Abstract P3-06-05: Importance of TGFβ-SMAD3 axis in resistance to anti-HER2 drugs
Authors: Shimoda M , Chihara Y , Kagara N , Naoi Y , Shimomura A , Shimazu K , Kim SJ , Noguchi S 
Published: May 2016

Abstract
Aim: The aim of this study was to elucidate the role of transforming growth factor β (TGFβ) in the resistance of HER2-positive breast cancer cells to anti-HER2 drugs including trastuzumab and lapatinib.
Methods: A HER2-positive breast cancer cell line, SKBR3, was cultured in the presence or absence of TGFβ for 14 days. Subsequently, TGFβ-treated cells were cultured for seven days with or without the anti-HER2 drugs. Sensitivity to trastuzumab and lapatinib was estimated by the WST-8 cell viability assay or absolute cell counts using In Cell Analyzer (GE Healthcare). Proportion of CD44+ CD24– breast cancer stem cells was estimated by flow cytometry of cells immunostained with anti-CD44 and anti-CD24 antibodies. For clinical study, 33 patients with HER2-positive breast cancer receiving neoadjuvant paclitaxel plus trastuzumab in our institution were analyzed. Among the cases, 27 biopsy samples obtained before any treatment from 27 patients who completed 12 cycles of weekly paclitaxel and trastuzumab were subjected to CD24 immunohistochemistry.
Results: SKBR3 cells cultured with TGFβ for 14 days exhibited decreased sensitivity to both trastuzumab and lapatinib. Time course study revealed that continuous stimulation for 14 days with TGFβ was required for the resistance to anti-HER2 drugs. Activation of SMAD3, a downstream target molecule of TGFβ, was enhanced over time, judged by the increase in phosphorylation and in nuclear translocation. During 14 day culture with TGFβ, proportion of CD44+ CD24– cells were dramatically increased, and mammosphere formation, another marker of breast cancer stem cells, was significantly enhanced compared to cells treated without TGFβ. Among four HER2-positive breast cancer cell lines, only SKBR3 cells showed increased proportion of CD44+ CD24– cells and resistance to the anti-HER2 drugs, while other two cell lines exhibited epithelial-mesenchymal transition (EMT) in response to TGFβ. To explore the possibility of targeting TGFβ-SMAD3 axis to overcome resistance to anti-HER2 therapy, we used SIS3, a specific inhibitor of SMAD3. Importantly, SIS3 completely restored the sensitivity to both trastuzumab and lapatinib of TGFβ-treated SKBR3 cells, with the decrease in the proportion of CD44+ CD24– cells. These in vitro results suggest that CD24 downregulation can be a surrogate marker of resistance to anti-HER2 therapy. To establish this, we evaluated the CD24 expression in tumor samples of breast cancer patients who received paclitaxel plus trastuzumab in the neoadjuvant setting. Weak CD24 expression in tumor cells in biopsy samples obtained before any treatment was significantly correlated with poorer response to the drugs.
Conclusion: These data clearly indicate the importance of TGFβ-SMAD3 axis in the acquired resistance to anti-HER2 drugs. Moreover, resistance to anti-HER2 therapy is associated with the property of breast cancer stem cells rather than EMT. Targeting TGFβ-SMAD3 axis warrants further investigation for overcoming resistance to anti-HER2 therapeutics.
Citation Format: Shimoda M, Chihara Y, Kagara N, Naoi Y, Shimomura A, Shimazu K, Kim SJ, Noguchi S. Importance of TGFβ-SMAD3 axis in resistance to anti-HER2 drugs. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-05.