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p53 haploinsufficiency and increased mTOR signalling define a subset of aggressive hepatocellular carcinoma

  • Yuan-Deng Luo
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Lei Fang
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Hong-Qiang Yu
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Jie Zhang
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Xiao-Tong Lin
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Xiao-Yu Liu
    Affiliations
    School of Medicine, Southern University of Science and Technology, Shenzhen, China
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  • Di Wu
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Gui-Xi Li
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Deng Huang
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Yu-Jun Zhang
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Shu Chen
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Yan Jiang
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Ling Shuai
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Yu He
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Lei-Da Zhang
    Correspondence
    Corresponding authors. Address: Prof. Chuan-Ming Xie (Lead contact), Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China 400038, China. Tel.: +86-23-68765809, fax: +86-23-68765809
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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  • Ping Bie
    Correspondence
    Corresponding authors. Address: Prof. Chuan-Ming Xie (Lead contact), Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China 400038, China. Tel.: +86-23-68765809, fax: +86-23-68765809
    Affiliations
    Department of Hepatobiliary and Pancreatic Surgery, The Third Affiliated Hospital of Chongqing Medical University (General Hospital), Chongqing, China
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  • Chuan-Ming Xie
    Correspondence
    Corresponding authors. Address: Prof. Chuan-Ming Xie (Lead contact), Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China 400038, China. Tel.: +86-23-68765809, fax: +86-23-68765809
    Affiliations
    Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China
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Open AccessPublished:July 29, 2020DOI:https://doi.org/10.1016/j.jhep.2020.07.036

      Highlights

      • Tsc1 deficiency facilitates p53 (haplo)insufficiency-mediated activation of the PTEN/Akt/mTOR axis to drive HCC tumorigenesis and metastasis.
      • Inhibiting mTOR activation is a potential therapeutic strategy for p53 insufficiency and Tsc1 insufficiency-driven hepatocarcinogenesis.
      • The oncogenic activity of the Akt/mTOR axis relies on Abcc4, which labels an aggressive subtype of human HCC.

      Background & Aims

      p53 mutations occur frequently in human HCC. Activation of the mammalian target of rapamycin (mTOR) pathway is also associated with HCC. However, it is still unknown whether these changes together initiate HCC and can be targeted as a potential therapeutic strategy.

      Methods

      We generated mouse models in which mTOR was hyperactivated by loss of tuberous sclerosis complex 1 (Tsc1) with or without p53 haplodeficiency. Primary cells were isolated from mouse livers. Oncogenic signalling was assessed in vitro and in vivo, with or without targeted inhibition of a single molecule or multiple molecules. Transcriptional profiling was used to identify biomarkers predictive of HCC. Human HCC materials were used to corroborate the findings from mouse models.

      Results

      p53 haploinsufficiency facilitates mTOR signalling via the PTEN/PI3K/Akt axis, promoting HCC tumorigenesis and lung metastasis. Inhibition of PI3K/Akt reduced mTOR activity, which effectively enhanced the anticancer effort of an mTOR inhibitor. ATP-binding cassette subfamily C member 4 (Abcc4) was found to be responsible for p53 haploinsufficiency- and Tsc1 loss-driven HCC tumorigenesis. Moreover, in clinical HCC samples, Abcc4 was specifically identified an aggressive subtype. The mTOR inhibitor rapamycin significantly reduced hepatocarcinogenesis triggered by Tsc1 loss and p53 haploinsufficiency in vivo, as well as the biomarker Abcc4.

      Conclusions

      Our data advance the current understanding of the activation of the PTEN/PI3K/Akt/mTOR axis and its downstream target Abcc4 in hepatocarcinogenesis driven by p53 reduction and Tsc1 loss. Targeting mTOR, an unexpected vulnerability in p53 (haplo)deficiency HCC, can be exploited therapeutically to treat Abcc4-positive patients with HCC.

      Lay summary

      Tsc1 loss facilitates the p53 (haplo)insufficiency-mediated activation of the PTEN/Akt/mTOR axis, leading to the elevated expression of Abcc4 to drive HCC tumorigenesis and metastasis in mice. Inhibition of mTOR protects against p53 haploinsufficiency and Tsc1 loss-triggered tumour-promoting activity, providing a new approach for treating an aggressive subtype of HCC exhibiting high Abcc4 expression.

