Tyrosine phosphatase SHP-2 mediates C-type lectin receptor-induced activation of the kinase Syk and anti-fungal TH17 responses.

Fungal infection stimulates the canonical C-type lectin receptor (CLR) signaling pathway via activation of the tyrosine kinase Syk. Here we identify a crucial role for the tyrosine phosphatase SHP-2 in mediating CLR-induced activation of Syk. Ablation of the gene encoding SHP-2 (Ptpn11; called 'Shp-2' here) in dendritic cells (DCs) and macrophages impaired Syk-mediated signaling and abrogated the expression of genes encoding pro-inflammatory molecules following fungal stimulation. Mechanistically, SHP-2 operated as a scaffold, facilitating the recruitment of Syk to the CLR dectin-1 or the adaptor FcRγ, through its N-SH2 domain and a previously unrecognized carboxy-terminal immunoreceptor tyrosine-based activation motif (ITAM). We found that DC-derived SHP-2 was crucial for the induction of interleukin 1ß (IL-1ß), IL-6 and IL-23 and anti-fungal responses of the TH17 subset of helper T cells in controlling infection with Candida albicans. Together our data reveal a mechanism by which SHP-2 mediates the activation of Syk in response to fungal infection.

Genomic and transcriptomic analyses of chemical hepatocarcinogenesis aggravated by oncoprotein loss.

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, Ikkß, or Ctnnb1/ß-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.

Pharmaceutical SH2 domain-containing protein tyrosine phosphatase 2 inhibition suppresses primary and metastasized liver tumors by provoking hepatic innate immunity.

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-ß 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.

Enhancing the therapeutic efficacy of programmed death ligand 1 antibody for metastasized liver cancer by overcoming hepatic immunotolerance in mice.

BACKGROUND AND AIMS: Immunotherapy with programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) blockade has shown low response rates in liver cancer patients, with the underlying mechanisms unclear. To decipher a specific impact of the liver microenvironment, we compared the effects of anti-PD-L1 antibody (αPD-L1) blockade on the same tumor grown s.c. or in the liver. APPROACH AND RESULTS: We generated s.c. tumors in mice by inoculating MC38 colorectal cancer (CRC) cells under the skin and metastatic liver tumors by portal vein or splenic injection of CRC cells. Tumor-bearing mice were treated by i.p. injection of αPD-L1, polyinosinic:polycytidylic acid (poly[I:C]), or both. αPD-L1 monotherapy significantly suppressed s.c. tumor growth, but showed no effect on metastatic liver tumors. However, the combination of αPD-L1 with poly(I:C), an innate immunity-stimulating reagent, robustly inhibited tumor progression in liver. The combination therapy effectively down-regulated myeloid-derived suppressor cells (MDSCs), but up-regulated ratios of M1/M2 macrophages, CD8/CD4, and CD8/regulatory T (Treg) cells infiltrated into liver tumors and whole liver. A group of long-lasting T-bet+ Eomes- PD-1- cytotoxic T cells was maintained in the combo-treated liver, leading to resistance to tumor recurrence. Depleting macrophages or blocking type Ⅰ interferon signaling abrogated the synergistic antitumor effect of αPD-L1 and poly(I:C), indicating a requirement of boosting innate immunity for optimized activation of cytotoxic T cells by PD-1/PD-L1 blockade. CONCLUSIONS: The poor response of liver cancers to αPD-L1 therapy is largely attributable to a unique hepatic immunotolerant microenvironment, independent of tumor origins or types. The success of a combinatorial immunotherapy relies on coordinated inhibition or activation of various innate and adaptive immune cell activities.

Improving the Efficacy of Liver Cancer Immunotherapy: The Power of Combined Preclinical and Clinical Studies.

Hepatocellular carcinoma (HCC) is a most deadly malignant disease worldwide, with no effective mechanism-based therapy available. Therefore, following the "miracle" outcomes seen in a few patients at the advanced stages of melanoma or lung cancer, the immune checkpoint inhibitors (ICIs) immediately entered clinical trials for advanced HCC patients without pre-clinical studies. Emerging data of clinical studies showed manageable toxicity and safety but limited therapeutic benefit to HCC patients, suggesting low response rate. Thus, one urgent issue is how to convert the liver tumors from cold to hot and responsive, which may rely on in-depth mechanistic studies in animal models and large scale data analysis in human patients. One ongoing approach is to design combinatorial treatment of different ICIs with other reagents and modalities. Indeed, a phase 3 clinical trial showed that combination of atezolizumab and bevacizumab achieved better overall and progression-free survival rates than sorafenib in unresectable HCC. This review highlights the value of animal models and the power of combining pre-clinical and clinical studies in efforts to improve HCC immunotherapy.

