Simvastatin interferes with cancer ‘stem-cell’ plasticity reducing metastasis in ovarian cancer

in Endocrine-Related Cancer
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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.

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  • 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.

 

      Society for Endocrinology

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    Identification, isolation and characterization of cancer-initiating cells (CICs) from HGS-ovC cell lines and primary tissue cultures both in vitro and in vivo. (A) Representative microphotography of morphologic aspects adopted by two ovarian cancer cell lines (HeyA8 (named as HEY in the figure) and UCI 101) and a primary tissue culture (established from ascites from a HGS-ovC) when growing under normal (2D conditions) or low-attachment conditions (3D conditions) (40×). (B and C) Relative mRNA and protein expression levels of stemness (NANOG, OCT4 and CD44) and EMT (N-cadherin, E-cadherin, TWIST and ZEB2) markers in HeyA8 cells growing under 2D and 3D conditions and measured by qPCR and WB. Graphs represent the results of three independent experiments. ACTB is shown as a loading control for WB. The bar graph at the right of blots shows the densitometry analyses. The asterisk indicates statistical significance (P < 0.05, Mann–Whitney test). The vertical bars indicate ±s.e.m. (D) The TP53 expression level in HeyA8 cells growing under 2D or 3D conditions and detected by IC. (E) Representative photographs of the whole abdominal cavities (upper panels) and segments of gut and mesentery of NSG mice 4 weeks after IP inoculation (1 × 106 cells) with either MOCK, non-selected, or HeyA8-derived CICs. The green arrows highlight metastatic foci highly vascularized either at the omentum or the mesentery. (F) Representative microphotographs of TP53, PAX8 and WT1 pattern and intensity staining as found in HGS-ovC (positive control) and in metastasis generated in the omentum by HeyA8-derived CIC intraperitoneal injection in the mouse model. The black bar in the microphotograph indicates 200 µm.

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    Effects of single and repeated doses of simvastatin on CIC-derived spheroid assembly, spheroid count, invasion and cell death. (A) Upper panels, HPF microphotograph (40×, contrast microscopy) showing the effect of a single pulse of vehicle (DMSO) or simvastatin (5 µM for 24 h) on HeyA8-derived and chemo-naïve primary culture-derived (patient UC172) spheroid integrity. Middle panels M, immunoblotting for PARP levels, as a cell death marker, upon the single pulse of vehicle or simvastatin (1–5 µM for 24 h). Lower panels, the graph represents the results of three separate and consistent experiments using a single pulse of vehicle or simvastatin on the total spheroid count. The vertical bars indicate ± s.e.m.. (B) Left panel, the effect of single simvastatin or vehicle pulse on HEY-derived CIC migration measured by Spheroid Migration Assay. The graph summarizes the results of three separate experiments (vertical bars indicate ± s.e.m.). The asterisk indicates statistical significance (P < 0.05, Mann–Whitney test). Right panel, representative microphotographs of the spheroid appearance at 0 h and 24 h of incubation upon MOCK or simvastatin treatment. (C) Effect of single simvastatin or vehicle pulse on HEY-derived CIC invasion measured by Matrigel Assays. The graph in the left summarizes the results of three separated experiments (vertical bars indicate ± s.e.m.). The asterisk indicates statistical significance (P < 0.05, Mann–Whitney test). At the right, a representative microphotography of cells trespassing to the reverse of the insert. (D) Effects on spheroid adherence and invasion in 3D-mesomimetic inserts. Red flags point spheroids on the obverse of the insert. Green flags indicate CICs trans-passing to the obverse of the insert. (E) The effect of repeated simvastatin pulses (four pulses, added after spheroid formation) on the number of spheroids formed from HeyA8 cells and primary tissue cultured cells (chemo-naïve patient UC172) after a week of incubation. Graphs represent the results of three separate experiments using repeated simvastatin pulses on the total spheroid count. The vertical bars indicate ± s.e.m. The asterisk indicates statistical significance (P < 0.05, Mann–Whitney test). (F) HPF microphotograph (40×, contrast microscopy) showing the effect of repeated pulses (up to four) of simvastatin (2–5 µM for 24 h) on HeyA8-derived and chemo-naïve primary culture-derived (patient UC172) spheroid integrity. (G) Detection of PARP cleavage by WB upon repeated pulses of simvastatin in HeyA8-derived and chemo-naïve primary culture-derived (patient UC172) spheroids. ACTB is shown as loading control of respective blots.