      Graphical abstract

      Keywords

      Introduction

      Despite remarkable progress in understanding the underlying molecular biology, HCC remains a fatal and treatment-refractory disease. The Cancer Genome Atlas (TCGA) database reported 13,657 mutations in 373 HCC samples with associated clinical outcomes. Among these mutations, approximately 50% involve abnormal upregulation of mammalian target of rapamycin (mTOR) expression.
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      The mTOR pathway in hepatic malignancies.
      mTOR forms 2 distinct signalling complexes, called mTORC1 and mTORC2. The mTORC1 complex is responsible for the control of cell growth and protein synthesis.
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      The mTORC2 complex controls the actin cytoskeleton and cell spreading.
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      Rac1 regulates the activity of mTORC1 and mTORC2 and controls cellular size.
      mTORC1 activity is positively regulated mainly by growth factors through the insulin/insulin-like growth factor 1-phosphoinositide-3 kinase (PI3K)-Akt pathway, Wnt-GSK3 pathway, and extracellular signal-regulated kinase/ribosomal S6 kinase cascade.
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      Tuberous sclerosis complex (Tsc) is a vital tumour suppressor of mTOR and is composed of Tsc1 and Tsc2,
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      which are also mutated in approximately 15% of patients with HCC.
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      Loss or impaired activity of Tsc decreases mTOR inhibition, leading to activation (also in HCC).
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      A decrease in the activity of mTOR, which is one of the master switches for proliferation, is therefore thought to potentially have a therapeutic effect on HCC. However, inhibiting mTOR does not achieve a desirable effect on some HCC subgroups.
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      In contrast, p53 plays critical roles in the induction of growth arrest and cell death.
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      The loss of functional p53 is a prerequisite for oncogenesis to promote growth and proliferation and is the most common anomaly in human cancers, including HCC.
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      Various stress signals stabilise and activate p53, which exerts a tumour-suppressive function.
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      Mutations in the p53 gene are found in 50% of human tumours,
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      highlighting the importance of p53 in tumour suppression. Many p53 target genes that regulate various processes involved in the prevention of tumorigenesis, such as the induction of cell cycle arrest, apoptosis, DNA repair, and senescence, have been identified.
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      • et al.
      Tumor suppression in the absence of p53-mediated cell-cycle arrest, apoptosis, and senescence.
      p53 mutations are considered to be major drivers of various cancers, including HCC.
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      • Bianchi J.
      • Wiman K.G.
      Targeting mutant p53 for efficient cancer therapy.
      ,
      • Zheng Y.
      • Lv P.
      • Wang S.
      • Cai Q.
      • Zhang B.
      • Huo F.
      LncRNA PLAC2 upregulates p53 to induce hepatocellular carcinoma cell apoptosis.
      The TCGA database indicates that p53 is uniformly mutated in 28.49% HCC. However, it is unclear whether p53 mutation can promote hyperactivated mTOR-driven HCC, and how to treat the subset of patients with HCC with mTOR activation and p53 mutations is also unresolved.
      We recently discovered that compared with Tsc1 loss or p53 haplodeficiency alone, concurrent Tsc1 loss and p53 haplodeficiency in mice led to a significant development of HCC. We then analysed the underlying mechanism in primary cancer cells and an in vivo transgenic mouse model and demonstrated that the loss of p53 further hyperactivated mTOR via the PTEN/Akt pathway in response to Tsc1 insufficiency. Furthermore, the primary cells and in vivo mouse model demonstrated that HCC cells with Tsc1 loss and p53 haplodeficiency were sensitive to treatment with the mTOR inhibitor rapamycin and sapanisertib. This activity could be further enhanced by dual inhibition of PI3K and mTOR. These observations suggest that mTOR inhibitors may have therapeutic benefits in HCC cases with p53 mutations (reduction or inactivation of p53) and Tsc1 loss (mTOR activation). In addition, ATP-binding cassette subfamily C member 4 (Abcc4) was identified as an oncogenic biomarker of hepatocarcinogenesis driven by the loss of both p53 and Tsc1.

      Materials and methods

      Patient data and tissue collection

      There were 106 cases of patients with HCC with clinical characteristics obtained in our hospital and 344 cases of patients with HCC with clinical characteristics obtained from the TCGA database. All HCC specimens determined by special pathologist must meet the following criteria. (1) Primary HCC only, cholangiocellular carcinoma, or mixed liver cancer was excluded. (2) Primary HCC without other primary cancers was a requirement. (3) Patients with primary HCC who died from other causes were excluded. (4) Patients from TCGA database without Abcc4 or p53 data were excluded. (5) Patients with key or many clinical data missing were excluded. (6) Patients with primary HCC who were unwilling to attend appointments for this research were excluded. The use of tissues for this research was approved by the local Ethics Committee, and written informed consent was obtained from the patients.

