Differential effect of TGF-β family members on proliferation and migration in primary liver cancer

Background and Aims:

Transforming Growth Factor Beta (TGF-β) belongs to a superfamily of cytokines that induces pleiotropic effects on different processes and cell types in the liver. While TGF-β signaling exerts tumor suppressive functions 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 epithelial-mesenchymal-transition (EMT) phenotype and acquire pro-metastatic properties.

Method:

Primary patient-derived (HCC & CCA) and established cell lines (PLC & HuCCT-1) were exposed to TGF-β1 and TGF-β2 (1 ng/ml; 5 ng/ml) for 72 hr. The effect of TGF-β on tumor-initiating potential was determined by side-population as well as colony and sphere formation assays. Invasive and migratory properties were determined using the wound healing invasion assays in vitro and in vivo. Selected stemness and EMT-related genes were assessed by qRT-PCR and Next Generation Sequencing was performed to explore differential gene expression patterns across the different treatments.

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 reduced in vitro tumorigenicity and spherogenicity a drastic effect on the putative tumor-initiating cell population was observed, reflected by a reduction in frequency of the side population and downregulation of stemness markers CD133 and EpCAM. Interestingly, a significant downregulation of epithelial marker E-cadherin and concomitant upregulation of mesenchymal markers such as Vimentin and SNAIL was exclusively observed after TGF-β1 treatment. Similarly, a significant increase in migratory and invasive properties of HCC and CCA cell lines was induced by TGF-β1 whereas TGF-β2 treated cells showed no effect. In addition, transcriptome profiling confirmed activation of gene sets involved in Cell Cycle:G1/S Checkpoint in response to both treatments (TGF-β1&β2) whereas enrichment in signaling pathways known to be involved in pro-metastatic properties resembling P13K, MAPK and WNT/β-Catenin pathway were predominantly associated with the TGF-β1 response.

Conclusion:

We here confirm the cytostatic effect of TGF- β1 and TGF- β2 in primary liver cancer. Further, TFG-β1 is an important regulator of EMT in progressed PLCs and induces migratory and invasive properties. These context-dependent dichotomic effects should be considered in TGF-β based therapeutic approaches.