Purpose/Objective(s)

Hepatocellular carcinoma (HCC) is a major global cause of mortality. The epithelial-mesenchymal transition (EMT) transcription factor TWIST1 has been implicated in the invasion and metastasis of HCC, but the mechanism is unclear. Integrated transcriptomic and proteomic analyses on primary liver tumors and lung metastases from a spontaneous Twist1 -dependent metastasis mouse model of MYC -induced HCC identified the hexosamine biosynthetic pathway (HBP) as a cancer cell autonomous mechanism for EMT-mediated invasion and metastasis, which was further validated in vitro and ex vivo .

Materials/Methods

RNA-sequencing and mass spectrometry-based proteomics were carried out on primary liver tumors, lung metastases, normal livers and normal lungs from a Twist1 -dependent metastasis mouse model of MYC -induced HCC (LMT); a metastatic MYC -induced HCC mouse model dependent on Twist1 phosphorylation mimetic mutant (LMT-DQD); a non-metastatic mouse model of MYC -induced HCC (LM); a non-metastatic MYC -induced HCC mouse model with the Twist box mutant (LMT-F191G); and wildtype mice. Data was analyzed in R. Organoids were derived from LM liver tumors, embedded into collagen and imaged using time-lapse DIC microscopy. Cell lines were derived from LM and LMT liver tumors, and along with human HCC cell lines Huh7, Hep3B and HepG2, were utilized for in vitro migration and invasion assays.

Results

91 genes were differentially expressed between lung metastases versus primary liver tumors; primary liver tumors versus normal liver; and normal lung versus normal liver with p <0.05. Gene Ontology and KEGG Pathway Enrichment analyses revealed an enrichment of metabolic processes. Gfpt2 , the gene encoding for the rate-limiting enzyme of the hexosamine biosynthetic pathway, was overexpressed >2 fold in metastases compared to primary tumor (p = 0.007). Kaplan-Meier estimates from cBioPortal show that HCC patients with genetic alterations in GFPT2 and/or its isoform GFPT1 have significantly worse overall median survival compared to patients without these alterations (27.5 months vs 83.24 months , p = 0.006). Functional validation studies demonstrated that increased O-GlcNAcylation by pharmacologic treatment with TMG or genetic manipulation by GFPT2 overexpression was sufficient to increase migration and invasion in vitro in a panel of human and murine HCC cell lines. Decreased HBP-O-GlcNAcylation by pharmacologic treatment with DON or genetic manipulation by GFPT2 shRNA knockdown inhibited migration and invasion in vitro . Similarly, using a novel organoid invasion assay, TMG treatment increased invasion of organoids derived from LM tumors ex vivo 2-fold (p <0.03) while DON treatment inhibited invasion 2-fold (p <0.03).

Conclusion

The hexosamine biosynthetic pathway is required and sufficient for invasion and metastasis in vitro in HCC cell lines and ex vivo in HCC tumor-derived organoids. The HBP may be a potential therapeutic target for advanced HCC patients.