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    Simvastatin effects on stemness and EMT marker expression and the activity of RhoA, Hippo/YAP/TAZ in HGS-ovC CICs. (A) Relative mRNA expression levels of HMGCR in ovarian cancer cell lines (UCI101, SKOV3, and HeyA8) and chemo-naïve and recurrent HGS-ovC primary cultures growing under 2D- or 3D-conditions as measured by qPCR (the figure summarizes three different experiments and vertical bars indicate ±s.e.m.). The asterisk indicates statistical significance (P < 0.05, Mann–Whitney test). (B) Changes in relative mRNA levels of stemness markers (CD44, OCT4, NANOG and SOX2) induced by a single pulse of simvastatin (1 µM per 24 h) compared to control in HeyA8-derived CICs as measured by qPCR. The graph summarizes the results of three separate experiments (vertical bars indicate ±s.e.m.). The asterisk indicates statistical significance (P < 0.05, Mann-Whitney test). (C) Representative gels of changes in protein levels of stemness (CD44, OCT4, NANOG, SOX2 and ALDH1A1) and EMT (N-cadherin) markers induced by the simvastatin pulse compared to control in CICs as measured by WB. (D) Representative gels of effects of a single pulse of simvastatin in the absence or presence of mevalonate (100 µM, upper panels) GGPP (10 µM, lower panels) on CD44 and inactive form of RhoA protein levels in HeyA8-derived CICs as measured by WB. (E) Representative gels of effects of simvastatin pulse on phospho-YAP, YAP and TAZ protein levels in HeyA8- and chemo-naïve primary culture-derived CICs (patient UC172, left and right panels, respectively) as measured by WB. ACTB and TUBB are shown as loading controls. (F) Representative microphotography of the changes in YAP cellular localization (The red-fluorescence staining corresponds to phospho-YAP; nuclei are stained in blue) in chemoresistant primary culture-derived CICs upon a single pulse as detected by IF (patient Ubib2). (G) Representative gels of effects of mevalonate and GGPP supplementation in simvastatin-induced changes in phospho-YAP levels in HeyA8-derived CICs. β-Actin is shown as a loading control. (H) Representative microphotographs of mevalonate supplementation in simvastatin-induced disassembly of HeyA8-derived spheroids. (I) Representative gels of effects of mevalonate supplementation in simvastatin-induced cell death in HeyA8-derived CICs (measured by detection of PARP cleavage). ACTB is shown as a loading control.

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    Effects of 4-week simvastatin treatment (2 mg/kg/day IP) on tumor growth, ascites, weight, metastasis distribution and expression of CD44, ALDH1A1 in HeyA8-derived CIC inoculated NSG mice. (A) Whole animal view (closed and opened abdomen) of a representative mouse for each condition (n = 3–4 per group, three experiments). The small circle under each right panel shows the amount of disease collected after debulking procedure. The green arrows highlight main disease foci. (B) Bar graphs showing the average effect of simvastatin on ascites volume (left panel) and body weight (right panel) compared with MOCK treatment. Vertical bars indicate ±s.e.m. The asterisk indicates statistical significance (P < 0.05, Mann–Whitney test). (C) Representative microphotographs of changes in the expression of CD44 (membrane), ALDH1A1 (cytoplasm), YAP (nucleus and cytoplasm) and p-YAP (cytoplasm) staining upon simvastatin treatment (panels in third and fourth rows) compared to MOCK treatment (panels in first and second rows) at the injection site (implantation site). The small black and blue frames included in the microphotographs (first and third rows, respectively) indicate the areas shown magnified under the corresponding one (second and fourth rows, respectively). The black bar in each microphotograph indicates 200 µm. The green arrows highlight the brown staining of the nucleus and reflect the nuclear localization of YAP.

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    Effects of 4-week simvastatin treatment (2 mg/kg/day IP) on KI67 (cell proliferation), TP53 expression (a reflex of metabolic adaptation), and vascular recruitment at metastatic foci (omentum) in HeyA8-derived CIC inoculated NSG mice. (A) Representative microphotographs of omental metastasis as captured under LPF (10×, upper panels in each condition) and HPF (40×, respective underlying panels). Left upper panels, shown hematoxylin-eosin staining (H&E); middle and right upper panels show TP53 and KI67 staining in IHC. Immediately underlying panels show magnified fields of equivalent areas included in the upper panels and using the same staining technique. (B) Mouse CD31 staining of any vascular structure detected at the injection or metastasis site (panels at the left). No staining suggestive of human CIC-derived neo-angiogenesis was detected (using huCD31 or huCD34 antibody). The table, at the right, summarizes the quantification of the vascular area under each condition. The *, #, and ^ signs indicate statistical significance for specific comparisons (P < 0.05, t-test).

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    Clinical impact of using statins among patients with HGS-ovC. (A and B) Comparison of progression-free survival (PFS) and overall survival (OS) curves among statin users (green line, n = 28) and non-users (violet line, n = 91). (C) Calculated systemic immune-inflammatory index (SII) among statin users and non-users. Box, line, bars and the asterisk in the graph indicate 25–75 interquartile, median, standard deviation, and statistical significance, respectively. (D) Cox proportional-hazards regression model including significant variables known for influencing OS in HGS-ovC.

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