      Mouse lines, breeding, and experiments

      Only male mice were used in this study. Alb-Cre (016833) mice express liver-specific Cre recombinase. p53mut/+ (002103) mice containing a p53 mutant allele were produced by a targeted neo insertion into the p53 locus and replacement of exons 2–6 (including the start codon). Tsc1fl/fl (005680) mice contain the floxed Tsc1 alleles. All mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Alb-cre and p53mut/+ mice were bred separately with Tsc1fl/fl mice to generate Tsc1fl/+;Alb-Cre and p53mut/+;Tsc1fl/+ mice, respectively. These 2 mice were then crossed to generate the desired genotypes Tsc1 knockout mice p53mut/+;Tsc1fl/fl;Alb-Cre and Tsc1fl/fl;Alb-Cre and validated by PCR using genomic DNA extracted from mouse tails and liver tissues (Fig. S1A–D). Wild-type C57BL/6 mice were purchased from the Chinese Academy of Medical Sciences (Beijing, China). Mouse breeding was performed at the specific-pathogen-free mouse facility at the Animal Center of Army Medical University. All mouse experiments were approved by the Institutional Animal Care and Use Committees of Army Medical University. HCC induction, evaluation, and rapamycin or 5-bromo-2′-deoxyuridine (BrdU) injection procedures are listed in the Supplementary Materials.

      Statistical analysis

      Either GraphPad Prism 8.0 software (GraphPad, San Diego, CA, USA) or IBM SPSS 24.0 software (Statistical Package for the Social Sciences; IBM, New York, NY, USA) was applied for statistical analysis. An unpaired Student's t test was performed to compare 2 groups. Comparisons between more than 2 groups were performed using ANOVA with Tukey's or Bonferroni's multiple comparisons test. The Pearson χ2 test was used to compare the distributions of categorical factors between groups. Survival curves were calculated using the Kaplan-Meier method and examined by the log-rank test. The Cox proportional hazards regression model was used to analyse the associations between clinicopathological variables and Abcc4 expression related to survival. Hazard ratios and 95% CIs were evaluated. Logistic regression was used to analyse the relationships between different factors. All experiments were repeated at least 3 times. Sample size for animal experiments was determined using power calculations. Many statistically significant effects were observed in the data, suggesting that the effective sample size was sufficient for studies. A p value of ≤0.05 was considered statistically significant (∗p ≤0.05; ∗∗p ≤0.01; and ∗∗∗p ≤0.001). The results are expressed as the mean ± SEM unless indicated otherwise.

      Further materials and methods

      A detailed Methods and Materials section is provided in the Supplementary Materials of this manuscript.

      Results

      p53 (haplo)insufficiency facilitates mTOR signalling, promoting HCC tumorigenesis

