Host CYP27A1 expression is essential for ovarian cancer progression

in Endocrine-Related Cancer
Correspondence should be addressed to E R Nelson: enels@illinois.edu
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There is an urgent need for more effective strategies to treat ovarian cancer. Elevated cholesterol levels are associated with a decreased progression-free survival time (PFS) while statins are protective. 27-Hydroxycholesterol (27HC), a primary metabolite of cholesterol, has been shown to modulate the activities of the estrogen receptors (ERs) and liver x receptors (LXRs) providing a potential mechanistic link between cholesterol and ovarian cancer progression. We found that high expression of CYP27A1, the enzyme responsible for the synthesis of 27HC, was associated with decreased PFS, while high expression of CYP7B1, responsible for 27HC catabolism, was associated with increased PFS. However, 27HC decreased the cellular proliferation of various ovarian cancer cell lines in an LXR-dependent manner. Intriguingly, ID8 grafts were unable to effectively establish in CYP27A1−/− mice, indicating involvement of the host environment. Tumors from mice treated with 27HC had altered myeloid cell composition, and cells from the marrow stem cell lineage were found to be responsible for the effects in CYP27A1−/− mice. While inhibition of CYP27A1 or immune checkpoint did not significantly alter tumor size, their combination did, thereby highlighting this axis as a therapeutic target.

Downloadable materials

  • Supplemental Table 1: Expression of key genes in cell models used in this study. Cq. refers to the threshold, as determined by the provided software. Expression refers to relative expression (delta-delta Ct method), and normalized to TBP expression. “Not expressed” refers to below detection limit.
  • Supplemental Table 2: Expression of key genes within tumors from this study. Above: MOE tumors corresponding to those in Fig.4A. Below: ID8-Luc tumors corresponding to those in Fig. 5A. Cq. refers to the threshold, as determined by the provided software. Expression refers to relative expression (delta-delta Ct method), and normalized to TBP expression. Please note that CYP7B1 amplified with two peaks, and thus this Cq value is overestimated.
  • Supplementary Figure 1: A high cholesterol diet increases the tumor growth rate of ID8 ovarian tumors. Mice were placed on a high cholesterol diet or control diet 4 weeks prior to tumor establishment. ID8-luc cancer cells were grafted into one ovary/bursa of C57BL/6 mice. (A) Mice on a high cholesterol diet did not gain weight as observed for mice on a control diet. Day 0 represents the day of the graft with ID8 cells. (B) Tumor growth was monitored over time by in vivo bioluminescence imaging. Linear regression analysis was used to fit the data and ANCOVA found significant differences between the slopes of the lines. Two-way ANOVA followed by a Bonferroni’s T test also found significant differences between the signal on the last day of imaging.
  • Supplementary Figure 2: Human Tumor Analysis. (A-B): similar analysis to Fig. 1. Data and statistical analysis were obtained with Kaplan-Meier Plotter, where patients with serous ovarian cancer and optimal debulking were selected. (A) Elevated expression of CYP27A1 (upper quartile), the enzyme responsible for 27HC synthesis, is associated with poor overall survival among patients diagnosed with early stage disease (stages 1 and 2) (n=79), (B) Elevated expression of CYP27A1 (upper tertile) within patients diagnosed with advanced stage disease (stages 3 and 4) is slightly protective (n=395). (C-D) Association of CYP27A1 with OS and PFS across several datasets, when CYP27A1 expression was considered as a continuous variable. (E-F) Association of CYP7B1 with OS and PFS across several datasets, when CYP7B1 expression was considered as a continuous variable. (G-H) Association of age at diagnosis with OS and PFS. (I) Association of tumor grade with OS. (J-K) Association of ESR1 (ERα) with OS and PFS. (L-M) Association of TFF1, a target gene of ERα, with OS and PFS. (N-O) Association of LXRα with OS and PFS. (P-Q) Association of ABCA1, an LXR target gene, with OS and PFS.
  • Supplementary Figure 3: 27HC has minimal impact on the proliferation of ER+ cell lines, or is even stimulatory in PEO4 cells. Indicated cell lines were treated with indicated ligands and proliferation was assessed by total DNA by Hoescht 33342 staining. PEO4 cells that express high levels of ER were stimulated to proliferate by estradiol (E2) or 27HC. This activity was inhibited by co-treatment with the ER antagonist, ICI 182 780 (ICI). Indicated doses of 27HC are log base 10.
  • Supplementary Fig. 4: Tumors fail to thrive in mice lacking CYP27A1. Luciferase tagged ID8 murine ovarian cancer cells (ID8-luc) were orthotopically grafted into the anterior left ovary/bursa area of C57BL/6 mice at Day 0. Tumor growth was monitored with bioluminescent imaging. (A) A representative image of these mice is depicted here, corresponding to quantified data in Fig. 5A. (B) Likewise, representative images of tumor ovary (left side), uterus and control ovary (right side) at the end of the experiment, corresponding to the quantified data in Fig. 5A. (C) Flow analysis of spleens isolated from mice on day 50, indicating decreased M-MDSCs and increased mature macrophages. Asterisks indicate statistical significance (P<0.05).
  • Supplementary Fig. 5: Ovarian cancer introduced by intraperitoneal graft fail to thrive in mice lacking CYP27A1. Luciferase tagged ID8 murine ovarian cancer cells (ID8-luc) were grafted by intraperitoneal injection into C57BL/6 mice at Day 0. Tumor growth was monitored with bioluminescence imaging. (A) A representative image of these mice is depicted here, corresponding to quantified data in Fig. 5G. (B) Quantification of biolumunescent imaging through time. Two-way ANOVA with Bonferroni multiple comparison test was performed. Asterisks indicate statistical significance (P<0.05). (C) Percent of mice with visible peritoneal tumors upon necropsy.
  • Supplementary Fig. 6: Immunophenotyping of cancer-naïve wildtype and CYP27A1-/- mice. Tissues were removed from female mice, dispersed or digested and stained with antibodies, and assessed by flow cytometry (similar to Fig. 6). (A) Bone Marrow. (B) Spleen. (C) Inguinal, mesenteric and para-aortic lymph nodes. Asterisks indicate statistical significance (P<0.05).

