Effects Of SOCS3 On Growth And Metastasis Of Lung Adenocarcinoma Cell Lines And Its Mechanism

Suppressor of cytokine signaling (SOCS) proteins was identified as negative regulators of cytokine signaling. For a long time SOCS has been regarded as a major negative regulator of JAK/STAT pathway. Recently there are emerging evidences indicating that SOCS3 regulates strength and duration of other multiple intracellular signaling pathways including ras/ERK1/2/MAPK, PI3-K/Akt in nonmalignant cells.Recently, hypermethylation-mediated sliencing of SOCS3 gene in a few tumor types led to speculation that SOCS3 might function as a tumor suppressor gene. In connection with SOCS3 and cancer, a majority of research focused on hypermethylation-mediated silencing of SOCS3 in different tumor types and regulatory mechanism of SOCS3 to STAT3. However, little is known about effect of SOCS3 on other intracellular signal cascades such an Erk1/2, Akt, which are also play critical role in tumorigenicity. Here, we examined the effect of SOCS3 protein on proliferation, migration and invasion of lung adenocarcinoma cell (LAC) lines (A549 and SPC-A1), and further explored the underlying mechanism, which provided a basis and targeted therapies for circumventing the process in tumor progression.Methods1. SOCS3 expression vectors and screening vectors were cotransfected into the LAC lines (A549 and SPC-A1) using lipofectmine 2000 in vitro. SOCS3-expressing cell clones were screened with G418. RT-PCR and immunocytofluorescence were used to analyze the expression of SOCS3 mRNA and protein in LAC lines.2 The Colony formation assay and MTT assay were used to determine the proliferation rate of cells. Growth of cell transplants in BALB/c nude mice was also investigated. The adhesion, scratch and Boyden chamber assay were employed to assess the migration and invasion ability of cells. Further, the responsiveness of LAC lines to cytotoxic drugs was evaluated by MTT assay.3. The phosphorylating level of STAT3, Akt and Erk1/2 (pSTAT3, pAkt, pErk1/2) was determined with corresponding phosphor-specific antibody by western blot.4. Western blotting was performed to measure the expression of cyclinD1, p21. The MMP2, 9 mRNA and activation of MMP2, 9 were assessed by semi-quantitive RT-PCR and gelatin zymography respectively.Results1. Expression of SOCS3 mRNA was not detected in A549 and SPC-A1 cells2. The positive cell clones cotransfected with SOCS3 gene expressing vectors and screening vectors were obtained after being screened with 500μg/ml G418. The expression of SOCS3 mRNA and protein was respectively confirmed by RT-PCR and immunocytofluorescence in cells stably transfected with SOCS3 gene.3. Compared with the control group, the colony numbers decreased by 73.8% and 55.9% in stably SOCS3-transfected A549 and SPC-A1 cells, respectively. The doubling time of stably SOCS3-transfected HLACs was significantly prolonged (64.5±1.6h vs. 49.2±0.9h in A549; 56.9±1.5h vs.44.8±1.0h in SPC-A1; P<0.01).4. SOCS3 increased the sensitivity to cisplatin, doxorubicin and paclitaxel by 3.1 and 3.4, 3.9 and 3.4, 7.3 and 6.4 fold in A549 and SPC-A1 cells respectively.5. The cell transplants of stably SOCS3-transfected cells in nude mice had lower volume and weight than those of mock group (0.33±0.08g vs.1.06±0.13g in A549; 1.28±0.31g vs. 2.96±0.69g in SPC-A1; P<0.01). The inhibitory rate of cell growth in xenograft model using A549 and SPC-A1 cells was 68.9% and 56.8% respectively.6. Compared with the control group, the adhesive rate of stably SOCS3-transfected A549 was lower in matrigel-coated and FN-coated filters respectively (41.2±2.2% vs. 73.5±8.1%; 38.8±4.6% vs. 66.8±6.5%; P<0.01). Similar results were seen in SPC-A1 cells (37.2±3.5% vs. 63.9±4.6%; 39.6±4.2% vs. 56.4±5.3; P<0.01). The number of the cells which crossed the no coated filters obviously decreased (19.3±2.1 vs. 41.7±3.5 in A549; 23.4±5.2 vs. 47.7±6.5 in SPC-A1; P<0.01). So was the number of the cells which crossed the matrigel-coated (12.3±2.5 vs. 23.0±4.0 in A549, P<0.05; 14.7±4.4 vs. 34.5±5.4 in SPC-A1; P<0.01).7. Compared with the mock group, the level of pSTAT3 in stably SOCS3-transfected A549 cells and SPC-A1 was hardly detected both in routine culture state (P<0.01), the level of pErk1/2, pAkt in stably SOCS3-transfected A549 cells was also obviously inhibited (P<0.05). Similar results were seen in stably SOCS3-transfected A549 cells exposed to EGF. However, the level of pAkt was not significantly inhibited in stably SOCS3-transfected SPC-A1 cells in EGF stimulatory state (P>0.05).8. The expression of cyclinD1 decreased (P<0.01 in A549; P<0.05 in SPC-A1 )and p21 increased significantly in stably SOCS3-transfected A549 and SPC-A1 cells (P<0.01).The mRNA expression and activities of MMP2,9 in A549 and SPC-A1 cells with SOCS3 stable expression were inhibited significantly (P<0.05).ConclusionHere, we found that SOCS3 in lung adenocarcinoma cell (LAC) lines (A549 and SPC-A1) was almost not detected. SOCS3 also enhanced the sensitiveity of LAC lines to cisplatin, adriamycin and paclitaxel by 3-7 fold. Restoration of SOCS3 suppressed growth, migration and invasion in LAC lines significantly. We demonstrated that growth-suppression effect of SOCS3 in LAC lines was associated with attenuating activation of Erk1/2, Akt and STAT3, accompanying by down-regulating cyclin D1, up-regulating p21 and decreasing the expression and activation of MMP2, MMP9. Thus, we suggested that SOCS3 modulated neoplastic process in LAC lines by acting on multiple signaling pathways and may be a good target for interference with cellular signal in lung cancer.