ILK correlates with HGSOC patient outcomes and OCSC phenotypes. Analysis of survival curves of advanced HGSOC patients (stage: 3 + 4, grade: 3) in KM database demonstrated that higher than median ILK expression correlated with poor PFS and OS after optimal debulking surgery (Fig. 1A,B, respectively), In addition, ILK expression was higher in HGSOC metastatic samples compared to primary tumor (TNMplot analysis; Fig. 1C), further supporting a role of ILK in OC progression. Regression analysis of HGSOC tumors in the TCGA demonstrated that ILK and ALDH1A1 levels were highly correlated (Fig. 1D), and co-occurring alterations between ILK and ALDH1A1 were observed (Fig. 1E). Moreover, higher than median ILK and ALDH1A1 combined expression levels were associated with worse progression-free survival (PFS) and overall survival (OS) after optimal debulking surgery in patients with advanced HGSOC (stage: 3 + 4, grade: 3) (Fig. 1F,G, respectively). Based on these strong correlations, we further examined ILK in OCSC phenotypes.
We measured p-ILKSer246 in human HGSOC cell lines and primary cells isolated from malignant ascites of HGSOC patients. Increased expression of p-ILKSer246 and ALDH1A1 was observed in OC cell lines and primary cells grown as spheroids compared to monolayers (Fig. 1H, I), as was increased expression of stemness-related markers ALDH1A1, Sox-2, Nanog and Oct-4 (Fig. S1A). Furthermore, expression of ILK (Fig. 1J) and stemness markers (Fig. S1B) was increased in OCSCs (ALDH+/CD133+) compared to non-OCSCs (ALDH−/CD133−). In primary tumors from patients diagnosed with HGSOC (stage: 3 + 4, grade: 3), p-ILKSer246 was greater compared to corresponding adjacent ovarian epithelium (Fig. 1H), demonstrating increased ILK activation, and p-ILKSer246 staining was greatest in metastatic tumors (Fig. 1K and Table S4). Taken together, these results support further examining ILK in OC progression and chemoresistance.
ILK inhibition inhibits OCSCs in vitro and prevents tumor-initiating capacity in vivo. To examine the functional role of ILK activation in OCSCs, ILK expression levels or activity were altered by using either sh-RNA mediated knockdown (KD) or pharmacological inhibition with compound 22 (cpd-22; specific cell-permeable ILK inhibitor (27)). ILK expression levels (Fig. S2A), spheroid formation (Fig. S2B) and colony formation capacity (Fig. S2C) were significantly decreased in sh-ILK transduced OC cells. Treatment of HGSOC primary cells with cpd-22 inhibited both spheroid and colony formation by (Fig. 2A,B), and flow cytometry analysis showed that cpd-22 treatment decreased the ALDH+/CD133+ population in HGSOC primary spheroids compared to DMSO vehicle control (Fig. 2C).
OCSCs are responsible for initiating tumor formation in vivo when injected in immunocompromised mice (31). ALDH+/CD133+ cells (1 x 10^5 cells) were cultured under stem cell conditions and treated with cpd-22 or DMSO for 7 days and then injected SC into the flanks of female nude mice. Tumor growth was inhibited by cpd-22 (Fig. 2D), and active-p-ILKSer246 and ALDH1A1 levels in the tumors were decreased by treatment with cpd-22 (Fig. 2E). In addition, single cells derived from cpd-22-treated tumors (Fig. 2F) were incapable of forming spheroids (Fig. 2G) and the ALDH+/CD133+ cell population was reduced compared to cells derived from vehicle treated tumors (Fig. 2H). These results support a key role for p-ILKSer246 activation in maintaining the OCSC phenotype and ILK as target for reducing OC tumor formation.
ILK modulates Fzd7 expression to maintain the OCSC phenotype. To further examine the underlying mechanism of action of ILK in OCSCs, FACS-sorted ALDH+/CD133+ cells were isolated, treated with cpd-22, grown as spheroids for 7 days and evaluated using a human CSC-focused gene array. Expression of genes related CSC maintenance (ALDH1A1, KIT, Prom1) and pluripotency (Myc, Nanog, Sox-2) was downregulated in cpd-22-treated cells compared to control cells (Fig. 3A, B; Table S5). Furthermore, we examined a key developmental pathway linked to cancer stemness in OC spheroids, the Wnt pathway, and ILK inhibition markedly downregulated several pathway members, including Fzd7 and β-catenin target gene c-Myc at mRNA levels (Fig. 3A, C; Table S4). WB of ILK-KD OC cells grown as spheroids showed decreased p-ILKSer246, Fzd7 and non-p-(active) β-CateninSer33/37/Thr41 (A-β-catenin) expression levels compared to shCtr cells (Fig. 3D). Consistently, p-ILKSer246, Fzd7 and A-β-catenin levels were reduced in HGSOC primary spheroids treated with cpd-22 (Fig. 3E). Moreover, Wnt-3A-treatment of OC spheroids with Fzd7-KD decreased p-ILKSer246 levels (Fig. 3F, Fig. S3B) and phosphorylation of GSK-3α/β at Ser21/9 was abrogated, preventing nuclear translocation of A-β-catenin spheres (Fig. 3G, Fig. S3C). Increased Fzd7 expression levels in the presence of Wnt-3A (Fig. 3F,G) confirmed direct β-catenin transcriptional regulation of this Wnt receptor (28).
