Traumatic brain injury (TBI) is a significant public health problem in the United States. Despite preclinical success of various drugs, to date all clinical trials investigating potential therapeutics have failed. Recently, sex steroid hormones have sparked interest as possible neuroprotective agents after traumatic injury. One of these is 17b-estradiol (E2), the most abundant and potent endogenous vertebrate estrogen. The goal of our study was to investigate the acute potential protective effects of E2 or the specific G protein-coupled estrogen receptor 1 (GPER) agonist G-1 when administered in an intravenous bolus dose 1 hour post-injury in the lateral fluid percussion (LFP) rodent model of TBI. The results of this study show that, when assessed at 24 hours post-injury, E2 or G-1 confers protection in adult male rats subjected to LFP brain injury. Specifically, we found that an acute bolus dose of E2 or G-1 administered intravenously 1 hour post-TBI significantly increases neuronal survival in the ipsilateral CA 2/3 region of the hippocampus and decreases neuronal degeneration and apoptotic cell death in both the ipsilateral cortex and CA 2/3 region of the hippocampus. We also report a significant reduction in astrogliosis in the ipsilateral cortex, hilus, and CA 2/3 region of the hippocampus. Finally, these effects were observed to be chiefly dose-dependent for E2, with the 5 mg/kg dose generating a more robust level of protection. Our findings further elucidate estrogenic compounds as a clinically relevant pharmacotherapeutic strategy for treatment of secondary injury following TBI, and intriguingly, reveal a novel potential therapeutic target in GPER.
Breast cancer brain metastases (BM) affect younger women disproportionally, including those lacking estrogen receptor (ER), progesterone receptor, and HER2 (known as triple-negative breast cancer; TNBC). Previous studies in preclinical models showed that pre-menopausal levels of estradiol (E2) promote TNBC-BM through incompletely understood mechanisms involving reactive astrocytes. Herein, a novel mechanism involving E2-dependent upregulation of brain-derived neurotrophic factor (BDNF) in astrocytes, and subsequent activation of tumor cell tropomyosin kinase receptor B (TrkB), is identified. E2 increased experimental BM of TNBC 4T1BR5 and E0771 cells by 21 and 3.6 fold, respectively, compared to E2-depleted mice. ERα + reactive astrocytes were found at early and late stages of BM, and E2 upregulated BDNF in ER + reactive astrocytes in vitro and in vivo. TrkB was expressed in TNBC brain-trophic cell lines, BM-patient-derived xenografts, and breast cancer BM. Conditioned media from E2-treated astrocytes (CM-E2) activated TrkB and downstream AKT, ERK, and PLC-γ signaling in TNBC cells, increasing their invasiveness and tumor-initiating capability in vitro. The promotion of BM by E2-activated astrocytes was found to be more complex, involving feedback loops and other receptor tyrosine kinases. In 4T1BR5 cells, there was a positive feedback loop whereby astrocytic BDNF induced cancer cell BDNF translation. Upregulation of cancer cell BDNF was required to promote full invasiveness of 4T1BR5 in response to CM-E2, and was observed in brain metastatic cells in E2-treated mice in vivo. Moreover, the non-competitive BDNF/TrkB inhibitor ANA-12 reduced E2-induced 4T1BR5 BM to levels similar to OVX mice. BDNF also activated EGFR in TrkB + EGFR + TNBC cells, suggesting that E2 action through astrocytes activates redundant pathways promoting BM. These findings have important therapeutic implications, as they provide a rationale to use E2-depletion therapies or TrkB inhibitors to prevent or delay development of BM in younger women.