      It is well known that the loss of Tsc1, the negative regulator of mTOR, results in the stimulation of p53 translation and apoptosis in response to stress, whereas the loss of p53 promotes cell proliferation and growth.
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      Heterozygous loss of TSC2 alters p53 signaling and human stem cell reprogramming.
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      A subset of metastatic pancreatic ductal adenocarcinomas depends quantitatively on oncogenic Kras/Mek/Erk-induced hyperactive mTOR signalling.
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      p53-mediated PI3K/AKT/mTOR pathway played a role in Ptox(Dpt)-induced EMT inhibition in liver cancer cell lines.
      However, the role of p53 loss in Tsc1 deficiency-driven hepatocarcinogenesis is still unclear. Therefore, we investigated whether p53 (haplo)insufficiency could facilitate mTOR signalling, promoting the development of HCC tumorigenesis. p53 haploinsufficient mice (mice with p53 haploinsufficiency are herein referred to as p53mut/+ or P mice) were designed. Knockout of the well-described tumour suppressor gene Tsc1 (Tsc1fl/fl) specifically in the liver was achieved under the control of the Alb promoter, and these mice were used as the base HCC model. We thus generated transgenic p53mut/+;Tsc1fl/fl;Alb-Cre mice (herein referred to as PTC mice). Previous studies showed that liver-specific p53 knockout mice developed liver tumours only after 14 months.
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      CRISPR/Cas9-mediated p53 and Pten dual mutation accelerates hepatocarcinogenesis in adult hepatitis B virus transgenic mice.
      Consistent with this finding, p53 haploinsufficiency mice only induced inflammation, which did not lead to the development of HCC after 320 days of monitoring (0/12) (Fig. 1A–D). In contrast, Tsc1fl/fl;Alb-Cre mice (herein referred to as TC mice) had a moderate tumour incidence rate (66.67%, 10/15), and PTC mice had a high tumour incidence rate (92.86%, 13/14, p <0.0001; Fig. 1C). The images show that the p53mut/+ mice developed no neoplasms. In contrast, compared with TC mice, PTC mice developed many more tumours, the largest tumours, and a high liver:body weight ratio (Fig. 1A, B). Consistent with those findings, N-nitrosodiethylamine (DEN)/carbon tetrachloride (CCl4)-induced HCC tumour numbers, the largest tumour size, and the liver:body weight ratio were higher in PTC than in P or TC mice (Fig. S2A–C). For further support of this observation, the proliferation of hepatocytes was determined by BrdU incorporation. Clearly, hepatocyte proliferation increased significantly in PTC mice compared with P and TC mice (Fig. S2D). In the P inflammation/tumour tissues, TC tumours, and PTC tumours, the haploinsufficiency status of p53 tumour suppressor and Tsc1 loss were detected by PCR and Western blotting (Figs. 1E, S1D, and S2E). To further investigate the role of p53 haploinsufficiency in Tsc1/mTOR-driven hepatocarcinogenesis, primary cells were isolated from 320-day-old P, TC, and PTC mice and used for cell proliferation evaluation. The results demonstrated that the PTC cells grew faster than the P or TC cells, which was similar to the observations in mouse models (Fig. 1F). In line with those findings, the levels of the proliferation markers Ki-67, Pcna, Ccnb1, and Ccnb2
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      were significantly increased at the mRNA levels in PTC tissue samples compared with P or TC tissue samples (Figs. 1G and S2F). To further validate those markers with high expression in liver tumours, Ccnb1 and Ccnb2 were detected by Western blotting. In addition, Ccnb2 and lipase C, a liver-specific protein,
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      were examined by immunohistochemistry (IHC). The results demonstrated that the cell cycle-related proteins Ccnb1 and Ccnb2 were significantly accumulated in PTC tumours compared with P or TC tissue samples (Figs. 1H and S2G). Furthermore, the fibrogenetic markers Col1a1, Col1a2, Acta2, Timp1, Pdgfr-β, and Pdgf-β
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      also showed significantly increased expression in PTC compared with P or TC tissues, which was supported by the strong Sirius Red staining signals in PTC, indicating high fibrosis in PTC mice (Figs. 1I and S2I). Taken together, these results indicated that p53 (haplo)insufficiency promoted mTOR-mediated HCC tumorigenesis.
      Figure thumbnail gr1
      Fig. 1p53 (haplo)insufficiency facilitates mTOR signalling, promoting the development of HCC tumorigenesis.
      (A) Representative tumorigenesis images of 320-day-old p53mut/+ mice (P), Tsc1fl/fl; Alb-Cre mice (TC), and p53mut/+; Tsc1fl/fl; Alb-Cre mice (PTC). (B) Quantification of the tumour number, largest tumour size, and liver:body weight ratio in P (n = 12), TC (n = 15), and PTC mice (n = 14). (C) Elevated tumour incidence rate in PTC mice (13/14) compared with P mice (0/12) and TC mice (10/15) (χ2 test, p <0.0001). (D) Representative H&E stain sections from P inflammation tissues, TC tumours, and PTC tumours. Images were obtained at 4× or 40× magnification; scale bar: 25 or 250 μm. Black box: The region shows high magnification. (E) Western blotting showing the reduction of p53 expression in liver of P and PTC mice, and loss of Tsc1 in liver of TC and PTC mice. (F) Representative proliferation curves for primary liver cells isolated from the whole liver of 320-day-old P, TC, and PTC mice. (G) qPCR showing elevated transcript levels for the proliferation markers Ki-67, Pcna, Ccnb1, and Ccnb2 in PTC mice. (H) The protein levels of Ccnb1 and Ccnb2 in P inflammation tissues, TC tumours, and PTC tumours were analysed by Western blotting. (I) qPCR showing elevated transcript levels for the fibrogenic markers Col1a1, Col1a2, Acta2, Timp1, Pdgfr-β, and Pdgf-β in PTC mice. Data are represented as the mean ± SEM. One-way ANOVA with Tukey's multiple comparisons test was used in (B), (F), (G), and (I). ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. mTOR, mammalian target of rapamycin; PTC, p53mut/+;Tsc1fl/fl;Alb-Cre; qPCR, quantitative PCR; Tsc1, tuberous sclerosis complex 1.

