The role of ETV4 in HCC: How transcription factors can inform immunotherapy combination treatments

FGF19/FGFR4-mediated elevation of ETV4 facilitates hepatocellular carcinoma metastasis by upregulating PD-L1 and CCL2Journal of HepatologyVol. 79Issue 1PreviewMetastasis remains the major reason for the high mortality of patients with hepatocellular carcinoma (HCC). This study was designed to investigate the role of E-twenty-six-specific sequence variant 4 (ETV4) in promoting HCC metastasis and to explore a new combination therapy strategy for ETV4-mediated HCC metastasis. Full-Text PDF See Article, pages 109–125 See Article, pages 109–125 Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer.1Siegel R.L. Miller K.D. Wagle N.S. Jemal A. Cancer statistics, 2023.CA Cancer J Clin. 2023; 73: 17-48Crossref PubMed Scopus (329) Google Scholar The molecular pathogenesis of HCC is highly heterogeneous due to its differing etiologies, which include viral hepatitis, alcohol consumption and metabolic disease.2Llovet J.M. Pinyol R. Kelley R.K. El-Khoueiry A. Reeves H.L. Wang X.W. Gores G.J. et al.Molecular pathogenesis and systemic therapies for hepatocellular carcinoma.Nat Cancer. 2022; 3: 386-401Crossref PubMed Scopus (35) Google Scholar Unfortunately, actionable mutations are found in only 25% of HCC tumors and most clinical trials using targeted therapies have failed.3Schulze K. Imbeaud S. Letouze E. Alexandrov L.B. Calderaro J. Rebouissou S. Couchy G. et al.Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.Nat Genet. 2015; 47: 505-511Crossref PubMed Scopus (1089) Google Scholar However, tumors often share a common trait: dysregulated transcriptional programs. Indeed, cancer cells are often addicted to specific transcription factors (TFs) that control oncogenic transcriptional programs. This addiction creates a vulnerability open for exploitation, as a loss of function of the TF would lead to cancer cell death.4Bradner J.E. Hnisz D. Young R.A. Transcriptional addiction in cancer.Cell. 2017; 168: 629-643Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar Aberrant TFs in the setting of cancer regulate a range of transcriptional programs including activation of intrinsic drivers of proliferation, but also regulation of immune escape mechanisms induced by crosstalk within the tumor microenvironment (TME). Taken together, TFs play a crucial role in regulating gene expression in cancer cells, influencing the TME and contributing to tumor progression. The TME plays a role at all stages of malignant progression, from initial transformation to invasion and metastasis.2Llovet J.M. Pinyol R. Kelley R.K. El-Khoueiry A. Reeves H.L. Wang X.W. Gores G.J. et al.Molecular pathogenesis and systemic therapies for hepatocellular carcinoma.Nat Cancer. 2022; 3: 386-401Crossref PubMed Scopus (35) Google Scholar,5de Visser K.E. Joyce J.A. The evolving tumor microenvironment: from cancer initiation to metastatic outgrowth.Cancer Cell. 2023; 41: 374-403Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar Metastases occur when cancer cells develop the ability to migrate, often through an epithelial to mesenchymal transition, and disseminate to other regions of the body.5de Visser K.E. Joyce J.A. The evolving tumor microenvironment: from cancer initiation to metastatic outgrowth.Cancer Cell. 2023; 41: 374-403Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar Despite advances in the understanding of the mechanisms underlying malignant progression, metastasis continues to significantly reduce survival in patients with HCC.6Yang B. Li M. Tang W. Liu W. Zhang S. Chen L. Xia J. Dynamic network biomarker indicates pulmonary metastasis at the tipping point of hepatocellular carcinoma.Nat Commun. 2018; 9: 678Crossref PubMed Scopus (127) Google Scholar Immunotherapy modulating the TME has revolutionized cancer treatment over the last decade. While single agent immunotherapy treatments have only modest benefit in HCC, combination immunotherapies show more promise.7Zhang T. Merle P. Wang H. Zhao H. Kudo M. Combination therapy for advanced hepatocellular carcinoma: do we see the light at the end of the tunnel?.Hepatobiliary Surg Nutr. 2021; 10: 180-192Crossref PubMed Google Scholar,8Cheng A.L. Hsu C. Chan S.L. Choo S.P. Kudo M. Challenges of combination therapy with immune checkpoint inhibitors for hepatocellular carcinoma.J Hepatol. 2020; 72: 307-319Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar Most notably, two such treatment combinations are now FDA approved as first-line treatments for advanced HCC; the combination of atezolizumab (a PD-LI monoclonal antibody) with bevacizumab (a VEGF monoclonal antibody) and tremelimumab (a CTLA-4 monoclonal antibody) with durvalumab (a PD-L1 monoclonal antibody).9Finn R.S. Qin S. Ikeda M. Galle P.R. Ducreux M. Kim T.Y. Kudo M. et al.Atezolizumab plus bevacizumab in unresectable hepatocellular carcinoma.N Engl J Med. 2020; 382: 1894-1905Crossref PubMed Scopus (2616) Google Scholar,10Abou-Alfa G.K. Lau G. Kudo M. Chan S.L. Kelley R.K. Furuse J. et al.Tremelimumab plus durvalumab in unresectable hepatocellular carcinoma.NEJM Evidence. 