HMGCR loss is synthetic lethal with PIK3CD inhibition in colorectal cancer cells

Abstract

BACKGROUND

Colorectal cancer (CRC) remains a prevalent malignancy with a high incidence globally. Conventional chemotherapy is limited by substantial toxicity, and the development of novel drugs faces considerable challenges. In this context, drug repurposing has emerged as a promising strategy for CRC treatment.

AIM

To explore the therapeutic potential of drug repurposing by evaluating the combined effect of atorvastatin and gefitinib in CRC.

METHODS

Integrated analysis of data from the SOLAD, CSSL, and SynLethDB databases was performed to identify synthetic lethal interactions involving PIK3CD, leading to the identification of HMGCR as a PIK3CD-interacting synthetic lethal partner. The anti-proliferative effects of atorvastatin (an HMGCR inhibitor) and gefitinib (an epidermal growth factor receptor-PIK3CD pathway inhibitor) were evaluated alone or in combination in SW480 and HCT116 CRC cells using the Cell Counting Kit-8 assay. Cell invasiveness was assessed by Transwell assay, and the expression of proteins related to the autophagy signaling pathway was measured via Western blot. An in vivo xenograft model was used to validate the combined antitumor effect.

RESULTS

Database screening revealed a synthetic lethal relationship between HMGCR and PIK3CD. Experimentally, the combination of atorvastatin and gefitinib exerted a synergistic inhibitory effect on the proliferation and invasion of CRC cells. Mechanistically, this synthetic lethality effect was mediated through the AMPK-SREBP-1 signaling pathway. In vivo, the combined treatment significantly suppressed tumor growth in a mouse xenograft model.

CONCLUSION

The combination of atorvastatin and gefitinib induces synthetic lethality in CRC via the AMPK-SREBP1 pathway, effectively inhibiting tumor proliferation and metastasis. This drug repurposing strategy presents a novel and translatable approach for CRC therapy.

Key Words: Colorectal cancer; Synthetic lethal; Atorvastatin; Gefitinib; Autophagy

Core Tip: Integrated analysis of data from the SOLAD, CSSL, and SynLethDB databases was performed to identify HMGCR as a PIK3CD-interacting synthetic lethal partner. Atorvastatin in combination with gefitinib synergistically inhibited the proliferation and metastasis of colorectal cancer (CRC) cells through a synthetic lethal effect via the AMP-activated protein kinase-sterol regulatory element-binding protein 1 signaling pathway. This study provides new research ideas for CRC treatment.

INTRODUCTION

Colorectal cancer (CRC), the third most prevalent malignancy globally, represents a major public health challenge. In recent years, its incidence has continued to rise annually[1,2]. China accounts for the highest number of new CRC cases and related deaths worldwide[3]. Studies have indicated that approximately 20% of CRC patients present with metastases at diagnosis, and the 5-year survival rate for metastatic CRC remains as low as 14%[4,5]. Although the comprehensive application of surgical resection, radiotherapy, chemotherapy, targeted therapy, and other treatment modalities has improved survival outcomes to some extent, therapeutic efficacy remains unsatisfactory for a subset of patients. Hence, there is an urgent need to develop safer and more effective precision treatment strategies.

Lipid metabolic reprogramming is closely associated with tumor growth, proliferation, and invasion during CRC progression[6,7]. Cholesterol metabolism, a key component of lipid metabolism, is critical for maintaining membrane fluidity and stability, thereby playing an important role in tumorigenesis and progression. Statins, a class of lipid-lowering agents widely used in cardiovascular disease, reduce cholesterol synthesis by inhibiting HMGCR and have attracted increasing attention due to their potential anticancer properties[8,9]. Several studies have demonstrated that statins can inhibit tumor growth by inducing cell cycle arrest, suppressing tumor angiogenesis and differentiation[10-13]. Therefore, the potential anticancer properties of statins are increasingly recognized.

Synthetic lethality has emerged as a promising anticancer strategy[14]. It refers to a genetic interaction wherein disruption of either of two genes alone is nonlethal, but simultaneous disruption of both results in cell death[15]. Drug development based on synthetic lethality has the potential to overcome limitations of conventional targeted therapies, such as systemic toxicity, drug resistance, and narrow target applicability, thereby providing a safer and more effective precision medicine approach[16]. For instance, PRMT5 inhibitors selectively eliminate MTAP-deficient tumor cells[17], while WRN inhibitors show promise for treating solid tumors with high microsatellite instability or mismatch repair deficiency[18]. Further elucidation of synthetic lethal interactions in cancer is expected to yield more effective clinical therapeutic strategies.

