126 - Radiotherapy Effects on CREB-1 Signaling, Membrane TROP-2, and Sacituzumab Govitecan Response in Triple-Negative Breast Cancer

03:50pm - 04:00pm ET

Room 204

Presenter(s)

Wen-Chi Yang, MD, PhD - National Taiwan University Hospital, Taipei, 100

W. C. Yang^1,2, M. F. Wei³, C. S. Huang⁴, and S. H. Kuo^2,3; ¹Department of Radiation Oncology, National Taiwan University Cancer Center, Taipei, Taiwan, ²Graduate Institute of Oncology, National Taiwan University College of Medicine, Taipei, Taiwan, ³Division of Radiation Oncology, Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan, ⁴Departments of Surgery, National Taiwan University Hospital, Taipei, Taiwan

Purpose/Objective(s):

Sacituzumab govitecan (SG) is a TROP-2 directed antibody drug conjugate (ADC) with proven anti-tumor activity in triple-negative breast cancer (TNBC). However, its clinical benefit may be limited in tumors with low baseline TROP-2 expression. We investigated whether radiotherapy (RT) enhances SG efficacy by upregulating membrane TROP-2 expression and sought to clarify the mechanistic role of CREB-1 in this RT-induced upregulation.

Materials/Methods:

Human TNBC cell lines (MDA-MB-231, MDA-MB-453, and MDA-MB-468) were used to evaluate RT–SG interactions. Cytotoxicity was assessed via MTT assay, while radiosensitization was evaluated by clonogenic assays. DNA damage and apoptosis were analyzed by western blotting (?H2AX and cleaved PARP) and flow cytometry (Annexin V/PI and surface TROP-2 staining). To interrogate the mechanism, CREB-1 signaling was assessed (p-CREB-1) after RT (8 Gy) with or without a CREB-1 inhibitor. Finally, MDA-MB-231 (low TROP-2) and MDA-MB-468 (high TROP-2) xenografts were established to evaluate tumor control and survival.

Results:

SG cytotoxicity varied across TNBC lines (IC50: 4.53 µM [MDA-MB-231], 315.7 nM [MDA-MB-453], and 126.6 nM [MDA-MB-468]), correlating with baseline TROP-2 levels. Clonogenic assays demonstrated synergistic growth inhibition with RT plus SG across all lines. RT dose-dependently (5–20 Gy) increased membrane TROP-2 expression at 24 h post-treatment. Combining RT and SG significantly heightened DNA damage and apoptosis compared to monotherapies. Notably, sequencing modulated efficacy: RT followed by SG (24 h interval) elicited more potent DNA damage and apoptosis than SG administered 1 h before RT, with significantly increased apoptotic fractions in MDA-MB-468 (57.60% vs. 32.47%, P < 0.01) and MDA-MB-231 (8.80% vs. 4.02%, P < 0.05).

Mechanistically, RT (8Gy) induced rapid CREB-1 activation, with p-CREB-1 increasing at 2–4 h post RT. A CREB-1 inhibitor (5 µM) suppressed both basal and RT induced p-CREB-1. CREB-1 inhibition abrogated TROP-2 upregulation. Flow cytometry showed that CREB-1 inhibition reduced the RT induced TROP-2 increase (MFI: 5.91 vs. 1.97 in MDA-MB-231, P < 0.0001; 5.77 vs. 2.75 in MDA-MB-468, P < 0.001), identifying CREB-1 as a key mediator. In vivo, RT followed by SG enhanced tumor suppression compared to control, SG alone, and RT alone in MDA-MB-468 (P < 0.001, P < 0.05, and P < 0.01) and MDA-MB-231 (P < 0.001, P < 0.001, and P < 0.01) xenografts. Furthermore, RT followed by SG significantly prolonged survival in the MDA-MB-231 model compared to all other groups (P < 0.001).

Conclusion:

Radiotherapy potentiates SG efficacy in TNBC by enhancing DNA damage and apoptosis through CREB-1–dependent membrane TROP-2 upregulation. These data provide a potential mechanistic rationale for sequencing RT before SG and suggest the CREB-1–TROP-2 axis as a therapeutic target to improve ADC responsiveness, particularly in tumors with low baseline TROP-2 expression.