      p53 (haplo)insufficiency facilitates mTOR signalling, promoting lung metastasis in HCC

      Lung metastasis is the main reason for cancer-related deaths in HCC.
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      The p53 gene is frequently deleted or mutated in HCC, and the loss of p53 function is closely related to tumour invasion.
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      Down-regulation of annexin A10 in hepatocellular carcinoma is associated with vascular invasion, early recurrence, and poor prognosis in synergy with p53 mutation.
      Thus, we compared lung metastases in our mouse models. We found the PTC mice had a higher incidence of lung metastases compared with TC mice, as indicated by the percentage of cases with metastasis and the number of observed metastases for each genotype (Fig. 2A–C). This finding was supported by high expression of the vascular invasion markers Icam1, Mmp9, Ccl2, and Vcam1 in PTC compared with P or TC mice
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      (Fig. 2D). To further validate lung metastasis, haematoxylin and eosin stain and IHC staining for lipase C were performed on lung tissue samples.
      • Feng D.
      • Huang Q.Y.
      • Liu K.
      • Zhang S.C.
      • Liu Z.H.
      Comparative studies of zebrafish Danio rerio lipoprotein lipase (lpl) and hepatic lipase (lipc) genes belonging to the lipase gene family: evolution and expression pattern.
      The results demonstrated that p53 haploinsufficiency dramatically promoted Tsc1 loss-driven HCC lung metastasis (Fig. 2E).
      Figure thumbnail gr2
      Fig. 2p53 (haplo)insufficiency facilitates mTOR signalling, promoting lung metastasis.
      (A) Macroscopic image of lung metastases was observed in PTC mice. (B) The lung metastasis rate was higher in PTC (10/13) than in P (0/12) or TC mice (3/10) (χ2 test, p = 0.024). (C) The number of observed metastases for each genotype was quantified. (D) qPCR showing the elevated expression of the vascular invasion markers Icam1, Mmp9, Ccl2, and Vcam1 in PTC mice. (E) Representative H&E and IHC staining images for lipase C showing distinct lung metastatic foci expressing lipase C. Images were obtained at 10× or 40× magnification; scale bar: 100 or 25 μm. Data are represented as the mean ± SEM. One-way ANOVA with Tukey's multiple comparisons test was used in (C) and (D). ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. IHC, immunohistochemistry; mTOR, mammalian target of rapamycin; PTC, p53mut/+;Tsc1fl/fl;Alb-Cre.

      p53 (haplo)insufficiency activates the PTEN/PI3K/Akt axis, enhancing mTOR signalling

