ZQL-4c exerts anti-tumor effects by specifically targeting SCD1 in triple-negative breast cancer both in vitro and in vivo

To the Editor: Breast cancer is a common cancer in women, and is a primary cause of cancer-associated mortality in China. Oleanolic acid, a pentacyclic triterpenoid isolated from various plants, has many biological properties including anti-inflammatory, anti-viral, and anti-cancer. Our previous study indicated that N-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-28-amide (ZQL-4c), an oleanolic acid derivative, inhibited the proliferation of breast cancer cells, particularly triple-negative breast cancer (TNBC). This study elucidated the potential inhibitory mechanisms of ZQL-4c in TNBC. Supplementary Figure 1A, http://links.lww.com/CM9/C427 illustrates ZQL-4c synthesis.

Herein, we evaluated the anti-tumor activity of ZQL-4c in multiple breast cancer cell lines. The effects of ZQL-4c on apoptosis and cell cycle progression in TNBC cells were detected using flow cytometry. Using a standard wound healing assay, the migratory behavior of TNBC cells was studied, and RNA sequencing and bioinformatic analyses were conducted to explore the potential targeting of ZQL-4c. The molecular interactions between ZQL-4c and stearoyl-CoA desaturase 1 (SCD1) were simulated using molecular docking, and these findings were further validated by Western blotting and Oil Red O staining. Finally, lentiviral transfection was employed to design cell models with reduced SCD1 expression. Western blotting was performed to detect the levels of proteins associated with apoptosis, cell cycle, cell migration, and the SCD1 signaling pathway. In addition, a mouse-based xenograft model was designed to verify the effects of ZQL-4c and SCD1.

To evaluate the cytotoxic potency of ZQL-4c, MDA-MB-231, MDA-MB-468, and MCF-10A mammary epithelial cells were treated with the different dosages of ZQL-4c (0, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 μmol/L) over different durations (12, 24, and 48 hours). The results of the Cell Counting Kit-8 (CCK-8) assay, used to determine cell viability are shown in Supplementary Figure 1B, http://links.lww.com/CM9/C427. The findings revealed time- and dose-dependent reductions in MDA-MB-231 and MDA-MB-468 cell viability post-ZQL-4c treatment. However, ZQL-4c exhibited limited toxicity toward MCF-10A mammary epithelial cells, implicating it as a potential anti-cancer agent specific to TNBC cells. The generated dose-response curves [Supplementary Figure 1C, http://links.lww.com/CM9/C427] indicated an IC₅₀ value of 0.74 ± 0.03 (MDA-MB-231) and 0.87 ± 0.04 μmol/L (MDA-MB-468) post-48 h of ZQL-4c treatment, whereas that of MCF-10A cells remained undetermined (>500 μmol/L). Thus, ZQL-4c is a promising therapeutic candidate for TNBC with minimal toxicity toward mammary epithelial cells.

To determine whether ZQL-4c-induced cell death is associated with apoptosis and if apoptosis accounts for ZQL-4c cytotoxicity within TNBC cells, we used several techniques including cell morphology, Annexin V fluorescein isothiocyanate/propidium iodide (FITC/PI) staining, and Western blotting. Supplementary Figure 2A, http://links.lww.com/CM9/C427 shows that as the ZQL-4c concentration increased, the morphological cellular alterations became more pronounced. In response, the cells detach and float in the medium, causing a spike in cell fragments and a substantial decrease in the number of adherent cells. After treatment with various ZQL-4c concentrations for 24 hours, the total apoptotic cell death rate increased in proportion to ZQL-4c concentration [Supplementary Figures 2B, C, http://links.lww.com/CM9/C427]. To further understand the molecular mechanisms underlying ZQL-4c-triggered apoptosis, Western blotting analysis was performed. Therefore, ZQL-4c treatment was associated with decreased Bcl-2 and Bcl-xL levels [Supplementary Figure 2E, http://links.lww.com/CM9/C427]. In contrast, the levels of Bax, cleaved caspase-3 and -9, and cytochrome c were increased. Furthermore, cytochrome c release from the mitochondria is negatively regulated by anti-apoptotic Bcl-2 family members (e.g., Bcl-2 and Bcl-xL) and positively regulated by pro-apoptotic Bcl-2 members (e.g., Bax and Bak). Cytochrome c has been shown to activate caspase-9 and -3 which induces apoptosis. Moreover, following ZQL-4c treatment, caspase-3 was dose-dependently activated, as evidenced by the caspase-3 activity assay [Supplementary Figure 2D, http://links.lww.com/CM9/C427], demonstrating a potential apoptosis mechanism. These findings align with previous research indicating that OA induces apoptosis in breast cancer cells primarily through the mitochondrial apoptosis pathway.^[1] Thus, the intrinsic/mitochondrial apoptotic pathway in TNBC may be activated.

