A Correlation Of Hedgehog Signaling Pathway With Cell Migration And Invasion In Ovarian Cancer

Background and PurposeOvarian cancer is one of the most lethal gynecologic malignancies worldwide. Due to the non-specific symptoms, most of ovarian cancer cases are presented with advanced stage disease and associate with the highest mortality rate.Both cell migration and invasion contribute the metastatic ability of the tumor cells and the genetic mechanism that regulates metastasis remains largely unknown. Therefore, it is of paramount importance to identify molecular mediators conferring the metastatic potential to ovarian cancer cells that may be used as tumor markers in predicting risk of ovarian cancer progression. Hedgehog (Hh) signaling pathway plays an important role in the process of cell fate decisions and cell proliferation. The pathway by Hh ligands, membrane receptors PTCH and transmembrane signal transduction protein Smo and the cytoplasmic protein involved in the Hh signal transduction complex, the last point of the cascade conduction is recognized as the zinc finger transcription factor Gli (glioma-associated oncogene transcription factors Gli), is a key node of the signaling pathway plays a pivotal role in signal transduction. Hh target genes are involved in cell proliferation, survival, differentiation, cell cycle, stem cells and invasion. Several reports indicate that the aberrant activation of Hh signaling is implicated in ovarian cancer, however, the exact role and regulatory mechanisms of Hh signaling for invasion and metastasis in ovarian cancer are not well understood. This project will discover new genes regulated by Hh signaling pathway. Using clinical research, cellular biological method, DNA tip technique, animal experiment, bioinformatics and other regular assays, we will illustrate the role and requlatory mechanisms of the new genes and further reveal the exact influence of Hh signaling pathway on tumorigenesis, invasion and matestasis of ovarian cancer. Our final objective is to offer theoretical guidance of new treatment strategies and drug development targeting to ovarian cancer. MethodsPart Ⅰ. The relation between the aberrant activation of Hh signaling pathway and clinicopathological parameters in ovarian cancer(1) To detect Gli2protein in65ovarian cancer samples by using two steps immunohistochemistry; then observed the relationship between the expression change of the key protein in Hedgehog signaling pathway and the clinicopathological paramenters.(2) Simultaneously, the expression of Shh, Gli1, Gli2(critical components of Hedgehog signaling pathway) mRNA and protein was assayed by real-time PCR and western-blot, respectively.Part Ⅱ. Blocking Hh signaling pathway decreases cell proliferation and migration in ovarian cancer(1) In order to explore the relationship of Hedgehog signaling pathway and cell proliferation and migration in ovarian cancer, Ovarian cancer cell lines (SKO-V3and ES-2) for the object of study, the transcription factor Gli-specific small molecule inhibitors GANT61processing the SKO-V3and ES-2cells, we detected Smo, Gli1, Gli2(critical components of Hedgehog signaling pathway) mRNA and protein in ovarian cancer cells (SKO-V3and ES-2) that were treated with DMSO or GANT61(30μim) by real-time PCR, western-blot and immunocytochemistry, respectively.(2) To detect cell viability and proliferation in ovarian cancer cells (SKO-V3and ES-2) that treated with DMSO or GANT61(30μm) by MTT assays and BrdU assays respectively;(3) To detect cell migration and cell colony formation in ovarian cancer cells (SKO-V3and ES-2) that treated with DMSO or GANT61(30μm) by wound-healing assays and soft agar assays, respectively. Part III. Blocking Hh signaling pathway changes cDNA microarrays genesexpression profiling in ovarian cancer cells(1) The transcription factor Gli-specific small molecule inhibitors GANT61processing the SKO-V3and ES-2cells, we detected the differentially expressed genes (DEGs) that regulated by Hedgehog signaling pathway in ovarian cancer cells (SKO-V3) that were treated with DMSO or GANT61(30μm) by cDNA microarrays gene expression profiling, and these differentially expressed genes were uploaded for GO pathway and KEGG pathway analysis by bioinformatics. we found that some genes are regulated by Hedgehog signaling pathway. Then, we validated the DEGs (such as ITGB4, FAK, CD24, MMP7, etc.) by real-time PCR and western-blot,respectively.