Human breast cancer metastases to the brain display GABAergic properties in the neural niche Josh Nemana, John Terminib, Sharon Wilczynskic, Nagarajan Vaidehid, Cecilia Choya,e, Claudia M. Kowolikc, Hubert Lid,e, Amanda C. Hambrechta,f, Eugene Robertsg,1, and Rahul Jandiala,f,1 Divisions of aNeurosurgery and cPathology, Departments of bMolecular Medicine, dImmunology, and gNeurobiochemistry, and eIrell and Manella Graduate School of Biological Sciences, City of Hope, Duarte, CA 91010; and fDepartment of Biology, University of Southern California, Los Angeles, CA 90089 Contributed by Eugene Roberts, November 27, 2013 (sent for review October 16, 2013) Dispersion of tumors throughout the body is a neoplastic process secondary sites (10–14). We previously showed that metastatic responsible for the vast majority of deaths from cancer. Despite cells have the ability to alter the cellular milieu of the brain disseminating to distant organs as malignant scouts, most tumor for growth advantage (3). cells fail to remain viable after their arrival. The physiologic mi- γ-Aminobutyric acid (GABA) was first identified in the croenvironment of the brain must become a tumor-favorable mi- mammalian brain over one-half a century ago and subsequent croenvironment for successful metastatic colonization by circulating studies have demonstrated its relevance to various medical and breast cancer cells. Bidirectional interplay of breast cancer cells and scientific paradigms (15–18). In addition to its role in neuro- native brain cells in metastasis is poorly understood and rarely transmission, GABA can act as a trophic factor during nervous studied. We had the rare opportunity to investigate uncommonly system development to influence cellular events including pro- available specimens of matched fresh breast-to-brain metastases liferation, migration, differentiation, synapse maturation, and tissue and derived cells from patients undergoing neurosurgical re- cell death (19, 20). Various cells can catabolize GABA to meet section. We hypothesized that, to metastasize, breast cancers may their metabolic needs. Under conditions that inhibit the tri- escape their normative genetic constraints by accommodating and carboxylic acid cycle, impair respiration, and enhance the accu- coinhabiting the neural niche. This acquisition or expression of brain- mulation of reactive oxygen intermediates, GABA can be used as like properties by breast cancer cells could be a malignant adaptation an energy source for growth through a pathway known as the required for brain colonization. Indeed, we found breast-to-brain GABA shunt (21). The expression of GABA in adult tissues is CELL BIOLOGY metastatic tissue and cells displayed a GABAergic phenotype similar to specific and tightly regulated. Abnormal GABA expression or that of neuronal cells. The GABAA receptor, GABA transporter, GABA GABAergic participation has been described in primary colon, transaminase, parvalbumin, and reelin were all highly expressed in gastric, ovarian, pancreatic, and breast cancers (22). + breast cancer metastases to the brain. Proliferative advantage was We obtained surgical specimens of HER2 and TN subtypes conferred by the ability of breast-to-brain metastases to take up and from patients undergoing neurological surgery. This offered catabolize GABA into succinate with the resultant formation of NADH a rare opportunity to examine the histological, cellular, and as a biosynthetic source through the GABA shunt. The results suggest molecular features of the tumor microenvironment that cannot that breast cancers exhibit neural characteristics when occupying the be recreated using highly passaged cell lines and derived xeno- brain microenvironment and co-opt GABA as an oncometabolite. grafts. Results showed that breast-to-brain metastatic (BBM) cells overexpressed integral proteins from the GABA-related brain metastasis | tumor microenvironment variables, including GABA transaminase (ABAT), GABAA receptor (GABAAR), glutamate decarboxylase (GAD67), GABA transporter, reelin, and parvalbumin. Brain metastases from both etastases are responsible for 90% of all cancer deaths, and + Mpatients diagnosed with brain metastases have a dismal types of breast cancer (HER2 , TN) metabolized GABA in vitro 20% probability of 1-y survival (1–3). The brain is increasingly the first site of recurrence after treatment of stage IV advanced Significance breast cancer, even when disease in other sites is in remission. This emerging clinical problem significantly limits the survival Breast cancer patients typically develop brain metastases years gains made from recent advances in systemic therapy for breast after their initial diagnosis. During this clinical latency, cancer cancer (4). Breast cancer metastasizes to the brain in ∼40% of cells must evolve and adapt to the neural microenvironment to + patients who have a tumor that is HER2 (>30% of tumor cells colonize. We hypothesized that breast cancer cells may assume have complete membrane staining for the tyrosine kinase re- brain-like properties to survive in the brain. Our results suggest ceptor erbB2) or triple negative (TN) (negative for the estrogen that metastases overexpress many variables related to the and progesterone receptors and have reduced expression of γ-aminobutyric acid (GABA) and were able to proliferate by + HER2 ) (5). Ninety percent of patients with these breast cancer metabolizing GABA as a biosynthetic energy source. The ex- subtypes will die of metastasis to the brain (1). Currently, pression of brain-like properties by breast cancer cells could be treatment options beyond radiotherapy and neurological surgery a malignant adaptation required for metastasis to the brain, are limited, underscoring the need for research into the biology which could potentially be exploited to develop new therapy for of these clinically recalcitrant tumors (6). breast cancer patients. Breast cancer patients typically develop brain metastases months to several years after their initial diagnosis (6). This Author contributions: J.N., J.T., E.R., and R.J. designed research; J.N., C.C., C.M.K., H.L., and A.C.H. performed research; J.N., N.V., and R.J. contributed new reagents/analytic unique clinical latency occurs despite the early presence of cir- tools; J.N., J.T., S.W., N.V., H.L., E.R., and R.J. analyzed data; and J.N., J.T., E.R., and R.J. culating tumor cells, often detectable at the time of primary di- wrote the paper. – agnosis (7 9). These observations suggest that the final step of The authors declare no conflict of interest. the metastatic cascade to colonize the brain may be the rate- Freely available online through the PNAS open access option. limiting biological step that determines whether or not clinical 1To whom correspondence may be addressed. E-mail: [email protected] or eroberts@coh. progression will occur. Primary cancers, especially those of org. breast origin, have numerous changes in transcriptome net- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. works that can lead to clones with additional survival gains at 1073/pnas.1322098111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1322098111 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 and used it as biosynthetic source to enhance cell growth. The expression of GABAergic features by BBM cells demonstrates that, despite their origins in distinct and disparate germ-line tis- sues, cancers have the ability to adapt to and colonize new microenvironments. Results Brain BBM Cells Exhibit GABA-Related Variables. The tumor and brain microenvironment is a highly specialized niche determined by its tissue-specific location and cell-derived contents. Unlike tumors originating within the brain (glioblastoma multiforme), meta- static breast tumors are noninfiltrating and retain their original tissue morphology. The demarcation of the brain–tumor in- terface can be clearly discerned histologically or clinically on magnetic resonance imaging (Fig. 1). In patients undergoing neurosurgical resection of BBMs, specimens from the tumor and brain interface demonstrated few healthy neurons. Metastatic Breast Metastasis breast cancer cells were juxtaposed to astrocytes expressing glial fibrillary acidic protein (GFAP) both in the peritumoral brain and tumor regions (Fig. 1). Furthermore, these reactive astro- cytes had a hypertrophic morphology comparable to the glial scars that form in response to traumatic brain injury (23). Resected BBM specimens were classified by their clinical + subtypes (HER2 and TN) and Bloom–Richardson pathological grade (24). Biopsies of brain metastases revealed ductal carci- nomas with high-grade Bloom–Richardson score (BRG3) (Fig. S1). Low-passage cells derived from metastatic brain tumor * resections maintained their original tissue morphology when grown in serum-free “brain-like” conditions and 3D Matrigel cultures (Fig. S1) (25). Within the intratumoral space in meta- static brain tissue, cells of glial origin interdigitated with the breast tumor cells (26). Investigating the tumor cell and stromal compartments separately revealed differences in the micro- environments (12). Staining with H&E combined with staining + for GFAP (expressed in the intratumoral glial cells) and HER2 (expressed in tumor cells) allowed us to definitively observe the brain–tumor boundary (Figs. S1 and S2). There was a large difference in volume between tumor nuclei (523 ± 43 μm3)and nontumor nuclei (58 ± 8 μm3). Cultured BBM cells organized