In this study, we analyzed synthetic lethal databases and identified a synthetic lethal interaction between HMGCR and PIK3CD. Based on this interaction, we subsequently treated CRC cells with a combination of atorvastatin (an HMGCR inhibitor) and gefitinib (an EGFR-PIK3CD signaling pathway inhibitor). Our results demonstrate that the combination of gefitinib and atorvastatin exerts synergistic antitumor effects, suppressing cell proliferation and metastasis while enhancing autophagy through the AMPK-SREBP-1 signaling pathway. These findings provide new insights for the development of combination therapeutic strategies for CRC treatment.

MATERIALS AND METHODS

Cell culture

The human CRC cell lines SW480 and HCT116 were procured from Wuhan Procell Biotechnology Co., Ltd. (Hubei Province, China). Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin. All cell cultures were incubated at 37 °C in a humidified atmosphere of 5% CO₂. The culture medium was refreshed 24 hours after seeding. Cells in the logarithmic growth phase were used for all experiments.

Cell viability (Cell Counting Kit-8) assay

SW480 and HCT116 cells were seeded into 96-well plates at a density of 2000 cells/well. After 24 hours, the cells were treated with a range of concentrations of gefitinib (0 μM, 2.5 μM, 5 μM, 10 μM, and 15 μM) or atorvastatin (0 μM, 2 μM, 4 μM, 8 μM, and 16 μM). Each treatment condition was performed in triplicate. Following 48 hours of incubation at 37 °C, the medium was replaced with 100 μL of DMEM containing 10% Cell Counting Kit-8 (CCK-8) reagent. The plates were incubated for an additional 3 hours, after which the absorbance was measured at a wavelength of 450 nm.

Colony formation assay

Cells were evenly seeded into 6-well plates at a density of 4000 cells/well and allowed to adhere overnight. Subsequently, the culture medium was replaced with 2 mL of fresh medium containing the designated drug concentrations. The medium was replaced every 2-3 days. After 7-10 days culture, the formed colonies were fixed with 4% paraformaldehyde for 15 minutes, washed three times with phosphate-buffered saline (PBS), and stained with a crystal violet solution for 20 minutes at room temperature. Excess stain was removed by washing with PBS, and the colonies were imaged and quantified.

Cell migration (Transwell) assay

Cell migration was assessed using Transwell chambers (Corning, United States) placed in 24-well plates. Serum-starved cells were harvested, resuspended, and adjusted to a density of 3 × 10⁴ cells/mL. A 200 μL aliquot of the cell suspension was added to the upper chamber, while the lower chamber was filled with 800 μL of medium containing 10% FBS as a chemoattractant. Cells were treated with gefitinib (5 μM), atorvastatin (8 μM), or their combination. After 24 hours of incubation, non-migrated cells on the upper surface of the membrane were carefully removed with a cotton swab. The migrated cells on the lower surface were washed with PBS, fixed with 4% formaldehyde for 30 minutes, and stained with 1% crystal violet for 20 minutes. The number of migrated cells was counted in five random fields per membrane under a light microscope, and ImageJ software was used for analysis. The experiment was independently repeated three times.

Western blot analysis

Following 24 hours of drug treatment, cells were collected, washed with cold PBS, and lysed on ice using RIPA lysis buffer for 40 minutes. The lysates were centrifuged at 12000 × g for 15 minutes at 4 °C, and the supernatant was collected. Protein concentration was determined using a bicinchoninic acid assay kit. Equal amounts of protein were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk for 1 hour at room temperature and then incubated with specific primary antibodies (1:1000 dilution) overnight at 4 °C. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence substrate.

5-ethynyl-2'-deoxyuridine assay

Cell proliferation was evaluated using a 5-ethynyl-2'-deoxyuridine (EdU) assay kit (C0075S, Beyotime, China). Briefly, cells were seeded into 24-well plates (5 × 10⁴ cells/well), treated for 24 hours, and then incubated with 200 μL of EdU working solution for 2 hours at 37 °C. The cells were fixed with 4% paraformaldehyde and processed according to the manufacturer's protocol.

Quantitative real-time PCR

Total RNA was extracted from cells after 24 hours of culture, and its concentration and purity were measured. Complementary DNA was synthesized by reverse transcription. Quantitative PCR was performed using SYBR Green Master Mix on a real-time PCR system. The relative mRNA expression levels were calculated using the 2^-ΔΔCt method. All primers were synthesized by Sangon Biotech (Shanghai, China).