ILK forms a complex and directly interacts with Fzd7 in OC spheroids. As active ILK may act as a Wnt/β-catenin co-activator through Fzd7, it was of interest to examine a possible interaction between ILK and Fzd7. First, OC spheroids isolated from two primary OC patient specimens and treated with Wnt-3A showed increased IF staining for Fzd7 and p-ILKSer246 compared to untreated spheroids (Fig. 4A). IF confocal and co-localization analysis showed that Fzd7 and p-ILKSer246 co-localized in HGSOC primary spheroids and that co-localization increased in the presence of Wnt-3A (Rcoloc = 0. 6, Fig. 4A). Second, to determine whether Fzd7-p-ILKSer246 complexes were detectable in human tumors, proximity ligation assay (PLA), a technique capable of identifying proteins localized within 40 nm distance in tissue, was utilized. Fzd7-p-ILKSer246 complex formation was detectable in spheroids derived from primary human malignant ascites and increased upon Wnt-3A treatment (Fig. 4B). Next, co-IP in cell lysates from OC spheroids and human recombinant Fzd7 and ILK proteins demonstrated that Fzd7 and ILK were detectable in complexes immunoprecipitated with anti-ILK and anti-Fzd7 Abs (Fig. 4C,D).
It was also of interest to evaluate OC patient specimens for a Fzd7 and p-ILKSer246 correlation. IHC analysis in primary tumors from patients diagnosed with HGSOC (stage: 3 + 4, grade: 3) revealed increased Fzd7 and active-p-ILKSer246 levels compared to corresponding adjacent normal ovarian epithelium (Fig. 4E), both Fzd7 and p-ILKSer246 staining was greatest in metastatic tumors (Fig. 4E and Tables S4 and S6). A regression analysis further revealed a high correlation between active ILK and Fzd7 levels in patient tumors (Fig. 4F; Pearson r = 0.31, P = 0.019), and a tendency of co-occurring alterations between ILK and Fzd7 in HGSOC tumors in the TCGA database (Fig. 4G,H). Furthermore, analysis of HGSOC tumor microarray data using OvMark demonstrated an increased estimated risk of death in patients with higher than median Fzd7 and ILK combined expression levels compared with patients with lower than median Fzd7 and ILK expression levels (Fig. 4I). Overall, the results support ILK, through its Fzd7 partner, as a direct Wnt/β-catenin co-activator and plays a key role in OC spheroid formation and OC metastasis.
ILK is a β-catenin target gene. Based on the observation that ILK was activated by Wnt-3A treatment (Fig. 3F), it was of interest to examine whether ILK is a direct Wnt target gene. As shown in Fig. S4A, ILK expression was decreased by siRNA-mediated β-catenin downregulation, supporting the hypothesis that ILK is transcriptionally regulated by β-catenin. By using a promoter motif searching software (PROMO), a potential TCF/LEF-responsive element was identified in the ILK promoter sequence (positions − 103 to − 96). To determine whether β-catenin interacts directly with the ILK promoter, chromatin pulled down by a β-catenin antibody were PCR amplified using primers specific for fragments corresponding to the TCF/LEF-responsive regions (Fig. S4B). No PCR product in chromatin immunoprecipitated with immunoglobulin G (IgG) was observed (Fig. S4B), demonstrating the specificity of the β-catenin antibody used for ChIP and that ILK is a direct Wnt/β-catenin target in OC cells.
ILK/Fzd7 predict response to chemotherapy and its targeting sensitizes OC to platinum. The majority of women diagnosed with advanced-stage epithelial OC experience tumor recurrence associated with chemoresistance (29). The above results indicated a role for both ILK and Fzd7 in platinum resistance. Tumor specimens from patients diagnosed with advanced (grade 3, stage 3) serous OC were divided in responders and non-responders to platinum–taxane therapy and analyzed in the ROC plotter database (30). Mann–Whitney test and ROC analysis demonstrated that higher than median expression of ILK, Fzd7, and/or combination of ILK-Fzd7 was associated with poor response to platinum–taxane therapy (Fig. 5A-C). KM analysis showed that higher than median expression levels for ILK, Fzd7, and/or combination of ILK-Fzd7, correlated with poor PFS in patients diagnosed with advanced (grade 3, stage 3 + 4) serous OC and received platinum–taxane therapy (Fig. 5D-F), further supporting a prognostic/predictive role of ILK and Fzd7 in HGSOC patients in response to chemotherapy.
