SOX18 Is a Novel Target Gene of Hedgehog Signaling in Cervical Carcinoma Cell Lines

Abstract

Although there is much evidence showing functional relationship between Hedgehog pathway, in particular Sonic hedgehog, and SOX transcription factors during embryonic development, scarce data are available regarding their crosstalk in cancer cells. SOX18 protein plays an important role in promoting tumor angiogenesis and therefore emerged as a promising potential target in antiangiogenic tumor therapy. Recently it became evident that expression of SOX18 gene in tumors is not restricted to endothelium of accompanying blood and lymphatic vessels, but in tumor cells as well.In this paper we have identified human SOX18 gene as a novel target gene of Hedgehog signaling in cervical carcinoma cell lines. We have presented data showing that expression of SOX18 gene is regulated by GLI1 and GLI2 transcription factors, final effectors of Hedgehog signaling, and that modulation of Hedgehog signaling activity in considerably influence SOX18 expression. We consider important that Hedgehog pathway inhibitors reduced SOX18 expression, thus showing, for the first time, possibility for manipulationwith SOX18 gene expression. In addition, we analyzed the role of SOX18 in malignant potential of cervical carcinoma cell line, and showed that its overexpression has no influence on cells proliferation and viability, but substantially promotes migration and invasion of cells in vitro. Pro-migratory effect of SOX18 suggests its role in promoting malignant spreading, possibly in response to Hedgehog activation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. The role of GLI transcription factors in the regulation of SOX18 promoter activity and SOX18 endogenous expression.

a) Expression analysis of selected HH signaling components in HeLa, SiHa and Ca Ski carcinoma cell lines.M-DNA ladder.b) Functional analysis of each GLI transcription factor overexpression on SOX18 promoter activity. Schematic illustration of putative biding sites for GLI transcription factors within SOX18 optimal promoter region represented by promoter construct 892pCAT6 is presented on upper panel. Normalized CAT activities were calculated as percentages of the corresponding reporter construct activity in cells co-transfected with empty pcDNA3.1 vector (which was set as 100%). Relative CAT activities were presented as the means ± SEM of at least four independent experiments. P values were calculated using Student’s t-test, *p ≤ 0.05, **p ≤ 0.01c)The effect of GLI’s overexpression on SOX18 gene expression detected by qRT-PCR. Relative SOX18 expression was presented as percentage of SOX18 expression in mock transfected cells that was set as 100%. Results were presented as the means ± SEM of at least three independent experiments performed in triplicates. P values were calculated using Student’s t-test, *p ≤ 0.05, **p ≤ 0.01.

Fig 2. The effect of GLI’s overexpression on SOX18 protein level in cervical carcinoma cell lines.

a) The effect of GLI’s overexpression on SOX18 protein leveldetected by Western blot. One representative blot was presented, and quantification of protein level was presented as histogram chart.α-tubuline was used as a loading control. The relative SOX18 protein level in HeLa, SiHa and Ca Ski cells upon transfection with GLI1-3 was calculated as a percentage of SOX18 level in mock transfected cells which was set as 100%. Data of three independent experiments are presented at histograms as the means ± SEM. Values of p≤0.05 are marked by *.b)The effect of GLI’s overexpression on SOX18 protein leveldetected by immunocytochemistry. Cells were cotransfected with EGFP-C1 (that was used as a marker of transfected cells) and eiherpcDNA-mock transfection (A-C), GLI1(D-F), GLI2 (G-I), or GLI3(J-L). Boxed regions in A-L are enlarged in the same figures. Cell nuclei were counterstained with DAPI (A, D, G and J). Scale bars: 50 μm.

Fig 3. EMSA with six different DNA probes deriving from SOX18 optimal promoter.