(2) In order to explore the molecular mechanisms by which Hh signaling pathway resulates cell invasion and metastasis in ovarian cancer, Gli-specific small molecule inhibitors GANT61, siRNA of Gli1and Hh signaling pathway ligand conditional medium (Shh-Med), were used to inhibit or activate the Hh signaling, The real-time PCR,western blot and cell invasion assay were performed to test cell migration and invasion of ovarian of cancer.Part IV. Blocking Hh signaling pathway reduces tumor growth in vivo(1) In order to test potential tumor growth inhibitory effects of Gli inhibitor GANT61in vivo, We establish the nude mice subcutaneous xenograft models employed human ovarian epithelial carcinoma tissue masses.(2) The nude mice subcutaneous xenograft models were treated with GANT61(50mg/5ml) or control. Tumor sizes were measured every other day. The volume and weight of tumor were compared each other.(3) Gli1and Gli2protein in xenografts were detected by western-blot. Simultaneously, ITGB4and p-FAK protein in xenografts were detected by immunohistochemistry. ResultsPart Ⅰ. The relation between the aberrant activation of Hedgehog signaling pathway and clinicopathological parameters in ovarian cancer(1) The expression of Shh, Gli1and Gli2protein in ovarian carcinoma was significantly higher than that in the adjacent tissues by western-blot, and the expression of Shh, Gli1and Gli2mRNA in ovarian carcinoma was significantly higher than that in the adjacent tissues by real-time PCR, the difference was statistically significant (p<0.01), The above data suggest that abnormal activation of the Hh signaling pathway was occured in ovarian cancer tissue.(2) Gli2protein expression was related with tumor TNM stage, the difference was statistically significant (p<0.05); and the expression of Gli2protein with patients age, histological type of tumor, the degree of tumor differentiation and lymph node matestasis were not significantly related (p>0.05).Part Ⅱ. Blocking Hedgehog signaling pathway decreases cell proliferation and migration in ovarian cancer(1) Gli-specific small molecule inhibitors GANT61induced down-regulation of transcription factor Gli and its downstream targets Ptch in protein level in ovarian cancer cells; GANT61induced down-regulation of transcription factor Gli1and Gli2in mRNA level in ovarian cancer cells, the difference was statistically significant (p<0.01); Gli2protein expression was significantly reduced in the cells treated with GANT61.(2) GANT61inhibited the viability of ovarian cancer cells, the difference was statistically significant (MTT assays; p<0.01); Also, GANT61inhibited the proliferation of ovarian cancer cells, the difference was statistically significant (BrdU assays; p<0.01);(3) GANT61inhibited cell migration of ovarian cancer cells, the difference was statistically significant (wound-healing assays; p<0.01); and GANT61inhibited the cell colony formation of ovarian cancer cells, the difference was statistically significant (soft agar assays; p<0.01).Part III. Blocking Hedgehog signaling pathway changes cDNA microarrays genes expression profiling in ovarian cancer cells(1) cDNA microarrays gene expression profiling and pathway network analysis indicated that the mapped DEGs in SKO-V3following GANT61treatment for60hr chiefly involved the focal adhesion pathway, including both up-and down-regulated genes. Of above genes, integrin and lamin are the main DEGs. Simultaneously, the mapped DEGs in SKO-V3are also accumulated in MAPK signaling pathway and most of those DEGs were up-regulated, several of those DEGs were down-regulated.(2) Real-time PCR and Western blot verified the microarray results at the mRNA and protein levels, the results indicated that the expression of ITGB4, COL1A1, COL5A1mRNA was significantly declined following GANT61treatment compared with DMSO treatment, the difference was statistically significant (p<0.01); However, the expression of LAMC2, ITGA5, LAMA3mRNA was significantly higher, the difference was statistically significant (p<0.01), consistent with cDNA microarrays. The expression of CD24, CD70, S100A2, MMP7mRNA were all declined,(p<0.01); The results were also consistent with cDNA microarrays; In addition, the expression of ITGB4, CD24, MMP7protein were all significantly declined following GANT61treatment compared with DMSO treatment, There consistent with real-time PCR assays.