br to examine angiogenesis in brain
to examine angiogenesis in beta-Nicotinamide mononucleotide tumours. As shown in Fig. 5J-K, tu-mours treated with BCF showed significant decrease in micro-vessel density, demonstrating that BCF decreases miR-1246 levels in exosomes to inhibit angiogenesis in brain microenvironment. In addition to its role in angiogenesis, miR-1246 was also reported to be involved in the Wnt pathway . As HMGA2 and β-catenin are downstream components of Wnt pathway, we tested the possibility that BCF modulates miR-1246 expression through either β-catenin or HMGA2. Therefore, we performed a gain of function approach by expressing HMGA2 or β-catenin in 231 and SKBr3 cells. As shown in Fig. 5L and Supplementary Fig. 4F, ectopic expression of β-catenin, but not HMGA2, significantly el-evated expression of miR-1246 in 231 and SKBr3 cells, indicating the role of β-catenin in regulation of miR-1246 expression. Collectively, these results demonstrate that BCF suppresses angiogenesis in the tu-mour microenvironment by suppressing β-catenin-miR-1246 signalling and that exosomal miR-1246 may serve as a potential biomarker of liq-uid biopsy to measure the effect of BCF in brain tumours.
3.5. Combination of radiation and BCF treatment suppress brain metastasis
Because radiation is the standard of care in the treatment of brain metastasis, and cancer stem cells are known to be involved in radiation resistance [6,40], we examined the effect of BCF on radio-resistant cell lines. We generated radiation resistant cell lines, 231BrM-RR and SKBrM3-RR (Fig. 6A). BCF treatment in these lines significantly sup-pressed their growth, CSC population, and colony formation ability, and this inhibition was rescued in the presence of Ethosuximide (Fig. 6B-F and Supplementary fig. 5A-B). This suppressive effect was ac-companied by the downregulation of HMGA2 (Fig. 6D). We then exam-ined the effect of the combination of BCF and radiation using the R2G2 mouse model. We found that BCF treatment combined with radiation significantly enhanced the anti-tumour effect compared to radiation alone, (Fig. 6G and supplementary 5B\\C), suggesting a potential syner-gistic effect of radiation and BCF in vivo.
Current therapeutic options for patients with brain metastatic dis-ease are quite limited mainly due to the Blood Brain Barrier (BBB), which blocks the entry of therapeutic drugs into the brain, and ironi-cally, tumour cells find the brain as a sanctuary. Radiation therapy ap-proaches are the standard of care for brain metastasis. However, they have limited efficacy and recurrence is inevitable. Accordingly, the sur-vival of patients with brain metastasis is dismal . The robust growth inhibitory effect of BCF observed in this study has established its efficacy in the treatment of primary as well as recurrent metastatic tumours in the brain. The tumour and tissue specificity of BCF is further illustrated by the absence of antiproliferative effects in both HMEC and MCF10A in addition to the absence of antiproliferative effects on two hepatocel-lular carcinoma cell lines . BCF mediated anti-proliferative effect on tumour cells through CACNA1H and the activation of p38 MAPK path-way. This demonstrates that low intensity RF EMF activate a specific transmembrane protein when modulated at specific frequencies, thus validating the hypothesis that BCF are demodulated by transmembrane
Fig. 4. BCF suppresses cancer stem cell population by decreasing HMGA2 expression. (A) Outline of RNA sequencing performed for 231-BrM and SKBrM3 cells exposed to Sham or BCF for 7 days (left panel). Expressions of 9 commonly up- or down-regulated genes are shown as a heat map on the right panel. (B) Overall relapse-free survival was examined by segregating breast cancer patients based on the HMGA2 expression level. Five GEO datasets were used to create a cohort with 710 patients. (C) HMGA2 expression was examined by western blot (upper panel) and qRT-PCR (lower panel) in 231-BrM and SKBrM3 cells after BCF treatment. (D-E) Brain tumour sections from Sham or BCF treated mice were examined for HMGA2 level by Immunohistochemistry, and the staining intensity was quantified. Representative images are shown in (E). (F) HMGA2 expression in patients with no metastasis (no-met), brain metastasis (Brain-met) and Lung metastasis (Lung-met) was examined in a combined cohort (see Methods section). (G) HMGA2 expression was examined in indicated cell lines by western blot (upper panel) and qRT-PCR (lower panel). (H) Cancer Stem Cells (CSC) population (CD44high/ESAhigh) was examined by FACS after treating 231-BrM, SKBrM3 and MCF7shXIST cells with Sham or BCF (n = 5/group). (I) Number of spheres was counted at day 7 after seeding SKBrM3 cells treated with Sham or BCF (n = 6/group). Representative pictures are shown in the right panel. (J) The CSC population in SKBr3 cells with or without overexpression of HMGA2 was quantified by FACS (n = 5/group). (K) CSC population was examined after treating SKBrM3 cells with Sham or BCF for 7 days (n = 5/group) in the presence of vehicle or ethosuximide. (L) 231BrM shScramble or shCav3.2 cells were treated with or without BCF for 7 days followed by western blot analysis to examine the level of HMGA2. (M) SKBrM3 cells were treated with Sham or BCF or BCF in the presence of KN93 (5 μM) and level of indicated proteins was examined by western blot. *, P-valueb.05, **, P-valueb.01 and ***, P-valueb.0001.