Overexpression of regulator of G protein signaling 11 promotes cell migration and associates with advanced stages and aggressiveness of lung adenocarcinoma

Regulator of G protein signaling 11 (RGS11), a member of the R7 subfamily of RGS proteins, is a well-characterized GTPase-accelerating protein that is involved in the heterotrimeric G protein regulation of the amplitude and kinetics of receptor-promoted signaling in retinal bipolar and nerve cells. However, the role of RGS11 in cancer is completely unclear. Using subtractive hybridization analysis, we found that RGS11 was highly expressed in the lymph-node metastatic tissues and bone-metastatic tumors obtained from patients with lung adenocarcinoma. Characterization of the clinicopathological features of 91 patients showed that around 57.1% of the tumor samples displayed RGS11 overexpression that was associated with primary tumor status, nodal metastasis and increased disease stages. Its high expression was an independent predictive factor for poor prognosis of these patients. Cotransfection of guanine nucleotide-binding protein beta-5 (GNB5) markedly increased RGS11 expression. Enhancement or attenuation of RGS11 expression pinpointed its specific role in cell migration, but not in cell invasion and proliferation. Signaling events initiated by the RGS11–GNB5 coexpression activated the c-Raf/ERK/FAK-mediated pathway through upregulation of the Rac1 activity. Consistently, increasing the cell invasiveness of the transfectants by additional cotransfection of the exogenous urokinase–plasminogen activator gene caused a significant promotion in cell invasion in vitro and in vivo, confirming that RGS11 functions in cell migration, but requires additional proteolytic activity for cell and tissue invasion. Collectively, overexpression of RGS11 promotes cell migration, participates in tumor metastasis, and correlates the clinicopathological conditions of patients with lung adenocarcinoma.


Generation of stable transfectants
To generate cells that stably overexpressed RGS11, CL1-0 cells were transfected with pcDNA-RGS11, pcDNA-GNB5, or both plasmids using Arrest-In (Open Biosystems, Huntsville, AL). The cells were then treated with appropriate antibiotics (400 mg/mL of G418 or 200 μg/mL hygromycin B; Thermo Scientific, Rockford, IL) and subjected to limiting dilution. The stable transfectants with coexpression of RGS11 and GNB5 were transfected with the pcDNA-His-uPA plasmid. The resultant cells were selected in medium containing 150 μg/mL Zeocin (Thermo Scientific) to generate stable cells expressing His-tagged uPA. CL1-5F4 cells were transfected with the pLKO-sh-RGS11 plasmid, which transcribes RGS11specific shRNA (Academia Sinica, Taipei, Taiwan) to generate transfectants with attenuated expression of RGS11, using Lipofectamine 2000 (Invitrogen). The cells were then treated with 2.5 μg/mL of puromycin (Thermo Scientific) and subjected to limiting dilution. Stable clones with high or knocked-down expression of RGS11 were determined by Western blot analysis. Simultaneously, their corresponding stable control cells were also established by transfecting either the pcDNA3.1 or pLKO.1 vector. FAK was attenuated in the RGS11-GNB5-transfected CL1-0 (R+G) cells by transfection with its specific sh-RNA construct (Academia Sinica, Taipei, Taiwan).

Subtraction hybridization by phenol emulsion reassociation
To identify metastasis-related genes, two pairs of fresh primary lung adenocarcinoma tissues and lymph-node metastatic counterparts were collected under approval of the Institutional Review Boards of the Chi-Mei Medical Center (IRB100-05-003). Genes differentially expressed between primary and lymphnode metastatic lung adenocarcinoma tissues were highly enriched by subtraction hybridization by phenol emulsion reassociation technique (PERT) [1,2], with some modifications. In brief, total RNA of the primary and metastatic tissues was purified using the RNeasy Mini kit (Qiagen, Hilden, Germany) and reversely transcribed using Superscript III reverse transcriptase (Invitrogen) and oligo-(dT) 18 as primer to generate firststranded cDNA. Synthesis of double-stranded cDNA for subtractive hybridization was performed using the Subtractor kit purchased from Invitrogen (Carlsbad, CA). The cDNA pool from the primary tissues was biotinylated using biotinylated nested primers (forward: 5'-biotin-GGACACTGACATGGACTGAAGGAGTA-3'; backward: 5'-biotin-CGCT-ACGTAACGGCATGACAGTG-3') by PCR and acted as driver cDNA in this case; on the other hand, the cDNA pool from the metastatic tissues was ligated with adaptors (forward: 5'-AATGCGGCCGCACTATAGCATGGACTG AAGGAGTA-3'; backward: 5'-CATTTAGGTGACACTA GTAACGGCATGACAGTG-3') and as tester cDNA. The cDNA pools were PCR amplified essentially as described by Wang and Brown [3]. Removal of unligated linkers and free nucleotides in PCR reactions were carried out using the GeneClean III system (Q·Biogene, Carlsbad, CA), as instructed in the manufacturer's manual. Subsequently, the biotinylated or adaptor-ligated cDNA pool was collected into water. The adaptor-ligated tester cDNA pool was mixed with 10-fold excess amount of biotinylated driver cDNA pool in a solution containing 40 mM Tris-HCl, pH8.0 / 4 mM EDTA, and denatured at 95°C for 5 min. Hybridization was carried out by PERT in a 500 μLsolution containing 2 M NaSCN / 8% phenol at room temperature (RT) for 24 h, as described by Kohne et al. [4], with continuous agitation on Vortex Mixer (Pittsburgh, PA). After hybridization, the solution was extracted with equal volume of chloroform/isoamyl alcohol (24:1). The aqueous layer was collected, added with 1 μL of 10 mg/ mL yeast tRNA / 50 μL of 8 M ammonium acetate / 300 μL of ice-cold absolute ethanol per 100 μL of the aqueous layer, and mixed well. The cDNA mixture was precipitated at -70°C for 30 min, centrifuged at 16,000 ×g, 4°C for 10 min, and washed with ice-cold 70% ethanol three times. After air dry, the cDNA pellet was resuspended in 10 mM HEPES/EDTA buffer containing 10 mg/mL streptavidin, mixed, and incubated for 10 min at RT. The biotinylated cDNA fragments were removed by repeated the phenol/ chloroform extraction procedure for three times. The adaptor-ligated resultant pool was re-precipitated as described above, washed with ice-cold 70% ethanol, and resuspended in water.
Construction of subtractive library and clonal analysis by PCR was performed as described by Zeng, et al. [2], with some modifications. After subtraction, the subtracted PCR products were amplified by random primers, cloned into TA vector and transformed into competent INVaF' cells (TA cloning kit, Invitrogen). For clonal analyses, white colonies were picked, and inserted genes were identified by direct sequencing.

Reverse transcription-polymerase chain reaction (RT-PCR)
To determine the gene expression profile of R7 RGS subfamily members, total RNAs of CL1-0 and CL1-5F4 cells were purified using the RNeasy Mini kit
Statistical analyses were performed using the SPSS 14 software package. The associations between various clinicopathological parameters and RGS11 expression status were evaluated using the chi-square test as appropriate. The end points analyzed were DSS and DMFS, which were calculated from the date of surgery until cancer death or development of distal metastasis, respectively, or the last follow-up appointment. The median period of follow-up was 21.3 months (range, 1-86.7). Univariate survival analysis was performed using Kaplan-Meier plots and the survival variables were compared using the log-rank test. A multivariate model using Cox proportional-hazards regression included the parameters with a univariate P-value < 0.05.