Brain metastases are an increasing burden among breast cancer patients, particularly for those with HER2+ and triple negative (TN) subtypes. Mechanistic insight into the pathophysiology of brain metastases and preclinical validation of therapies has relied almost exclusively on intracardiac injection of brain-homing cells derived from highly aggressive TN MDA-MB-231 and HER2+ BT474 breast cancer cell lines. Yet, these well characterized models are far from representing the tumor heterogeneity observed clinically and, due to their fast progression in vivo, their suitability to validate therapies for established brain metastasis remains limited. The goal of this study was to develop and characterize novel human brain metastasis breast cancer patient-derived xenografts (BM-PDXs) to study the biology of brain metastasis and to serve as tools for testing novel therapeutic approaches. We obtained freshly resected brain metastases from consenting donors with breast cancer. Tissue was immediately implanted in the mammary fat pad of female immunocompromised mice and expanded as BM-PDXs. Brain metastases from 3/4 (75%) TN, 1/1 (100%) estrogen receptor positive (ER+), and 5/9 (55.5%) HER2+ clinical subtypes were established as transplantable BM-PDXs. To facilitate tracking of metastatic dissemination using BM-PDXs, we labeled PDX-dissociated cells with EGFP-luciferase followed by reimplantation in mice, and generated a BM-derived cell line (F2-7). Immunohistologic analyses demonstrated that parental and labeled BM-PDXs retained expression of critical clinical markers such as ER, progesterone receptor, epidermal growth factor receptor, HER2, and the basal cell marker cytokeratin 5. Similarly, RNA sequencing analysis showed clustering of parental, labeled BM-PDXs and their corresponding cell line derivative. Intracardiac injection of dissociated cells from BM-E22-1, resulted in magnetic resonance imaging-detectable macrometastases in 4/8 (50%) and micrometastases (8/8) (100%) mice, suggesting that BM-PDXs remain capable of colonizing the brain at high frequencies. Brain metastases developed 8–12 weeks after ic injection, located to the brain parenchyma, grew around blood vessels, and elicited astroglia activation characteristic of breast cancer brain metastasis. These novel BM-PDXs represent heterogeneous and clinically relevant models to study mechanisms of brain metastatic colonization, with the added benefit of a slower progression rate that makes them suitable for preclinical testing of drugs in therapeutic settings.
Introduction: Triple negative breast cancers (TNBC, which lack estrogen receptor (ER), progesterone receptor and HER2), occur three times more frequently in premenopausal women and metastasize expeditiously to brain and lungs. Clinical and experimental evidence shows that estrogen, the main premenopausal ovarian hormone, promotes metastases of TNBC, through their action on the tumor microenvironment. We have shown that 17-β-Estradiol (E2) promotes experimental brain metastases (BM) of TNBC cells by inducing estrogen-receptor positive (ER+) astrocytes in the brain niche to secrete growth factors that promote cancer cell migration, invasion and growth. Given that 53% of pre-menopausal women with TNBC develops BM and 80% die within a year, it is now critical to define whether blocking estrogen signaling could have therapeutic value to prevent or delay progression of BM. Results: To assess whether E2-depletion could prevent brain metastatic colonization, brain trophic sublines of human TNBC (231BR), murine TNBC (4T1BR5), a non-selected TN cell line syngeneic to C57BL mice (E0771-GFP-luc) were intracardially (ic) injected two-days post-endocrine initiation, in ovariectomized (OVX) female mice treated with a) E2 (levels equivalent to those found in pre-menopausal women), b) placebo (OVX), or c) the aromatase-inhibitor (AI) letrozole (OVX + Letrozole) to block brain and peripheral E2-synthesis. BM were quantified histologically, via MRI or ex-vivo imaging of brains at euthanasia. E2-treated mice injected with E0771-GFP-luc cells