Lens differentiation is controlled by the balance between PDGF and FGF signaling.

How multiple receptor tyrosine kinases coordinate cell fate determination is yet to be elucidated. We show here that the receptor for platelet-derived growth factor (PDGF) signaling recruits the p85 subunit of Phosphoinositide 3-kinase (PI3K) to regulate mammalian lens development. Activation of PI3K signaling not only prevents B-cell lymphoma 2 (BCL2)-Associated X (Bax)- and BCL2 Antagonist/Killer (Bak)-mediated apoptosis but also promotes Notch signaling to prevent premature cell differentiation. Reducing PI3K activity destabilizes the Notch intracellular domain, while the constitutive activation of Notch reverses the PI3K deficiency phenotype. In contrast, fibroblast growth factor receptors (FGFRs) recruit Fibroblast Growth Factor Receptor Substrate 2 (Frs2) and Rous sarcoma oncogene (Src) Homology Phosphatase 2 (Shp2) to activate Mitogen-Activated Protein Kinase (MAPK) signaling, which induces the Notch ligand Jagged 1 (Jag1) and promotes cell differentiation. Inactivation of Shp2 restored the proper timing of differentiation in the p85 mutant lens, demonstrating the antagonistic interaction between FGF-induced MAPK and PDGF-induced PI3K signaling. By selective activation of PI3K and MAPK, PDGF and FGF cooperate with and oppose each other to balance progenitor cell maintenance and differentiation.

A tumorigenic index for quantitative analysis of liver cancer initiation and progression.

Primary liver cancer develops from multifactorial etiologies, resulting in extensive genomic heterogeneity. To probe the common mechanism of hepatocarcinogenesis, we interrogated temporal gene expression profiles in a group of mouse models with hepatic steatosis, fibrosis, inflammation, and, consequently, tumorigenesis. Instead of anticipated progressive changes, we observed a sudden molecular switch at a critical precancer stage, by developing analytical platform that focuses on transcription factor (TF) clusters. Coarse-grained network modeling demonstrated that an abrupt transcriptomic transition occurred once changes were accumulated to reach a threshold. Based on the experimental and bioinformatic data analyses as well as mathematical modeling, we derived a tumorigenic index (TI) to quantify tumorigenic signal strengths. The TI is powerful in predicting the disease status of patients with metabolic disorders and also the tumor stages and prognosis of liver cancer patients with diverse backgrounds. This work establishes a quantitative tool for triage of liver cancer patients and also for cancer risk assessment of chronic liver disease patients.

Disrupting phosphatase SHP2 in macrophages protects mice from high-fat diet-induced hepatic steatosis and insulin resistance by elevating IL-18 levels.

Chronic low-grade inflammation plays an important role in the pathogenesis of type 2 diabetes. Src homology 2 domain-containing tyrosine phosphatase-2 (SHP2) has been reported to play diverse roles in different tissues during the development of metabolic disorders. We previously reported that SHP2 inhibition in macrophages results in increased cytokine production. Here, we investigated the association between SHP2 inhibition in macrophages and the development of metabolic diseases. Unexpectedly, we found that mice with a conditional SHP2 knockout in macrophages (cSHP2-KO) have ameliorated metabolic disorders. cSHP2-KO mice fed a high-fat diet (HFD) gained less body weight and exhibited decreased hepatic steatosis, as well as improved glucose intolerance and insulin sensitivity, compared with HFD-fed WT littermates. Further experiments revealed that SHP2 deficiency leads to hyperactivation of caspase-1 and subsequent elevation of interleukin 18 (IL-18) levels, both in vivo and in vitro Of note, IL-18 neutralization and caspase-1 knockout reversed the amelioration of hepatic steatosis and insulin resistance observed in the cSHP2-KO mice. Administration of two specific SHP2 inhibitors, SHP099 and Phps1, improved HFD-induced hepatic steatosis and insulin resistance. Our findings provide detailed insights into the role of macrophagic SHP2 in metabolic disorders. We conclude that pharmacological inhibition of SHP2 may represent a therapeutic strategy for the management of type 2 diabetes.