      p53 dysfunction has been proposed to induce HCC through Akt and Erk,
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      p53-mediated PI3K/AKT/mTOR pathway played a role in Ptox(Dpt)-induced EMT inhibition in liver cancer cell lines.
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      2 kinases that are major upstream regulators of the Tsc/mTOR cascade, which receives signals from growth factors.
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      • Sabatini D.M.
      mTOR signaling in growth control and disease.
      All PTC and TC mice developed macroscopically visible liver tumours, whereas p53mut/+ mice developed only inflammation or fibrosis. These lesions were analysed by IHC, which demonstrated that p-AktSer473 (7/9, 77.78%), p-Erk1/2Thr202/Tyr204 (2/9, 22.22%), and p-mTORSer2448 (p-4EBP1Thr37/46 and p-S6Ser235/236, 9/9, 100%) levels were significantly upregulated in PTC mice compared with TC mice (n = 9), suggesting that the Akt-mTOR axis but not the Erk/mTOR axis plays a critical role in p53 haploinsufficiency-mediated HCC tumorigenesis in response to Tsc1 deficiency (Fig. S3A, B). Consistent with these findings, in the DEN/CCl4-induced liver cancer mouse model, phosphorylation of Akt at Ser473 but not Erk1/2 at Thr202/Tyr204 was also significantly increased in PTC mice (n = 6) compared with TC mice (n = 8), which is in line with the activation of Akt-mTOR axis by p53 haploinsufficiency (Fig. 3A). Furthermore, we found that the Akt-mTOR pathway in those lung metastases still had high activity and were not influenced by microenvironmental factors in the lung (Fig. S3C).
      Figure thumbnail gr3
      Fig. 3p53 (haplo)insufficiency activates the PTEN/PI3K/Akt axis, enhancing oncogenic Tsc1/mTOR signalling.
      (A) Representative consecutive IHC images of Akt/mTOR and Erk/mTOR signals are shown in P tumours, TC tumours, and PTC tumours. Mice were treated with DEN followed by 14 injections of CCl4. Cells positive for p-AktSer473, p-Erk1/2Thr202/Tyr204, p-mTORSer2448, p-4EBP1Thr37/46, or p-S6Ser235/236 signals were counted among a total of 500 cells on average from 3 independent tumours derived from 3 mice per group. Images were obtained at 10× or 40× magnification; scale bar: 100 or 25 μm. (B) Western blotting analysis demonstrated the phosphorylation levels of p-AktSer473, p-Erk1/2Thr202/Tyr204, p-mTORSer2448, p-S6Ser235/236, and p-4EBP1Thr37/46 in P mice (#27, 28, and 109), TC mice (#82, 87, and 89), and PTC mice (#47, 88, and 84). The intensity of each band was quantified using ImageJ and normalised to β-Actin. Data were expressed as relative changes. (C) Western blotting analysis showing the phosphorylation levels of p-AktSer473, p-Erk1/2Thr202/Tyr204, p-mTORSer2448, p-S6Ser235/236, and p-4EBP1Thr37/46 in 2 different PTC primary cell lines (#1017 and 1018) after pharmacological inhibition of Mek (PD98059, 20 μM), PI3K (GDC-0326, 1 μM), mTOR (rapamycin, 100 nM), PI3K + mTOR, or Mek + mTOR for 48 h. The intensity of each band was quantified using ImageJ and normalised the β-Actin. (D) Western blotting analysis showing the phosphorylation levels of PTEN, p-AktSer473, p-Erk1/2Thr202/Tyr204, p-mTORSer2448, p-S6Ser235/236, and p-4EBP1Thr37/46 in p53+/+ (#64, 65, and 66) and p53mut/+ mice (#16, 17, and 18). The intensity of each band was quantified using ImageJ and normalised to β-Actin. (E) Proliferation curves for 2 different PTC primary cell lines (#1017 and 1018) were generated after pharmacological inhibition of PI3K (GDC-0326, 1 μM), mTOR (rapamycin, 100 nM), Mek (PD98059, 20 μM), PI3K + mTOR, or Mek + mTOR for 24 or 48 h. Data are represented as the mean ± SEM. One-way ANOVA with Tukey's multiple comparisons test was used in (A) and (E). ∗∗p <0.01; ∗∗∗p <0.001. CCl4, carbon tetrachloride; DEN, N-nitrosodiethylamine; IHC, immunohistochemistry; mTOR, mammalian target of rapamycin; n.s., not significant; PI3K, phosphoinositide-3 kinase; PTC, p53mut/+;Tsc1fl/fl;Alb-Cre; Tsc1, tuberous sclerosis complex 1.
      To further validate whether Akt or Erk plays a critical role in the activation of mTOR signalling, primary cells were generated from PTC tumours (#47, 88, and 84), TC tumours (#82, 87, and 89), and P inflammation tissues (#27, 28, and 109). As shown in Fig. 3B, p53 reduction in Tsc1-deficient cells compared with Tsc1-deficient cells further activated Akt, Erk, and mTOR. To further substantiate these activation patterns, we investigated the influence of pharmacologically inhibiting Mek (using PD98059), PI3K (using GDC-0326), and/or mTOR (using rapamycin) signalling in 2 different PTC cell lines (#1017 cells and 1018 cells). Single inhibition of PI3K/Akt could significantly reduce the activation of mTOR and its downstream targets p-4EBP1Thr37/46 and p-S6Ser235/236, whereas single inhibition of Erk did not change the p-mTORSer2448 level, suggesting that p53 loss promoted mTOR activation via Akt signalling but not Erk signalling in HCC tumorigenesis (Fig. 3C). In line with these findings, dual inhibition of PI3K and mTOR strongly significantly reduced p-S6Ser235/236 and p-4EBP1Thr37/46 levels (Fig. 3C).
      p53 is a well-known transcription factor for PTEN, Tsc2, Phlda3, Ampkb1, and Igfbp3 that control PI3K/Akt pathway, among which PTEN is more sensitive to downregulation by p53 haploinsufficiency in liver tissues (Fig. S4). PTEN negatively regulates Akt activation.
      • Stambolic V.
      • MacPherson D.
      • Sas D.
      • Lin Y.
      • Snow B.
      • Jang Y.
      • et al.
      Regulation of PTEN transcription by p53.
      Here, we confirmed that p53 haploinsufficiency activated Akt by downregulating PTEN expression at both the mRNA and protein levels (Figs. 3D and S4). In addition, reduction of p53 increased p-Erk1/2Thr202/Tyr204 and p-mTORSer2448 levels as well as p-4EBP1Thr37/46 and p-S6Ser235/236 (Fig. 3D). To further demonstrate that p53 (haplo)insufficiency facilitates mTOR signalling via PTEN/PI3K/Akt, cell proliferation was analysed in PTC cells in the presence or absence of the indicated inhibitors. Consistent with the effects of the inhibitors on mTOR activity observed in PTC cells, inhibition of Mek/Erk weakly decreased cell proliferation, whereas inhibition of PI3K/Akt dramatically inhibited cell proliferation. Furthermore, dual inhibition of PI3K and mTOR completely blocked cell proliferation, suggesting that PI3K/Akt but not Mek/Erk is involved in hepatocarcinogenesis driven by the loss of p53 and Tsc1 (Fig. 3E).

