Aims

Hepatic angiomyolipoma (AML) often shows epithelioid morphology with inconspicuous fat. Epithelioid component can mimic hepatocellular adenoma (HCA) or carcinoma (HCC). The aims of this study were to examine the expression of commonly used markers for HCA or HCC in hepatic AML and highlight pitfalls in diagnosis.

Methods and results

Resected hepatic AMLs (n = 16) were reviewed; reticulin stain, immunohistochemistry for glutamine synthetase (GS), β-catenin and liver fatty acid binding protein (LFABP) were performed along with Sanger sequencing of exon 3 of CTNNB1 and next-generation sequencing (NGS). Predominant epithelioid component (≥50%) was seen in 80% of cases. Foamy macrophage was present in 33% of cases. High-risk histological features were often present in tumours with benign outcome: marked atypia (19%), mitoses (20%) and necrosis (33%). GS staining (≥10% of tumour) was seen in epithelioid components in 13 (87%) cases, and was diffuse (>50% of tumour) in six (40%) cases. LFABP staining or nuclear β-catenin staining was not seen in any case. Sanger sequencing and NGS did not reveal CTNNB1 mutation in any tested case. NGS demonstrated TSC2 mutations in all five cases tested.

Conclusions

The predominance of epithelioid component resembling HCA or HCC is common in hepatic AML. Absence of LFABP and presence of fat can be mistaken for HNF1α-inactivated HCA. Diffuse GS staining can be mistaken for β-catenin-activated HCA or HCC. Diffuse GS expression is not related to CTNNB1 mutation. All tested cases showed TSC2 mutation, supporting this as the driving genetic event for hepatic AML.

Background & aims

Gain of function (GOF) mutations in the CTNNB1 gene are one of the most frequent genetic events in hepatocellular carcinoma (HCC). T-box transcription factor 3 (TBX3) is a liver-specific target of the Wnt/β-catenin pathway and thought to be an oncogene mediating activated β-catenin-driven HCC formation.

Methods

We evaluated the expression pattern of TBX3 in human HCC specimens. Tbx3 was conditionally knocked out in murine HCC models by hydrodynamic tail vein injection of Cre together with c-Met and ΔN90-β-catenin (c-Met/β-catenin) in Tbx3^flox/flox mice. TBX3 was overexpressed in human HCC cell lines to investigate the functions of TBX3 in vitro.

Results

A bimodal expression pattern of TBX3 in human HCC samples was detected: high expression of TBX3 in GOF CTNNB1 HCC and downregulation of TBX3 in non-CTNNB1 mutant tumors. High expression of TBX3 was associated with increased differentiation and decreased expression signatures of tumor growth. Using Tbx3^flox/flox mice, we found that ablation of Tbx3 significantly accelerates c-Met/β-catenin-driven HCC formation. Moreover, Tbx3(-) HCC demonstrated increased YAP/TAZ activity. The accelerated tumor growth induced by loss of TBX3 in c-Met/β-catenin mouse HCC was successfully prevented by overexpression of LATS2, which inhibited YAP/TAZ activity. In human HCC cell lines, overexpression of TBX3 inhibited HCC cell growth as well as YAP/TAZ activation. A negative correlation between TBX3 and YAP/TAZ target genes was observed in human HCC samples. Mechanistically, phospholipase D1 (PLD1), a known positive regulator of YAP/TAZ, was identified as a novel transcriptional target repressed by TBX3.

Conclusion

Our study suggests that TBX3 is induced by GOF CTNNB1 mutants and suppresses HCC growth by inactivating PLD1, thus leading to the inhibition of YAP/TAZ oncogenes.

Lay summary

TBX3 is a liver-specific target of the Wnt/β-catenin pathway and thought to be an oncogene in promoting liver cancer development. Herein, we demonstrate that TBX3 is in fact a tumor suppressor gene that restricts liver tumor growth. Strategies which increase TBX3 expression and/or activities may be effective for HCC treatment.