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Page 10 of 13 Morgan et al. J Cancer Metastasis Treat 2018;4:46 I http://dx.doi.org/10.20517/2394-4722.2018.19

the increased number of cells invading through an artificial basement membrane matrix. However, in our
cell proliferation studies, significant increases in cell proliferation were seen at 10 ng/mL of HGF while the
number of cells invading through the artificial membrane only significantly increased at 50 ng/mL. Thus, we
believe that if the results of the invasion assay could be influenced by an increase in cell proliferation then
we would have observed a significant number of cells invading the basement membrane at 10 ng/mL Thus,
unlike in previous studies that use cell lines that already have metastatic capabilities, we have shown, for
the first time that treating non-invasive PCa cells with HGF can induce an invasive phenotype. Building on
from this finding future work would will need to expanded the range of cell types used to include more non-
[43]
invasive PCa cell lines to achieve a more rounded representation of “PCa” .

ARF6 is a member of the Ras superfamily and can be activated by various growth factors, in particular, by
[28]
HGF . It functions in a range of biological activities and has be shown to play roles in adherens junction
[26]
[44]
[45]
disassembly , cell migration and cell proliferation . While there is very little information on the pres-
ence or activation of ARF6 in PCa we have recently shown that ARF proteins are over-expressed in PCa
[31]
tissue compared to normal control tissue . In this study, gene expression analysis revealed that ARF6 is
expressed in all the prostate cells. Analysis showed that there was no significant difference in expression
between cells derived from normal prostate epithelium and localised, non-invasive PCa but a significant
difference in expression between non-invasive and invasive cancer cells was evident. ARF6 levels were sig-
nificantly higher in LNCaP, DU145 and PC-3 cells compared to CAHPV-10 but there was no significant dif-
ference between the invasive cell lines regardless of their differing invasive capacities. Protein analysis con-
firmed the gene expression data and showed that ARF6 protein was detectable in all prostate cells. Like the
[28]
majority of GTPases, ARF6 cycles between an inactive GDP-bound form and an active GTP-bound form .
Inactive ARF6 localises to the cytosol and endosomes and when activated it translocates to the plasma mem-
[46]
brane . Immunofluorescence revealed a strong defined signal at the periphery of the cells in both DU145
and PC-3 indicating a close association of ARF6 and the cell membrane while staining intensity appeared to
be markedly reduced in both CAHPV-10, and PNT2. This data would appear to suggest that activated ARF6
increases with the aggressiveness of the cancer. To validate the immunofluorescence data, Western blotting
and densitometry analysis of ARF6-GTP and total ARF6 were performed. ARF6-GTP was shown to be evi-
dent in the invasive cell lines but very low levels in the non-invasive cell lines. The ratio between ARF6-GTP
and total levels of ARF6 were also calculated and highlighted the fact that levels of ARF6-GTP were higher
in all the cells derived from invasive tumours. In particular, in the cell line PC-3, which is a cell line derived
from a highly aggressive PCa that has metastasised to the bone and is representative of the main metastatic
site for PCa, ARF6-GTP levels were found to be 3.5 and 2.7 times higher than the levels found in normal and
non-invasive cells respectively.

[26]
It has been documented that HGF can activate ARF6 in epithelial cells . As neo-adjuvant ADT can in-
crease the levels of HGF, we wanted to determine whether HGF stimulation could activate ARF6 in the non-
invasive cell line CAHPV-10. Using 50 ng/mL for 48 h we found that activated ARF6 levels increased com-
pared to untreated CAHPV-10 cells. ARF6 plays an essential role in epithelial and endothelial cell migration
and elevated levels of activated ARF6 have been found to increase the invasive capacity of melanoma cells
[29]
[28]
both in vitro and in vivo , while silencing ARF6 has been shown to inhibit the ability of breast cancer
[30]
cells to invade through an artificial basement membrane . Whether HGF stimulated activation of ARF6 is
responsible for the increased proliferation, motility and invasiveness observed following HGF stimulation of
CAHPV-10 cells now needs to be determined.
In conclusion, ADT is increasingly being used in the neo-adjuvant setting. With the importance of HGF and
cMet in PCa documented, it has been hypothesised that while current ADT may inhibit AR mediated cell
proliferation and survival, it may abolish its suppressive role on the HGF/c-Met pathway, unintentionally
driving tumour progression. We have shown that HGF enhanced cell proliferation, induced scattering and