Upper panel represents schematic illustration of putative GLI binding sites. Positions relative to tsp are indicated above scheme, and relative positions of corresponding probes are presented by lines and names below. Lower panel represents EMSA reactions; NE HeLa-HeLa nuclear extracts; Unlab.probe- unlabeled corresponding probe in 100-fold molar excess; Cons.GLI- unlabeled oligonucleotide probe with GLI consensus binding site in 100-fold molar excess. Completely faded complexes in competition reaction with GLI consensus probe are marked with black arrows, and partially faded with red arrows.

Fig 4. EMSA with “enriched” proteins and supershift assay.

a) EMSA with whole cell lysates prepared from HeLa cells transfected with pcDNA3.1 empty vector (WCL HeLa) or cells transfected with either GLI1 (WCL HeLa+GLI1) or GLI2 expression vector construct (WCL HeLa+GLI2). b)Supershift assay with anti-GLI1 antibody (GLI1Ab). “Enriched” complexes are marked by asterisks,supershifts i.e. fading of complexes are marked by arrows and ss.

Fig 5. The effect of HH pathway inhibition on proliferation, viability, migration and SOX18 expression in HeLa cells.

a) The inhibitory effect of cyclopamine on GLI1 and PTCH expression. b) Proliferation curve of HeLa cells. Cells were treated with 10μM cyclopamine or tomatidine as a negative control, and counted after 1, 3 and 5 days of continuous treatment. Results were presented as the means ± SEM of at least three independent experiments. c) MTT viability assay performed after 1, 3 and 4 days of treatment with 10 μM cyclopamine or tomatidine. Relative cell viability was calculated as a percentage of HeLa cells viability after tomatidine treatment that was set as 100%. Results were presented as the means ± SEM of at least three independent experiments. P values were calculated using Student’s t-test, *p ≤ 0.05, **p ≤ 0.01.d) The effect of cyclopamine on cell’s migration, wound-scratch migration assay. Cells migration was quantified 24 h after scratching in constant presence of cyclopamine or tomatidine,by measuring the difference in gap closure where gap wide at 0 h was set as 100%. Results were presented as the means ± SEM of at least three independent experiments. e) Relative SOX18 expression after cyclopamine treatment detected by qRT-PCR. Relative SOX18 expression was presented as percentage of SOX18 expression in cells treated with tomatidine that was set as 100%. Results were presented as the means ± SEM of at least three independent experiments performed in triplicates. P values were calculated using Student’s t-test, *p ≤ 0.05, **p ≤ 0.01.f) The effect of cyclopamine on SOX18 protein level. Proteins were isolated after three independent treatments together with adequate controls, followed by Western blot. One representative blot was presented. α-tubuline was used as a loading control. The relative SOX18 protein level in HeLa cells upon treatment with cyclopamine was calculated as a percentage of SOX18 level in cells trated with tomatidine which was set as 100%. Data of three independent experiments are presented at histograms as the means ± SEM. Values of p≤0.01 are marked by **.g) The effect of GANT61 on SOX18 protein level. Proteins were isolated after three independent treatments together with adequate controls, followed by Western blot. One representative blot was presented. α-tubuline was used as a loading control.

Fig 6. The effect of HH pathway activation on proliferation, viability, migration and SOX18 expression in HeLa cells.

a) Stimulatory effect of purmorphamine on GLI1 and PTCH expression. b) Proliferation curve of HeLa cells. Cells were treated with 10μM purmorphamine or DMSO as a control, and counted after 1 and 3 days. Results were presented as the means ± SEM of at least three independent experiments. c) MTT viability assay performed after 1 and 3 days of treatment with 10μM purmorphamine or DMSO. Relative cell viability was calculated as a percentage of HeLa cells viability after DMSO treatment that was set as 100%. Results were presented as the means ± SEM of at least five independent experiments. P values were calculated using Student’s t-test, *p ≤ 0.05, **p ≤ 0.01.d) The effect of purmorphamine treatment on cell migration, wound-scratch migration assay. Cells migration was quantified 24 h after scratching in constant presence of purmorphamine or DMSO, by measuring the difference in gap closure where gap wide at 0 h was set as 100%. Results were presented as the means ± SEM of at least three independent experiments. e) Relative SOX18 expression after purmorphamine treatment detected by qRT-PCR. Relative SOX18 expression was presented as percentage of SOX18 expression in cells treated with DMSO that was set as 100%. Results were presented as the means ± SEM of at least three independent experiments performed in triplicates. P values were calculated using Student’s t-test, *p ≤ 0.05, **p ≤ 0.01.f) The effect of purmorphamine treatment on SOX18 protein leveldetected by immunocytochemistry. Cell nuclei were counterstained with DAPI (A, and C). Scale bars: 50 μm.

