Rap1 mediates galanin receptor 2-induced proliferation and survival in squamous cell carcinoma

Abstract

Previously we showed that galanin, a neuropeptide, is secreted by human squamous cell carcinoma of the head and neck (SCCHN) in which it exhibits an autocrine mitogenic effect. We also showed that rap1, a ras-like signaling protein, is a critical mediator of SCCHN progression. Given the emerging importance of the galanin cascade in regulating proliferation and survival, we investigated the effect of GAL on SCCHN progression via induction of galanin receptor 2 (GALR2)-mediated rap1 activation. Studies were performed in multiple SCCHN cell lines by inducing endogenous GALR2, by stably overexpressing GALR2 and by downregulating endogenous GALR2 with siGALR2. Cell proliferation and survival, mediated by the ERK and AKT signaling cascades, respectively, were evaluated by functional and immunoblot analysis. The role of rap1 in GALR2-mediated proliferation and survival was evaluated by modulating expression. Finally, the effect of GALR2 on tumor growth was determined. GALR2 stimulated proliferation and survival via ERK and AKT activation, respectively. Knockdown or inactivation of rap1 inhibited GALR2-induced, AKT and ERK-mediated survival and proliferation. Overexpression of GALR2 promoted tumor growth in vivo. GALR2 promotes proliferation and survival in vitro, and promotes tumor growth in vivo, consistent with an oncogenic role for GALR2 in SCCHN.

Figure 1. Endogenous GALR2 promotes proliferation and survival in SCCHN

(A) qPCR (left panel) shows the mRNA level of GALR2 in HOK, UM-SCC-1, OSCC3, UM-SCC-(22B and 11A) cells. Data were analyzed by relative quantification and expressed as fold change relative to HOK. Whole cell lysates from these cells were blotted with GALR2 and GAPDH antibodies (right panel). (B and C) GALR2 promotes proliferation via ERK activation. UM-SCC-1 cells were pre-treated with 10μM of U0126 or vehicle control for 1h followed by 300nM of [D-Trp²]galanin-(1–29) for 24h. Total cell number was determined (B; p≤0.05). ERK activation was evaluated by immunoblot analysis of whole cell lysates with phospho-ERK and total ERK (C). (D and E) GALR2 enhances cell survival via PI3K/AKT pathway. UM-SCC-1 cells were pre-incubated for 1h with 25μM of LY294002 followed by 300nM of [D-Trp²]galanin-(1–29) stimulation for 2h prior to etoposide treatment. The percentage of apoptotic cells was determined by staining with AnnexinV-FITC and propidium iodide followed by flow cytometry (D). Data are expressed as percent of the corresponding DMSO-treated control. AKT activation was evaluated by immunoblot analysis of whole cell lysates with phospho-AKT and total AKT (E).

Figure 2. Downregulation of endogeneous GALR2 by siGALR2 downregulates ERK1/2 and Akt signaling pathway and inhibits proliferation

(A & B) GALR2 was downregulated in UM-SCC-1 (A) and OSCC3 (B) cells by two siRNAs, siGALR2-7, and siGALR2-10 or with non target siRNA(NT). GALR2 knockdown was verified by immunoblot analysis. ERK and AKT downregulation was also verified by phospho-ERK and phospho-AKT antibodies (Fig 2A left). Total ERK and total AKT was used as loading controls. GALR2 was downregulated in UM-SCC-1 (Fig. 2A right) and OSCC3 (Fig. 2B right) cells with two GALR2 specific siRNA, siGALR2-7 and siGALR2-10. Viable cells were counted on days 1, 3 and 5^th post-seeding. Data are representative of three independent experiments, each with three replicates (*p<0.001).

