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.
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).