2022; 1Crossref Google Scholar Considering this triumph, the mechanistic insights regarding other combinatorial regimens warrant evaluation. In this issue of Journal of Hepatology, Xie et al.11Xie M. Lin Z. Ji X. Luo X. Zhang Z. Sun M. Chen X. et al.FGF19/FGFR4-mediated elevation of ETV4 facilitates hepatocellular carcinoma metastasis by upregulating PD-L1 and CCL2.J Hepatol. 2023; 79: 109-125Abstract Full Text Full Text PDF Google Scholar describe a paradigm for establishing combinatorial therapies targeting both upstream and downstream signaling pathways of TFs, such as ETV4, in HCC metastasis. ETV4 is an E-twenty-six (ETS) TF that is upregulated in all stages of HCC. The expression of ETV4 is inversely correlated with overall survival, a fact comprehensively validated by the authors. ETV4 regulates multiple pro-tumorigenic genes, including matrix metalloproteinase 1, urokinase-type plasminogen activator receptor, and Wnt/β-catenin pathway members.12Sun T. Zhang J. ETV4 mediates the Wnt/beta-catenin pathway through transcriptional activation of ANXA2 to promote hepatitis B virus-associated liver hepatocellular carcinoma progression.J Biochem. 2021; 170: 663-673Crossref PubMed Scopus (5) Google Scholar, 13Kim E. Kim D. Lee J.S. Yoe J. Park J. Kim C.J. Jeong D. et al.Capicua suppresses hepatocellular carcinoma progression by controlling the ETV4-MMP1 axis.Hepatology. 2018; 67: 2287-2301Crossref PubMed Scopus (57) Google Scholar, 14Yang Q.X. Zhong S. He L. Jia X.J. Tang H. Cheng S.T. Ren J.H. et al.PBK overexpression promotes metastasis of hepatocellular carcinoma via activating ETV4-uPAR signaling pathway.Cancer Lett. 2019; 452: 90-102Crossref PubMed Scopus (55) Google Scholar Recent work also suggests an interplay between ETV4 and Yes-associated protein 1 in promoting growth of liver cancer cells and altering the TME by decreasing T-cell infiltration and increasing infiltration of myeloid-derived suppressor cells (MDSCs).15Xu X. Wang B. Liu Y. Jing T. Xu G. Zhang L. Kun J. et al.ETV4 potentiates nuclear YAP retention and activities to enhance the progression of hepatocellular carcinoma.Cancer Lett. 2022; 537215640Crossref PubMed Scopus (6) Google Scholar Finally, in HBV-related HCC, the HBV X protein activates ETV4 to promote HCC progression.16Zheng C. Liu M. Ge Y. Qian Y. Fan H. HBx increases chromatin accessibility and ETV4 expression to regulate dishevelled-2 and promote HCC progression.Cell Death Dis. 2022; 13: 116Crossref PubMed Scopus (7) Google Scholar Collectively, multiple studies suggest that ETV4 plays a key role in influencing the TME and consequently HCC progression. Herein, the authors further characterize the complicated signaling network in ETV4-elevated HCC metastasis and report on combinatorial drug regimens that are effective in preventing metastasis in HCC mouse models. First, the authors established ETV4 overexpression in orthotopic mouse models that lead to increased tumor size, higher number of lung metastases, and a decrease in tumor-infiltrating CD8+ T cells, accompanied by an increase in tumor-infiltrating MDSCs and tumor-associated macrophages. By modulating signaling pathways in the context of the ETV4 overexpression model, the authors identify direct targets of ETV4 including metastasis-related genes such as FGFR4, as well as genes that contribute to an immunosuppressive TME such as CD274 (PD-L1) and CCL2 (Fig. 1). These genes were further confirmed as regulatory targets of ETV4 by ChIP-sequencing and luciferase promoter binding assays. Using several methods, including ablation of PD-L1 or CCL2, as well as deletion of MDSCs using clodronate-encapsulated liposome or anti-Gr-1, the authors demonstrate the direct ETV4-target PD-L1 suppresses CD8+ T-cell infiltration, while CCL2 recruits MDSCs to the TME. This confirms that poor survival and metastasis mainly depends on the capacity of ETV4 to induce an immunosuppressive TME. The authors also describe the mechanisms by which the direct ETV4-target FGFR4 leads to further activation of ETV4. FGF19 binding to FGFR4 activates the ERK1/2 signaling pathway, inducing the phosphorylation of ELK1 and consequential upregulation of ETV4 (Fig. 1). As mentioned above, ETV4 induces the expression of FGFR4, creating a FGF19-ETV4-FGFR4 positive feedback loop. As many signaling pathways are able to activate ERK1/2, the authors tested several other inhibitors and discovered that the HGF-cMET-ERK1/2 pathway also contributes to ETV4 upregulation. Lastly, the authors tested combinatorial drug treatments, using an FGFR4 inhibitor previously validated in a phase I study (BLU-554)17Kim R.D. Sarker D. Meyer T. Yau T. Macarulla T. Park J.W. Choo S.P. et al.First-in-Human phase I study of fisogatinib (BLU-554) validates aberrant FGF19 signaling as a driver event in hepatocellular carcinoma.Cancer Discov. 