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Hunt et al. Extracell Vesicles Circ Nucleic Acids 2020;1:57-62 I http://dx.doi.org/10.20517/evcna.2020.04 Page 59

tumors found within in vivo mouse models. Thus, these data suggest that miR-34a, and other similarly
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acting miRNAs, prevent neuritogenesis and that, when lost in p53 EVs, the surrounding neurons exhibit
increased neuritogenesis. In concert with this hypothesis, the group next inhibited miR-34a by transfecting
cultured neurons with an antagomiR that specifically inhibits miR-34a. This experiment resulted in
increased neuritogenesis in the antagomiR-34a transfected neurons when compared to neurons transfected
WT
with scrambled antagomiRs, thus confirming the hypothesis that miR-34a from p53 -derived EVs acts to
prevent neuritogenesis.

To complement these findings, they next identified candidate miRNAs that drive neuritogenesis. Towards
this effort, they uncovered that antagomiR-mediated inhibition of miR-21 and miR-324 resulted in
decreased neuritogenesis in transfected neurons when compared to those transfected with scramble
antagomiRs. Conversely, transfection of neurons directly with a combination of miR-21 and miR-324
resulted in a robust increase in neuritogenesis when compared to neurons transfected with scramble
miRNA molecules. Moreover, adding miR-34a to this combination of miR-21 and miR-324 decreased
neuritogenesis, demonstrating that cancer-driven neuritogenic processes lie in a delicate balance, dictated,
at least in part, by these miRNAs.

The researchers noticed that these newly formed neurites stained positively for the adrenergic marker
tyrosine hydroxylase (TH), demonstrating thereby that the responding neurons were adrenergic in nature.
However, upon exposure to EVs derived from p53 OCSCC tumors, they found that the number of
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adrenergic neurons increased dramatically in both the in vitro and in vivo models, suggesting that p53 -
derived EVs were promoting an adrenergic state. Conversely, exposure to p53 -derived EVs decreased the
WT
number of intratumoral TH-positive adrenergic neurites, suggesting that these wildtype EVs inhibit cancer-
associated adrenergic neuritogenesis. At this point, the researchers were uncertain about the origin of these
[18]
adrenergic neurites. Amit et al. wanted to know whether these adrenergic neurites arose from previously
existing adrenergic neurons or whether these newly-formed adrenergic neurites arose from another neuron
type. They subsequently found that transfection of trigeminal sensory neuronal cultures with miR-21 and
miR-324 resulted in increased adrenergic staining, suggestive of neurotype switching from a sensory to a
sympathetic nature. However, when miR-34a was added to the combination of miR-21 and miR-324, the
effect was lost, demonstrating that along with inhibiting neuritogenesis, miR-34a activity also inhibits neo-
adrenergic neurotype switching. These findings were subsequently bolstered by transcriptomic analysis,
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which demonstrated that neurons incubated with EVs derived from p53 OCSCC tumors expressed
increased levels of catecholamine biosynthesis-related genes and decreased levels of sensory neuron
signaling genes. Specifically, the transcription factors POU5F1 and KLF4 were found to be upregulated in
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trigeminal neurons following incubation with EVs derived from p53 OCSCC tumors. These transcription
factors are sufficient to drive neuronal differentiation of adult neural stem cells. Additionally, these two
factors are directly regulated by miR-34a activity. NEUROG2 and ASCL1 are two additional factors that
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were upregulated in trigeminal neurons following incubation with p53 OCSCC EVs. These factors are
also candidate targets of miR-34a regulation, and their activity drives neuronal differentiation, specifically
to an adrenergic fate. These expression changes illustrate a neurotype switching event in sensory neurons
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adjacent to p53 OCSCC tumors. Moreover, the combination of the findings described thus far suggest
that depletion of miR-34a in EVs released by p53 OCSCC tumors is the mechanism by which these
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tumors drive increased neo-adrenergic innervation of the tumor body.

Knowing that the tumor-innervating neurons that drive neurotropic tumor growth are adrenergic in nature
opens up avenues for the use of readily available therapies to treat patients with OCSCCs. Beta adrenergic
receptor blocking medications, for example, are already approved for the treatment of hypertension, heart
[19]
arrhythmias, angina, migraines and other illnesses, and are widely available . Additional data from
published clinical trials support the use of anti-adrenergic approaches to treating several types of cancers,