An Efficient Combination Immunotherapy for Primary Liver Cancer by Harmonized Activation of Innate and Adaptive Immunity in Mice.

Immunotherapy with checkpoint inhibitors for liver cancer, while active in many clinical trials worldwide, may have uncertain outcomes due to the unique immunotolerant microenvironment of the liver. In previous experiments, we unexpectedly identified a robust liver tumor-preventive effect of a synthetic double-stranded RNA, polyinosinic-polycytidylic acid (polyIC), in mice. Herein we further demonstrate that polyIC given at the precancer stage effectively prevented liver tumorigenesis by activating natural killer cells, macrophages, and some T-cell subsets; no inhibitory effect was observed on tumor progression if injected after tumor initiation. Nevertheless, polyIC administration potently induced programmed death ligand 1 (PD-L1) expression in liver sinusoid endothelial cells, which prompted us to test a combined treatment of polyIC and PD-L1 antibody (Ab). Although injecting PD-L1 Ab alone did not show any therapeutic effect, injection of polyIC sensitized the hepatic response to PD-L1 blockade. Combination of polyIC and PD-L1 Ab resulted in sustained accumulation of active cluster of differentiation 8 cytotoxic T cells and robust liver tumor suppression and conferred a survival advantage in mice. These preclinical data in animal models suggest that, despite the low efficacy of PD-L1/PD-1 blockade alone, careful design of mechanism-based combinatorial immunotherapeutic protocols may shift the paradigm in liver cancer treatment by coordinating maximal activation of multiple innate and adaptive immune functions. Conclusion: We provide proof of principle for the development of an efficient prevention strategy of liver tumorigenesis and a powerful combination immunotherapy for primary liver cancer.

Hepatic Autophagy Deficiency Compromises Farnesoid X Receptor Functionality and Causes Cholestatic Injury.

Autophagy is important for hepatic homeostasis, nutrient regeneration, and organelle quality control. We investigated the mechanisms by which liver injury occurred in the absence of autophagy function. We found that mice deficient in autophagy because of the lack of autophagy-related gene 7 or autophagy-related gene 5, key autophagy-related genes, manifested intracellular cholestasis with increased levels of serum bile acids, a higher ratio of tauromuricholic acid/taurocholic acid in the bile, increased hepatic bile acid load, abnormal bile canaliculi, and altered expression of hepatic transporters. In determining the underlying mechanism, we found that autophagy sustained and promoted the basal and up-regulated expression of farnesoid X receptor (Fxr) in the fed and starved conditions, respectively. Consequently, expression of Fxr and its downstream genes, particularly bile salt export pump, and the binding of FXR to the promoter regions of these genes, were suppressed in autophagy-deficient livers. In addition, codeletion of nuclear factor erythroid 2-related factor 2 (Nrf2) in autophagy deficiency status reversed the FXR suppression. Furthermore, the cholestatic injury of autophagy-deficient livers was reversed by enhancement of FXR activity or expression, or by Nrf2 deletion. Conclusion: Together with earlier reports that FXR can suppress autophagy, our findings indicate that autophagy and FXR form a regulatory loop and deficiency of autophagy causes abnormal FXR functionality, leading to the development of intracellular cholestasis and liver injury.

Alx4 relays sequential FGF signaling to induce lacrimal gland morphogenesis.

The sequential use of signaling pathways is essential for the guidance of pluripotent progenitors into diverse cell fates. Here, we show that Shp2 exclusively mediates FGF but not PDGF signaling in the neural crest to control lacrimal gland development. In addition to preventing p53-independent apoptosis and promoting the migration of Sox10-expressing neural crests, Shp2 is also required for expression of the homeodomain transcription factor Alx4, which directly controls Fgf10 expression in the periocular mesenchyme that is necessary for lacrimal gland induction. We show that Alx4 binds an Fgf10 intronic element conserved in terrestrial but not aquatic animals, underlying the evolutionary emergence of the lacrimal gland system in response to an airy environment. Inactivation of ALX4/Alx4 causes lacrimal gland aplasia in both human and mouse. These results reveal a key role of Alx4 in mediating FGF-Shp2-FGF signaling in the neural crest for lacrimal gland development.

Nuclear Shp2 directs normal embryo implantation via facilitating the ERα tyrosine phosphorylation by the Src kinase.

