EGFL6 is an important growth factor involved in endometrial cancer tumorigenesis (073)

Abstract

Objectives: Epidermal growth factor-like domain 6 (EGFL6) has been linked with obesity and implicated in the tumorigenesis of multiple tumor types. Endometrial cancer (EnCa) is most strongly linked with obesity; however, little is known about the role of EGFL6 in the tumorigenesis of EnCa. The objective of this study was to investigate the role of EGFL6 in EnCa tumorigenesis. Methods: Cell proliferation, tumor spheroid, transwell migration, and scratch assays were performed using three established human endometrial cancer cell lines (HEC-1-A, Ishikawa, and MFE-280). Cell lines were treated with recombinant EGFL6 (rEGFL6), and Western blot analysis was performed to examine EGFL6 signaling pathways. A lentivirus transduction system was used to overexpress EGFL6 in a low-EGFL6 expressing cell line (HEC-1-A). shRNA was used to knock down EGFL6 in two cell lines with intermediate (Ishikawa) and high (MFE-280) EGFL6 baseline expression. Tumor xenografts of overexpression and knockdown EGFL6 clones and their respective vector controls were injected in NSG mice. Tumor volume was monitored twice weekly, and tumor weights were obtained at the time of euthanasia. Comparisons between groups were made with the student’s t-test (two-tail, p<0.05). One-way ANOVA was used to analyze differences in mean tumor volume across groups. EGFL6 overexpressing mice were evaluated for obesity and uterine tumor growth. The Human Protein Atlas dataset was analyzed for the impact of EGFL6 expression on overall survival in EnCa. Results: EGFL6 overexpression and treatment with rEGFL6 of low and intermediate EGFL6-expressing cell lines resulted in significantly increased total spheroid number (p<0.05). Parental cell lines exposed to rEGFL6 and EGFL6-overexpressing clones demonstrated increased migration compared to unstimulated cells (p<0.05) and vector control (p=0.009), respectively. EGFL6 knockdown clones showed decreased total tumor spheroid number and migration compared to vector controls (p<0.05). Western blot analysis demonstrated an increase in phosphorylated MAPK at 7.5 minutes after treatment with rEGFL6 compared to no-treatment controls. Tumor xenograft studies showed that EGFL6 overexpression significantly increased tumor growth with EGFL6 overexpression (p<0.0001), whereas EGFL6 downregulation significantly decreased tumor growth when compared to controls (p<0.001). Transgenic EGFL6 overexpressing mice developed massive obesity, uterine hypertrophy, and in some cases, uterine tumors. Consistent with these results, we found that patients whose EnCa express high levels of EGFL6 had significantly worse overall survival than patients whose tumors express low levels of EGFL6 (67% vs 80%, p=0.004). Conclusions: Our findings indicate EGFL6 plays an important role in EnCA growth. Importantly, EGFL6 may represent a biological link between obesity and the development of EnCa. Finally, these results indicate that EGFL6 represents a potential therapeutic target in endometrial cancer. Objectives: Epidermal growth factor-like domain 6 (EGFL6) has been linked with obesity and implicated in the tumorigenesis of multiple tumor types. Endometrial cancer (EnCa) is most strongly linked with obesity; however, little is known about the role of EGFL6 in the tumorigenesis of EnCa. The objective of this study was to investigate the role of EGFL6 in EnCa tumorigenesis. Methods: Cell proliferation, tumor spheroid, transwell migration, and scratch assays were performed using three established human endometrial cancer cell lines (HEC-1-A, Ishikawa, and MFE-280). Cell lines were treated with recombinant EGFL6 (rEGFL6), and Western blot analysis was performed to examine EGFL6 signaling pathways. A lentivirus transduction system was used to overexpress EGFL6 in a low-EGFL6 expressing cell line (HEC-1-A). shRNA was used to knock down EGFL6 in two cell lines with intermediate (Ishikawa) and high (MFE-280) EGFL6 baseline expression. Tumor xenografts of overexpression and knockdown EGFL6 clones and their respective vector controls were injected in NSG mice. Tumor volume was monitored twice weekly, and tumor weights were obtained at the time of euthanasia. Comparisons between groups were made with the student’s t-test (two-tail, p<0.05). One-way ANOVA was used to analyze differences in mean tumor volume across groups. EGFL6 overexpressing mice were evaluated for obesity and uterine tumor growth. The Human Protein Atlas dataset was analyzed for the impact of EGFL6 expression on overall survival in EnCa. Results: EGFL6 overexpression and treatment with rEGFL6 of low and intermediate EGFL6-expressing cell lines resulted in significantly increased total spheroid number (p<0.05). Parental cell lines exposed to rEGFL6 and EGFL6-overexpressing clones demonstrated increased migration compared to unstimulated cells (p<0.05) and vector control (p=0.009), respectively. EGFL6 knockdown clones showed decreased total tumor spheroid number and migration compared to vector controls (p<0.05). Western blot analysis demonstrated an increase in phosphorylated MAPK at 7.5 minutes after treatment with rEGFL6 compared to no-treatment controls. Tumor xenograft studies showed that EGFL6 overexpression significantly increased tumor growth with EGFL6 overexpression (p<0.0001), whereas EGFL6 downregulation significantly decreased tumor growth when compared to controls (p<0.001). Transgenic EGFL6 overexpressing mice developed massive obesity, uterine hypertrophy, and in some cases, uterine tumors. Consistent with these results, we found that patients whose EnCa express high levels of EGFL6 had significantly worse overall survival than patients whose tumors express low levels of EGFL6 (67% vs 80%, p=0.004). Conclusions: Our findings indicate EGFL6 plays an important role in EnCA growth. Importantly, EGFL6 may represent a biological link between obesity and the development of EnCa. Finally, these results indicate that EGFL6 represents a potential therapeutic target in endometrial cancer.