showed an average of 6.6± 3.5 metastatic clusters per mice, compared to 3.6 ± 3.2 and 1.8 ± 1.8 in OVX mice alone or OVX + letrozole treated mice (a 3.6 fold increase, P=0.0013, E2 vs OVX + Let). Imaging of excised brains showed comparable results, with a mean total photon flux of 5.1 × 106 ± 5.3 × 106 in E2 vs 1.1 × 106 ± 1.5 × 106 in OVX + letrozole (P=0.0056). Similar preventive effects of E2-depletion were found in 4T1BR5 and 231BR-cell injected mice, using histological quantification and MRI as endpoints. To assess whether E2-depletion therapies could decrease brain metastatic progression in a therapeutic model mimicking the clinical setting, E0771-GFP-luc cells were injected in OVX-mice that had been supplemented with E2 and metastasis allowed to develop for 6 days. Mice were then randomized to a) E2, b) E2-withdrawal (E2WD) or c) E2WD+ letrozole, and then all mice were irradiated (whole brain irradiation, single-dose, 15 Gy) to mimic current standard of care for BMs. All mice were euthanized 10 days later, when E2-treated mice showed signs of CNS impairment. Mice that received E2WD+letrozole showed the lowest BM burden compared to mice that remained in E2-treatments, as assessed by histological quantification (0.7 ± 1.2 vs 6.5 ± 5.3 metastatic clusters, respectively, P=0.0132) and ex-vivo brain imaging (mean total-photon flux 3.4 × 105 ± 4.1 × 105 in E2WD + let vs 3.1 × 106 ± 1.4 × 106 in E2, P=0.037). Brain metastasis in TNBC often occur in women who also present with systemic metastatic disease, and E0771-GFP-luc cells can colonize multiple organs following ic injection. Thus, non-brain metastatic burden (systemic metastasis) was measured by in vivo imaging before euthanasia in this cohort. Unlike brain metastasis, E2WD or E2WD+letrozole showed no statistically significant effect on systemic metastatic burden (mean total flux 1.41 × 106 ± 0.9 × 106 in E2, vs 0.54 × 106 ± 2.6 × 106 in E2WD vs 0.79 × 106 ± 1.0 × 106 in E2WD+let, P=0.416), supporting the notion that brain-specific rather than systemic mechanisms drive progression of brain metastasis in response to E2. Conclusions: These studies have important clinical implications as they provide pre-clinical evidence that ovarian suppression in combination with AIs can prevent and delay progression of established brain metastasis in younger women with TNBC. Citation Format: Diana M. Cittelly, Maria J. Contreras-Zarate, Ricaurte A. Marquez-Ortiz, Nicole Day, Bebhinn Nagle, Ryan Ormond, Virginia F. Borges, Peter Kabos. Estrogen-depletion therapies prevent and delay progression of brain metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-20-03.
Introduction: Interleukin-13 receptor alpha 2 (IL13Rα2) is a high affinity receptor for IL-13, best known as a decoy receptor that lacks intracellular kinase activity. Yet, IL13Rα2 has been shown to play an important role in the pathobiology of glioblastomas, colon, ovarian and pancreatic cancer, through the recruitment and activation of FAK, SRC and other intracellular signaling pathways. In breast cancer, the function of IL13Rα2 remains controversial. Increased IL13Rα2 expression is associated with poor prognosis for high grade tumors but not low-grade tumors, while was also reported to be associated with poor prognosis in luminal subtypes only. Increased levels of IL13Rα2 were reported in breast cancer cells with brain metastatic tropism, yet the function of IL13Rα2 during brain metastatic colonization remains unexplored. Since IL-13 is expressed by various cells within the brain niche, this study tests the hypothesis that ligand dependent and independent mechanisms modulate IL13Rα2 function during brain metastatic progression. Methods and results: qPCR, Western blot and IHC analysis demonstrated that IL13Rα2 was expressed at various levels in HER2+ and TN brain-trophic breast cancer cell lines (231BR, JmT1BR3, 4T1BR5, F2-7), brain-metastasis patient-derived xenografts (BM-PDXs) and a cohort of 26 clinical breast cancer brain metastasis (BCBM) samples. To assess how IL13Rα2 influences pro-metastatic abilities independent of