dc.description.abstract | p16INK4a (p16) traps the cell at the restriction point of the cell cycle by binding to cyclin-dependent kinase 4/6 thus preventing the phosphorylation of the
retinoblastoma protein (pRB). As p16 accumulates the cell stops dividing and
becomes senescent. This study investigates the modulation of p16 function by
the putative membrane protein ABCB5 and a group of five putative oncogenic
microRNAs (oncomiRs).
ABCB5 is a poorly characterised member of the B-subfamily of human ATP
Binding Cassette transporters. ABCB5 is reportedly transcribed into four
transcripts, one of which could potentially encode a full-length transporter
(ABCB5fl) whilst a second could encode a half-transporter (ABCB5β). The other
two transcripts (ABCB5α and ABCB5γ) could only encode short polypeptides.
Exogenous expression of ABCB5fl and ABCB5β was achieved in HEK293T
cells, but the recombinant protein expressed poorly and localised to the
endoplasmic reticulum. Point mutations introduced into the ATP catalytic
domain failed to improve expression levels suggesting that protein function was
not deleterious to the cell. Exogenous expression in HEK293T cells also allowed
commercial antibodies purportedly raised against ABCB5 isoforms to be tested.
Several were found not to recognise ABCB5 necessitating re-interpretation of
published data. However, one antibody recognised both ABCB5fl and
ABCB5β, and was subsequently used to evaluate protein expression levels in
other cell types.siRNA knockdown of ABCB5 in human mammary epithelial cells (HMECs)
caused a concomitant reduction in p16 expression and an increase in cellular
proliferation. Differential siRNAs and RT-qPCR analyses demonstrated ABCB5β
to be the relevant transcript with respect to the reduction in p16 expression;
however, no native ABCB5β protein was detected in HMECs. Together these
data lead to the hypothesis that the ABCB5β transcript may act as a long noncoding
RNA to regulate p16.
Exogenous expression of each of five distinct putative oncomiRs in HMECs was
found to increase cellular proliferation and, surprisingly, increase p16
expression. These results mirror a phenotype commonly observed in p16-positive basal-like breast cancer (BLBC), an aggressive form of breast cancer with poor prognosis and few treatment options. Bioinformatic analysis of the predicted
target genes for these oncomiRs identified multiple transcriptional regulators of
pRB. These predictions, together with the work performed in a cellular model of
p16-positive BLBC, suggest that the oncomiRs may cause unrestricted cell
proliferation by indirectly reducing transcription of the pRB gene, RB1. In the
absence of pRB, p16 expression is induced via a previously reported oncogeneinduced
senescence-like positive feedback loop. These data, and previously
published observations, suggest that a similar mechanism may explain the basis | en_US |