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Poorly differentiated hepatocellular carcinoma (HCC) is an aggressive disease with poor prognosis. Here the authors show that GDF1, a member of the TGF-β superfamily, is highly expressed in high-grade poorly differentiated HCC and is associated with tumor plasticity, and that GDF1-induced reexpression of cancer testis antigens could render tumors sensitive to immune checkpoint inhibition.

Summary: Here, we present a detailed protocol for the identification of potential oncofetal targets for hepatocellular carcinoma (HCC) patients through a hepatocyte differentiation model and a sorafenib refractory cell-line-derived xenograft model. We describe the procedures of tumor sphere formation, organoid generation, and subcutaneous tumor formation for functional studies. We then detail the procedures of immunohistochemistry and immunofluorescence for examination of changes in lineage-specific markers. Finally, we describe the development of antibody-based therapeutics targeting tumor lineage plasticity in HCC.For complete details on the use and execution of this protocol, please refer to Kong et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Hepatocellular carcinoma (HCC) is one of the most common human malignancies worldwide with very poor prognosis. Resistance to targeted therapeutic drugs such as sorafenib remains one of the major challenges in clinical treatment. In the present study, PARP1 was found to be highly expressed in human embryonic stem cells, but progressively decreased upon specified hepatic differentiation. Reactivation of PARP1 expression was also detected in HCC residual tumors after sorafenib treatment in xenograft mouse model, indicating the potential important roles of PARP1 in stem cell pluripotency and HCC sorafenib treatment resistance. Overexpression of PARP1 was frequently observed in HCC patients, and closely associated with poor clinical outcome. Treatment of Sorafenib induced activation of DNA damage repair signaling, which is highly active and essential for maintenance of stem cell pluripotency in HCC residual tumors. PARP inhibitor Olaparib extensively suppressed the DNA damage repair signaling, and significantly inhibited the global pluripotent transcriptional network. The repression of key pluripotent transcriptional factors and DNA damage repair signaling by Olaparib was mainly through CHD1L-mediated condensation of the chromatin structure at their promotor regions. The global reshaping of the pluripotent transcriptome by Olaparib might reinforce Sorafenib in eliminating HCC residual tumors and enhance therapeutic efficiency.

Abstract Background Gradual loss of terminal differentiation markers and gain of stem cell-like properties is a major hall mark of cancer malignant progression. The stem cell pluripotent transcriptional factor SOX family play critical roles in governing tumor plasticity and lineage specification. This study aims to establish a novel SOX signature to monitor the extent of tumor dedifferentiation and predict prognostic significance in hepatocellular carcinoma (HCC). Methods The RNA-seq data from The Cancer Genome Atlas (TCGA) LIHC project were chronologically divided into the training (n = 188) and testing cohort (n = 189). LIRI-JP project from International Cancer Genome Consortium (ICGC) data portal was used as an independent validation cohort (n = 232). Kaplan-Meier and multivariable Cox analyses were used to examine the clinical significance and prognostic value of the signature genes. Results The SOX gene family members were found to be aberrantly expressed in clinical HCC patients. A five-gene SOX signature with prognostic value was established in the training cohort. The SOX signature genes were found to be closely associated with tumor grade and tumor stage. Liver cancer dedifferentiation markers (AFP, CD133, EPCAM, and KRT19) were found to be progressively increased while hepatocyte terminal differentiation markers (ALB, G6PC, CYP3A4, and HNF4A) were progressively decreased from HCC patients with low SOX signature scores to patients with high SOX signature scores. Kaplan-Meier survival analysis further indicated that the newly established SOX signature could robustly predict patient overall survival in both training, testing, and independent validation cohort. Conclusions An oncogenic dedifferentiation SOX signature presents a great potential in predicting prognostic significance in HCC, and might provide novel biomarkers for precision oncology further in the clinic.

Clinical observation of the association between cancer aggressiveness and embryonic development stage implies the importance of developmental signals in cancer initiation and therapeutic resistance. However, the dynamic gene expression during organogenesis and the master oncofetal drivers are still unclear, which impeded the efficient elimination of poor prognostic tumors, including human hepatocellular carcinoma (HCC). In this study, human embryonic stem cells were induced to differentiate into adult hepatocytes along hepatic lineages to mimic liver development in vitro. Combining transcriptomic data from liver cancer patients with the hepatocyte differentiation model, the active genes derived from different hepatic developmental stages and the tumor tissues were selected. Bioinformatic analysis followed by experimental assays was used to validate the tumor subtypespecific oncofetal signatures and potential therapeutic values. Hierarchical clustering analysis revealed the existence of two subtypes of liver cancer with different oncofetal properties. The gene signatures and their clinical significance were further validated in an independent clinical cohort and The Cancer Genome Atlas database. Upstream activator analysis and functional screening further identified E2F1 and SMAD3 as master transcriptional regulators. Small-molecule inhibitors specifically targeting the oncofetal drivers extensively down-regulated subtype-specific developmental signaling and inhibited tumorigenicity. Liver cancer cells and primary HCC tumors with different oncofetal properties also showed selective vulnerability to their specific inhibitors. Further precise targeting of the tumor initiating steps and driving events according to subtype-specific biomarkers might eliminate tumor progression and provide novel therapeutic strategy. [ABSTRACT FROM AUTHOR]