Cell plasticity of ‘stem-like’ cancer-initiating cells (CICs) is a hallmark of cancer, allowing metastasis and cancer progression. Here, we studied whether simvastatin, a lipophilic statin, could impair the metastatic potential of CICs in high-grade serous ovarian cancer (HGS-ovC), the most lethal among the gynecologic malignancies. qPCR, immunoblotting and immunohistochemistry were used to assess simvastatin effects on proteins involved in stemness and epithelial-mesenchymal cell plasticity (EMT). Its effects on tumor growth and metastasis were evaluated using different models (e.g., spheroid formation and migration assays, matrigel invasion assays, 3D-mesomimetic models and cancer xenografts). We explored also the clinical benefit of statins by comparing survival outcomes among statin users vs non-users. Herein, we demonstrated that simvastatin modifies the stemness and EMT marker expression patterns (both in mRNA and protein levels) and severely impairs the spheroid assembly of CICs. Consequently, CICs become less metastatic in 3D-mesomimetic models and show fewer ascites/tumor burden in HGS-ovC xenografts. The principal mechanism behind statin-mediated effects involves the inactivation of the Hippo/YAP/RhoA pathway in a mevalonate synthesis-dependent manner. From a clinical perspective, statin users seem to experience better survival and quality of life when compared with non-users. Considering the high cost and the low response rates obtained with many of the current therapies, the use of orally or intraperitoneally administered simvastatin offers a cost/effective and safe alternative to treat and potentially prevent recurrent HGS-ovCs.
Supplementary Figure 1. Effects of simvastatin exposure in spheroid assembly and count, invasion and migration, cell death, stemness and EMT marker expression levels, and Hippo/YAP/TAZ activity, and the rescue effect of mevalonate supplementation in UCI 101-derived CICs. A) Representative microphotographies of the effects of a single pulse of simvastatin (1uM to 5uM each 24h) in CIC clustering (assembly). The bar graph, at its right, summarizes the simvastatin effects on total sphere count of three separate experiments. Vertical bars indicate ± SEM (standard error of the mean). Below the microphotographs and the bar graph, a representative gel of the effect of a single simvastatin pulse on CIC cell death is shown (measured by detection of PARP cleavage). B) Bar graph showing the average simvastatin effect on the spheroid area as seen in ten replicates at 24h exposure (three different experiments). Vertical bars indicate ± SEM. The asterisk indicates statistical significance (p < 0.05, Mann-Whitney test). Next to the graph, representative microphotographs of single spheroid migration at 24h of exposure to simvastatin or MOCK treatment. The light blue circle on each of the right panels overlaps the time 0h spheroid area, highlighting the change in spheroid size experienced over time. The small pictures under each microphotograph correspond to the magnification of the spheroid edge. The red arrows highlight the bright halo containing the initiator migratory buds. C) The bar graph shows the effect of simvastatin on spheroid migration (three different experiments). Vertical bars indicate ± SEM. The asterisk indicates statistical significance (p < 0.05, Mann-Whitney test). At the right, representative microphotographs of separate experiments are presented showing CIC migration upon simvastatin compared with MOCK treatment in matrigel Boyden chamber assays. D.1) Representative microphotographies of the effect of repeated simvastatin pulses (four pulses, 1uM to 5uM, each 24h) in CIC clustering (spheroid assembly). D.2) Representative gel of the effect of same number of pulses on CIC cell death (measured by detection of PARP cleavage). ß-actin is shown as loading control. D.3) Bar graph showing the effect of repeated simvastatin pulses (1 uM to 5 uM) on the total spheroid count after a week of incubation (three separate experiments). Vertical bars indicate ± SEM. The asterisk indicates statistical significance (p < 0.05, Mann-Whitney test). E) Representative gels of the effects of single simvastatin pulse (1uM for 24h) in protein levels of stemness (e.g., CD44, Oct-4, and SOX-2) and EMT markers (e.g., N-cadherin), and F) Hippo/YAP/TAZ activity (by detecting the phosphorylated form of YAP) in CICs, as measured by W-B. G) Representative microphotographs of the effect of mevalonate supplementation on simvastatin-induced spheroid disassembly H) Representative gels of the effect of mevalonate supplementation in simvastatin-induced CIC cell death (measured by detection of PARP cleavage). -actin is shown as a loading control.
Supplementary Figure 2. Effects of repeated simvastatin pulses on clustering and spheroid formation (when pre-incubated) and in the RhoA activity in HeyA8-derived CICs, and characterization of the aggregates isolated from ascites in mouse xenografts. A) Representative microphotographies of the effect of repeated pulses of simvastatin (four pulses, 1uM to 5uM, each 24h) in CIC clustering (assembly) and B) spheroid count when started before its formation. The bar graph summarizes the results of three separate experiments. Vertical bars indicate ± SEM (standard error of the mean). The asterisk indicates statistical significance (p < 0.05, Mann-Whitney test). C) Representative gel of active and inactive RhoA protein levels after pull-down as detected by W-B. Leptin (100 ng/ml for 30 min) was used to induce RhoA activity. D) Panels showing aggregate characterization. The left panel shows the negative control and the middle and right panels show the CD44 and ALDH1A1 staining detected in the aggregates by immunocytochemistry.
Supplementary Figure 3. Effects of simvastatin in a case of recurrent high-grade serous ovarian cancer heavily treated and resistant to chemotherapy (patient UC02). A) Original histology at diagnosis time (H-E, 10X, left), the cytological aspect of aggregates isolated from ascites at last recurrence (40X, middle) and CD44 staining of same aggregates as detected IC (40X, right) before starting simvastatin PO. B) Bar graph showing the in vitro sensitivity (MTS assay) of CICs, isolated from ascites at the time of recurrence, to different potential chemotherapeutic schemes (administered either IV or PO). Each assay was carried out in quintuplicate, and the dotted line indicate the consensus umbral between resistant and sensitive. Notice that CICs were sensitive only to schemes including taxanes, a drug discontinued in this patient due to severe neurotoxicity. C) Effects of repeated simvastatin pulses (1 to 5 M) on spheroid count (left panel) and cell death (detected by PARP cleavage, right panel) in CICs isolated from this case, as measured under HPF contrast microscopy and WB, respectively. The graph summarizes the results of three separate and consistent experiments (vertical bars indicate ± SEM [standard error of the mean]). The asterisk indicates statistical significance (p < 0.05, Mann-Whitney test). D) Evolution of CA125 and cholesterol levels through different periods of disease treatment in the same case. Arrows indicate the starting point of different therapies (black arrow points chemotherapy and red arrow points simvastatin treatment). Yellow rectangles indicate the period under statin use. Violet rectangle indicates anticoagulant treatment period. Blue bar, its size, and spacing represent each paracentesis, ascites volume, and interval among procedures, respectively. E) Relative Oct-4, CD44 and Nanog mRNA levels in ascites aggregates collected from this case before and after two weeks of simvastatin treatment (10 mg/day PO), as measured by qPCR.
Supplementary Table 1. Primers used to assess stemness/EMT markers and HMGCR mRNA levels by qPCR in ovarian cancer-initiating cells.
Supplementary Table 2. Demographics and clinical differences among statin user and non-users in a cohort of high-grade serous ovarian cancers.