturally overexpressed, such as cell lines, tumors and transgenic or knockout mice (Xu and Li, 2003[ 40 ]). Although the effect of RAC3 overexpression in non-tumoral cells has not been deeply investigated up to date, we have previously demonstrated that RAC3 overexpression as a unique change, in the non-tumoral human embryonic kidney cell line (HEK293) gives to these cells the ability to grow in soft agar forming colonies (Rubio et al, 2012[ 30 ]) and to induce cancer stem cells (CSC) (Panelo et al, 2018[ 25 ]).…”