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Page 579 Loh et al. Extracell Vesicles Circ Nucleic Acids 2023;4:568-87 https://dx.doi.org/10.20517/evcna.2023.34

CPE/NF-α1
CPE is a prohormone processing enzyme expressed in endocrine cells and neurons, as well as in many
cancer cells [118-120] . It also has neurotrophic activity and was given an alternative name, NF-α1 . CPE/NF-α1
[121]
is a RSP protein that has many non-enzymatic roles. Its transmembrane form acts as a sorting receptor at
the TGN to target prohormones to the RSP vesicles . Within the secretory vesicle, soluble CPE acts as a
[34]
prohormone processing enzyme. The transmembrane form of CPE has a cytoplasmic tail that interacts with
microtubule motors to transport the vesicles to the release site . CPE mediates intercellular
[122]
communication upon secretion from RSP vesicles by binding to the human membrane serotonin receptor,
HTR1E, to activate the extracellular signal-regulated kinase (ERK)-B-cell lymphoma 2 (BCL2) signaling
pathway to promote cell survival in neurons and cancer cells [117,123,124] . Interestingly, CPE and Cpe mRNA
have been found in sEVs in many types of cancer cells, including human hepatocellular carcinoma (HCC)
cells . Cpe mRNA level in serum-derived, immunoaffinity purified sEVs from cancer patients with various
[112]
types of cancer has been shown to be correlated with the metastatic state of the disease. Hence, sEV CPE is a
potential prognostic cancer biomarker .
[112]
Studies have been carried out on PC12 (a pheochromocytoma neuroendocrine cell line) and HCC cells to
examine the effect of soluble CPE, secreted from secretory vesicles into the cell culture medium, or
exogenously applied, on the proliferation and survival of these cells. Secreted CPE and exogenously applied
CPE promoted survival of pheochromocytoma (PC12) cells under nutrient starvation, and HCC cells under
hypoxic conditions, respectively. HCC cells treated with CPE under hypoxic conditions activated the ERK
signaling pathway to up-regulate pro-survival genes BCL2, tumor necrosis factor (TNF), nuclear factor κB
(NF-κB), and inhibitor of NF-κB (I-κB) alpha to promote cell survival . This pro-survival effect via ERK-
[117]
BCL2 signaling on HCC cells is consistent with similar actions of CPE in neurons, mediated through
[124]
[120]
binding to HTR1E receptor , also present in HCC cells . However, exogenously applied recombinant
CPE had no effect on HCC cell proliferation and no change in the expression of cell cycle regulatory
genes . In contrast, when isolated sEVs secreted from high metastatic HCC cells which contain CPE
[115]
protein were co-incubated with low metastatic HCC cells, they promoted proliferation and invasion of the
[112]
cells . sEVs isolated from high metastatic HCC cells treated with shCPE to suppress expression had no
effect on promoting proliferation or invasion in low metastatic HCC cells, indicating this effect is dependent
on sEV-CPE. The differential effects of soluble free CPE versus sEV-CPE on proliferation also support the
presence of CPE in the sEVs, rather than being a contaminant in the EV preparation. Interestingly, when
HEK293 cell sEVs loaded with CPE-shRNA were incubated with high metastatic HCC cells, these recipient
cells exhibited downregulation in expression of CPE and cyclin D1, a cell cycle regulatory gene, and a
decrease in proliferation. In summary [Figure 8], these HCC cell studies showed that CPE is trafficked to
sEVs. Moreover, soluble CPE secreted from DCVs promoted stress-induced cell survival but not
proliferation, while CPE in sEVs increased proliferation and invasion in recipient cells. These observations
suggest that CPE released by the two different mechanisms may have distinct functions as mediators of
intercellular communication.

BDNF
In neurons, pro-BDNF and mature BDNF are packaged into classical secretory vesicles in the RSP and
[32]
released upon stimulation into the synaptic cleft . BDNF binds to tropomyosin receptor kinase B (TrKB)
receptors in the post-synaptic neurons to activate various signaling pathways that mediate functions such as
neurodevelopment, neuroprotection, and synaptic plasticity [125-127] . In addition, BDNF is also expressed in
the gut and other tissues and plays a role in energy homeostasis including regulating glucose metabolism to
prevent β-cell exhaustion . Recently, BDNF has been found in sEVs isolated from the umbilical cord
[128]
blood of neonates. The level of sEV BDNF was inversely correlated with cord ferritin levels and maternal
iron deficiency . In contrast, plasma BDNF was reported to be lowered in the umbilical cord blood of
[129]