Bruceine D suppresses CAF-promoted TNBC metastasis under TNF-α stimulation by inhibiting Notch1-Jagged1/NF-κB(p65) signaling

Background : Triple-negative breast cancer (TNBC) has a poor prognosis because of its high degree of malignancy and the lack of effective treatment options. Cancer-associated?fibroblasts (CAFs)?comprise?the most abundant stromal cells in the tumor microenvironment?(TME), leading to functional impairments and facilitating tumor metastasis. Excessive TNF-α further promotes cross-talk between different cells in TME. Therefore, there is an urgent need to develop more effective therapies and potential drugs that target the key factors that promote TNBC metastasis. Purpose : The study aimed to evaluate the efficacy of Bruceine D, an active compound derived from the Chinese herb Brucea javanica , in inhibiting metastasis and elucidate the underlying mechanism of action in TNBC. Methods : In vitro , the clonogenic and the Transwell assays were used to assess?the effects of Bruceine?D?on the proliferation, migration and invasion abilities of co-cultured CAFs and MDA-MB-231 (4T1) cells under TNF-α stimulation. TNF-α, IL-6, CXCL12, TGF-β1, and MMP9 levels in the supernatant of co-cultured cells were determined using ELISA. Western blotting was utilized to detect?the?expression levels?of proteins related to the Notch1-Jagged1/NF-κB(p65) pathway. In vivo , the anti-tumor growth and anti-metastatic effectiveness of Bruceine D was evaluated by determining tumor weight, number of metastatic lesions, and pathological changes in the tumor and lung/liver tissues. The inhibitory effect of Bruceine D on α-SMA + CAFs activation and CAF-medicated extracellular matrix remodeling was accessed using immunohistochemistry, immunofluorescence, and Masson and Sirius Red staining. The expression levels of Notch1, Jagged1 and p-NF-κB(p65) proteins in the primary tumors were measured by immunohistochemistry and western blotting. Results : In vitro , Bruceine D significantly inhibited the migration and invasion of co-cultured CAFs and MDA-MB-231 (4T1) cells under TNF-α stimulation, reduced the expression of tumor-promoting and matrix-remodeling cytokines secreted by CAFs, and hindered the mutual activation of Notch1-Jagged1 and NF-κB(p65). In vivo , Bruceine D significantly suppressed tumor growth and the formation of lung and liver metastases by decreasing TNF-α stimulated α-SMA + CAFs activation, collagen fibers, MMPs production, and inhibited Notch1-Jagged1/NF-κB(p65) signaling in TNBC-bearing mice. Conclusion : Bruceine D effectively weakened the "tumor-CAF-inflammation" network by inhibiting the mutual activation of Notch1-Jagged1 and NF-κB(p65) and thereby suppressed TNBC metastasis. This study first explored whether Bruceine D disrupts the cross-talk between CAFs and tumor cells under TNF-α stimulation to inhibit the metastasis of TNBC, and highlighted the potential of Bruceine D as therapeutic agent for suppressing tumor metastasis.

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