Background:
With a worldwide prevalence of 14 million patients, hepatocellular carcinoma (HCC)
is the most common primary malignant neoplasia of the liver (85 – 90%) and displays
the second highest tumor related mortality. Although possibly curable at an early
stage, most tumors are diagnosed not until late to end stages since only then tumors
will present distinct symptoms, correlating with a poor prognosis. The insulin like
growth factor (IGF) system regulates growth, cell division and cell survival via IGF
binding to the IGF receptor. Insulin like growth factor binding proteins (IGFBPs)
control IGF availability by competitive binding of IGF, thus preventing it from binding
to the IGF receptor, therefore limiting proliferative IGF effects. We previously identified
IGFBP2 as a target gene of p53 and especially p73, members of the p53 family of transcription
factors, in HCC. This family of tumor suppressors replies to cellular stress signals
by the induction of senescence or apoptosis. However, physiologic functions of p53-family
induced IGFBP2 in HCC are so far unknown. Thus, effects of IGFBP2 on HCC cell viability,
proliferation and migration were evaluated and IGFBP2 induction by HCC-relevant therapeutics
was studied.
Methods:
IGF secretion and surface levels of IGF receptors were analyzed in the human HCC cell
line Hep3B. To evaluate IGFBP2-mediated effects, cells were cultured with recombinant
IGFBP2 (100 – 1000 ng/ml) for up to 96 hours. Proliferation was measured by flow cytometry,
cell viability was determined by MTS assay and cell migration was analyzed using a
wound healing model. Furthermore, effects of HCC-relevant therapeutics on IGFBP2 and
p73 were analyzed by Western blot, qPCR and ELISA.
Results:
Hep3B cells displayed an intact IGF system, indicated by IGF secretion and surface
expression of insulin receptor and IGF receptor 1. Generally, in the presence of IGFBP2
cell viability of Hep3B cells increased in a time-dependent fashion, where most pronounced
effects were observed using concentrations between 250 and 500 ng/ml. Regarding cell
proliferation, highest induction rates were detected using IGFBP2 levels between 100
and 250 ng/ml with a 14- and 16-fold increase at 48h and decreasing thereafter. Concordantly,
IGFBP2 doses between 100 and 500 ng/ml resulted in enhanced cell migration. Interestingly,
treatment of Hep3B cells with the drugs bleomycin, doxorubicin and regorafenib resulted
in an induction of TP73 and – slightly delayed – also IGFBP2.
Conclusion:
Although being regarded as growth-limiting factor within the IGF-system, IGFBP2 exerted
proliferative effects on HCC cells when applicated in a recombinant manner. However,
IGFBP2 was shown to be a p53 family target gene and was induced by HCC-relevant therapeutics
together with the tumor suppressor p73. We therefore hypothesize, that p53-family-mediated
mechanisms must exist which redirect IGFBP2-dependent signaling towards growth inhibition.
Thus, detailed elucidation of signal transduction on the p73-IGFBP2-axis is indispensable
to develop novel diagnostic and therapeutic options for HCC.