      Abcc4 is a biomarker for PI3K/Akt/mTOR-driven hepatocarcinogenesis

      We compared transcriptional profiles among a panel of liver tissue samples from P, TC, and PTC mice. The overlapping genes in those 3 genotypes were considered potential biomarkers for patients with HCC with functional loss of both p53 and Tsc1.
      Using fragments per kilobase of transcript per million mapped reads >1, log2(read count) >1, log2(fold change) >1.5, and a statistical cut-off for the false discovery rate of <0.05, 507 genes were found to be differentially expressed between P and PTC mice, and 95 genes were found between TC and PTC mice, among which there were 19 overlapping genes (Fig. 4A, B). To explore the functional relevance of these genes in HCC, we analysed the poor survival of patients with HCC using the TCGA database. Six genes (Abcc4, Entpd2, Ubqln2, Cul7, Glg1, and Igf1) were found to be significantly associated with poor survival in patients with HCC. Furthermore, quantitative real-time PCR was used to analyse liver expression levels of those genes in wild-type P, TC, and PTC mice (Fig. 4C). The results were consistent with the RNAseq data and demonstrated that Abcc4 and Entpd2 exhibited much more dramatically elevated expression than other genes in PTC mice (PI3K/Akt-mTOR signature) compared with TC mice (mTOR signature) (Fig. 4B, C). In line with this finding, in the DEN/CCl4-induced HCC mouse model, we found that compared with other selected genes, Abcc4 and Entpd2 again exhibited dramatically increased expression, but only Abcc4 expression was significantly increased in p53 haplodeficiency mice (PI3K/Akt signature) compared with wild-type mice, suggesting that Abcc4 was associated with PTC mice (PI3K/Akt-mTOR signature) (Fig. S5A). In vivo validation of these molecular signatures was carried out using IHC studies for Abcc4 and 3 other proteins, Entpd2, Ubqln2, and Cul7, which exhibited substantial changes in the top 19 genes between PTC mice (PI3K/Akt-mTOR signature) and TC (mTOR signature) or P (PI3K/Akt signature) mice. In agreement with the RNAseq results, compared with those from P or TC mice, Abcc4, Entpd2, Ubqln2, and Cul7 in cancer cells from PTC mice showed positively increased expression, in which Abcc4 expression was stronger than Entpd2, Ubqln2, or Cul7 expression, implying that Abcc4 is associated with PI3K/Akt/mTOR-driven hepatocarcinogenesis (Figs. 4D and S5B).
      Figure thumbnail gr4
      Fig. 4Abcc4 is a biomarker for PI3K/Akt/mTOR-driven hepatocarcinogenesis in response to p53 insufficiency.
      (A) Venn diagram showing significantly changed genes at the mRNA level in liver tissue samples between P and PTC mice and between TC and PTC mice. (B) Heatmap illustrating the top 19 gene signatures representing different groups. (C) qPCR showing the relative expression of Abcc4, Entpd2, Ubqln2, Cul7, Glg1, and Igf1 in different groups. (D) IHC demonstrating the expression of Abcc4, Entpd2, Ubqln2, and Cul7 in different groups. Cells positive for Abcc4, Entpd2, Ubqln2, and Cul7 signals were counted among a total of 500 cells on average from 3 independent tumours derived from 3 mice per group. Images were obtained at 40× magnification; scale bar: 25 μm. Data are represented as the mean ± SEM. One-way ANOVA with Tukey's multiple comparisons test was used in (C) and (D). ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. Abcc4, ATP-binding cassette subfamily C member 4; IHC, immunohistochemistry; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide-3 kinase; PTC, p53mut/+;Tsc1fl/fl;Alb-Cre; qPCR, quantitative PCR.

