Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model

Abstract

BackgroundPreviously we have shown that oncogenic Ha-Ras stimulated in vivo metastasis through RalGEF-Ral signaling. RalA and RalB are highly homologous small G proteins belonging to Ras superfamily. They can be activated by Ras-RalGEF signaling pathway and influence cellular growth and survival, motility, vesicular transport and tumor progression in humans and in animal models. Here we first time compared the influence of RalA and RalB on tumorigenic, invasive and metastatic properties of RSV transformed hamster fibroblasts.MethodsRetroviral vectors encoding activated forms or effector mutants of RalA or RalB proteins were introduced into the low metastatic HET-SR cell line. Tumor growth and spontaneous metastatic activity (SMA) were evaluated on immunocompetent hamsters after subcutaneous injection of cells. The biological properties of cells, including proliferation, clonogenicity, migration and invasion were determined using MTT, wound healing, colony formation and Boyden chamber assays respectively. Protein expression and phosphorylation was detected by Westen blot analysis. Extracellular proteinases activity was assessed by substrate-specific zymography.ResultsWe have showed that although both Ral proteins stimulated SMA, RalB was more effective in metastasis stimulation in vivo as well as in potentiating of directed movement and invasion in vitro. Simultaneous expression of active RalA and RalB didn't give synergetic effect on metastasis formation. RalB activity decreased expression of Caveolin-1, while active RalA stimulated MMP-1 and uPA proteolytic activity, as well as CD24 expression. Both Ral proteins were capable of Cyclin D1 upregulation, JNK1 kinase activation, and stimulation of colony growth and motility. Among three main RalB effectors (RalBP1, exocyst complex and PLD1), PLD1 was essential for RalB-dependent metastasis stimulation.ConclusionsPresented results are the first data on direct comparison of RalA and RalB impact as well as of RalA/RalB simultaneous expression influence on in vivo cell metastatic activity. We showed that RalB activation significantly more than RalA stimulates SMA. This property correlates with the ability of RalB to stimulate in vitro invasion and serum directed cell movement. We also found that RalB-PLD1 interaction is necessary for the acquisition of RalB-dependent high metastatic cell phenotype. These findings contribute to the identification of molecular mechanisms of metastasis and tumor progression.

RalB directly triggers invasion downstream Ras by mobilizing the Wave complex

“…In HEK-HTRasV12 cells, the silencing of RalB impaired Transwell invasion of approximatively 60%, the silencing of RalA had not effect, and the silencing of both RalB and RalA reached up to 90% invasion inhibition ( Figure 5C ) (see Figure 5—figure supplement 1A for depletion efficiencies), indicating that Ras-dependent RalB activation substantially contributes to the invasive phenotype of Ras-mutated cells, and that RalA is dispensable, but it might partially compensate when RalB is absent. The specific role of RalB, with respect to RalA, for cell motility and invasion is in perfect agreement with several previous works ( Oxford et al, 2005 ; Rossé et al, 2006 ; Lim et al, 2006 ; Rybko et al, 2011 ). The RalB requirement for in vitro invasion of HEK-HTRasV12 cells was confirmed using the Inverted Invasion assay with collagen ( Figure 5D ) (see Figure 5—figure supplement 1B for depletion efficiency up to 5 days post-siRNA transfection).…”

“…RalB, but not RalA, is required for the contractility-driven invasion of lung cancer cells (A549, K-Ras mutated) ( Biondini et al, 2015 ). Moreover, in vivo metastasis assays in mice (tail vein injection) ( Ward et al, 2001 ; Lim et al, 2006 ) and in hamsters (subcutaneous injection) ( Rybko et al, 2011 ) supported a function of RalB pathway in the formation of tumor metastasis, both in Ras-mutated and Rous sarcoma virus-transformed cells. Besides the cancer context, we previously found that RalB, but not RalA, controls the mesenchymal migration of normal cells (NRK, HEK-HT, wild-type for Ras) by mobilizing its effector exocyst ( Rossé et al, 2006 ; Parrini et al, 2011 ; Biondini et al, 2016 ), which is an octameric protein complex involved in the tethering of secretory vesicles to the plasma membrane prior to SNARE-mediated fusion ( Wu and Guo, 2015 ).…”

Effects of RAL signal transduction in KRAS- and BRAF-mutated cells and prognostic potential of the RAL signature in colorectal cancer

“…Diminishing GTPase activity by RALA knock-down in HT-29 cells had no significant effect on proliferation supporting the notion that proliferation is mainly triggered by RAF/MAPK signaling. The migratory potential of these colorectal cancer cell lines was not affected by knocking-down RALA expression in contrast to other types of cancer cells [ 31 ]. This suggests that the migratory potential requires mechanisms controlled by other effector pathways.…”

Arf6 promotes cell proliferation via the PLD‐mTORC1 and p38MAPK pathways

“…Pointed features make this model especially attractive for studying influence of different proteins on SMA. We have shown previously metastasis stimulating activity of some other G‐proteins, such as Ha‐Ras, RalA [Tchevkina et al, 2005], and RalB (Rybko et al, 2011, in press) using this cell line. However, we did not reveal significant effect of Arf6 on metastatic activity of HET‐SR cells.…”