The inflammatory cytokine TNF contributes with RAC3-induced malignant transformation

Abstract

RAC3 is a coactivator of steroid receptors and NF-κB. It is usually overexpressed in several tumors, contributes to maintain cancer stem cells and also to induce them when is overexpressed in non-tumoral cells. In this work, we investigated whether the inflammatory cytokine TNF may contribute to the transforming effects of RAC3 overexpression in the non-tumoral HEK293 cell line. The study model included the HEK293 tumoral transformed cell line constitutively overexpressing RAC3 by stable transfection and control non-tumoral cells transfected with an empty vector. The HeLa and T47D tumoral cells that naturally overexpress RAC3 were used as positive control. We found that TNF potentiated RAC3-induced mesenchymal transition, involving an increased E-Cadherin downregulation, Vimentin and SNAIL upregulation and enhanced migratory behavior. Moreover, concerning the molecular mechanisms by which TNF potentiates the RAC3 transforming action, they involve the IKK activation, which in addition induced the β-Catenin transactivation. Our results demonstrate that although RAC3 overexpression could be a signal strong enough to induce cancer stem cells, the inflammatory microenvironment may be playing a key role contributing to the migratory and invasive phenotype required for metastasis and cancer persistence.

Keywords: RAC3; TNF-malignant transformation; cancer stem cells; mesenchymal cells.

Figure 1. TNF contributes to RAC3 overexpression-induced mesenchymal transformation of HEK293 cells. qPCR analysis for Vimentin and E-Cadherin (A), Snail (C) and RAC3 (D) of total RNA from cells EV (empty vector) and RAC3 (overexpressing RAC3) HEK293 stimulated during 24 h with: vehicle (Basal), TNF 20 ng/ml (TNF), sulfasalazine 250 μM (SZ) and TNF plus sulfasalazine (TNF+SZ). In B, western blot for Vimentin and E-Cadherin showing one representative image and the relative densitometric units (R.D.U) from three independent experiments.

a: p < 0.05 respect to basal EV, b: p < 0.05 respect to basal RAC3, c: p < 0.05 respect to TNF stimulated RAC3, d: p < 0.05 respect to cells transfected with empty vector

Figure 2. TNF stimulation potentiates RAC3 overexpression-induced cell migration. MMP2 expression was determined by qPCR (A) and activity by zimography assays (B), where the down panel image corresponds to one representative assay from three. The diagram bars show the average +/- SD of wound coverage compared to time 0 and relative to basal EV in HEK293 (C), HeLa (D) and T47D (E). qPCR assays show a significant inhibition of RAC3 expression levels in HeLa (F) and T47D (G) stably expressing shRAC3.

a: p < 0.05 respect to basal EV, b: p < 0.05 respect to basal RAC3, c: p < 0.05 respect to TNF stimulated RAC3, d: p < 0.05 respect to basal scramble (SC), e: p < 0.05 respect to TNF stimulated SC, f: p < 0.05 respect to TNF stimulated shRAC3, *p < 0.01 respect to SC.

Figure 3. TNF stimulation together with RAC3 overexpression induce the β-catenin nuclear translocation and transcriptional activity of TCF/β-catenin complex

A. Inmunofluorescence analysis of β-Catenin in HEK293 (upper panels) and HeLa (bottom panels) stimulated during 24 h with: vehicle (Basal), TNF 20 ng/ml (TNF), sulfasalazine 250 μM (SZ) and TNF plus sulfasalazine (TNF+SZ). B and C-Reporter assays for TCF/β-Catenin-Luc. The bar diagram shows the average relative light units (RLU) of HEK293 (B) and HeLa (C) stimulated with vehicle (Basal), TNF 20 ng/ml, sulfasalazine 250 μM (SZ), TNF plus sulfasalazine (TNF+SZ) and normalized respect to the β-galactosidase activity at each case. a: p < 0.05 respect to basal RAC3, b: p < 0.05 respect to TNF stimulated RAC3, c: p < 0.05 respect to basal SC, d: p < 0.05 respect to TNF stimulated SC.

Figure 4. The interplay of RAC3 overexpression and TNF as part of a positive feedback contributes to tumor development

The increase of RAC3 that could be induced by inflammatory cytokines is a mesenchymatic transforming signal that increases Vimentin, donwregulates E-Cadherin expression, enhances β-catenin-dependent transcriptional activity and cell migration. These effects are potentiated by TNF and some of them are dependent of NF-κB. However, additional cytoplasmic RAC3 actions or NF-κB independent effects of this coactivator could not be excluded.