Previous data indicate that Tankyrase inhibitors exert anti-growth functions in many cancer cell lines due to their ability to inactivate the YAP protooncogene. In the present manuscript, we investigated the effect of Tankyrase inhibitors on the growth of hepatocellular carcinoma (HCC) cell lines and the molecular mechanisms involved. For this purpose, we performed cell proliferation assay by colony-forming ability in seven human HCC cells subjected to XAV-939 and G007-LK Tankyrase inhibitors. Noticeably, the two Tankyrase inhibitors suppressed the HCC cell growth in a dose-dependent manner. Furthermore, we found that Tankyrase inhibitors synergized with MEK and AKT inhibitors to suppress HCC cell proliferation. At the molecular level, Tankyrase inhibitors significantly decreased YAP protein levels, reduced the expression of YAP target genes, and inhibited YAP/TEAD luciferase reporter activity. In addition, Tankyrase inhibitors administration was accompanied by upregulation of Angiomotin-like 1 (AMOTL1) and Angiomotin-like 2 (AMOTL2) proteins, two major negative regulators of YAP. Altogether, the present data indicate that XAV-939 and G007-LK Tankyrase inhibitors could suppress proliferation of hepatocellular carcinoma cells and downregulate YAP/TAZ by stabilizing AMOTL1 and AMOTL2 proteins, thus representing new potential anticancer drugs against hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a deadly form of liver malignancy with limited treatment options. Amplification and/or overexpression of c-MYC is one of the most frequent genetic events in human HCC. The mammalian target of Rapamycin Complex 1 (mTORC1) is a major functional axis regulating various aspects of cellular growth and metabolism. Recently, we demonstrated that mTORC1 is necessary for c-Myc driven hepatocarcinogenesis as well as for HCC cell growth in vitro. Among the pivotal downstream effectors of mTORC1, upregulation of Fatty Acid Synthase (FASN) and its mediated de novo lipogenesis is a hallmark of human HCC. Here, we investigated the importance of FASN on c-Myc-dependent hepatocarcinogenesis using in vitro and in vivo approaches. In mouse and human HCC cells, we found that FASN suppression by either gene silencing or soluble inhibitors more effectively suppressed proliferation and induced apoptosis in the presence of high c-MYC expression. In c-Myc/Myeloid cell leukemia 1 (MCL1) mouse liver tumor lesions, FASN expression was markedly upregulated. Most importantly, genetic ablation of Fasn profoundly delayed (without abolishing) c-Myc/MCL1 induced HCC formation. Liver tumors developing in c-Myc/MCL1 mice depleted of Fasn showed a reduction in proliferation and an increase in apoptosis when compared with corresponding lesions from c-Myc/MCL1 mice with an intact Fasn gene. In human HCC samples, a significant correlation between the levels of c-MYC transcriptional activity and the expression of FASN mRNA was detected. Altogether, our study indicates that FASN is an important effector downstream of mTORC1 in c-MYC induced HCC. Targeting FASN may be helpful for the treatment of human HCC, at least in the tumor subset displaying c-MYC amplification or activation.

Background & aims

Yes-associated protein (YAP) and its paralog transcriptional co-activator with post synaptic density protein, drosophila disc large tumor suppressor and zonula occludens-1-binding motif (TAZ) are 2 co-activators downstream of Hippo tumor-suppressor cascade. Both have been implicated in the development of hepatocellular carcinoma (HCC). However, whether YAP and TAZ have distinct or overlapping functions during hepatocarcinogenesis remains unknown.

Methods

Expression patterns of YAP and TAZ were analyzed in human HCC samples. The requirement of Yap and/or Taz in protein kinase B (Akt)/ neuroblastoma RAS viral oncogene homolog (NRas) -driven liver tumorigenesis was analyzed using conditional Yap, Taz, and Yap;Taz knockout mice. Transcriptional programs regulated by YAP and/or TAZ were identified via RNA sequencing.

Results

We found that in human HCC samples, an almost ubiquitous activation of YAP or TAZ occurs, underlying their role in this tumor type. Intriguingly, 70% of HCC samples showed only nuclear YAP or TAZ immunoreactivity. In the Akt/NRas liver tumor model, where nuclear Yap and Taz can be detected readily, deletion of Yap or Taz alone only mildly delayed liver tumor development, whereas their concomitant ablation strongly inhibited tumor cell proliferation and significantly suppressed Akt/NRas-driven hepatocarcinogenesis. In HCC cell lines, silencing of either YAP or TAZ led to decreased expression of both overlapping and distinct sets of genes, with the most prominent gene signatures related to cell-cycle progression and DNA replication.

Conclusions

YAP and TAZ have overlapping and distinct roles in hepatocarcinogenesis. HCCs may display unique activation of YAP or TAZ, thus relying on either YAP or TAZ for their growth.

Background & aims

Mounting evidence implicates the Hippo downstream effectors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) in hepatocellular carcinoma (HCC). We investigated the functional contribution of YAP and/or TAZ to c-MYC-induced liver tumor development.

