Tiffany Nicole Seagroves

Oncogenic cell signaling, including the activation of cellular stress or cytokine-induced pathways, is a hallmark of cancer. In triple-negative breast cancer (TNBC), p38 MAPK phosphorylates glucocorticoid receptors (GR) at Ser134 in response to cytokines such as TGFβ1. This activated Phospho-Ser134-GR (pSer134-GR) regulates genes promoting cancer cell migration, invasion, and altered metabolism. Glucocorticoids also activate the functionally and structurally-related mineralocorticoid receptors (MR) whose ligand, aldosterone, mediates hypertension, inflammation and fibrosis. We and others have previously shown the role of GR in the advanced phenotypes exhibited by TNBC, but the potential importance of GR-MR crosstalk and the specific contribution of MR remains unknown. Interestingly, our new analyses of MR expression in TNBC tumors revealed elevated MR transcript levels relative to luminal breast cancer subtypes, which are predictive of worse overall survival. Cytoplasmic MR-GR complexes that are formed upon treatment of TNBC cells with TGFβ1 required both p38 MAPK signaling and pSer134-GR. In contrast, nuclear MR-GR complexes predominated in response to dexamethasone and/or aldosterone. MR antagonists (spironolactone, finerenone) significantly reduced aldosterone- or TGFβ1-induced migratory and stemness properties and blocked MR-pGR and MR-GR interactions; MR knockdown similarly attenuated these advanced cancer phenotypes. MR expression was essential for both a functional p38 MAPK module and pSer134-GR downstream of TGFβ1 receptor activation. Crucially, MR-deficient models exhibited reduced lung metastasis following mouse tail-vein injection, phenocopying cells harboring phospho-mutant S134A-GR. As with p-Ser134-GR, we define a novel role for MR-GR cooperation downstream of TGFβ1 for regulation of TNBC cell migration, stemness, and in vivo lung colonization.

Supplementary Figure 2. Randomization schemes to assign treatment cohorts, based on mouse body weight and whole head total photon flux.

Breast cancer brain metastasis (BCBM) remains a clinical challenge marked by limited therapeutic options and poor survival rates. Approximately 30% of all patients with metastatic breast cancer develop BCBM, with the highest incidence in patients with aggressive molecular subtypes, including triple-negative breast cancer (TNBC). Patients with TNBC with brain metastasis experience rapid disease progression and significantly reduced survival times due to a lack of targeted treatments that can penetrate the blood–brain barrier (BBB) and effectively control metastatic expansion. Current treatment options, such as whole-brain radiotherapy and chemotherapy, offer limited efficacy and are associated with significant toxicities, underscoring the urgent need for novel therapeutics that can target BCBM directly. We developed innovative colchicine binding site inhibitors (CBSI) targeting tubulin, SB-216 and SP-1-39, that show potent preclinical efficacy against brain and extracranial metastases in BCBM models. Both CBSIs cross the BBB, inhibit cell growth and migration, and induce apoptosis with low nanomolar potencies, similar to Azixa (MPC-6827), another CBSI previously evaluated in clinical trials. SB-216 reduced brain and concurrent extracranial metastases in a preventive dosing paradigm, extending overall survival. SB-216 also suppressed the expansion of preestablished brain lesions. In a taxane-refractory patient-derived TNBC model, SP-1-39 therapy markedly reduced brain and extracranial tumor burden. Together, these results highlight the promising therapeutic potential of SB-216 and SP-1-39 in treating taxane-sensitive or -resistant BCBM, filling a critical gap in TNBC management by offering targeted treatments that can cross the BBB and combat chemorefractory disease.

Supplementary Figure 1. SB-216 and SP-1-39 potently inhibit tumor cell growth in TNBC TxR sublines, and TxR cells up-regulate P-gp (ABCB1) expression at the mRNA and protein levels.

Supplementary Figure 4. Maximum tolerated dose (MTD) study for SP-1-39 HCl salt when administered by intraperitoneal (IP) injection.

Supplementary Figure 6: Brain vacuolization was observed in all mice with end-stage brain metastases.

Supplementary Figure 3. Bioimaging data for example mice from the SB-216 overall survival study through day 21 of treatment.

Supplementary Figure 5: No gross changes in pathology were observed between vehicletreated and CBSI-treated mice at treatment study endpoints.

Supplementary Table 1. The BBB score, which accounts for multiple structural properties of compounds to predict the likelihood of activity in the central nervous system, was calculated in the Schrodinger Modeling software using the equation described in (50): ∑T0 [Aro_R, HA, (1.5) MWHBN, 2 (TPSA), (0.5) pka].