As cell cycle arrest may be a critical mechanism for the inhibition of cancer cell growth,^[2] PI staining was used to examine cell cycle distribution via flow cytometry. MDA-MB-231 and MDA-MB-468 cells were treated with escalating doses (0, 0.8, 1.2, and 1.6 μmol/L) of ZQL-4c for 24 hours, after which the DNA content was analyzed. Therefore, following ZQL-4c treatment, we observed a dose-dependent accumulation of these cells in the G2/M phase [Supplementary Figures 3A, B, http://links.lww.com/CM9/C427]. Furthermore, Western blotting was used to assess the effects of ZQL-4c on cell cycle checkpoint proteins [Supplementary Figure 3C, http://links.lww.com/CM9/C427], revealing that the protein levels of cyclins B1, D1, and E1 decreased, whereas those of P21 and P27 increased in a dose-dependent manner in MDA-MB-231 and MDA-MB-468 cells. Thus, ZQL-4c-induced cell cycle arrest at the G2/M phase inhibited the normal cell cycle and prevented cell proliferation, which positively correlated with apoptosis.

Impact of ZQL-4c’s on TNBC cellular migration and invasion and epithelial-mesenchymal transition (EMT) was explored [Supplementary Figures 3D and E, http://links.lww.com/CM9/C427]. As ZQL-4c’s concentration increased, the number of invading cells in each visual field progressively and significantly decreased. Western blotting was used to verify the significance of ZQL-4c-based EMT inhibition [Supplementary Figure 3F, http://links.lww.com/CM9/C427]. Moreover, ZQL-4c triggered a dose-dependent reduction in the protein levels of matrix metalloprotease-2 (MMP-2) and MMP-9, which play crucial roles in tumor metastasis [Supplementary Figure 3F, http://links.lww.com/CM9/C427]. Therefore, ZQL-4c is essential to the inhibition of TNBC cell migration and invasion.

To identify the potential targets of ZQL-4c, we performed RNA sequencing and bioinformatics analyses, revealing that differentially expressed genes (DEGs) distinguished by RNA sequencing of MDA-MB-231 cells treated with or without ZQL-4c, yielded 578 DEGs, divided into two categories: 268 upregulated and 310 downregulated genes [Supplementary Figure 4A, http://links.lww.com/CM9/C427]. Furthermore, by employing a threshold of |log fold change (FC)| >2 and log₁₀ false discovery rate (FDR) >2, 44 DEGs were identified. Notably, SCD1 was the most downregulated gene in response to ZQL-4c treatment [Supplementary Figure 4B, http://links.lww.com/CM9/C427]. Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were utilized to show that SCD1 had significant associations with metabolic reprogramming, membrane system dynamics, and adenosine monophos phate (AMP)-activated protein kinase (AMPK) pathway [Supplementary Figure 4C, http://links.lww.com/CM9/C427]. To expand our research, we utilized the STRING database to elucidate protein interactions among the 44 DEGs and construct a protein–protein interactions network. We identified two submodels with eight proteins: SCD1, HMGCR, ABCG1, NFATC2, IDO1, EGF, MMP7, and PAG1 [Supplementary Figure 4D, http://links.lww.com/CM9/C427]. Of these eight proteins, SCD1 had the highest score. Further studies showed that compared to HER2-positive (SKBR3 and MDA-MB-453) and luminal cell lines (MCF-7 and T47D), the TNBC cell lines (MDA-MB-231 and MDA-MB-468) had higher SCD1 expression levels [Supplementary Figure 4E, http://links.lww.com/CM9/C427].

Molecular docking reveals ZQL-4c stabilizes in SCD1 binding cavity via an inward “e-loop” conformation, with critical polar interactions: M36 hydrogen-bonds to C33 methyl, K176 carbonyl spatially complements C25, and N106 amide N-H directs hydrogen bonding, collectively immobilizing the ligand [Supplementary Figure 4F, http://links.lww.com/CM9/C427].

SCD1, a key lipogenic enzyme, catalyzes saturated fatty acid desaturation to monounsaturated species that are esterified into triglycerides and sequestered in lipid droplets. To further investigate the effect of ZQL-4c on SCD1, we used Oil Red O staining to examine the presence of lipid droplets in cells. This method revealed that ZQL-4c treatment triggered a significant reduction in lipid droplets in MDA-MB-231 and MDA-MB-468 cells [Supplementary Figure 4G, http://links.lww.com/CM9/C427]. In addition, using Western blotting, we analyzed the expression levels of SCD1-related lipid metabolism pathway proteins, including SCD1, sterol regulatory element binding protein 1, and fatty acid synthase [Supplementary Figure 4H, http://links.lww.com/CM9/C427]. Significant decreases in the expression levels of these proteins were observed. In conclusion, SCD1 may be a target of ZQL-4c.