Methods

The requirement for YAP and/or TAZ in c-Myc-driven hepatocarcinogenesis was analyzed using conditional Yap, Taz, and Yap;Taz knockout (KO) mice. An hepatocyte-specific inducible TTR-CreER^T2 KO system was applied to evaluate the role of YAP and TAZ during tumor progression. Expression patterns of YAP, TAZ, c-MYC, and BCL2L12 were analyzed in human HCC samples.

Results

We found that the Hippo cascade is inactivated in c-Myc-induced mouse HCC. Intriguingly, TAZ mRNA levels and activation status correlated with c-MYC activity in human and mouse HCC, but YAP mRNA levels did not. We demonstrated that TAZ is a direct transcriptional target of c-MYC. In c-Myc induced murine HCCs, ablation of Taz, but not Yap, completely prevented tumor development. Mechanistically, TAZ was required to avoid c-Myc-induced hepatocyte apoptosis during tumor initiation. The anti-apoptotic BCL2L12 gene was identified as a novel target regulated specifically by YAP/TAZ, whose silencing strongly suppressed c-Myc-driven murine hepatocarcinogenesis. In c-Myc murine HCC lesions, conditional knockout of Taz, but not Yap, led to tumor regression, supporting the requirement of TAZ for c-Myc-driven HCC progression.

Conclusions

TAZ is a pivotal player at the crossroad between the c-MYC and Hippo pathways in HCC. Targeting TAZ might be beneficial for the treatment of patients with HCC and c-MYC activation.

Lay summary

The identification of novel treatment targets and approaches for patients with hepatocellular carcinoma is crucial to improve survival outcomes. We identified TAZ as a transcriptional target of c-MYC which plays a critical role in c-MYC-dependent hepatocarcinogenesis. TAZ could potentially be targeted for the treatment of patients with c-MYC-driven hepatocellular carcinoma.

Different pyruvate kinase isoforms are expressed in a tissue-specific manner, with pyruvate kinase M2 (PKM2) suggested to be the predominant isoform in proliferating cells and cancer cells. Because of differential regulation of enzymatic activities, PKM2, but not PKM1, has been thought to favor cell proliferation. However, the role of PKM2 in tumorigenesis has been recently challenged. Here we report that increased glucose catabolism through glycolysis and increased pyruvate kinase activity in c-MYC-driven liver tumors are associated with increased expression of both PKM1 and PKM2 isoforms and decreased expression of the liver-specific isoform of pyruvate kinase, PKL. Depletion of PKM2 at the time of c-MYC overexpression in murine livers did not affect c-MYC-induced tumorigenesis and resulted in liver tumor formation with decreased pyruvate kinase activity and decreased catabolism of glucose into alanine and the Krebs cycle. An increased PKM1/PKM2 ratio by ectopic PKM1 expression further decreased glucose flux into serine biosynthesis and increased flux into lactate and the Krebs cycle, resulting in reduced total levels of serine. However, these changes also did not affect c-MYC-induced liver tumor development. These results suggest that increased expression of PKM2 is not required to support c-MYC-induced tumorigenesis in the liver and that various PKM1/PKM2 ratios and pyruvate kinase activities can sustain glucose catabolism required for this process. Cancer Res; 77(16); 4355-64. ©2017 AACR.

Objective

Increased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC).

Design

We investigated the functional contribution of fatty acid synthase (Fasn)-mediated de novo FA synthesis in a murine HCC model induced by loss of Pten and overexpression of c-Met (sgPten/c-Met) using liver-specific Fasn knockout mice. Expression arrays and lipidomic analysis were performed to characterise the global gene expression and lipid profiles, respectively, of sgPten/c-Met HCC from wild-type and Fasn knockout mice. Human HCC cell lines were used for in vitro studies.

Results

Ablation of Fasn significantly delayed sgPten/c-Met-driven hepatocarcinogenesis in mice. However, eventually, HCC emerged in Fasn knockout mice. Comparative genomic and lipidomic analyses revealed the upregulation of genes involved in cholesterol biosynthesis, as well as decreased triglyceride levels and increased cholesterol esters, in HCC from these mice. Mechanistically, loss of Fasn promoted nuclear localisation and activation of sterol regulatory element binding protein 2 (Srebp2), which triggered cholesterogenesis. Blocking cholesterol synthesis via the dominant negative form of Srebp2 (dnSrebp2) completely prevented sgPten/c-Met-driven hepatocarcinogenesis in Fasn knockout mice. Similarly, silencing of FASN resulted in increased SREBP2 activation and hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (HMGCR) expression in human HCC cell lines. Concomitant inhibition of FASN-mediated FA synthesis and HMGCR-driven cholesterol production was highly detrimental for HCC cell growth in culture.

Conclusion

Our study uncovers a novel functional crosstalk between aberrant lipogenesis and cholesterol biosynthesis pathways in hepatocarcinogenesis, whose concomitant inhibition might represent a therapeutic option for HCC.