EGFL6 Regulates the Asymmetric Division, Maintenance, and Metastasis of ALDH+ Ovarian Cancer Cells

Abstract

Little is known about the factors that regulate the asymmetric division of cancer stem-like cells (CSC). Here, we demonstrate that EGFL6, a stem cell regulatory factor expressed in ovarian tumor cells and vasculature, regulates ALDH⁺ ovarian CSC. EGFL6 signaled at least in part via the oncoprotein SHP2 with concomitant activation of ERK. EGFL6 signaling promoted the migration and asymmetric division of ALDH⁺ ovarian CSC. As such, EGFL6 increased not only tumor growth but also metastasis. Silencing of EGFL6 or SHP2 limited numbers of ALDH⁺ cells and reduced tumor growth, supporting a critical role for EGFL6/SHP2 in ALDH⁺ cell maintenance. Notably, systemic administration of an EGFL6-neutralizing antibody we generated restricted tumor growth and metastasis, specifically blocking ovarian cancer cell recruitment to the ovary. Together, our results offer a preclinical proof of concept for EGFL6 as a novel therapeutic target for the treatment of ovarian cancer. Cancer Res; 76(21); 6396-409. ©2016 AACR.

Figure 1. Expression of EGFL6 in ovarian tumors and normal tissue

A. Expression of EGFL6 across ovarian cancers histologies in the (i) Hendrix dataset (ii) TCGA, GTEx datasets. B. IHC of EGFL6 in the indicated tissues. C. Immunofluorescence of EGFL6 (red) and CD31 (green) in human tumor vasculature (blue is DAPI staining of nucleus). D. qRT-PCR analysis of EGFL6 expression in ovarian cancer cell lines, MCF7 (breast cancer), HEK293 (kidney), and hemangioma stem cells (HemSC) controls. E. Kaplan Meier curves for recurrence-free and overall survival for ovarian cancer patients with or without vascular EGFL6 expression in primary debulking specimens. F. GSEA demonstrating EGFL6-correlated genes are associated with invasive ovarian cancer phenotype and an Embryonic Stem Cell core signature. See Suppl.Fig1C for ES scores, p-values, and FDR q-values.

Figure 2. Effect of EGFL6 on ovarian cancer cell proliferation

A(i). Western blot of EGFL6 following control or EGFL6 transfection of HEK293 cells, (ii) Coomassie stain of the steps of EGFL6 purification. B. Total cell number for EGFL6 and vehicle control-treated SKOV3 cells and primary tumor cells (PT112 and PT122). C. Cell-cycle analysis of EGFL6-treated SKOV3 cells showing, (i) Summary of 3 independent analyses and, (ii) Representative cell cycle profile. Experiments were performed in duplicate. Error bars represent standard deviations.

Figure 3. EGFL6 promotes ALDH⁺ cell asymmetric division

A. Summary of 3 replicate experiments demonstrating EGFL6 treatment is associated with, (i) increasing total cell numbers, (ii) decreasing percentages of ALDH⁺ cells, but (iii) no change in absolute ALDH⁺ cell number. B. Percentages of ALDH⁺ primary ovarian cancer cells following treatment with EGFL6 or vehicle. C. Single cell microfluidic culture showing, (i) Representative immunofluorescence images demonstrating initial ALDEFLUOR stain (ALDH⁺ green, ALDH⁽⁻⁾ gray) in captured single cells and the observed types of cell division outcomes for ALDH⁽⁻⁾ vs. ALDH⁺ cells after capture, (ii–iii) Summary of percentages of division events and average number of progeny/microfluidic well with EGFL6 or vehicle treatment of (ii) SKOV3 cells and (iii) 3 primary patient samples. SKOV3 cells were analyzed in 3 independent experiments. Primary samples were analyzed in 2 independent experiments.