Hematoxylin and eosin staining

Paraffin-embedded tissue sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) following standard protocols. After dehydration and mounting, the sections were examined under a microscope for histological analysis.

Immunofluorescence (Ki67) staining

Tissue sections were fixed, permeabilized with 0.3% Triton X-100, and blocked with bovine serum albumin. The sections were then incubated overnight at 4 °C with an anti-Ki67 primary antibody (9449T, Cell Signaling Technology, United States). After washing, the sections were incubated with a fluorescently labeled secondary antibody, counterstained with 4',6-diamidino-2-phenylindole, and mounted for imaging.

In vivo xenograft model

Female BALB/c nude mice (4-6 weeks old, weighing 23-25 g) were purchased from Silaike Jingda Laboratory Animal Co., Ltd. (Hunan Province, China). A single-cell suspension of HCT116 or SW480 cells was prepared at a density of 5 × 10⁷ cells/mL in PBS. Each mouse was subcutaneously injected with 200 μL of the cell suspension into the right flank. Tumor size was measured weekly with calipers, and tumor volume was calculated using the formula: (Length × width²)/2. Four weeks post-inoculation, the mice were humanely euthanized. Euthanasia was performed by intravenous injection of 3% sodium pentobarbital, followed by cervical dislocation after the loss of consciousness and pain reflex was confirmed. The terminal tumors were excised, and their size, weight, and volume were recorded. The maximum tumor volume did not exceed 1.5 cm³. All animal experiments were approved by the Animal Care and Use Committee of the Third Hospital of Xiangya University and conducted in strict accordance with relevant guidelines and regulations, including the ARRIVE guidelines.

Statistical analysis

All statistical analyses were performed using SPSS 20.0 and GraphPad Prism 8.0 software. Quantitative data are presented as the mean ± SD. Comparisons between two groups were analyzed using the Student's t-test, while comparisons among multiple groups were evaluated by one-way analysis of variance (ANOVA). A P value of less than 0.05 was considered statistically significant.

RESULTS

HMGCR loss was synthetic lethal with PIK3CD inhibition in CRC cells

Bioinformatic analysis using the SOLAD, CSSL, and SynLethDB databases predicted a synthetic lethal interaction between HMGCR and PIK3CD in CRC (Figure 1A). To validate this prediction, SW480 and HCT116 CRC cells were treated with gefitinib and atorvastatin. CCK-8 assays demonstrated that the combination of gefitinib and atorvastatin synergistically inhibited cell viability in a concentration-dependent manner (Figure 1B and C). Consistently, colony formation assays revealed a gradual decrease in the number of colonies with increasing concentrations of both drugs (Figure 1D and E).

Figure 1 HMGCR loss is synthetic lethal with PIK3CD inhibition in colorectal cancer cells. A: As indicated in the SOLAD, CSSL and SynLethDB online databases, HMGCR is synthetic lethal with PIK3CD in colorectal cancer (CRC); B: Cell Counting Kit-8 (CCK-8) results show that gefitinib enhanced the toxicity of CRC SW480 and HCT116 cells, and cell viability decreased in a concentration-dependent manner; C: CCK-8 results show that atorvastatin enhanced the toxicity of CRC SW480 and HCT116 cells, and cell viability decreased in a concentration-dependent manner; D and E: Clone formation assay showed that gefitinib or atorvastatin alone can enhance the toxicity of CRC SW480; F and G: HMGCR depletion significantly attenuated the anti-proliferative effects of the gefitinib-atorvastatin combination in both cell lines; H and I: Compared with atorvastatin or gefitinib alone, the combination of atorvastatin and gefitinib increased inhibition of the activity of SW480 cells and HCT116 cells; J: 5-ethynyl-2'-deoxyuridine assay revealed that compared with atorvastatin or gefitinib alone, the combination of atorvastatin and gefitinib increased inhibition of the activity of SW480 cells and HCT116 cells. Data are presented as the mean ± SD. ^aP < 0.001, ^bP < 0.01. OD: Optical density.

The critical role of HMGCR in this synthetic lethal interaction was confirmed by knockdown experiments. HMGCR depletion significantly attenuated the antiproliferative effects of gefitinib based combination treatment in both cell lines (Figure 1F and G). Compared with single-agent treatments, the combination of atorvastatin and gefitinib exerted a superior inhibitory on the proliferation of SW480 and HCT116 cell (Figure 1H and I). This enhanced antiproliferative effect was further corroborated by EdU assays (Figure 1J). Based on the concentration-dependent inhibitory profiles, the median lethal concentrations (5 μM gefitinib and 8 μM atorvastatin) were determined and selected for subsequent combination therapy experiments.

