Deciphering the Role of Transforming Growth Factor Beta (TGF-β1&2) during Tumor Promotion and Suppression in Primary Liver Cancer

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.