Methods: To address this, we utilized our HA hydrogel platform to study the relationship between matrix rigidity-induced dormancy and drug resistance of BMBC spheroids. BMBC spheroids were prepared using MDA-MB-231Br, a triple negative human-derived BMBC cell line, or BT474Br3, a human epidermal growth factor receptor 2 positive (HER2 +) human-derived BMBC cell line. Spheroids were cultured on soft (~0.4 kPa) or stiff (~4.5 kPa) HA hydrogels for 5 days and treated with either Paclitaxel (PTX) or Lapatinib (LAP). Proliferation and apoptosis assays were conducted 2 days post-treatment to measure the spheroids’ drug response. Gene expression analysis was performed using qRT-PCR for molecules associated with glucocorticoid receptor (GR) signaling, which has been previously implicated in the development of drug resistance in breast cancer. To further confirm the role of GR signaling, particularly serum/ glucocorticoid-regulated kinase 1 (SGK1), in mediating drug resistance, an inhibitor of SGK1 (GSK650394) was used and the response to the drug treatment was measured.
Results: Spheroids cultured on soft HA hydrogels exhibited a dormant state, with low proliferation, while spheroids cultured on stiff HA hydrogels exhibited enhanced proliferation. Further, spheroids in the soft HA hydrogel condition displayed resistance to treatment with PTX or LAP, evidenced by the lack of significant change in proliferation and apoptosis. Conversely, spheroids on stiff HA hydrogels exhibited decreased proliferation and increased apoptosis in response to PTX or LAP treatment. Moreover, GR and SGK1 expression was upregulated in the soft HA hydrogel condition for both cell types. However, in MDA-MB-231Br spheroids, RANBP1 (Ran-specific binding protein 1), downstream of SGK1, was upregulated in the soft HA hydrogel condition in response to treatment with PTX while, in BT474Br3 spheroids, GSK-3β (glycogen synthase kinase 3β) and β-catenin, was upregulated in response to treatment with LAP. This data provided evidence that drug resistance was mediated by, in part, by GR signaling via SGK1 and RANBP1 in triple negative BMBC cells or SGK1, β-catenin, and GSK-3β in HER2+ BMBC cells. Accordingly, SGK1 inhibition alleviated drug resistance in spheroids on soft HA hydrogels indicating SGK1 as a potential therapeutic target to mediate drug resistance in BMBC.
Conclusions: Overall, our HA hydrogel system demonstrates the effects of matrix rigidity-induced dormancy on drug resistance. We provide evidence that GR signaling, in part, mediates dormancy associated drug resistance in both triple negative and HER2+ BMBC cells. The HA hydrogel system can serve as a platform to further understand the mechanisms of dormancy and drug resistance in the context of BMBC.