Previous research has highlighted that SCD1 is associated with poor prognosis in diverse tumor types due to its cell-protective properties against apoptosis and ferroptosis.^[3] MDA-MB-231 and MDA-MB-468 cells were transiently transfected with SCD1 to further elucidate the role of SCD1 in ZQL-4c-induced apoptosis and cell cycle arrest [Supplementary Figure 5A, http://links.lww.com/CM9/C427]. Following SCD1 transfection, cells were treated with ZQL-4c over 24 hours. These results revealed that compared to the empty vector group, SCD1 overexpression compensated for the ZQL-4c-induced cell viability inhibition in the ZQL-4c treatment group [Supplementary Figures 5B, C, http://links.lww.com/CM9/C427]. Moreover, flow cytometry results indicated that the ratio of apoptotic cells to G2/M phase cells was restored by SCD1 overexpression [Supplementary Figures 5D, E, G, H, http://links.lww.com/CM9/C427]. This was confirmed by observing the corresponding apoptosis-regulated and cell cycle-associated proteins [Supplementary Figure 5F, I, http://links.lww.com/CM9/C427]. We also explored the role of SCD1 in ZQL-4C cell migration and invasion. As depicted in Supplementary Figures 5J and K, http://links.lww.com/CM9/C427, in the empty vector (EV) group, the number of invasive cells per field decreased significantly post-ZQL-4c treatment. Although the number of invasive cells per field was decreased in the OE-SCD1 group, the difference was significantly lower than that observed in the EV group. This was confirmed by examining EMT and transfer-related proteins [Supplementary Figure 5L, http://links.lww.com/CM9/C427]. Therefore, SCD1 overexpression reversed the ZQL-4c-induced cell invasion inhibition, suggesting that SCD1 may be a poor prognostic factor for TNBC. Moreover, SCD1 overexpression, which was highlighted in previous studies in relation to breast cancer, may indicate poor prognosis in patients with breast cancer.^[4]

Mika et al^[5] results were consistent with our findings, that is, that impeding multiple lipid metabolism regulatory genes, including SCD1, reduces the lipidomic characterization and viability of breast cancer cells. Consequently, SCD1 is considered a therapeutic target in cancer, predominantly, because of its high expression in various cancers and central role in stimulating cancer cell growth by fatty acid metabolism regulation.

The anti-tumor efficacy of ZQL-4c was assessed in an MDA-MB-231 xenograft tumor model, which highlighted its capacity to inhibit tumor growth. Significant reductions in tumor volume and weight were observed after 14 days of treatment with a 5 mg/kg dose of ZQL-4c, with no evident decrease in body weight [Supplementary Figures 6A–D, http://links.lww.com/CM9/C427]. Furthermore, intratumoral biomarkers associated with apoptosis and cell cycle were analyzed using Western blotting. These findings were consistent with the in vitro study results [Supplementary Figures 6E–G, http://links.lww.com/CM9/C427]. In addition, using immunohistochemical staining, we investigated Ki-67 and SCD1 expression post-ZQL-4c treatment and found that their expressions were significantly decreased. Various organs were harvested, sectioned, and stained with hematoxylin and eosin to assess the in vivo side effects of ZQL-4c. No histological differences in the lung, liver, spleen, kidney, or heart were found between the ZQL-4c treatment groups, indicating no notable toxicity [Supplementary Figure 6I, http://links.lww.com/CM9/C427]. To evaluate the role of SCD1 in the anti-tumor effects of ZQL-4c, SCD1 was overexpressed in an MDA-MB-231 xenograft tumor model. Therefore, SCD1 overexpression may have diminished ZQL-4c efficacy in vivo [Supplementary Figure 6J–L, http://links.lww.com/CM9/C427]. Based on our research, we propose that ZQL-4c inhibits SCD1, which affects the permeability of the endoplasmic reticulum and mitochondrial membrane, thereby triggering mitochondria-dependent apoptosis [Supplementary Figure 7, http://links.lww.com/CM9/C427]. However, certain potential issues require further examination. Specifically, although we identified SCD1 as a ZQL-4c target, its precise mechanism remains unknown.

In conclusion, this study demonstrated that ZQL-4c exhibits anti-tumor activity against TNBC both in vitro and in vivo. Moreover, ZQL-4c inhibits cell migration and invasion, induces apoptosis, and promotes G2/M phase cell cycle arrest in TNBC cells. Furthermore, ZQL-4c may mediate these effects by SCD1 targeting, offering insights into additional molecular mechanisms that enhance our comprehension of ZQL-4c-induced cell death, potentially anteceding the development of new therapeutic agents for TNBC treatment.

Funding

This work was supported by grants from the Natural Science Foundation of Liaoning Province (Nos. 2023-MS-057 and 2024JH2/102500058), Beijing Medical Award Foundation (No. CORP-239-N27), and Fundamental Research Funds for Central Universities (No. LD2023024).

Conflicts of interest

None.

Supplementary Material

cm9-138-2359-s001.pdf ^{(1.1MB, pdf)}