Gen-Sheng Feng

Background and Aims: So far, there is no effective mechanism-based therapeutic agent tailored for liver tumors. Immune checkpoint inhibitors (ICIs) have demonstrated limited efficacy in liver cancer, often associated with severe adverse effects. Although poly-inosinic:cytidylic acid (polyIC) has shown an adjuvant effect when combined with anti-PD-L1 antibody (αPD-L1) in treating liver tumors in animal models, its systemic toxicity limits its clinical utility. To address this challenge, the original goal of this study was to develop a liver-targeted strategy to minimize toxicity. Methods and Results: We constructed lipid nanoparticles (LNPs) encapsulating polyIC for selective delivery to the liver and evaluated the tumor-suppressive effects of polyIC-LNPs, free polyIC, and/or αPD-L1 in multiple murine liver tumor models. We also analyzed changes in the hepatic immune microenvironment using single-cell RNA sequencing and flow cytometry. Surprisingly, polyIC-LNPs alone robustly suppressed both primary and metastatic liver tumors, independent of αPD-L1. Even a single dose of polyIC-LNPs was sufficient to control liver tumor progression. Primarily taken up by hepatocytes, polyIC-LNPs induced sustained type I interferon signaling, reshaped the hepatic immune landscape, and promoted CD8⁺ T cell infiltration and activation by enhancing the maturation of conventional dendritic cells (cDC1), ultimately resulting in a potent anti-tumor response. Conclusion: Contrary to our original goal, we unexpectedly found that polyIC-LNPs function as an efficacious monotherapy tailored for liver cancer, capable of coordinately modulating antitumor immunity. This novel approach eliminates the need for ICIs, thereby addressing key limitations of current immunotherapies.

Background: Immune checkpoint inhibitors combined with antiangiogenic therapy have become the standard of care for advanced HCC, albeit with limited therapeutic benefit. Our previous studies demonstrated the immunomodulatory and antitumor effects of polyIC, a synthetic dsRNA. Here, we compared the efficacy of anti-programmed death ligand 1 (αPD-L1) plus polyIC versus αPD-L1 plus anti-vascular endothelial growth factor (αVEGF) in mouse tumor models. Methods: We established a primary liver tumor model using hydrodynamic tail vein injection of Ras/Myc oncogenes and a metastasized tumor model via intrasplenic injection of colon cancer cells. Flow cytometry and gene expression analysis were performed to assess immune profiles across treatment groups. Key factors contributing to antitumor efficacy were explored. Results: In both models, αPD-L1 plus polyIC demonstrated superior antitumor effects relative to αPD-L1 plus αVEGF. Unlike αVEGF, polyIC enhanced the immune response to αPD-L1 by increasing T cell infiltration, T effector memory CD8⁺ T cells, CD8⁺ to CD4⁺ T cell ratio, and CD8⁺ T cell function. This combination also promoted apoptosis in tumors and the accumulation of conventional dendritic cells and invariant natural killer T cells. In addition, αPD-L1 plus polyIC treatment led to upregulation of cytokines and chemokines, with CCL5 blockade partially reducing the CD8⁺ to CD4⁺ T cell ratio and attenuating polyIC-driven antitumor effects. Conclusions: This preclinical study identifies polyIC as an efficacious adjuvant of αPD-L1 treatment in liver cancer, providing a better strategy to improve immunotherapy outcomes.

The metabolic landscape of cancer greatly influences antitumor immunity, yet it remains unclear how organ-specific metabolites in the tumor microenvironment influence immunosurveillance. We found that accumulation of primary conjugated and secondary bile acids (BAs) are metabolic features of human hepatocellular carcinoma and experimental liver cancer models. Inhibiting conjugated BA synthesis in hepatocytes through deletion of the BA-conjugating enzyme bile acid–CoA:amino acid N -acyltransferase (BAAT) enhanced tumor-specific T cell responses, reduced tumor growth, and sensitized tumors to anti–programmed cell death protein 1 (anti–PD-1) immunotherapy. Furthermore, different BAs regulated CD8 ⁺ T cells differently; primary BAs induced oxidative stress, whereas the secondary BA lithocholic acid inhibited T cell function through endoplasmic reticulum stress, which was countered by ursodeoxycholic acid. We demonstrate that modifying BA synthesis or dietary intake of ursodeoxycholic acid could improve tumor immunotherapy in liver cancer model systems.

Background and Aims: The chemical carcinogen diethylnitrosamine (DEN) is often used to induce HCC in mice. Curiously, several labs have reported that the removal of oncoproteins from hepatocytes exacerbated DEN-induced HCC, with mechanisms unknown. This study aimed at deciphering molecular mechanisms underlying the tumor suppressive effect of oncoproteins. Approach and Results: We generated mutant mouse lines with hepatocyte-specific deletions of Met, Ptpn11/Shp2, Ikkβ, or Ctnnb1/β-catenin and assessed DEN-induced tumorigenesis in the wild-type and mutant mice. To systematically examine genetic and molecular signaling alterations, we performed whole exome and RNA-sequencing on liver samples collected at the pre-cancer and established cancer stages. Although the mutational profiles of DEN-induced tumors were barely different in wild-type and mutant mice, oncoprotein ablation increased DEN-induced mutational burdens, especially in Shp2-deficient tumors. RNA-sequencing revealed multiple changes in signaling pathways, in particular, upregulated epithelial-mesenchymal transition, cell migration, and tumor metastasis, as well as downregulated small molecule metabolism that was affected by oncoprotein ablation. We identified key molecules and pathways that are associated with hepatic innate immunity and implicated in liver tumorigenesis. In addition, we unveiled markedly changed expression of a few miRNAs in the human HCC database. Conclusions: The aggravation of DEN-induced HCC progression seen on oncoprotein ablation could be caused by common and distinct genomic and signaling alterations. This study reveals a new level of complexity in hepatocarcinogenesis and elucidates molecular mechanisms underlying tumor evolution and recurrence.