Estrogen and progesterone coupled with locally produced signaling molecules are essential for embryo implantation. However, the hierarchical landscape of the molecular pathways that governs this process remains largely unexplored. Here we show that the protein tyrosine phosphatase Shp2, a positive transducer of RTK signaling, is predominately localized in the nuclei in the periimplantation mouse uterus. Uterine-specific deletion of Shp2 exhibits reduced progesterone receptor (PR) expression and progesterone resistance, which derails normal uterine receptivity, leading to complete implantation failure in mice. Notably, the PR expression defects are attributed to the limited estrogen receptor α (ERα) activation in uterine stroma. Further analysis reveals that nuclear Shp2, rather than cytosolic Shp2, promotes the ERα transcription activity. This function is achieved by enhancing the Src kinase-mediated ERα tyrosine phosphorylation, which facilitates ERα binding to Pgr promoter in an ERK-independent manner in periimplantation uteri. Besides uncovering a regulatory mechanism, this study could be clinically relevant to dysfunctional ERα-caused endometrial disorders in women.

A Src family kinase-Shp2 axis controls RUNX1 activity in megakaryocyte and T-lymphocyte differentiation.

Hematopoietic development occurs in complex microenvironments and is influenced by key signaling events. Yet how these pathways communicate with master hematopoietic transcription factors to coordinate differentiation remains incompletely understood. The transcription factor RUNX1 plays essential roles in definitive hematopoietic stem cell (HSC) ontogeny, HSC maintenance, megakaryocyte (Mk) maturation, and lymphocyte differentiation. It is also the most frequent target of genetic alterations in human leukemia. Here, we report that RUNX1 is phosphorylated by Src family kinases (SFKs) and that this occurs on multiple tyrosine residues located within its negative regulatory DNA-binding and autoinhibitory domains. Retroviral transduction, chemical inhibitor, and genetic studies demonstrate a negative regulatory role of tyrosine phosphorylation on RUNX1 activity in Mk and CD8 T-cell differentiation. We also demonstrate that the nonreceptor tyrosine phosphatase Shp2 binds directly to RUNX1 and contributes to its dephosphorylation. Last, we show that RUNX1 tyrosine phosphorylation correlates with reduced GATA1 and enhanced SWI/SNF interactions. These findings link SFK and Shp2 signaling pathways to the regulation of RUNX1 activity in hematopoiesis via control of RUNX1 multiprotein complex assembly.

High-fat feeding reprograms maternal energy metabolism and induces long-term postpartum obesity in mice.

BACKGROUND: Excessive gestational weight gain (EGWG) closely associates with postpartum obesity. However, the causal role of EGWG in postpartum obesity has not been experimentally verified. The objective of this study was to determine whether and how EGWG causes long-term postpartum obesity. METHODS: C57BL/6 mice were fed with high-fat diet during gestation (HFFDG) or control chow, then their body composition and energy metabolism were monitored after delivery. RESULTS: We found that HFFDG significantly increased gestational weight gain. After delivery, adiposity of HFFDG-treated mice (Preg-HF) quickly recovered to the levels of controls. However, 3 months after parturition, Preg-HF mice started to gain significantly more body fat even with regular chow. The increase of body fat of Preg-HF mice was progressive with aging and by 9 months after delivery had increased 2-fold above the levels of controls. The expansion of white adipose tissue (WAT) of Preg-HF mice was manifested by hyperplasia in visceral fat and hypertrophy in subcutaneous fat. Preg-HF mice developed low energy expenditure and UCP1 expression in interscapular brown adipose tissue (iBAT) in later life. Although blood estrogen concentrations were similar between Preg-HF and control mice, a significant decrease in estrogen receptor α (ERα) expression and hypermethylation of the ERα promoter was detected in the fat of Preg-HF mice 9 months after delivery. Interestingly, hypermethylation of ERα promoter and low ERα expression were only detected in adipocyte progenitor cells in both iBAT and WAT of Preg-HF mice at the end of gestation. CONCLUSIONS: These results demonstrate that HFFDG causes long-term postpartum obesity independent of early postpartum fat retention. This study also suggests that HFFDG adversely programs long-term postpartum energy metabolism by epigenetically reducing estrogen signaling in both BAT and WAT.

ß-catenin deficiency in hepatocytes aggravates hepatocarcinogenesis driven by oncogenic ß-catenin and MET.