Figure 4. EGFL6 signaling requires Integrin-mediated phosphorylation of SHP2

A. SKOV3 cell numbers after 72 hours of treatment with EGFL6 or EGFL6^RGE. B. qRT-PCR demonstrating increased expression of Integrin β₃, but not β₁ or β₅, mRNA levels in ALDH⁺ vs. ALDH⁽⁻⁾ ovarian cancer cells. C. FACS plot demonstrating the Integrin β1/β3 competitive inhibitor Echistatin inhibits EGFL6-mediated reduction in ALDH⁺ cell percentages. D. Western blot analysis of the indicated proteins with and without EGFL6 treatment demonstrating (i) SHP2 is preferentially phosphorylated in ALDH⁺ cells and EGFL6 further increases SHP2 activation in ALDH+ cells. EGFL6 treatment is associated with increased p-ERK in both ALDH⁺ and ALDH⁽⁻⁾ cells, (ii) EGFL6^RGE mutant does not increase p-SHP2 or p-ERK, (iii) EGFL6-mediated SHP2 and ERK phosphorylation is suppressed by Echistatin and anti-EGFL6 treatment. Bar graphs below graphs indicate densitometric quantification of p-SHP2. E (i) SHP2 western blot of three independent SHP2 shRNA (Sh-SHP2), (ii) ALDH percentage and (iii) proliferation in Sh-SHP2 cells treated with EGFL6 or EGFL6^RGE. F(i) ALDH⁺ cell percentage and (ii) cell proliferation in SHP2 inhibitor treated cells. All experiments were performed at least twice. Error bars indicate standard deviations.

Figure 5. EGFL6 expression in tumor cells promotes ovarian tumor growth

A(i). Tumor growth curves and tumor weights of EGFL6 and control vector-transfected ovarian cancer cells (n=10/group in two independent experiments), (ii) IHC analysis and quantification of ALDH1A1 expression in EGFL6 vs. control tumors. B (i). qRT-PCR analysis of EGFL6 expression in control and EGFL6 shRNA knockdown (Sh-EGFL6) NIHOVCAR3 cells, (ii) ALDH FACS and (iii) Western blot analysis in control and Sh-EGFL6 cells, (iv and v) Tumor growth curves and overall survival for OVCAR3 control (n=10) and Sh-EGFL6 cells (n=6/group). C(i–ii) Tumor growth curves and weights, and (iii–iv) ALDH+ cell percentages and absolute number for NIHOVCAR3 tumors mock-treated or treated with anti-EGFL6 (EGFL6Ab, n=10/group). Error bars indicate standard deviation.

Figure 6. Vascular EGFL6 promotes tumor growth

A. EGFL6 Western of control and EGFL6-lentivirally-transduced infantile hemangioma stem cells (HemSC^EGFL6). B. Co-IF of human CD31 (hCD31) and EGFL6 in tumor vessels in SKOV3:HemSC^EGFL6 tumor xenografts. C. Tumor growth curves of SKOV3:HemSC^EGFL6 tumors vs. SKOV3:HemSC^Control tumors (n=10/group in two independent experiments). D. IHC analysis of Ki67 expression in EGFL6-expressing vs. control tumors. E. Tumor growth curves of freshly isolated primary patient cells co-injected with HemSC^EGFL6 or HemSC^Control (n=4 patients with 2 tumors each). F. H&E and Ki67 IHC of tumors generated with primary patient cells co-injected with HemSC^EGFL6 or HemSC^Control (n=6/group). G. (i) Tumor growth curve, (ii) weights, (iii) ALDH⁺ cell percentage, and (iv) absolute cell number, for control and anti-EGFL6-treated SKOV3 (non-EGFL6 expressing cells) flank tumors (n=10/group in two separate experiments). Error bars indicate standard deviations.

Figure 7. The role of EGFL6 in ovarian cancer metastasis

A. GSEA demonstrating EGFL6 expression correlated with metastatic gene signatures in endometrial ovarian cancer and melanoma. (See Suppl.Fig1C for ES scores, p-values, and FDR q-values.) B(i) Immunofluorescent GFP labeled ALDH⁺ SKOV3 cells after capture (top) and migration (bottom) in microfluidic migration device. Control cells (bottom left) have no gradient vs. EGFL6 gradient (bottom right), (ii) summary of distance migrated for the indicated cells from replicate experiments. C (i) Percentage of mice with identifiable metastases when SKOV3 cells or primary human ovarian cancer cells were grown subcutaneously (SQ) combined with HemSC^Control or HemSC^EGFL6, and (ii) IHC demonstrating ALDH⁺ cells in SKOV3-HemSC^EGFL6 tumor vessels. D. Percentage of metastasis to the indicated body sites in mice injected intraperitoneally with SKOV3 mock-treated (control) or treated with anti-EGFL6. E. Percentage of mice with metastasis to the indicated body sites in mice injected intravenously with SKOV3 cells and mock-treated (control) or anti-EGFL6-treated cells (n=10/group in 2 separate experiments).