Triple-negative breast cancer (TNBC) is challenging to treat due to its aggressive nature. Its lack of hormone receptors renders conventional therapies less effective. This study assessed the efficacy of a novel compound, compound 2, in modulating TNBC cell behaviour. We used in vitro assays with MDA-MB-468 and MDA-MB-231 cell lines. Methods included annexin V apoptosis assay, flow cytometry for cell cycle and qRT-PCR for gene expression. Clonogenic, adhesion and wound healing assays were used for phenotypic characterization. Cytokine and chemokine levels in MDA-MB-468 cells were also measured using a Luminex assay. Compound 2 increased both early and late apoptosis in cancer cells, particularly MDA MB 468 cells. It also upregulated pro-apoptotic genes while downregulating anti-apoptotic genes. Additionally, it induced G1-phase arrest in MDA MB 468 cells with downregulation in Ki67 expression. Compound 2 also reduced cancer stem cell populations, suppressed colony formation, and impaired cell migration at IC50 concentrations. Significant changes in gene expression profiles for EMT-related genes were observed. Compound 2 decreased IL4 and IL8 levels and increased CCL2 and CXCL1. However, it did not significantly affect the levels of IL6, IL10, CXCL2, CCL5, TNF-α, IFN-γ, IL-1β, and IL2. Compound 2 thus exhibited a multifaceted anticancer profile, suggesting its potential in preventing cancer relapse and limiting cell proliferation which makes it a promising candidate for TNBC targeted therapy. This study lays the groundwork for further in vivo studies and potential clinical applications to explore full therapeutic potential of compound 2 in aggressive breast cancer types.

Breast cancer (BC) is a prevalent malignancy that affects women worldwide. In 2022, the number of reported BC cases reached 2.3 million, resulting in 670,000 deaths. Similar to any other disease, these figures represent only reported cases, and the actual number of individuals affected by breast cancer may be higher, as some cases may go undiagnosed or unreported. The disease encompasses various subtypes with distinct molecular characteristics, prognosis, and treatment responses.

Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous subtype of breast cancer, accounts for approximately 10-15% of all breast cancer cases globally and presents significant challenges due to its aggressive behaviour, high relapse rates, and limited treatment options. Although chemotherapy remains the primary approach for TNBC, it faces obstacles such as drug resistance and cytotoxicity to healthy cells. Also, the molecular landscape of TNBC contributes to its varying responses to chemotherapy. Targeted therapies aimed at specific pathways may enhance sensitivity to chemotherapy but can also suppress the immune system, posing additional complexities in finding effective TNBC treatments. Thus, there is an urgent need to identify novel and effective therapeutic strategies for TNBC management.

Standard chemotherapy for TNBC typically includes anthracyclines, alkylating agents, taxanes, and fluorouracil. This regimen is administered as neoadjuvant therapy, followed by surgery for early-stage cases. However, in cases of relapsed or refractory TNBC, there is no established standard treatment. Responses to existing options such as capecitabine, gemcitabine, eribulin, and platinum-based agents are often short-lived, and metastases remain common. Current research focuses on discovering new drugs and repurposing existing ones for TNBC treatment. Targeted therapies that block specific molecules and pathways associated with TNBC are gaining prominence.Small-molecule compounds have become crucial in drug discovery, especially in the battle against cancer. They can block cancer-promoting pathways, trigger cell death in cancer cells, and alter the tumour environment to boost the effectiveness of other treatments. Furthermore, advances in medicinal chemistry and computational biology have enhanced the design and optimisation of small molecules and increased selectivity while reducing adverse effects. Three small molecules (compounds 1, 2 and 3) previously identified to target Leishmania. Our previous studies utilized cytotoxicity screening and computer-aided drug discovery method to assess the anticancer potential of three compounds, based on the previously established connection between anticancer and antileishmanial drugs as well as the core scaffolds of these compounds (chromen-based and triazolopyridazine-based). Initial cytotoxicity and selectivity assays were performed using four cancer cell lines: MDA-MB-468, MDA-MB-231, DLD-1, PC3 and the normal prostate epithelial line PNT2 to evaluate compound 2's selectivity. Compound 2 which is a chromen-based compound, (PubChem ID: STK610045) emerged as the lead molecule, exhibiting potent and selective cytotoxicity against TNBC cell lines, particularly MDA-MB-468 (IC = 9.46 µM) and MDA-MB-231 (IC = 18.30 µM), while sparing normal prostate epithelial cells (PNT2), indicating a favourable selectivity index. Network pharmacology, docking, and molecular dynamics simulations identified AKT1, EGFR and VEGFR2 as compound 2's most likely targets. Compound 2 bound strongly to the ATP-binding site (Lys745) and DFG motif (Asp855) of EGFR, as well as the ATP-binding site (Cys919) of VEGFR2, with both interactions showing favourable binding free energy. These findings support the hypothesis that compound 2 exerts its anticancer effects via targeted inhibition of EGFR signalling and related oncogenic pathways.

Chromen-based compounds have been found to exhibit anticancer characteristics because they disrupt multiple cellular processes that contribute to cancer growth. They can cause apoptosis, slow cell development, and disrupt the cell cycle of cancer cells. Furthermore, chromen-based compounds have also been shown to influence the tumour microenvironment and improve the efficacy of other therapies. As researchers continue to investigate the various modes of action of chromen-based compounds, these chemicals are promising candidates for developing innovative cancer therapeutics. Systematic in vitro screening of compound 2 was therefore necessary to elucidate its potential anticancer mechanisms and validate its utility as a therapeutic candidate for aggressive breast cancers like TNBC.

In this study, we conducted further in vitro studies built upon previous findings to validate the biological activity of compound 2 in TNBC cells and explore its downstream effects on apoptosis, cell cycle, and EMT. We showed that Compound 2 caused a G1-cell arrest in MDA-MB-468 cells and triggered significant late apoptosis in most cell populations. Furthermore, it affected EMT markers gene expression, cell migration, and cancer stem markers all of which have implications for cancer metastasis. Our findings further show the promise compound 2 holds as a potential therapeutic target for cancer, especially, TNBCs.