ligand, inducible-shRNAs and CRISPR/cas9 were used to downregulate IL13Rα2 expression in 231BR cells. Knockdown of IL13Rα2 reduced the ability of 231BR cells to migrate (41.7% ± 4.5 vs 71.5% ± 9.7; p=0.001) and invade (49.5% ± 6.5 vs 73.1% ± 2.9; p=0.001) compared to the empty vector (EV) control cells. Furthermore, inducible-IL13Rα2 downregulation decreased significantly the proliferation (-22.1% at 96 hours, p<0.0001) and migration (-10.6% at 24 hours, p<0.0001) as compared to EV controls. Consistently, inducible overexpression of IL13Rα2 in HER2+ BT474 cells (which lack endogenous IL13Rα2) increased proliferation by 22.8% (p<0.0005, at 5 days) compared to parental cells. Knockdown of IL13Rα2 in 231BR cells impaired their ability to colonize the brain in mice following intracardiac injection as compared to EV 231Br cells (median number of metastatic clusters: 11.8 ± 5.5 vs 30.5 ± 13.9, respectively, p=0.2671), suggesting that upregulation of IL13Rα2 facilitates early brain metastatic colonization. To assess whether levels of IL13Rα2 remain high at late stages of brain metastasis, a publicly available transcriptome profile of 21 matched primary tumor and brain metastases was analyzed. Surprisingly, high IL13RA2 mRNA expression in brain metastasis (but not in primary tumors) predicted better brain-metastasis free survival (n=21, p=0.0026). Since the brain has several sources of IL-13, we assessed whether ligand-dependent mechanisms alter IL13Rα2 function, leading to a loss of expression in more aggressive brain metastasis. Western blot and IF analyses showed IL-13 expressed in reactive astrocytes. IL-13 treatment of serum-deprived IL13Rα2+ 231BR and JmT1Br cells resulted in decreased proliferation, a moderate increase in invasion, and phosphorylation of FAK and SRC. Knockdown of IL13Rα2 blocked the ability of IL-13 to phosphoactive FAK and SRC, and prevented the antiproliferative effect of IL-13 on 231BR cells. Conclusions: These results suggest that ligand-dependent and independent mechanisms modulate IL13Rα2 through brain metastatic colonization. Ligand-independent IL13Rα2 promotes proliferation and invasion of cancer cells, favoring early dissemination and brain colonization. Loss of IL13Rα2 at later stages of BM, may prevent IL-13-induced blockage of proliferation and promote aggressiveness of established BM. Citation Format: Ricaurte Alejandro Marquez-Ortiz, Maria J Contreras-Zarate, Nicole L Day, Ryan Ormond, Virginia F Borges, Diana M Cittelly. Ligand dependent and independent roles of interleukin-13 receptor alpha 2 in breast cancer brain metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-04.
Epidemiological data indicate that drug abuse rates increase following traumatic brain injury (TBI), but the underlying reasons remain unclear. There is overlap in reward pathways and regions commonly damaged in TBI suggesting that TBI could alter risk for drug abuse. Adult male Sprague Dawley rats underwent sham (control) or lateral fluid percussion injury of moderate severity followed by assesment of tolerance development and intravenous self ‐administration behavior. The antinociceptive effects of oxycodone were evaluated using hot plate‐induced paw withdrawal and warm water tail withdrawal models both acutely and following chronic administration of oxycodone. The reinforcing effects of oxycodone were examined using acquisition of oxycodone self‐administration behavior. No consistent differences have been detected in the antinociceptive effects of oxycodone between TBI subjects and sham injured subjects. However the data suggest that the high (0.03 mg/kg/infusion) and intermediate (0.01 mg/kg/infusion) doses of oxycodone resulted in a faster rate and a higher total percentage of TBI subjects acquiring self‐administration. Additionally, the TBI subjects appeared to be less sensitive to oxycodone's effects, self‐administering the highest number of infusions at the 0.03 mg/kg/infusion dose as compared to sham injured subjects. Research supported by DoD grant W81XWH‐11–1‐0374.
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