2019; 9: 1696-1707Crossref PubMed Scopus (124) Google Scholar or a MAPK inhibitor (trametinib) in combination with anti-PD-L1 and observed significant improvement of antitumoral effects compared to each drug treatment alone.11Xie M. Lin Z. Ji X. Luo X. Zhang Z. Sun M. Chen X. et al.FGF19/FGFR4-mediated elevation of ETV4 facilitates hepatocellular carcinoma metastasis by upregulating PD-L1 and CCL2.J Hepatol. 2023; 79: 109-125Abstract Full Text Full Text PDF Google Scholar Overall, the paradigm described by the authors of targeting both upstream activators and downstream targets of ETV4, and thereby blocking both intrinsic and extrinsic drivers of tumor progression, may prove to be a powerful therapeutic approach for advanced HCC. More work is still needed to validate the utility of ETV4 levels as a biomarker for patient stratification or its efficiency as a target for precision treatment strategies, but these preclinical results provide the basis for an approach to target aberrant TFs in this devastating disease. This approach can be used for future studies aiming to understand the complex role of TFs in cancer. The complexity of TF signaling networks and the implications toward altering the TME shown here by Xie et al.11Xie M. Lin Z. Ji X. Luo X. Zhang Z. Sun M. Chen X. et al.FGF19/FGFR4-mediated elevation of ETV4 facilitates hepatocellular carcinoma metastasis by upregulating PD-L1 and CCL2.J Hepatol. 2023; 79: 109-125Abstract Full Text Full Text PDF Google Scholar open multiple avenues for future research (Fig. 1). First, the authors show that ETV4 binds mostly to transcription start site regions, possibly regulating dozens to hundreds of genes directly and indirectly. Considering the direct role of ETV4 in regulating MYC, MTA1 and VEGFA, among other metastasis-related genes, one can wonder about other potential combinatorial treatments that could be used for ETV4-dependent HCC. Considering the heterogeneity of HCC, not all patients will develop a dependency on ETV4 and thus some will be resistant to the proposed combination treatments. Evaluation of larger cohorts is needed to show that elevated ETV4 expression is a common event leading to HCC progression and metastasis in patients. To incorporate combinatorial treatments targeting TF signaling networks into the precision medicine schema, a complete repertoire of TFs that drive HCC metastasis will need to be described as well as the mechanisms by which patients can be stratified by their TF dependencies. In this regard, activated ERK1/2 is responsible for phosphorylating many oncogenic ETS factors in prostate cancer cells in vitro,18Nicholas T.R. Strittmatter B.G. Hollenhorst P.C. Oncogenic ETS factors in prostate cancer.Adv Exp Med Biol. 2019; 1210: 409-436Crossref PubMed Scopus (23) Google Scholar and this could be an avenue for future exploration to complement the mechanisms described here. This is also important considering TFs work cooperatively and may, in the right context, compensate for each other to maintain cancer progression in the case of a treatment-induced loss-of-function. Further, once combinatorial treatments begin eliminating the dominant cancer cell population, resistant cancer cells that have different TF dependencies and use different growth signaling pathways may flourish. Non-invasive biomarkers may be key to track these dependency changes in patients over time.19Singal A.G. Reig M. Villanueva A. Emerging tools for hepatocellular carcinoma surveillance.Am J Gastroenterol. 2022; 117: 1948-1951Crossref PubMed Scopus (1) Google Scholar In conclusion, Xie et al.11Xie M. Lin Z. Ji X. Luo X. Zhang Z. Sun M. Chen X. et al.FGF19/FGFR4-mediated elevation of ETV4 facilitates hepatocellular carcinoma metastasis by upregulating PD-L1 and CCL2.J Hepatol. 2023; 79: 109-125Abstract Full Text Full Text PDF Google Scholar provide a framework for comprehensive profiling of TF dependencies in HCC by studying the role of ETV4 in promoting metastasis. By focusing on new combination therapy strategies targeting both upstream and downstream signaling pathways of aberrant TFs, especially pathways that create an immunosuppressive TME, they demonstrate an avenue for future studies in our tireless attempt to inhibit HCC progression and metastasis. The authors received no financial support to produce this manuscript. The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details. A.J.C. and M.A.D.: wrote the manuscript. The following are the supplementary data to this article: Download .pdf (.22 MB) Help with pdf files Multimedia component 1