Hyeonji Hwang

Combination therapies are key to overcoming resistance to androgen receptor (AR) signaling inhibitors in prostate cancer. Current paradigms for developing synergistic therapies, however, remain chronically inefficient and resource‐intensive due to the prohibitive scale of the combinatorial screening space and a lack of computational frameworks for navigating signaling crosstalk. This work introduces a hybrid in silico and in vitro lead discovery platform that integrates knowledge‐augmented large language models (LLMs) with an oxygen‐supplemented 3D spheroid system. By comparing compound mechanism‐of‐action annotations with disease‐relevant signaling crosstalk, the LLM framework nominates drug pairs with predictive performance and interpretable, pathway‐based rationales. This computational pipeline is complemented by an engineered 3D spheroid model that utilizes oxygen supplementation to mitigate artifactual necrosis, a common confounder that masks synergistic signals in standard screening. Using this approach to screen 3,592 natural products, we identified berberine‐enzalutamide as an in vitro combination hit that re‐sensitized resistant prostate cancer cells to AR blockade. Molecular and transcriptomic profiling revealed changes in mTORC1 and AMPK signaling that were consistent with the pathway‐based hypothesis generated by LLM. These results establish proof of concept that mechanism‐informed language modeling coupled with 3D screening can prioritize and experimentally evaluate drug‐combination hypotheses at an early stage of discovery.

Castration-resistant prostate cancer (CRPC) is an advanced form of therapy-resistant prostate cancer with limited therapeutic options and a leading cause of prostate cancer-related mortality. TROP2, a transmembrane glycoprotein, is highly expressed in CRPC and has emerged as a promising therapeutic target. Here, we found that SLC4A7, a sodium/bicarbonate cotransporter, physically interacts with TROP2 and is also widely expressed in advanced prostate cancer. Loss of SLC4A7 significantly decreases TROP2-driven cell growth and invasion in vitro, as well as TROP2-mediated tumor growth and metastasis in vivo, indicating that SLC4A7 plays a major role in TROP2-mediated prostate cancer progression. Mechanistically, downregulation of SLC4A7 decreases intracellular pH levels, leading to reduced NOTCH1 activity and diminished expression of NOTCH1 downstream targets, HES1 and HEY1. Overexpression of TROP2 activates NOTCH signaling, suggesting that TROP2 regulates NOTCH signaling via SLC4A7-mediated intracellular pH regulation. Altogether, our findings present a novel TROP2-SLC4A7-NOTCH signaling axis driving prostate cancer progression and suggest this pathway as a potential therapeutic target for advanced prostate cancer.

Supplementary Figure S2 shows that treatment with recombinant TECD enhances migration ability of prostate cancer cells.

Supplementary Figure S1 shows that Shed TECD is detected in the serum of patients with advanced and clinically significant prostate cancer.

Supplementary Figure S5 shows that TECD regulates pathways involved in metastasis.

Supplementary Figure S3 shows that overexpression of secreted TECD increases migration and invasion ability.

Supplementary Table 1 shows the summary of clinical characteristics of patients for sera TROP2 pull-down.

Supplementary Figure S4 shows that treatment with recombinant TECD enhances the extracellular matrix in prostate cancer.

Metastasis is the main cause of prostate cancer–associated deaths, highlighting the urgent need to determine the mechanisms underlying prostate cancer progression. TROP2 (also known as tumor-associated calcium signal transducer 2) is an oncogenic transmembrane surface protein that is highly expressed in metastatic prostate cancer. Naturally occurring cleavage of TROP2 leads to a release of the TROP2 extracellular domain (TECD) into the extracellular environment. In this study, we identified an important functional role of TECD in prostate cancer metastasis. TECD was detectable in media from prostate cancer cells and serum from patients with clinically significant prostate cancer. Although shed TECD did not affect prostate cancer cell proliferation and tumor growth, it increased cell migration and invasion in vitro and promoted metastatic colonization and spontaneous metastasis in vivo. TECD interactome and proteomic studies revealed that TECD binds to EGFR and shed TECD modulates a set of proteins associated with invasion, migration, mTOR signaling, and epithelial-to-mesenchymal transition. Furthermore, elevated shed TECD increased EGFR phosphorylation, resulting in the activation of the EGFR–PI3K–AKT–mTOR pathway in prostate cancer. EGFR inhibitors suppressed the invasive ability of prostate cancer cells driven by TECD overexpression, further supporting the key role of EGFR in TECD-mediated prostate cancer progression. This study uncovers a function of TECD in driving prostate cancer progression and provides mechanistic insights into TECD signaling through EGFR.Significance:Shed extracellular domain of TROP2 binds to and activates EGFR and stimulates the PI3K-AKT-mTOR signaling cascade to promote prostate cancer metastasis, providing potential biomarkers and therapeutic targets.

Metastasis is the main cause of prostate cancer–associated deaths, highlighting the urgent need to determine the mechanisms underlying prostate cancer progression. TROP2 (also known as tumor-associated calcium signal transducer 2) is an oncogenic transmembrane surface protein that is highly expressed in metastatic prostate cancer. Naturally occurring cleavage of TROP2 leads to a release of the TROP2 extracellular domain (TECD) into the extracellular environment. In this study, we identified an important functional role of TECD in prostate cancer metastasis. TECD was detectable in media from prostate cancer cells and serum from patients with clinically significant prostate cancer. Although shed TECD did not affect prostate cancer cell proliferation and tumor growth, it increased cell migration and invasion in vitro and promoted metastatic colonization and spontaneous metastasis in vivo. TECD interactome and proteomic studies revealed that TECD binds to EGFR and shed TECD modulates a set of proteins associated with invasion, migration, mTOR signaling, and epithelial-to-mesenchymal transition. Furthermore, elevated shed TECD increased EGFR phosphorylation, resulting in the activation of the EGFR–PI3K–AKT–mTOR pathway in prostate cancer. EGFR inhibitors suppressed the invasive ability of prostate cancer cells driven by TECD overexpression, further supporting the key role of EGFR in TECD-mediated prostate cancer progression. This study uncovers a function of TECD in driving prostate cancer progression and provides mechanistic insights into TECD signaling through EGFR. Significance: Shed extracellular domain of TROP2 binds to and activates EGFR and stimulates the PI3K-AKT-mTOR signaling cascade to promote prostate cancer metastasis, providing potential biomarkers and therapeutic targets.