Both activating and inactivating mutations in catenin ß1 (ctnnb1), which encodes ß-catenin, have been implicated in liver tumorigenesis in humans and mice, although the underlying mechanisms are not fully understood. Herein, we show that deletion of endogenous ß-catenin in hepatocytes aggravated hepatocellular carcinoma (HCC) development driven by an oncogenic version of ß-catenin (CAT) in combination with the hepatocyte growth factor receptor MET proto-oncogene receptor tyrosine kinase (MET). Although the mitogenic signaling and cell cycle progression was modestly impaired after CAT/MET transfection, the ß-catenin-deficient livers displayed changes in transcriptomes, increased DNA damage response, expanded Sox9+ cells, and up-regulation of protumorigenic cytokines, including interleukin-6 and transforming growth factor ß1. These events eventually exacerbated CAT/MET-driven hepatocarcinogenesis in ß-catenin-deficient livers, featured by up-regulation of extracellular signal-regulated kinase (Erk), protein kinase B (Akt), and Wnt/ß-catenin signaling and cyclin D1 expression. The resultant mouse tumors showed similar transcriptomes to human HCC samples with concomitant CTNNB1 mutations and MET overexpression. CONCLUSION: These data argue that while dominantly activating mutants of ß-catenin are oncogenic, inhibiting the oncogenic signaling pathway generates a pro-oncogenic microenvironment that may facilitate HCC recurrence following a targeted therapy of the primary tumor. An effective therapeutic strategy must require disruption of the oncogenic signaling in tumor cells and suppression of the secondary tumor-promoting stromal effects in the liver microenvironment. (Hepatology 2018;67:1807-1822).

Stress Conditions Induced by Locoregional Therapies Stimulate Enrichment and Proliferation of Liver Cancer Stem Cells.

PURPOSE: This study tested the hypothesis that stress conditions that simulated percutaneous thermal ablation (PTA), transarterial embolization (TAE), or transarterial chemoembolization stimulated enrichment of hepatocellular carcinoma (HCC) cancer stem cells (hCSCs) and that hCSC inhibitors can suppress this effect. MATERIALS AND METHODS: Human HCC cell lines HepG2 and PLC/PRF/5 were subjected to a 46.5°C heat bath for 10 minutes or to 1% hypoxia for 72 hours without fetal bovine serum and with or without doxorubicin. Cells were then treated with a ß-catenin inhibitor (FH535 or XAV939), a PI3 kinase inhibitor (Ly294002), or niclosamide, a US Food and Drug Administration-approved antihelminthic drug that acts as a mitochondrial decoupler and mixed inhibitor. Surviving cells were analyzed for hCSC markers by flow cytometry, for stemness by colony-forming assay or sphere-forming assay, and for proliferative capacity by MTT assay (where MTT is 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide). Expression of proteins related to CSC renewal and proliferation were analyzed by immunoblotting and immunostaining. RESULTS: Conditions that simulated PTA, TAE, and transarterial chemoembolization resulted in an enrichment of cells bearing hCSC markers (CD133, CD44, and EpCAM). Cells surviving heat stress exhibited higher colony- or sphere-forming capacity and a greater proliferative state. These effects could be suppressed by niclosamide and inhibitors of ß-catenin and PI3 kinase. CONCLUSIONS: Stress conditions induced by locoregional therapies stimulated hCSC enrichment and proliferation, which could be suppressed by niclosamide and inhibitors of pathways important for hCSC renewal. Future studies will determine whether combining locoregional therapies with adjuvant hCSC inhibitors reduces HCC recurrence.

Targeting of Ras-mediated FGF signaling suppresses Pten-deficient skin tumor.

Deficiency in PTEN (phosphatase and tensin homolog deleted on chromosome 10) is the underlying cause of PTEN hamartoma tumor syndrome and a wide variety of human cancers. In skin epidermis, we have previously identified an autocrine FGF signaling induced by loss of Pten in keratinocytes. In this study, we demonstrate that skin hyperplasia requires FGF receptor adaptor protein Frs2α and tyrosine phosphatase Shp2, two upstream regulators of Ras signaling. Although the PI3-kinase regulatory subunits p85α and p85ß are dispensable, the PI3-kinase catalytic subunit p110α requires interaction with Ras to promote hyperplasia in Pten-deficient skin, thus demonstrating an important cross-talk between Ras and PI3K pathways. Furthermore, genetic and pharmacological inhibition of Ras-MAPK pathway impeded epidermal hyperplasia in Pten animals. These results reveal a positive feedback loop connecting Pten and Ras pathways and suggest that FGF-activated Ras-MAPK pathway is an effective therapeutic target for preventing skin tumor induced by aberrant Pten signaling.

Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting.

The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adipose-derived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling.

Shp2 deletion in hepatocytes suppresses hepatocarcinogenesis driven by oncogenic ß-Catenin, PIK3CA and MET.

BACKGROUND & AIMS: Shp2 is an SH2-tyrosine phosphatase acting downstream of receptor tyrosine kinases (RTKs). Most recent data demonstrated a liver tumor-suppressing role for Shp2, as ablating Shp2 in hepatocytes aggravated hepatocellular carcinoma (HCC) induced by chemical carcinogens or Pten loss. We further investigated the effect of Shp2 deficiency on liver tumorigenesis driven by classical oncoproteins c-Met (receptor for HGF), ß-catenin and PIK3CA. METHODS: We performed hydrodynamic tail vein injection of two pairs of plasmids expressing c-Met and ΔN90-ß-catenin (MET/CAT), or c-Met and PIK3CAH1047R (MET/PIK), into WT and Shp2hep-/- mice. We compared liver tumor loads and investigated the pathogenesis and molecular mechanisms involved using multidisciplinary approaches. RESULTS: Despite the induction of oxidative and metabolic stresses, Shp2 deletion in hepatocytes suppressed hepatocarcinogenesis driven by overexpression of oncoproteins MET/CAT or MET/PIK. Shp2 loss inhibited proliferative signaling from c-Met, Wnt/ß-catenin, Ras/Erk and PI3K/Akt pathways, but triggered cell senescence following exogenous expression of the oncogenes. CONCLUSIONS: Shp2, acting downstream of RTKs, is positively required for hepatocyte-intrinsic tumorigenic signaling from these oncoproteins, even if Shp2 deficiency induces a tumor-promoting hepatic microenvironment. These data suggest a new and more effective therapeutic strategy for HCCs driven by oncogenic RTKs and other upstream molecules, by inhibiting Shp2 and also suppressing any tumor-enhancing stromal factors produced because of Shp2 inhibition. LAY SUMMARY: Primary liver cancer is a malignant disease with poor prognosis, largely because there are limited systemic therapies available. We show here that a cytoplasmic tyrosine phosphatase Shp2 is required for liver tumorigenesis. This tumorigenesis is driven by two oncoproteins that are implicated in human liver cancer. This, together with our previous studies, uncovers the complexity of liver tumorigenesis, by elucidating the pro- and anti-tumor effects of Shp2 in mouse models. This data can be used to guide new therapies.

Gab2 mediates hepatocellular carcinogenesis by integrating multiple signaling pathways.

Our previous studies have found that Growth factor receptor-bound protein 2-associated binding protein 2 (Gab2)-a docking protein-governs the development of fatty liver disease. Here, we further demonstrate that Gab2 mediates hepatocarcinogenesis. Compared with a faint expression in para-carcinoma tissue, Gab2 was highly expressed in ∼60-70% of human hepatocellular carcinoma (HCC) specimens. Deletion of Gab2 dramatically suppressed diethylnitrosamine-induced HCC in mice. The oncogenic effects of Gab2 in HepG2 cells were promoted by Gab2 overexpression but were rescued by Gab2 knockdown. Furthermore, Gab2 knockout in HepG2 cells restrained cell proliferation, migration and tumor growth in nude mice. Signaling pathway analysis with protein kinase inhibitors demonstrated that oncogenic regulation by Gab2 in hepatic cells involved multiple signaling molecules, including ERK, Akt, and Janus kinases (Jaks), especially those that mediate inflammatory signaling. IL-6 signaling was increased by Gab2 overexpression and impaired by Gab2 deletion via regulation of Jak2 and signal transducer and activator of transcription 3 phosphorylation and the expression of downstream genes, such as Bcl-2 (B-cell lymphoma 2), c-Myc, MMP7 (matrix metalloproteinase-7), and cyclin D1in vitro and in vivo These data indicate that Gab2 mediates the pathologic progression of HCC by integrating multiple signaling pathways and suggest that Gab2 might be a powerful therapeutic target for HCC.-Cheng, J., Zhong, Y., Chen, S., Sun, Y., Huang, L., Kang, Y., Chen, B., Chen, G., Wang, F., Tian, Y., Liu, W., Feng, G.-S., Lu, Z. Gab2 mediates hepatocellular carcinogenesis by integrating multiple signaling pathways.