(3) In order to observe the differences in gene expression changes. Shh ligand conditioned medium (Shh-Med) is used to activate the Hh signaling pathway, we found that the expression of ITGB4protein and mRNA in SKO-V3was significantly higher following Shh-Med treatment compared with control treatment, the difference was statistically significant (p<0.01). In addition, cell invasion assay indicated that the motility and invasiveness of ovarian cancer cells caused by Shh-Med can be blocked by the anti-ITGB4,(p<0.01).(4) In order to confirm the specific role of GANT61to the transcription factor Gli, Gli1siRNA(miR-Gli1-720) was used to interfere the expression of Gli, Western blot results indicated that Gli1and ITGB4protein expression levels of SKO-V3cells transfected with miR-Gli1-720were significantly decreased, consistent with the inhibitory effect of GANT61.(5) In order to clarify the biological significance of Hh signaling pathway affect1TGB4expression, eithor Shh-Med or GANT61was used to treat SKO-V3cells, the results suggested that compared with control treatment, the expression of p-FAK protein in SKO-V3was significantly higher following Shh-Med treatment, the difference was statistically significant (p<0.01); But the difference in the expression of FAK protein was not statistically significant (p>0.05); However the expression of p-FAK levels were significantly decreased following GANT61treatment (p<0.01). Western blot results suggested that increase in p-FAK levels of SKO-V3cell caused by stimulation of Shh-Med can be blocked by the anti-ITGB4,(p<0.01). the above data confirmed that the Hh signaling pathway can promote FAK phosphorylation.(6) Next, we employed immunofluorescence technique to examine the effects of GANT-61on the expression of p-FAK. SKO-V3cells were treated with GANT61, and the expression of p-FAK-Y397was observed. GANT61inhibited the expression of p-FAK-Y397. In addition, we found that cytoskeleton of ovarian cancer cell was destroyed following GANT61treatment for48hr, and cell pseudopodia were significantly lessen.Part Ⅳ. Blocking Hedgehog signaling pathway reduces tumor growth(1) The nude mice subcutaneous xenograft models employed human ovarian epithelial carcinoma tissue masses were successfully established.(2) The volumes of the nude mice subcutaneous xenografts were significantly reduced following GANT61treatment compared with control treatment, especially at the ninth day. The tumor weight of the nude mice was also significantly reduced following GANT61treatment compared with control treatment, the difference was statistically significant (p<0.01).(3) Western-blot assays indicated that the expression of Glil and Gli2protein in xenografts that treated with GANT61was significantly lower than that treated with vehicle, the difference was statistically significant (p<0.01); While immunohisto chemistry assays indicated that the expression of ITGB4and p-FAK protein in xenografts that treated with GANT61was significantly lower than that treated with vehicle.Conclusion1. The aberrant activation of Hh signaling pathway was implicated in ovarian cancer. Gli2as the important component of Hh signaling, its protein expression was related with tumor stage of ovarian cancer.2. GANT61decreased the expression of Gli1, Gli2and Ptch protein, Simultaneously, it inhibited cell viability, proliferation, migration and colony formation.3. Some DEGs were screened by cDNA micrearrays following GANT61treatment, and of these genes, several ones related to cell adhesion and migration were verified in protein and mRNA level. The results were consistent with cDNA micrearrays. Down-regulation of Gli decreased the expression of ITGB4protein and mRNA, and anti-ITGB4inhibited Shh mediated cell migration and invasion of ovarian cancer by destroying ITGB4/FAK pathway. We found that Hh pathway regulated cell migration and invasion of ovarian cancer, which may was mediated by ITGB4/FAK signaling.4. GANT61reduced tumor volume and weight of xenografts in the nude mice, and the expression of Gli1,Gli2, ITGB4and p-FAK protein all declined following GANT61treatment, Gli as the final transcription factor of Hh pathway, it may become the new promising target of ovarian cancer, and it will offer theoretical guidance of treatment strategies and drug development targeting to ovarian cancer.