Figure 3. GALR2 induces proliferation and survival

(A & B) UM-SCC-1 (A, left panel) and OSCC3 (B, left panel) cells were stably transfected with pcDNA or pcDNA-GALR2. GALR2 expression was verified by immunoblot analysis. ERK and AKT activation were detected with phospho-ERK and phospho-AKT antibodies, respectively. Total ERK and total AKT were used to verify total protein. GAPDH was used as a loading control. GALR2 promotes proliferation. UM-SCC-1 (A, middle panel) and OSCC3 (B, middle panel) cells stably transfected with control vector and GALR2 were seeded in triplicate at 20,000 cells/well. Cell number was determined at days 1 through 4 after seeding. Values represent mean of cell number (*p<0.004). GALR2 promotes survival. UM-SCC-1 (A, right panel) and OSCC3 (B, right panel) cells stably transfected with control vector or GALR2 were treated with U0126 or LY294002 or combined were stained with AnnexinV-FITC and propidium iodide. Apoptosis was measured by flow cytometry. Data represent total apoptotic cells and are expressed as percentage of the corresponding vehicle-treated control cells. Data represent mean and SEM of three independent experiments (p≤0.05).

Figure 4. GALR2 induces rap1, ERK and AKT activation

(A) UM-SCC-1 cells were stimulated with 300nM of [D-Trp²]galanin-(1–29) for 24h and rap1 activation was evaluated (left panel). UM-SCC-1 cells were transfected with non target siRNA or siGALR2-7 followed by stimulation with 300nM of [D-Trp²]galanin-(1–29) for 24h and rap1 activation was evaluated (middle panel). Rap1 activation was also determined in UM-SCC-1cells overexpressing GALR2 compared to control cells (right panel). Total rap1 was used as loading controls. Active, GTP-bound rap1 was quantified by densitometry and normalized to total rap1. Data are representative of two independent experiments with similar results. (B) Dose response of GAL-mediated activation of rap1, ERK and AKT. UM-SCC-1 control cells (left panel) and GALR2 overexpressing cells (right panel) were stimulated with varying concentrations of GAL, for 1h. Whole cell lysates were blotted with phospho-ERK and phospho-AKT antibodies followed by total ERK and total AKT antibodies respectively. Rap1 activation was evaluated by the rap1 pull down assay and total rap1 was used as loading control. Active rap1, Phospho-ERK and phospho-AKT signals were normalized to corresponding total rap1, ERK and AKT, respectively and then to pcDNA.

Figure 5. Rap1 mediates GALR2-induced cell proliferation and survival by ERK and AKT signalling pathways

(A) Rap1GAP inactivates rap1. UM-SCC-1 cells were stably transfected with empty vector (pcDNA) or FLAG-tagged rap1GAP and whole cell lysates were blotted with FLAG, rap1, and GAPDH antibodies. Rap1 inactivation in rap1GAP transfected cells was confirmed by pull down assay. (B) Rap1 mediates ERK and AKT activation. UM-SCC-1 cells stably transfected with pcDNA and FLAG-tagged-rap1GAP were stimulated with 500 nM GALP for 24h. Active ERK and AKT were evaluated with respect to total ERK and AKT. (C) UM-SCC-1 cells overexpressing GALR2 were transfected with rap1 siRNA and were stimulated with 5 nM GAL for 10 minutes. Active and total ERK, AKT, rap1 and GAPDH were determined. (D) GALR2 promotes proliferation via rap1. UM-SCC-1-GALR2 cells transfected with rap1 siRNA or non target siRNA were seeded in triplicate. Viable cells were counted on days 1, 3 and 5 (*p<.001). (E) GALR2 mediates cell survival via rap1. Upper panel: UM-SCC-1-GALR2 cells transfected with siRap1 or non target siRNA were stimulated with 10 nM GAL for 24h and were stained with AnnexinV-FITC and propidium iodide. The percentage of apoptotic cells was determined by flow cytometry and compared to UM-SCC-1-pcDNA with UM-SCC-1-GALR2 as 100% (*p<0.038). Lower panel: UM-SCC-1-GALR2 cells transfected with siRap1 or non target siRNA were incubated with U0126, LY294002 or both inhibitors and the percentage apoptotic cells was determined by flow cytometry and compared to their corresponding controls (*p< 0.04).

Figure 6. GALR2 promotes tumor growth

(A & B) OSCC3 cells stably transfected with pcDNA or pcDNA-GALR2 were injected subcutaneously in mice. After 2 weeks, the tumors were harvested and tumor volume (A) (*p<0.02) and weight (B) (**p<0.01) were determined. (C) Proposed model: GALR2 mediates tumor growth via rap1-induced, ERK- and AKT-mediated proliferation and cell survival. ERK also regulates cell survival.