br Radiation BCF br No Radiation
Radiation + BCF
No Radiation control
Days after irradiation
VGCCs play major roles in tumour progression . Specifically, T-type channels are known for both oncogenic and tumour suppressive functions in a tumour-type and context-dependent manner [49–51]. Our finding that AM RF EMF results in tumour inhibition through Cav3.2 T-type VGCC is consistent with a report in which drug treatment-induced calcium flux through Cav3.2 T-type VGCC inhibited breast cancer cell growth , suggesting that activation of this channel may play tumour suppressive role in breast cancer. However, studies in other tumour types suggest that inhibition of T-type VGCCs suppresses stem cell population and cell growth [50,53]. It should be noted that these studies used pan-T-type VGCC inhibitors and did not differentiate the isoform-specific effect in tumour inhibition. Calcium entry through T-type VGCCs are differentially regulated at each phase of cell cycle, and individual isoforms may play a specific role in a tumour-type de-pendent manner [54,55]. It is plausible that BCF alters the calcium oscil-lations from the Cav3.2 T-type channel at different stages of Adriamycin to slow down transition from one stage to another. Therefore, BCF possibly disrupt breast cancer specific calcium oscillation to induce cell cycle arrest.
Multiple previous findings have revealed that exposure to EMFs can influence specific biological processes in cells such as cellular metabo-lism, morphology and differentiation . Interestingly, some of these studies have shown that EMF treatment affects signal transduction pathways by regulating intracellular levels of c-AMP and calcium [49,56]. Other studies using mammalian models have shown that amplitude-modulated EMF affect calcium flux at specific frequencies, the so-called window effect, while unmodulated EMF do not result in any biological activity [42,43,57]. Similarly, exposure to pulsed electric field has been shown to increase calcium influx by decreasing plasma membrane integrity . Our findings indicate that intracellular cal-cium flux through CACNA1H plays a crucial role in the suppressive effect of BCF. Furthermore, we also demonstrate that BCF induced calcium in-flux activates p38MAPK via CAMKII to inhibit cell growth. To our knowl-edge, this is the first report to identify CAMKII-p38 MAPK axis as a major player in EMF's biological effect on tumour cells (Fig. 6H). It was previ-ously shown that p38 is activated by intracellular flux of calcium and in-duces cellular stasis or apoptosis [59,60]. However, we did not observe cell death in cells treated with BCF, suggesting that intermittent calcium flux through CACNA1H induced by BCF exposure activates p38 signal-ling only to mediate tumour suppressive effects without triggering cell death. It is also possible that CACNA1H functionally couples to putative microdomains to activate CAMKII-p38 signalling restricting calcium mobilisation to other cellular compartments.
We have also shown that BCF decreased the gene expression of HMGA2 to suppress cancer stem cell property of brain metastatic cells. The oncogenic function of HMGA2 was reported by several groups and mainly involves activation of stem cell programing in the tumour cells [32,33,61]. In fact, HMGA2 is known to be the downstream target of two major pathways that induce stemness, TGF-β- and Wnt – pathways [62,63] and a prognostic indicator of reduced relapse-free survival . Therefore, it is conceivable that BCF reduces stem cell abil-ity and cell proliferation via downregulation of HMGA2 gene expres-sion. Importantly, our results suggest that the decrease in HMGA2 expression by BCF treatment was mediated through CACNA1H and CAMKII mediated β-catenin phosphorylation. The decrease in CSC ex-plains the striking and long-lasting responses to BCF treatment for treatment-refractory metastatic patients which was observed in our previous feasibility trial .
Brain metastasis is a complex disease due to the unique environ-ment of the organ. We and several other groups have shown that brain metastatic cells are molecularly rewired to communicate and modify brain microenvironment for tumour progression in the brain [14,65,66]. It was also reported that angiogenesis plays a prominent role in intracranial tumours arising from breast cancer, and inhibiting VEGF receptor tyrosine kinase suppressed tumour growth in the brain [67,68]. In fact, metastatic growth occurs in close proximity or by direct contact to the blood vessels in the brain . In this report, we observed a significant decrease in exosomal miR-1246 level in BCF-treated cells. miR-1246 was previously found to promote tumour progression by augmenting angiogenesis . Indeed, exosomes from BCF-treated cells suppressed angiogenic ability of brain microvascular cells in this study. Consistently, BCF treatment decreased CD31+ microvessel den-sity in tumours in the brain. Importantly, treatment of tumour bearing mice with BCF decreased the serum miR-1246 level, demonstrating the potential utility of this miRNA in predicting response to BCF in pa-tients. Our results also indicate that brain-tropic variant cells shed higher levels of miR-1246 in exosomes. Therefore, miR-1246 may po-tentially serve as a biomarker of brain metastasis. Taken together, these results strongly suggest that miR-1246 orchestrate the angiogenic niche in the brain and that BCF disrupts miR-1246 signalling to suppress angiogenesis in the brain microenvironment. Last but not least, we pro-vide the first evidence that the SAR level delivered by intrabuccal ad-ministration enables treatment of breast cancer brain metastasis.