Aims:
TGF-β is a major signaling pathway of the liver with pleiotropic effects on different
processes and cell types. During cancer progression while TGF-β signaling exerts tumor
suppressor effects at pre-neoplastic and early tumor stages, cytostatic effects of
TGF-β are often lost in progressed stages due to (epi-) genetic disruption of several
members of the signaling pathway. This progressed stage is characterized by activation
of a “late TGF-β signature” which promotes the phenotypic switch from tumor suppressor
to promoter of cancer. Consequently, cancer cells display an EMT phenotype by acquiring
invasive and pro-metastatic properties.
Methods:
Several established (PLC & HuCCT-1) and newly generated primary (HCC & CCA) cell lines
were exposed to of TGF-β1 and TGF-β2 for 72hr. Effect of TGF-β on tumor-initiating
potential was estimated by colony and sphere formation assays. Further, selected stemness
markers were evaluated using flow cytometry. Invasive and migratory properties were
determined using the wound healing assay and molecular changes were estimated by next
generation sequencing analysis.
Results:
Treatment with TGF-β1 and TGF-β2 led to a significant reduction in colony and spheroid
forming ability in all investigated cell lines. Consistent with the reduced in vitro tumorigenicity and spherogenicity, a drastic effect of TGF- β on the putative tumor-initiating
cell population was observed, reflected by the downregulation of stemness markers
CD133 and EpCAM. Interestingly, treatment with TGF-β1 led to a significant increase
in the expression of CD44 accompanied as well as activation of established epithelial-mesenchymal-transition
(EMT) transition markers. Accordingly, a significant downregulation of E-Cadherin
paralleled by upregulation in SNAIL transcription factor was seen. Consequently, enhanced
migratory and invasive properties of HCC and CCA were observed evidenced by increased
wound healing and invasion. In addition, pathways enriched with differentially expressed
genes known to be involved in EMT (P13K, WNT/β-Catenin pathway) were identified by
NGS analysis.
Conclusions:
In conclusion, we here confirm the cytostatic effect of TGF-β1 and TGF-β2 by reducing
the frequency of stem-like cancer cells in both HCC and iCCA. Further, TFG-β1 seems
to be an important regulator of EMT as well as invasive properties in progressed PLCs.
These context-dependent dichotomic effects should be considered in TGF-β based therapeutic
approaches. We would further validate the obtained findings in vivo using a battery
of different xenotransplantation models representing the observed in vitro findings.
