Targeting Squalene Epoxidase Interrupts Homologous Recombination via the ER Stress Response and Promotes Radiotherapy Efficacy

Abstract

Over 50% of all patients with cancer are treated with radiotherapy. However, radiotherapy is often insufficient as a monotherapy and requires a nontoxic radiosensitizer. Squalene epoxidase (SQLE) controls cholesterol biosynthesis by converting squalene to 2,3-oxidosqualene. Given that SQLE is frequently overexpressed in human cancer, this study investigated the importance of SQLE in breast cancer and non-small cell lung cancer (NSCLC), two cancers often treated with radiotherapy. SQLE-positive IHC staining was observed in 68% of breast cancer and 56% of NSCLC specimens versus 15% and 25% in normal breast and lung tissue, respectively. Importantly, SQLE expression was an independent predictor of poor prognosis, and pharmacologic inhibition of SQLE enhanced breast and lung cancer cell radiosensitivity. In addition, SQLE inhibition enhanced sensitivity to PARP inhibition. Inhibition of SQLE interrupted homologous recombination by suppressing ataxia-telangiectasia mutated (ATM) activity via the translational upregulation of wild-type p53-induced phosphatase (WIP1), regardless of the p53 status. SQLE inhibition and subsequent squalene accumulation promoted this upregulation by triggering the endoplasmic reticulum (ER) stress response. Collectively, these results identify a novel tumor-specific radiosensitizer by revealing unrecognized cross-talk between squalene metabolites, ER stress, and the DNA damage response. Although SQLE inhibitors have been used as antifungal agents in the clinic, they have not yet been used as antitumor agents. Repurposing existing SQLE-inhibiting drugs may provide new cancer treatments.

Significance: Squalene epoxidase inhibitors are novel tumor-specific radiosensitizers that promote ER stress and suppress homologous recombination, providing a new potential therapeutic approach to enhance radiotherapy efficacy.

Figure 1.. SQLE is overexpressed and correlated with poor survival in BC and NSCLC.

A, SQLE expression was elevated in BC. B, The median SQLE IHC score (Immunoreactive score, IRS) of the BC tumors was significantly higher than that of the ANTs. IRS was determined from the staining intensity (SI) and percentage of positive cells (PP): IRS = SI × PP. An IRS ≥ 3 score was classified as SQLE positive. Groups were compared with the Mann–Whitney test. C, Representative IHC images of matched BC and ANT tissue. Magnification 40×. D, Association of SLQE expression and BC subtypes. E, High SQLE expression correlates with poor survival in a BC population on adjuvant therapy. Kaplan-Meier survival analysis of our cohorts. F, TCGA data analysis showed that high SQLE expression correlates with poor survival in a BC population administered radiation therapy (GSE10374). G, SQLE expression was elevated in NSCLC. SQLE protein expression in NSCLC and ANT was determined using IHC. H, The median SQLE staining of NSCLC tumors was significantly higher than in the ANTs. I, Representative IHC images of NSCLC tumors with more than or less than 3-year survival. Magnification 40×. J, The percentage of positive SQLE staining in NSCLC tumors of different TNM stages. K, High SQLE expression correlated with poor survival in NSCLC. L, Multivariate cox regression analysis of OS in our cohort of lung cancer. Data in (A, D, G and H) was evaluated by chi-square test and in (B and H) was evaluated by Mann-Whitney test. Survival in (E, F and K) were determined using the Kaplan-Meier method, and differences between groups were tested using the log-rank test. Multivariate analysis in (L) was performed with the cox proportional hazard regression model.*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 2.. SQLE inhibition enhanced radiosensitivity in vitro and in vivo.