The nonreceptor tyrosine phosphatase SHP2 has been at the center of cell signaling research for three decades. SHP2 is required to fully activate the RTK/RAS/ERK signaling cascade, although the underlying mechanisms are not completely understood. PTPN11 , which encodes SHP2, is the first identified proto-oncogene that encodes a tyrosine phosphatase, with dominantly activating mutations detected in leukemias and solid tumors. However, SHP2 has pro- and antioncogenic effects, and the most recent data reveal opposite activities of SHP2 in tumor cells and microenvironment cells. Allosteric SHP2 inhibitors show promising antitumor effects and overcome resistance to inhibitors of RAS/ERK signaling in animal models. Many clinical trials with orally bioactive SHP2 inhibitors, alone or combined with other regimens, are ongoing for a variety of cancers worldwide, with therapeutic outcomes yet unknown. This review discusses the multifaceted functions of SHP2 in oncogenesis, preclinical studies, and clinical trials with SHP2 inhibitors in oncological treatment.

CD133 (prominin 1) is widely viewed as a cancer stem cell marker in association with drug resistance and cancer recurrence. Herein, we report that with impaired RTK-Shp2-Ras-Erk signaling, heterogenous hepatocytes form clusters that manage to divide during mouse liver regeneration. These hepatocytes are characterized by upregulated CD133 while negative for other progenitor cell markers. Pharmaceutical inhibition of proliferative signaling also induced CD133 expression in various cancer cell types from multiple animal species, suggesting an inherent and common mechanism of stress response. Super-resolution and electron microscopy localize CD133 on intracellular vesicles that apparently migrate between cells, which we name ‘intercellsome.’ Isolated CD133⁺ intercellsomes are enriched with mRNAs rather than miRNAs. Single-cell RNA sequencing reveals lower intracellular diversity (entropy) of mitogenic mRNAs in Shp2-deficient cells, which may be remedied by intercellular mRNA exchanges between CD133⁺ cells. CD133-deficient cells are more sensitive to proliferative signal inhibition in livers and intestinal organoids. These data suggest a mechanism of intercellular communication to compensate for intracellular signal deficit in various cell types.

CD133 (prominin 1) is widely viewed as a cancer stem cell marker in association with drug resistance and cancer recurrence. Herein we report that with impaired RTK-Shp2 Ras-Erk signaling, heterogenous hepatocytes form clusters that manage to divide during liver regeneration. These hepatocytes are characterized by upregulated CD133 while negative for other progenitor cell markers. Pharmaceutical inhibition of proliferative signaling also induced CD133 expression in various cancer cell types, suggesting an inherent and common mechanism of stress response. Super-resolution and electron microscopy localize CD133 on intracellular vesicles that apparently migrate between cells, which we name “intercellsome”. Isolated CD133+ intercellsomes are enriched with mRNAs rather than miRNAs. Single-cell RNA sequencing reveals lower intracellular diversity (entropy) of mitogenic mRNAs in Shp2-deficient cells, which may be remedied by intercellular mRNA exchanges between CD133+ cells. CD133-deficient cells are more sensitive to proliferative signal inhibition in livers and intestinal organoids. These data suggest a mechanism of intercellular communication to compensate intracellular signal deficit in various cell types.

Background and Aims: SH2 domain–containing protein tyrosine phosphatase 2 (Shp2) is the first identified pro‐oncogenic tyrosine phosphatase that acts downstream of receptor tyrosine kinases (RTKs) to promote Ras–extracellular signal–regulated kinase signaling. However, this phosphatase was also shown to be antitumorigenic in HCC. This study is aimed at deciphering paradoxical Shp2 functions and mechanisms in hepatocarcinogenesis and at exploring its value as a pharmaceutical target in HCC therapy. Approaches and Results: We took both genetic and pharmaceutical approaches to examine the effects of Shp2 inhibition on primary liver cancers driven by various oncogenes and on metastasized liver tumors. We show here that the catalytic activity of Shp2 was essential for relay of oncogenic signals from RTKs in HCC and that chemical inhibition of Shp2 robustly suppressed HCC driven by RTKs. However, in contrast to a tumor‐promoting hepatic niche generated by genetically deleting Shp2 in hepatocytes, treatment with a specific Shp2 inhibitor had a tumor‐suppressing effect on metastasized liver tumor progression. Mechanistically, the Shp2 inhibitor enhanced antitumor innate immunity by down‐regulating inflammatory cytokines, suppressing the chemokine (C‐C motif) receptor 5 signaling axis, but up‐regulating interferon‐β secretion. Conclusions: These results unveil complex mechanisms for the tumor‐suppressing effect of pharmaceutical Shp2 inhibition in the liver immune environment. We provide a proof of principle for clinical trials with specific Shp2 inhibitors in patients with primary and metastasized liver cancer.