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SRC-mediated activation of PIK3/AKT/mTOR signaling, we treated BBM1 cells with 100 nmol/L of AC93253
for 48 h, followed by Western blot analysis of the total protein extract. Our analysis showed that inhibition
of SRC leads to inhibition of PIK3CA and mTOR phosphorylation [Figure 5D]. As our analysis indicated
that SRC is an upstream regulator of ERBB2 and the PIK3-AKT/mTOR pathway in BBM1 cells and that
inhibition of both SCR and ERBB2 induces BBM1 cell death, we analyzed the combined effect of SRC and
ERBB2 inhibitors on BBM1 cell viability. We treat the cells with 50 nmol/L of AC93253 and 50 nmol/L of
Lapatinib, separately and in combination, for 48 h. Our analysis showed robust inhibition of BBM1 cells
in the presence of either inhibitor alone, however, the effect was greatest in presence of both [Figure 5E].
Similar to combinatorial SRC/ERBB2 treatment, concurrent inhibition of ERBB2 and PIK3CA showed a
significant additive inhibitory effect on BBM1 cell survival [Figure 5E].

We also evaluated the combinatorial treatment of ERBB2 and SRC inhibitors with a GABA agonist.
BBM1 cells were treated with either 100 nmol/L of Isoguvacine alone or in combination with Lapatinib
or AC93253 for 48 h. Cell viability and apoptosis analyses showed that the combination of Isoguvacine
with both Lapatinib and AC93253 induced higher inhibition of cell viability compared to independent
treatments [Figure 6A and B]. Our analysis showed that the expression of GABA receptors and subunits was
significantly higher in nBrain compared to PT, MT, and nBreast [Figure 6C, Supplementary Figure 5].

DISCUSSION
Brain metastases are the most common and complicated central nervous system disease in adults. The
incidence of brain metastases is increasing due to both improved diagnosis and increased cancer patient
survival through advanced systemic treatments. Outcomes of patients remain disappointing and treatment
options are limited, usually involving multimodality approaches of radiation and palliative chemotherapy.
Brain metastases represent an unmet clinical challenge in caring for solid tumor, especially in breast cancer,
where the incidence of brain metastases are frequent and that result in impaired quality of life.

BBMs are common in patients with the HER2-positive and TNBC breast cancer subtypes, and the natural
course of BBM is strongly influenced by the biology of the primary tumor subtype. Although the biology of
BBM according to tumor subtypes is still poorly understood, recent breakthroughs have been achieved in
the identification of specific mediators of BBM and in the development of preclinical models for therapeutic
studies. However, the use of established cell lines and comparative analysis of unrelated tissue samples
often identify factors that eventually fail to represent as a key regulator in preclinical and clinical stage. To
overcome this issue we obtained both primary and metastasis tissue from same individual patients (PT1/
MT2 and PT2/MT2). Due to the inherent difficulty in the procurement of such tissue pairs we have used
limited numbers of tissue samples. Our RNA-seq analysis revealed that ERBB2-mediated activation of
PIK3CA and its downstream AKT/mTOR pathway plays a critical role in the survival and proliferation of
breast to brain metastatic cells. Inhibition of ERBB2 or its downstream targets suppresses BBM cell viability
to different degrees. The critical function of ERBB2 in HER2+ primary and metastatic breast cancer has been
well established, and the ERBB2 inhibitor Lapatinib and trastuzumab have been used as chemotherapeutic
agents targeting HER2+ breast cancer [25-28] . In addition, our earlier studies revealed the contribution of neural
factors, such as the astrocyte-secreted brain-derived neurotrophic factor, in the activation of HER2-TrkB
[25]
signaling that leads to AKT1 activation and BBM1 cell proliferation . Several studies have also reported
mutations and/or copy number changes in PIK3CA in hormone receptor-positive breast cancers [29-31] . Indeed,
targeting PIK3-AKT/mTOR signaling to inhibit breast cancer has been evaluated extensively, and there is
phase I to III preclinical trial data demonstrating that inhibition of this pathway leads to regression of solid
tumors and breast cancer [32-37] .

Recent gene expression analyses of BBM samples identifed cyclooxygenase (COX)-2, EGFR ligands, and a
[38]
sialyltransferase, as mediators of cancer cell passage through the BBB . In contrast to COX-2 and EGFR,