A, The IC₅₀s of TF in the BC cell lines (MCF-7 and HCC-38) and also NSCLC H1299 (24 h treatment). Blue dash lines indicate the dose of 50 μM TF that did not significantly inhibit cell viability and was used for the colony formation assay. SF, Survival fraction. B, TF sensitized BC and NSCLC to ionizing radiation (IR) in the in vitro colony formation assay. Sensitizer enhanced ratio (SER) was showed in each cell lines. TF was given 24 h prior to irradiation. Data in (B) are presented as mean ± SD. Statistical significance was evaluated by two-way ANOVA and the Bonferroni post-hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. C and D, TF enhanced IR efficacy. Vehicle (DMSO) or SQLE inhibitor TF (60 mg/kg) were administered by intraperitoneal injection daily for two weeks. Local fractionated x-ray radiation (2 Gy) was administered for five days, beginning on day 5. Data are presented as the mean tumor volume ± SEM. Statistical significance was evaluated by one-way ANOVA and the Bonferroni post-hoc test; ****, P < 0.0001 (C). Kaplan-Meier analysis was used for overall survival, and the statistical significance was examined using the Mantel-Cox test; *, P < 0.05 (D). E, SQLE inhibition had no impact on body weight. One-way ANOVA did not show significant differences between groups. F, SQLE inhibition did not affect organ weights (one-way ANOVA). G, Representative serum biochemistry profiling indicated no toxicity to the liver (ALT and AST), heart (CK), kidneys (CREAT and BUN) or lipid metabolism (CHOL and TRIG) in the TF-treated or TF combined IR groups. For one-way ANOVA, ns, not significant; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatinine kinase; CREAT, creatinine; BUN, blood urea nitrogen; CHOL, cholesterol; TRIG, triglyceride. H, Representative histological images of skin and intestine. Magnification, 40×.

Figure 3.. SQLE inhibition impaired HR activity and delayed RT-induced DSB repair.

A and B, SQLE inhibition by TF or NB-598 promoted sensitivity to PARP inhibitor (AZD2281) in MCF-7 and HCC-38 cell lines. C, SQLE inhibition impaired HR. The percentage of I-SceI-induced GFP-positive cells was significantly decreased in MCF-7 and H1299 cells treated with 50 μM TF. All data are normalized with negative control. D, SQLE inhibition by TF abrogated IR-induced RAD51 foci formation in MCF-7 and HCC-38 cells, demonstrated by IF. The percentage of cells with positive RAD51 foci were presented. The cells with ≥10 foci were counted as positive. E, Representative IF images of IR-induced Rad51 foci in MCF-7 treated with or without 50 μM TF (magnification, 63× oil. IR: 4 Gy). F, SQLE inhibition led to the retention of IR-induced γ-H2AX in TF-treated MCF-7 and HCC-38 cells (IR, 4 Gy). The percentage of cells with positive γ-H2AX foci were presented. The cells with ≥10 foci were counted as positive. G, Representative γ-H2AX foci in MCF-7 cells (magnification, 63× oil). H, SQLE inhibition by TF promoted IR-induced DSBs by the comet assay (IR, 4 Gy). OTM, olive tail moment. I, Representative comet assay images used to quantify the olive tail moment in (H) (magnification, 20×). TF was given 24 h prior to irradiation (D-I). Data are presented as mean ± SD. Statistical significance was evaluated by one-way (C and D) or two-way (A, B, F and H) ANOVA and Bonferroni’s post-hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 4.. SQLE inhibition impaired the WIP1-ATM axis.

A and B, SQLE inhibition led to a decrease in IR-induced p-ATM (S1981) levels and phosphor-ATM-1981 foci (30 min post-2Gy IR) in MCF-7 and HCC-38 cells, as detected by WB and immunofluorescence staining. TF was given 24 h prior to irradiation. The percentage of cells with positive p-ATM (S1981) foci were presented. The cells with ≥10 foci were counted as positive. C, SQLE inhibition by TF decreased IR-induced p-ATM (S1981) levels at different time points after IR, as measured by immunofluorescence staining of MCF-7 cells. TF was given 24 h prior to irradiation. D, Representative immunofluorescence images of p-ATM (S1981) foci (magnification, 63× oil). E, ATM inhibition (10 μM) abrogated the TF-induced reduction in HR activity in MCF-7 and H1299 cells, as detected by the HR-DrGFP reporter assay. F-H, SQLE knockdown or pharmaceutical inhibition increased WIP1 protein expression in cells with or without wild-type. I and J, SQLE inhibition by SQLE knockdown or inhibitors did not affect WIP1 mRNA expression. K, Schematic representation of sucrose gradient preparation, polysome fractionation, and fraction analysis. L, Polysomes profiling. M, SQLE inhibition by TF increased polysome-associated WIP1 mRNA in MCF-7 cells. WIP1 mRNA was detected by qRT-PCR. The TF-treated MCF-7 cells showed significant increments in the WIP1 mRNA-associated polysomes. TF was given 24 h prior to irradiation (A-D). Data are presented as the mean ± SD in (B, C, E, I and J) and as the mean ± SEM in (M). All data in (I and J) are normalized with shCon or Con group. Statistical significance was determined by two-way (C) or one-way ANOVA (B, E, I and J) followed by Bonferroni’s post-hoc analysis for multiple comparisons. The Student’s t-test was used for the data in (M) for each group comparison. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Figure 5.. SQLE inhibition-induced alterations to the WIP1-ATM axis and radiosensitivity was dependent on squalene.

