Abstract P1-10-09: AQP4 inhibition prevents cytotoxic edema of AQP4+ astrocytes but promotes tumor growth of AQP4+ breast cancer brain metastasis
Authors: Contreras-Zarate Maria J. , ALvarez-Eraso Karen , Tsuji Nicole , Kabos Peter , Ormond D.Ryan , Karam Sana , Cittelly Diana 
Published: March 2023

Abstract
Brain edema is a complication of radiation used to treat brain metastasis (BM) in which the brain parenchyma accumulates fluid and ions, often leading to the suspension of systemic anticancer treatment. While brain edema is often attributed to disruption of the Blood Brain Barrier (BBB), RTx induces cytotoxic-edema, a premorbid cellular process whereby extracellular Na+ and other cations enter into neurons and astrocytes and accumulate intracellularly, resulting in osmotic expansion of the cells and necrotic cell death. Aquaporin 4 (AQP4) is a main regulator of osmotic expansion (water intake) in astrocytes and we have shown that RTx upregulates AQP4 in astrocytes and leads to astrocytic swelling in vitro. However, whether pharmacological modulation of AQP4 could be used to prevent cytotoxic brain edema in RTx-treated BM and its impact on metastatic tumor progression remains unknown. Goal: To determine if the FDA-approved drug Topiramate (TPM), an anti-epileptic drug able to inhibit AQP4) can prevent astrocytic swelling in vitro, reduce RTx-induced brain edema and modulate brain metastatic progression. Results: Electron microscopy of brain cortex from mice treated with 35 Gy RTx showed acute astrocytic end-feet swelling and increase in AQP4 expression compared with non-irradiated mice. A single 8 Gy dose increased astrocytic cell area of human astrocytes by 4.8 fold compared with non-irradiated cells 24 h after RTx. This increased cell-swelling did not result from senescence-associated cellular hypertrophy, as staining of senescent β-galactosidase positive (SA-β-Gal+) cells showed that Rtx-induced astrocytic area only increased significantly in non-senescent (SA-β-Gal- cells). shAQP4s reduced AQP4 levels by 60% and 50%, respectively, and significantly reduced RTx-induced astrocytic swelling. Since there are no FDA-approved AQP4 inhibitors, we tested whether the AQP4-blocking function of TPM could be sufficient to prevent cytotoxic edema, prevent BBB dysfunction and protect from necrotic cell death in vitro. TPM pretreatment did not alter radiation-induced ERK1/2 or AKT activation (a known maker of radioprotection) in astrocytes, but TPM decreased radiation-induced PARP-cleavage, pP38 and pJNK levels. TPM prevented loss of Trans-electric epithelial resistance (TEER) of Rtx-treated astrocytes, but was not able to protect astrocytes from ultimate cell death. Immunohistochemical analysis of a cohort of breast cancer BM showed heterogeneous AQP4 expression in cancer cells ranging from 1.6% to 91% AQP4+ tumoral areas and from 0.6% to 86.9% in stroma. AQP4 inhibition using shRNAs decreased proliferation and survival of AQP4 + 231BR, and EO711 cells in vitro. However, TPM did not alter survival of AQP4+ or AQP4- cells in vitro, suggesting that while AQP4 expression is important for survival of AQP4+ cells in vitro, the inhibition of AQP4 function by TPM is not sufficient to decrease their growth. To determine if TPM could decrease brain edema without negatively impacting tumor progression, female NSG mice were injected intracardially with JmT1BR3 AQP4-cells and ten days later randomized to (1) RTx + vehicle, (2) RTx + TPM (2 days prior to irradiation), (3) Non-RTx + vehicle, and (4) Non-RTx + TPM. TPM decreased brain-water content (a marker of brain edema) in irradiated mice as compared with vehicle-treated mice, without alteration of metastatic burden 21 days post-injection. However, a similar study using AQP4+ E0711 cells in C57Bl6 mice showed TPM was less effective in decreasing brain water content and resulted in a significant increase in extracranial metastatic tumor burden, suggesting that TPM can promote tumor progression by non-tumor intrinsic mechanisms. Conclusions: while TPM shows promise in preventing RTx-induced brain edema, our results show a potential pro-tumorigenic mechanism for TPM that warrants further investigation.
Citation Format: Maria J. Contreras-Zarate, Karen ALvarez-Eraso, Nicole Tsuji, Peter Kabos, D.Ryan Ormond, Sana Karam, Diana Cittelly. AQP4 inhibition prevents cytotoxic edema of AQP4+ astrocytes but promotes tumor growth of AQP4+ breast cancer brain metastasis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-10-09.