Combined atorvastatin and gefitinib suppressed metastasis and promoted autophagy in CRC cells

We next investigated the effect of the combination treatment on metastasis and autophagy. Transwell assays showed that the combination of atorvastatin and gefitinib inhibited the migration of SW480 and HCT116 cells than either agent alone (Figure 2A and B). Western blotting indicated that the combination treatment upregulated the epithelial marker E-cadherin and downregulated the mesenchymal marker vimentin, with effects more pronounced than those of single agent treatments (Figure 2C and D), suggesting enhanced suppression of epithelial-mesenchymal transition.

Figure 2 Atorvastatin combined with gefitinib had an enhanced inhibitory effect on colorectal cancer cell metastasis and promotes autophagy. A and B: Transwell assay showed that compared with atorvastatin or gefitinib alone, atorvastatin combined with gefitinib increased the inhibition of SW480 cells and HCT116 cell metastasis; C and D: Western blotting results showed that after atorvastatin and gefitinib treatment, the expression of E-cadherin increased, and the expression of vimentin decreased, compared with atorvastatin or gefitinib alone. The combination of atorvastatin and gefitinib had a stronger effect on protein expression; E: Compared with atorvastatin or gefitinib alone, the combination of atorvastatin and gefitinib showed more autophagy spots; F and G: Western blotting showed that compared with atorvastatin or gefitinib alone, the combination of atorvastatin and gefitinib increased the expression of autophagy-related genes LC3-II/I and Beclin-1, and decreased the expression of P62. Data are presented as the mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001; NS: Not significant.

Combination therapy significantly promoted autophagy. Immunofluorescence staining revealed a greater increase in LC3 puncta formation in cells treated with both drugs, indicating enhanced autophagosome formation (Figure 2E). Western blotting of autophagy-related markers consistently showed that the combination treatment increased the LC3-II/I ratio and Beclin-1 expression, while decreasing P62 expression, to a greater extent than single-agent treatments (Figure 2F and G).

Combined atorvastatin and gefitinib inhibited the AMPK-SREBP1 signaling pathway

To explore the underlying mechanism, we examined the AMPK-SREBP1 pathway. Western blotting demonstrated that the combination of atorvastatin and gefitinib reduced the levels of p-AMPK and SREBP1 more effectively than either drug alone (Figure 3A and B). Similarly, the combination of HMGCR knockdown (sh-HMGCR) with gefitinib also led to a greater reduction in p-AMPK and SREBP1 levels compared with individual interventions (Figure 3C and D). These results indicated that the combination therapy inhibited the AMPK-SREBP1 signaling pathway.

Figure 3 Atorvastatin combined with gefitinib inhibited the AMPK-SREBP1 signaling pathway. A and B: Western blotting analysis revealed that, after combination treatment with atorvastatin and gefitinib, the expression of p-AMPK and SREBP1 decreased compared with that in the single-agent atorvastatin or gefitinib groups; C and D: Western blotting analysis revealed that, after combination treatment with sh-HMGCR and gefitinib, the expression of p-AMPK and SREBP1 decreased compared with that in the single-agent sh-HMGCR or gefitinib groups. Data are presented as the mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Combined atorvastatin and gefitinib potentiated inhibition of tumorigenesis in vivo

Antitumor efficacy of the combination was evaluated using a mouse xenograft model. Compared with the control or single-agent groups, cotreatment with atorvastatin and gefitinib significantly reduced tumor volume and weight (Figure 4A-C). H&E staining of major organs (heart, lungs, and liver) revealed no obvious pathological damage (Figure 4D). Serum biochemical analysis showed no significant differences in liver function markers (alanine aminotransferase, aspartate aminotransferase) or CREA levels between the control and treatment groups, confirming the favorable safety profile of the combination (Figure 4E).

Figure 4 Atorvastatin combined with gefitinib enhanced the inhibition of tumorigenesis in mice. A: The photograph shows that compared with atorvastatin or gefitinib alone, the combination of atorvastatin and gefitinib inhibited tumor formation in mice; B: Compared with atorvastatin or gefitinib alone, the combination of atorvastatin and gefitinib inhibited tumor weight; C: Compared with atorvastatin or gefitinib alone, the combination of atorvastatin and gefitinib inhibited tumor volume; D: The hematoxylin and eosin staining results showed that there was no obvious damage to the heart, lungs and liver of mice; E: There were no significant differences in liver function markers (alanine aminotransferase, aspartate aminotransferase) or creatinine levels between the control and treatment groups, indicating a favorable safety profile; F: KI67 staining showed that the combination of atorvastatin and gefitinib inhibited tumor proliferation; G: Immunofluorescence results showed that the number of LC3 fluorescence spots increased and P62 reduced following atorvastatin and gefitinib treatment. Data are presented as the mean ± SD. ^aP < 0.05, ^bP < 0.001. NS: Not significant; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; CREA: Creatinine.

