Bufalin Inhibits Tumorigenesis, Stemness, and Epithelial–Mesenchymal Transition in Colorectal Cancer through a C-Kit/Slug Signaling Axis

Abstract

Colorectal cancer (CRC) is a major source of morbidity and mortality, characterized by intratumoral heterogeneity and the presence of cancer stem cells (CSCs). Bufalin has potent activity against many tumors, but studies of its effect on CRC stemness are limited. We explored bufalin’s function and mechanism using CRC patient-derived organoids (PDOs) and cell lines. In CRC cells, bufalin prevented nuclear translocation of β-catenin and down-regulated CSC markers (CD44, CD133, LGR5), pluripotency factors, and epithelial–mesenchymal transition (EMT) markers (N-Cadherin, Slug, ZEB1). Functionally, bufalin inhibited CRC spheroid formation, aldehyde dehydrogenase activity, migration, and invasion. Network analysis identified a C-Kit/Slug signaling axis accounting for bufalin’s anti-stemness activity. Bufalin treatment significantly downregulated C-Kit, as predicted. Furthermore, overexpression of C-Kit induced Slug expression, spheroid formation, and bufalin resistance. Similarly, overexpression of Slug resulted in increased expression of C-Kit and identical functional effects, demonstrating a pro-stemness feedback loop. For further study, we established PDOs from diagnostic colonoscopy. Bufalin differentially inhibited PDO growth and proliferation, induced apoptosis, restored E-cadherin, and downregulated CSC markers CD133 and C-Myc, dependent on C-Kit/Slug. These findings suggest that the C-Kit/Slug axis plays a pivotal role in regulating CRC stemness, and reveal that targeting this axis can inhibit CRC growth and progression.

c-Kit Receptors as a Therapeutic Target in Cancer: Current Insights

“…148 Many published studies have confirmed the supportive role of c-KIT in colonic cancer stem cells (CSCs), as it promotes migration, invasiveness, and EMT. 56 , 149 In this context, Ding et al proposed that bufalin has anti-stemness activity by blocking the C-Kit/Slug signaling axis, and therefore it could be a potent treatment for CRC patients. 56 Although c-KIT has been reported in many recently published studies to have an important role in CRC carcinogenesis, imatinib has not yet been approved for the treatment of CRC cases.…”

“… 56 , 149 In this context, Ding et al proposed that bufalin has anti-stemness activity by blocking the C-Kit/Slug signaling axis, and therefore it could be a potent treatment for CRC patients. 56 Although c-KIT has been reported in many recently published studies to have an important role in CRC carcinogenesis, imatinib has not yet been approved for the treatment of CRC cases. 59 Accordingly, many clinical trials have been performed to assess the therapeutic effect of imatinib on CRC-CMS4.…”

“… 20 , 33 Activation mutations in exons 11, 13, 9, and 17 34–38 Imatinib, nilotinib, dasatinib sunitinib, sorafenib, masitinib 39 Research was conducted on the combination of TKIs with other therapeutic modalities 40–43 Breast cancer (BC) Loss-of-function mutations. 22 , 44 , 45 Activation mutations in TNBC and adenoid cystic carcinoma 46–53 Lapatinib Approved for HER2-positive BC, as it also targets EGFRs 53 , 54 Approved by FDA in 2007 Nilotinib A potent drug for TNBC treatment 53 Colorectal carcinoma (CRC) Activation mutations in the consensus molecular subtype 4 (CMS4) subtype 55 Bufalin Blocks the C-Kit/Slug signaling axis 56 Approved by Chinese FDA Imatinib Mitigates the aggressive behavior of CRC-CMS4, and induces genes responsible for CMS-switching 57–60 Renal cell carcinoma (RCC) Activation mutations 61–65 Sorafenib, sunitinib, pazopanib, axitinib, cabozantinib, lenvatinib Considered the standard of care in patients with advanced RCC 66–69 Mastocytosis (MC) Missense mutations affecting exons 17, 18, 11, 8, and 9 5 , 70–72 Imatinib Approved by US FDA for systemic MC 73 Midostaurin Multikinase/KIT inhibitor against D816V mutant MC 74 ...…”

Influence of Different Ratios of DSPE-PEG2k on Ester Prodrug Self-Assembly Nanoparticles for Cell Migration and Proliferation Suppression

“…4 The anticancer mechanism of BFL involves the modulation of multiple signaling pathways, including PI3K/Akt, NF-κB, and Wnt, thereby culminating in suppression of tumor cell growth and metastasis. [5][6][7][8] Additionally, BFL exerts its antitumor effects through mechanisms such as cell cycle regulation, angiogenesis inhibition, and immune modulation. 9,10 Furthermore, bufalin could reverse different tumor cell resistance through multiple mechanisms and have a good synergistic therapeutic effect with other clinical chemotherapy drugs such as cisplatin, paclitaxel, sorafenib and gefitinib.…”