 

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    Enzymes responsible for 27HC synthesis and catabolism are implicated in overall and progression free survival of ovarian cancer patients. Data and statistical analysis were obtained with Kaplan–Meier Plotter, an online tool for genome-wide validation of survival-associated biomarkers, which curated TCGA, GEO and EGA databases. Among ovarian cancer patients with optimal debulking: (A) Elevated expression of CYP27A1 (upper quartile), the enzyme responsible for 27HC synthesis, is associated with poor overall survival among patients diagnosed with early stage disease (stages 1 and 2) (n = 102). (B) Elevated expression of CYP27A1 (upper tertile) within patients diagnosed with advanced stage disease (stages 3 and 4) is slightly protective (n = 595). (C) Very low CYP27A1 expression (lower quartile) is protective against cancer recurrence (n = 696). (D and E) Elevated expression of CYP7B1 (lower tertile), the enzyme responsible for 27HC catabolism is not associated with overall survival among patients diagnosed with either (D) early stage disease (stages 1 and 2, n = 102) or (E) advanced stage disease (stages 3 and 4, n = 595). (F) Among all ovarian cancer patients, elevated expression of CYP7B1 (upper 57%) is associated with an increased progression-free survival time. For this analysis (F) the auto-calculated best cutoff was selected with follow up threshold of 5 years (n = 1435). P values as determined by the Mantel–Cox or Gehan–Breslow–Wilcoxon test are as indicated in the figure.

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    27HC displays anti-proliferative activity in ovarian cancer cells. (A and B) Inhibition of CYP27A1 with the small-molecule inhibitor GW273297X (GWX, 10 μM) does not significantly alter the proliferation of ID8 or HEY A8 ovarian cancer cells in vitro. Cells were treated for 6 days with either vehicle or GWX prior to assessment of total DNA by Hoescht 33342 staining. (C) siRNA-mediated knockdown of CYP27A1 does not significantly alter the proliferation of ID8 cells in vitro. (D, E and F) Treatment with exogenous 27HC at indicated doses significantly decreases proliferation of ID8, MOE or HEY A8 cells in vitro. (G, H, I and J) Dose–response curves of 27HC at indicated doses on inhibition of ID8, MOE, HEY A8 or ES2 proliferation. Non-linear regression was used to fit the curve. Statistics for: A-C unpaired T test. D-F two-way ANOVA followed by Bonferroni T test, asterisks indicating significant differences from vehicle (P < 0.05).

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    27HC decreases ovarian cancer cell proliferation via activation of the LXRs and subsequent cholesterol efflux. (A, B, C and D) Similar to 27HC, a synthetic LXR agonist, GW3965, also decreases proliferation of ID8, MOE, HEY A8 and ES2 cells. (E) 27HC or GW3965 do not significantly alter cell viability as assessed by an MTT assay. (F) Heat map indicating gene expression changes of ID8 cells as assessed by quantitative PCR (QPCR). Left Panel: ID8 cells were transfected with a control vector, or one encoding a constitutively active nuclear SREBP-2 form (nSR2, nSREBP-2) and treated with either vehicle or 27HC (V, 27). Middle Panel: ID8 cells were treated with the SREBP-2 activators haloperidol (H, Hal), clozapine (C, Clo), or the inhibitor Betulin (B, Bet), in the presence or absence of 27HC. Right Panel: ID8 cells were transfected with control siRNA (SiC), siRNA against LXRα/β (siLXR) or siRNA against ERα (siER), and then treated with either vehicle or 27HC. A scale for the heat map is located on the far right. Classic SREBP-2 target genes include HMGCR, LDLR, HMGCS, Insig-1 and SQLE. Classic LXR target genes include ABCA1, ABCG1 and SREBP-1c. (G, H and I) Proliferation of ID8 cells under the same experimental conditions as described for (F) indicates that the effects of 27HC depend on the expression of the LXRs. (J, K, L and M, N, O) Parallel experiments were performed on MOE and HEY A8 cells respectively, yielding similar results. (P) Inhibition of proliferation by 27HC is attenuated by co-treatment with the LXR antagonist GSK2033 in HEY A8 cells. (Q) Inhibition of proliferation by 27HC is attenuated by supplementation with exogenous LDL cholesterol. One-way ANOVA followed by student Newman–Keul’s test was used for statistical analysis, with different letters indicating significant differences between groups (P<0.05, as in a is different than b, but ab is not different from either a or b).