Immunohistochemical staining for Ki67 showed that the combination treatment suppressed tumor cell proliferation more effectively than either monotherapy (Figure 4F). Additionally, immunofluorescence staining of tumor tissues confirmed enhanced autophagy in the combination group in vivo, as evidenced by increased LC3 puncta and decreased P62 expression (Figure 4G). Collectively, these findings illustrated that the combination atorvastatin and gefitinib exerted potent antitumor activity and clinical potential.

DISCUSSION

Cancer treatment strategies are contingent on many factors, including tumor origin (sporadic or genetic), molecular profile, patient age, and cancer stage[19,20]. Conventional modalities, such as surgery, chemotherapy, radiotherapy, comprehensive therapy, and immunotherapy remain the cornerstone of clinical management[21,22]. In addition, advanced approaches, including gene therapy, cell therapy, and targeted immunotherapy, are still under investigation and development[23,24]. Despite these options, current therapies are often hampered by limitations such as tumor recurrence, off-target effects, and drug resistance. Among potential therapeutic targets, the EGFR-PI3K pathway represents a critical vulnerability in a subset of CRC[25]. Although gefitinib is currently approved for lung cancer, its primary target (EGFR) is also highly relevant in CRC[26,27], with approximately 70%-80% of cases showing EGFR overexpression. Accordingly, EGFR tyrosine kinase inhibitors such as gefitinib have been increasingly explored in clinical trials for CRC and other malignancies[28]. These considerations underscore the continued need to develop and optimize novel treatment strategies for CRC.

Statins are widely used for dyslipidemia management, and are now being evaluated in clinical studies for cancer prevention and treatment, both as monotherapy and in combination with chemotherapy[29,30]. Their established long-term safety profile supports the investigation of high-dose regimens in oncology. Moreover, accumulating evidence indicates that statins may reduce cancer risk, tumor recurrence, and cancer-related mortality, positioning them as promising, well-tolerated agents for chemoprevention and adjunctive therapy[31,32]. In the present study, we demonstrated that the combination of atorvastatin and gefitinib exerts a synthetic lethal effect in CRC cells, leading to synergistic antitumor activity through the suppression of proliferation and metastasis, along with the induction of autophagy. However, our in vivo study had limitations, including a short treatment duration and small sample size. While the results are encouraging, future investigations with extended follow-up and larger cohorts are warranted to confirm the long-term efficacy and durability of this combination regimen.

Our findings regarding the synthetic lethal interaction between atorvastatin and gefitinib should be interpreted within the evolving context of statin repurposing in oncology. Previous studies have examined combinations such as atorvastatin with celecoxib, which co-targets the mevalonate and cyclooxygenase-2 pathways to concurrently inhibit proliferation and inflammation[33,34]. Although promising, this strategy follows a conventional parallel-pathway rationale. In contrast, the gefitinib-atorvastatin strategy introduces several novel aspects. First, it originates from a computational prediction of synthetic lethality, rather than extrapolation from known pathway crosstalk. Second, it mechanistically targets a unique axis-converging mevalonate pathway inhibition with off-target PI3Kδ (PIK3CD) suppression-a vulnerability previously unexploited in CRC. Third, celecoxib-based combinations modulate inflammatory pathways, and our approach drives concerted inhibition of the AMPK-SREBP1 signaling pathway, representing a distinct mechanistic outcome. Thus, we propose a new conceptual and therapeutic framework for statin-based combinations in CRC, shifting from anti-inflammatory synergy toward a targeted synthetic lethality paradigm.

CONCLUSION

Gefitinib and atorvastatin individually inhibited proliferation, invasion, and autophagy induction in SW480 and HCT116 cells. However, their combination resulted in significantly enhanced inhibitory effects. This study elucidated the antitumor mechanism of the gefitinib-atorvastatin combination from an autophagic perspective and provides an experimental foundation for its further development. The combined regimen increased the sensitivity of CRC cells to gefitinib and exhibited a favorable safety profile without significant cardiac, pulmonary, or hepatic toxicity. These findings support the clinical translational potential of this treatment strategy.