To investigate the underlying mechanism of the Fzd7/ILK axis in chemoresistance, cell lines derived from a patient diagnosed with HGSOC (PEA1, platinum sensitive (S)) and from the same patient with acquired platinum resistance (PEA2, platinum resistant (R)) were utilized. In addition, OC-R cell lines (OVCAR-3-R and HEY-A8-R) were generated by continuous in vitro exposure to platinum at IC50 concentrations (IC50 = 4.2 µM for OVCAR-3 and IC50 = 6.1 µM HEY-A8 cells) (Fig. S5A). Increased expression of p-ILKSer246 and the active ILK-target p-AKTSer473 were observed in PEA2, OVCAR-3-R and HEY-A8-R cells compared to the S cell counterparts (Fig. 5G, Fig. S5A). Furthermore, Fzd7 expression and A-β-catenin levels were increased in the R compared to the S cells (Fig. 5H, Fig. S5B). In addition, ILK inhibition by cpd-22 or Fzd7-KD blocked spheroid proliferation and colony formation (Fig. 5I,J) and increased the sensitivity to platinum compared to controls (Fig. 5K,L).
To investigate the mechanism underlying Fzd7/ILK axis in chemoresistance, anti-apoptotic signaling through AKT, one of the known ILK targets (21), was examined. Carboplatin treatment increased p-ILKSer246, p-AKTSer473, and Fzd7 levels in OC spheroids compared to controls (Fig. 6A-C), which were inhibited by cpd-22 treatment or Fzd-7-KD (Fig. 6A-C). Activation of p-AKTSer473 inhibits the pro-apoptotic activity of Bad by promoting phosphorylation at Ser136 (31). Cpd-22 and Fzd7-KD decreased p-BADSer136 levels in platinum treated OC spheroids and increased cleaved caspase 3 (Fig. 6B,C), enhancing the pro-apoptotic signaling prompted by platinum treatment.
ILK inhibition targets the platinum resistant OCSC population and improves response to chemotherapy. In OC, spheroids contribute to ip dissemination and metastasis (5, 32). We examined the effects of cpd-22 on tumor formation and dissemination alone or in combination with platinum in an ip OC xenograft model. Tumor volume, weight, and the number of peritoneal implants were significantly decreased in xenografts treated with either drug alone compared to control cells (Fig. 7A-D). However, the combination of cpd-22 plus carboplatin was significantly more effective in decreasing tumor volume and weight and inhibiting the number of implants compared to carboplatin alone (Fig. 7A-D). Cpd-22 was well tolerated, indicated by no overt signs of toxicity or loss of final body weight at time of sacrifice compared to initial body weight (Fig. S6), in agreement with a previous study (27).
Next, tumors from mice treated with either vehicle, carboplatin, or cpd-22 alone or combination of cpd-22 with carboplatin were dissociated to single-cell suspension at the end of treatment and cells were analyzed for Aldefluor positivity, CSC markers, survival signaling pathways and spheroid formation ability (Fig. 7E). Compared to vehicle-treated mice, single cells isolated from carboplatin treated mice showed increased spheroid formation (Fig. 7F) and expression of stemness markers (ALDH1A1, Nanog, Oct-4, Sox-2) (Fig. 7G,H). Interestingly, decreased spheroid proliferation and CSC signature genes were observed in cells isolated from mice treated with cpd-22 alone compared to vehicle or carboplatin alone (Fig. 7F-H). Spheroid formation ability (Fig. 7F) and expression of the CSC markers (Fig. 7G, H) were further decreased by the combination of cpd-22 plus carboplatin compared to vehicle alone.
To examine the functional consequence of blocking outside in signaling by ILK on the OCSC population, cells grown as spheroids from carboplatin treated tumors were examined. Increased levels of Fzd7, active p-ILKSer246, and active p-AKTSer473 were observed compared to vehicle (Fig. 7I). Both cpd-22 alone and moreover the combination of cpd-22 plus carboplatin decreased p-ILKSer246, p-AKTSer473, Fzd7 and the activation of pro-apoptotic pathways, demonstrated by decreased phosphorylation of Bad at Ser136, loss of Bad inhibition as determined by increased cleaved caspase-3 levels (Fig. 7I). Collectively, our data support the targeting of ILK in combination with chemotherapy as promising intervention to decrease the chemoresistant OCSC population and improve response to platinum.