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    27HC increases peritoneal spread of ovarian cancer and imparts resistance to carboplatin. (A) Primary tumor weight of MOE grafts at an early stage, being treated with a low dose of carboplatin (16 mg/kg every 4 days). (B) Relative peritoneal nodules resulting from MOE tumors in (A). (C) Primary tumor weight of MOE grafts at a late stage, being treated with a higher dose of carboplatin (40 mg/kg every 4 days). (D) Relative peritoneal spread resulting from MOE tumors in (C). Different letters denote statistical significance (P < 0.05, one-way ANOVA followed by student Newman–Keuls test).

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    Tumors fail to thrive in mice lacking CYP27A1. (A) Luciferase tagged ID8 murine ovarian cancer cells (ID8-luc) were orthotopically grafted into the anterior left ovary/bursa area of C57BL/6 mice at Day 0. Tumor growth was monitored with bioluminescent imaging. A representative image of these mice is depicted in Supplementary Fig. 4. (B) The experiment in (A) was repeated in a separate cohort of mice, yielding similar results. (C) Tumor burden at Day 61 as assessed by bioluminescent imaging, from the experiment in (B). (D) Representative images of luciferase expression at Day 61, from the experiment in (B). (E) Representative images of tumor ovary (left side), uterus and control ovary (right side) at the end of the experiment (Day 63), from the experiment in (B). (F) Primary tumor weight at experiment endpoint (Day 63), from the experiment in (B). (G) ID8 grafts fail to thrive when introduced by intraperitoneal (i.p.) injection. ID8 cells were injected into the peritoneal space and followed through time by bioluminescent imaging. Bioluminescence at day 49 post-graft is depicted here. The corresponding time course is included in Supplementary Fig. 5. (H) CYP27A1−/− mice were supplemented with 27HC (20 mg/kg/day) starting 5 days prior to tumor engraftment. At Day 0, ID8-luc murine ovarian cancer cells were grafted into one ovary/bursa of the mice. At day 74, mice were randomized into two groups with the 27HC group continuing to receive 27HC while 27HC Withdraw group received vehicle instead. The final tumor mass was determined at day 99. Statistical analysis for A and B was performed with two-way ANOVA followed by a Bonferroni multiple comparison test or Student T-test. Two-tailed T tests were used for C, and F–H. Asterisks indicate statistical significance (P < 0.05).

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    27HC alters immune cell populations within ovarian tumors and promotes the differentiation of M-MDSCs. (A) Tumors grown in wildtype mice had significantly more CD11B+ myeloid cells compared to ovarian tissue from CYP27A1−/− mice. (B, C, D, E, F and G) 27HC treatment resulted in alterations of the indicated immune cell populations. (H) 27HC stimulated the in vitro differentiation of M-MDSCs from bone marrow progenitors in the presence of IL6 and GM-CSF. (I) Microarray expression data for 551 ovarian cancer patients was accessed and parsed based on median expression of CYP27A1, into low and high groups. Expression of CD33, a human marker of M-MDSCs was plotted. Asterisks indicate statistical significance (P < 0.05).

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    Wildtype bone marrow transplant is sufficient to allow ID8 tumors to grow in CYP27A1−/− mice. CYP27A1−/− mice were irradiated and transplanted with wildtype (+/+) or CYP27A1−/− bone marrow. After allowing sufficient time for donor bone marrow to reconstitute all cell lineages, luciferase tagged ID8 murine ovarian cancer cells (ID8-luc) were orthotopically grafted into one ovary/bursa of mice. (A) Tumor growth was monitored by bioluminescent imaging throughout the study, and (B) peritoneal colonization was assessed at necropsy (day 74). Statistical analysis was performed with two-way ANOVA with Bonferroni multiple comparison test (A) or Fisher’s exact test (B). Asterisks indicate statistical significance (P < 0.05).

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    CYP27A1 inhibition in combination with immune checkpoint inhibition reduces primary tumor volume. MOE murine ovarian cancer cells were orthotopically grafted into one ovary/bursa of FVB/N mice on Day 0. Starting from Day 7, mice were treated intraperitoneally with Veh or 100 mg/kg CYP27A1 inhibitor (GW273297X) daily. IgG2b control or 200 µg anti-PDL1 were injected intraperitoneally on days 10, 12, 14, 16, 18, 20, 22. A waterfall plot is presented here, with each mouse represented by a bar. The grey line indicates the chosen cutoff indicating a positive response (tumors less than 300 mg). A Fisher’s exact test was used to determine whether there were differences in response as assessed by tumors less than 300 mg.

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