Systematic Analysis of Cytostatic TGF-Beta Response in Mesenchymal-Like Hepatocellular Carcinoma Cell Lines

Abstract

Background Hepatocellular carcinoma (HCC) is one of the most challenging malignancies, with high morbidity and mortality rates. The transforming growth factor-β (TGF-β) pathway plays a dual role in HCC, acting as both tumor suppressor and promoter. A thorough understanding of the mechanisms underlying its opposing functions is important. The growth suppressive effects of TGF-β remain largely unknown for mesenchymal HCC cells. Using a systematic approach, here we assess the cytostatic TGF-β responses and intracellular transduction of the canonical TGF-β/Smad signaling cascade in mesenchymal-like HCC cell lines. Methods Nine mesenchymal-like HCC cell lines, including SNU182, SNU387, SNU398, SNU423, SNU449, SNU475, Mahlavu, Focus, and Sk-Hep1, were used in this study. The cytostatic effects of TGF-β were evaluated by cell cycle analysis, BrdU labeling, and SA-β-Gal assay. RT-PCR and western blot analysis were utilized to determine the mRNA and protein expression levels of TGF-β signaling components and cytostatic genes. Immunoperoxidase staining and luciferase reporter assays were performed to comprehend the transduction of the canonical TGF-β pathway. ResultsWe report that mesenchymal-like HCC cell lines are resistant to TGF-β-induced growth suppression. The vast majority of cell lines have an active canonical signaling from the cell membrane to the nucleus. Three cell lines had lost the expression of cytostatic effector genes. Conclusion Our findings reveal that cytostatic TGF-β responses have been selectively lost in mesenchymal-like HCC cell lines. Notably, their lack of responsiveness was not associated with a widespread impairment of TGF-β signaling cascade. These cell lines may serve as valuable models for studying the molecular mechanisms underlying the loss of TGF-β-mediated cytostasis during hepatocarcinogenesis.

The Bright and the Dark Side of TGF-β Signaling in Hepatocellular Carcinoma: Mechanisms, Dysregulation, and Therapeutic Implications

“…The increase in p15 INK4b and p21 CIP1 levels by TGF-β critically thwarts the activities of cyclin D-CDK4/6 and cyclin E-CDK2 complexes, respectively, leading to pRb hypophosphorylation and the subsequent inhibition of E2F activity [ 71 ]. It is worth noting that these responses are specific to well-differentiated hepatoblast-like HCC cell lines, while poorly-differentiated mesenchymal-like HCC cell lines are generally resistant to TGF-β-mediated cytostasis [ 64 , 72 ]. In addition, TGF-β may, potentially, reduce cyclin D expression while increasing hypophosphorylated pRb levels via a Smad-interacting β-spectrin adaptor protein, SPTBN1, formerly known as embryonic liver fodrin or ELF [ 73 ].…”

“…Accumulating evidence suggests that epigenetic modifications in TGF-β signaling mediators, regulators, and effectors may result in the dysregulation of TGF-β/Smad signaling and in defective cytostatic responses. In this regard, a common mechanism that interrupts TGF-β-induced cytostasis is the inactivation of the INK4 locus (encodes p15 INK4b and p16 INK4a ) by deletion or epigenetic silencing via promoter methylation [ 72 , 200 , 201 ]. Another mechanism that impairs the antiproliferative effects of the TGF-β pathway in HCC is based on tristetraprolin (TTP), a negative post-transcriptional regulator of c-Myc with downregulated expression in HCC tumor tissues.…”