Fig 7. The role of SOX18 in the regulation of HeLa cells proliferation, viability and migration.

a) Proliferation chart. HeLa cells were seeded day prior to transient transfection with empty pCI vector, wt or DN SOX18 expression constructs and counted 24 h after transfection. Results were presented as the means ± SEM of at least four independent experiments. b) MTT viability assay. HeLa cells were transiently transfected while seeded in microplate and MTT test was performed 24 h later. Relative cell viability was calculated as a percentage of HeLa cells viability without transfection that was set as 100% Results were presented as the means ± SEM of at least four independent experiments. c)Semiquantitative RT-PCR. Cells were transiently transfected with empty pCI vector, wt or DN SOX18 expression constructs followed by RNA isolation and RT-PCR analysis for the expression level of cyclin D1. The level of wt or DN SOX18 transcription upon transfection was also evaluated. d) The effect of wt or DN SOX18 overexpression on HeLa cell’s migration, wound-scratch migration assay. Representative images of cells migration were presented at left panel. Graphs presented at right panels quantify the migration of transfected cells 24 h after scratching. The changes in migration distance were quantified by measuring the difference in gap closure where gap wide at 0 h was set as 100%. Results were presented as the means ± SEM of at least three independent experiments. The differences in number of single cells that migrated into the empty area was measured by counting the number of single cells in empty scratched area in two different fields in at least three independent experiments and presented as scatter chart. P values were calculated using Student’s t-test, *p ≤ 0.05, **p ≤ 0.01.e) Transwell migration and invasion assays on HeLa cells transfected with empty pCI vector, wt SOX18 or DN SOX18. Representative images of transwell migration/invasion assays were presented.The relative change in cells migration/invasion was calculated as a percentage of HeLa cells migration/invasion in mock transfection that was set as 100%. Cells were counted from five fields and averages were calculated. Results were presented as the means ± SEM of at least three independent experiments performed in duplicates and P values were calculated using Student’s t-test, *p ≤ 0.05, **p ≤ 0.01.f) The influence of DN SOX18 overexpression on GLI1/GLI2-mediated HeLa cell’s migration, wound-scratch migration assay. Graph quantifies the migration of transfected cells 24 h after scratching. Table describes combination of expression vectors used in each experiment. The changes in migration distance were quantified by measuring the difference in gap closure where gap wide at 0 h was set as 100%. Results were presented as the means ± SEM of at least three independent experiments. P values were calculated using Student’s t-test comparing group 1 (cells transfected with GLI1 or GLI2) with group 2 (cells co-transfected with GLI1 or GLI2 together with DN SOX18);*p ≤ 0.05, **p ≤ 0.01.

Fig 8. The role of SOX18 in the regulation of GLI1-3 and PTCHexpression.

HeLa cells were transiently transfected with either empty pCI vector or wtSOX18 or DNSOX18 and the effect of their overexpression on GLI1-3 and PTCH genes expression was analyzed by qRT-PCR. Relative gene expression was presented as percentage of gene expression in cells transfected with empty, pCI vector that was set as 100%. Results were presented as the means ± SEM of at least three independent experiments performed in triplicates.P values were calculated using Student’s t-test, *p ≤ 0.05, **p ≤ 0.01.