A and B, SQLE inhibition led to increased squalene levels in BC and lung cancer cells. Squalene levels were detected by LC-MS and normalized to control or shCon values. C, SQLE inhibition by 50 μM TF or 8 μM NB-598 increased SQLE protein in MCF-7 and HCC-38 cells. D, Blocking squalene synthesis for 48 h with TAK-475 (10 μM) reversed the effects of TF (50 μM) and NB-598 (8 μM) effects on WIP1 expression in MCF-7 and HCC-38 cells. E, Relative mRNA level of FDFT1 in MCF-7 and HCC-38 with or without FDFT1 knockdown. F and G, Squalene depletion by FDFT1 knockdown abrogated the increased WIP1 levels induced by TF (50 μM) or NB-598 (8 μM) in MCF-7, HCC-38, and H1299 cells. H, TAK-475 (10 μM) for 48 h restored the SQLE inhibition (50 μM TF)-induced reduction in HR in MCF-7 cells. All data are normalized with negative control. I, TAK-475 (10 μM) restored the SQLE inhibition (50 μM TF)-induced reduction in IR-induced p-ATM (S1981) foci in MCF-7 cells. J and K, TAK-475 (10 μM) eliminated SQLE inhibition-induced radiosensitivity in MCF-7 cells or HCC-38 cells. TF was given 24 h prior to irradiation (I-K). Data are presented as the mean ± SD. All data in (A, B, E and H) are normalized with shCon or Con group. Statistical significance was evaluated with one-way (A, B, E, H and I) or two-way (J and K) ANOVA and Bonferroni’s post-hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Figure 6.. SQLE inhibition-induced ER stress was dependent on squalene.

A, SQLE knockdown increased GRP78 expression in MCF-7 and HCC-38 cells. B, SQLE inhibition by TF (50 μM) or NB-598 (8 μM) increased GRP78 expression, as detected by western blotting. C, SQLE knockdown increased GRP78 transcript levels in MCF-7 and HCC-38 cells, as detected by qRT-PCR. D, SQLE inhibition by TF (50 μM) or NB-598 (8 μM) increased GRP78 mRNA levels in MCF-7 and HCC-38 cells. E and F, SQLE inhibition by shRNA knockdown or TF (50 μM) and NB-598 (8 μM) triggered GRP78 protein and transcript expression in H1299 cells. G, SQLE inhibition by TF (50 μM) or NB-598 (8 μM) increased GRP78 transcript levels in H1299 cells, as detected by qRT-PCR. H-J, Squalene was required for SQLE inhibition-induced GRP78 expression. Squalene depletion by the 10 μM TAK-475 inhibitor for 48 h or FDFT1 knockdown abrogated the effects of TF (50 μM) and NB-598 (8 μM) on GRP78 expression in BC and H1299 cells. Data are presented as the mean ± SD. All data are normalized with shCon or Con group. Statistical significance was evaluated with one-way ANOVA (C, D, F and G) and Bonferroni’s post-hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Figure 7.. SQLE inhibition activated the PERK- eIF2α pathway.

A and B, SQLE inhibition by knockdown or inhibitors (50 μM TF and 8 μM NB-598) increased p-eIF2α and ATF4 levels in MCF-7 and HCC-38 cell lines. C, CHOP transcription was upregulated by 50 μM TF or 8 μM NB-598 in MCF-7 and HCC-38 cells. All data are normalized with Con group. D, eIF2α downregulation abrogated TF-induced WIP1 expression in MCF-7 and HCC-38 cells. E and F, SQLE inhibition promoted the expression of p-eIF2a and ATF4 in H1299 cell. G, CHOP expression was upregulated following the treatment of 50 μM TF or 8 μM NB-598 in H1299 cell. H, eIF2α knockdown eliminated TF-induced WIP1 expression in H1299 cells. I, A schematic model for radiosensitization by SQLE inhibition. Figure created using BioRender (https://biorender.com/). The data in C and G are presented as mean ± SD. Statistical significance was evaluated with one-way ANOVA and Bonferroni’s post-hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.