Hedyotis diffusa injection suppresses breast cancer progression and lung metastasis through dual targeting of PTK2B and CCN2.

BACKGROUND: Lung metastasis is a leading cause of mortality in patients with breast cancer, especially triple-negative breast cancer (TNBC), where current therapies are limited and toxic. Hedyotis diffusa injection (HDI) is used clinically as an adjuvant, but its efficacy and mechanisms against lung metastasis remain unclear.

PURPOSE: To investigate the antitumour and antimetastatic activities of HDI and its active constituent, asperulosidic acid (ASPA), identify its molecular targets, and assess its potential synergy with gefitinib.

METHODS: The antiproliferative, proapoptotic, antimigratory, and anti‑invasive effects of HDI and ASPA were evaluated in 4T1, MDA‑MB‑231, and MCF‑7 cells via CCK‑8, colony formation, flow cytometry, wound‑healing, and Transwell assays. Anti‑tumor efficacy, anti‑lung metastatic effects, and systemic toxicity were assessed in a BALB/c 4T1 orthotopic breast tumor model. Mechanistic studies employed data‑independent acquisition (DIA) proteomics, Western blotting, immunohistochemistry, UPLC-MS, network pharmacology, molecular docking, and microscale thermophoresis. The synergistic activity of HDI with gefitinib was examined in TNBC cells.

RESULTS: UPLC-MS identified 1112 constituents in HDI, with deacetylasperulosidic acid, D-(+)-malic acid, and asperulosidic acid among the most abundant compounds. HDI dose- and time-dependently inhibited the proliferation of 4T1, MDA-MB-231, and MCF-7 cells, reduced colony formation, induced apoptosis, and suppressed migration and invasion. In the orthotopic 4T1 breast cancer model, HDI significantly decreased primary tumor growth and lung metastatic burden in a dose-dependent manner, while showing lower systemic toxicity than cisplatin. DIA proteomics identified PTK2B and CCN2 as key downregulated proteins and revealed suppression of focal adhesion-related signaling. Mechanistic validation showed that HDI inhibited PTK2B phosphorylation and CCN2 expression, blocked KRAS-p38 MAPK signaling, reversed EMT, and regulated extracellular matrix remodeling by increasing TIMP2 and decreasing MMP2. Functional activation or overexpression of PTK2B and CCN2 partially weakened the inhibitory effects of HDI, supporting their involvement in HDI action. Asperulosidic acid, a major iridoid glycoside in HDI, reproduced the antitumor and antimetastatic effects of HDI, directly bound PTK2B with a Kd of 56.7 μM, and suppressed the PTK2B/CCN2-KRAS-p38 axis. HDI also synergized with gefitinib in MDA-MB-231 cells and further inhibited migration, invasion, EMT, and PTK2B/CCN2-associated signaling.

CONCLUSION: HDI inhibits breast cancer growth and lung metastasis by dual targeting of PTK2B and CCN2, thereby suppressing KRAS-p38 MAPK signaling, reversing EMT, and modulating TIMP2/MMP2-mediated extracellular matrix remodeling. Asperulosidic acid is identified as an important active constituent contributing to these effects. The favorable safety profile of HDI and its synergistic interaction with gefitinib support its potential application as a multitarget adjuvant therapy for metastatic breast cancer, particularly TNBC.