Differential effects of modulation of SOCS3 expression and STAT3 inhibition on HNSCC migration.

<p>(A) Representative phase-contrast images of the in vitro wound area in the different cell cultures (100×magnification) for the gain of function experiments by transfection of a CMV-driven SOCS3 expression plasmid or the empty vector control. STAT3 biochemical inhibitor was used to assess possible effects of SOCS3 that are independent of modulation of STAT3 activity. The distance between the edges of the wound was measured on digital images captured adjacent to the reference notch made in the cell culture plastic (black area on the right of the images) Non-neoplastic HaCAT cells were transfected with the empty vector to control for non-specific effects of the transfection procedure on cell migration. Bars represent averages and vertical lines standard deviation of three independent wounding experiments, measured in triplicate (*p<0.05 indicates significant difference in comparison to the distance between edges of the wound at the same time period in empty-vector transfected cells). (B) Representative phase-contrast images of the in vitro wound area in the loss of function experiments by transfection of SOCS3 siRNA or non-targeting siRNA in OSCC3 cells. Quantitation of cell migration was performed as described in (A) and the graph represent average and standard deviations of three independent wounding experiments measured in triplicate.</p

Gain of function of SOCS3 reduces HNSCC invasion in vitro.

<p>(A) Representative images of lower aspect of the transwell membrane after removal of the matrigel on its upper aspect and staining of the cells with the Diff-Quick kit (200×magnification). The non-neoplastic epithelial cell line (HaCAT) was used as a control and did not invade the matrigel layer. (B) Invading cells in the lower part of the transwell were counted in 6 random high-power fields (400X) by an examiner that was blind to the experiment purposes. Three independent experiments were performed and the bars indicate averages and vertical lines the standard deviations (*p<0.05 in comparison to empty vector-transfected cells, *p<0.05 between indicated groups).</p

Modulation of SOCS3 expression in gain and loss of function experiments in HNSCC only reduces proliferation of cells without (UM-SCC-22B) detectable levels of endogenous SOCS3.

<p>(A) Immunoblot analysis of whole cell extracts shows that transfection of the SOCS3 expression plasmid resulted in increased levels of SOCS3 after 24 and 48 h in OSCC3 and UM-SCC-22B cells. Pre-treatment of both cell lines with the indicated concentrations of the biochemical inhibitor effectively decreased phosphorylation of STAT3 30 min after stimulation with IL-6 (25 ng/mL). (B) Subcellular localization of SOCS3 in the overexpression experiments was confirmed by immunofluorescence analysis. Images are representative of three independent experiments assessing SOCS3 transgene expression 48 h after plasmid transfection. (C) Cell proliferation was determined by direct counting the cells and the trypan blue dye exclusion test 24 and 48 h after transfection. (D) Cell proliferation was also determined in loss of function experiments by direct counting of cells using trypan blue exclusion test 24 and 48 h after siRNA transfection in OSCC3 cells. Graphs (C and D) represent growth curves (number of viable cells) over time, according to the experimental condition (in C: transfection of empty vector, SOCS3 expressing vector, or treatment with STAT3 inhibitor; in D: reagent control, SOCS3 siRNA and non-target control siRNA). Vertical lines standard deviations of three independent experiments and * indicates p<0.05 for comparison with vehicle control (empty vector or non-targeting siRNA in gain and loss of function experiments, respectively). (E) Immunoblot analysis verifying the efficiency of siRNA-mediated inhibition of endogenous SOCS3 expression 24, 48 and 72 h after transfection. Images are representative of three to five independent experiments.</p

Decrease of SOCS3 expression is an early event in head and neck cancer.

<p>Immunohistochemical staining for SOCS3 in four different tissue microarrays was analyzed with a digital slide scanner system. The intensity of positive staining was normalized to the area of positive staining to account for the larger area occupied by epithelial cells in the tumor samples. (A) Representative images of the results for immunohistochemical staining of SOCS3 are shown for 4 of the squamous cell carcinoma (SCC) cases that had both neoplastic and non-neoplastic samples with the indicated pathological information (location, TNM staging) (400X). Below are representative images of the classification system with the slide scanner. The protocol designed recognizes positive staining (in red), non-stained tissue (in yellow), cell nuclei (in blue) and empty spaces (in gray) (100X). (B) Results for the normalized intensity of SOCS3 expression according to tumor staging classification. The number of cases analyzed in each condition is indicated above the graphs (‘n’). This number varies because some TMAs did not have all the staging information and because some spots were lost during the staining process. Bars represent averages and vertical lines the standard deviations. Different letters above the columns indicate a statistically significant difference (p<0.05).</p

Subcellular distribution of constitutive and IL-6-induced SOCS3 in four different HNSCC cell lines and a non-neoplastic epithelial cell line (HaCAT).

<p>(A) Immunoblot of nuclear and cytoplasmic fractions of protein isolated from three HNSCC cell lines and a non-neoplastic epithelial cell line (HaCAT). HNSCC cell lines that still presented some level of SOCS3 expression and the non-neoplastic HaCAT cells were stimulated with IL-6 (25 ng/mL) for 18 hours. These results support the immunofluorescence findings of a nuclear localization of SOCS3 in HNSCC cells, in contrast to the predominantly cytoplasmic localization in HaCAT cells. Expression of nuclear Lamin A/C and cytoplasmic GAPDH were used as controls for the purity of the different protein fractions, whereas beta-actin was used as a loading control for a mixture of equal concentrations of nuclear and cytoplasmic proteins. (B) The indicated cell lines were plated on chamber slides and treated with IL-6 (25 ng/mL) for 18 h to induce SOCS3 expression. After fixation and permeabilization, cells were stained with a rabbit polyclonal antibody against SOCS3 followed with Alexa488-conjugated anti-rabbit IgG secondary antibody. The nuclei of the cells were counterstained with DAPI. Images from random fields were obtained at 600×magnification and are representative of three independent experiments.</p

Constitutive and cytokine-induced SOCS3 expression in HSNCC cell lines.

<p>OSCC3, UM-SCC-11B, UM-SCC-22A and UM-SCC-22B and one non-neoplastic human epithelial cell line (HaCAT) were evaluated for expression of SOCS3 upon IL-1β and IL-6 stimulation for 18 hours. (A) RT-qPCR results of SOCS3 mRNA normalized to GAPDH. Bars represent mean and vertical lines the standard deviation of 3 independent experiments. (B) Immunoblot results for SOCS3 protein expression and STAT3 activation after stimulation with IL-1 or IL-6. Images are representative of three independent experiments.</p