      Abcc4 labels an aggressive subset of human p53 mutated HCC

      Since Abcc4 is a biomarker of PI3K/Akt/mTOR-driven hepatocarcinogenesis in response to p53 deficiency, which promotes tumour growth and metastasis in vivo, we attempted to determine whether this biomarker was also relevant in humans. We downloaded 344 cases of HCC from the TCGA database, of which 98 cases were p53mutant and 246 were p53wild type. An unpaired Student's t test showed that patients with HCC with p53mutant phenotype had high Abcc4 expression (unpaired Student's t test, p = 0.0004) compared with p53 wild type (Fig. 5A). Consistent with this finding, the expression of Abcc4 was also increased in the liver of p53mut/+ compared with p53+/+ mice (Fig. S6A). Logistic regression analysis indicated that the expression of Abcc4 was positively correlated with p53 mutation (regression coefficient 0.167, odds ratio 1.182, p = 0.002; Table S1). Furthermore, Kaplan-Meier analysis demonstrated that high expression of Abcc4 was positively associated with poor survival in patients with HCC with mutated p53 (the cut-off value for Abcc4 was set as 1.6 by the median value of the p53 mutant patient cohort; Figs. 5B and S6B), whereas no significant association was found for p53 wild-type patients with HCC (the cut-off value for Abcc4 was set as 0.8 by the median value of the p53 wild-type patient cohort; Figs. 5B and S6C), indicating that high Abcc4 expression has poor survival, especially in an aggressive subtype of HCC with mutated p53.
      Figure thumbnail gr5
      Fig. 5Abcc4 labels an aggressive subset of human p53 mutated HCC.
      (A) HCC sample data were obtained from the TCGA database. Abcc4 expression levels were higher in p53mutant (n = 98) than in p53wild-type patients (n = 246) (unpaired Student's t test, p = 0.0004). (B) Kaplan-Meier analysis of the overall survival (OS) of p53-mutant patients with HCC with high (n = 47) or low Abcc4 expression (n = 51), and p53 wild-type patients with HCC with high (n = 122) or low Abcc4 expression (n = 124). (C) Representative images showing Abcc4 expression in primary HCC and adjacent non-cancerous liver tissue samples. (D) Kaplan-Meier analysis of the OS of patients with high (n = 65) or low Abcc4 (n = 41) expression. (E) Scatter plots of the largest tumour size in patients with high or low Abcc4 expression (unpaired Student's t test, p = 0.019). (F–H) Distribution of Abcc4 according to (F) tumour differentiation, (G) TNM stage, or (H) vascular thrombosis analysed by IHC. (I and J) HCC cell line Huh7 and PTC primary cells were transfected with a non-targeting siRNA control (siCont) or siRNA targeting Abcc4 (si-Abcc4) for different time intervals. (I) The efficiency of si-Abcc4 was analysed by Western blotting. (J) Cell proliferation was analysed by a CCK-8 assay. (K) Huh7 cells transfected with siCont or si-Abcc4 for 24 h and analysed by the transwell assay. Images were obtained at 4× magnification; scale bar: 250 μm. Data are represented as the mean ± SEM. One-way ANOVA with Tukey's multiple comparisons test was used in (J) and (K). ∗p <0.05; ∗∗∗p <0.001. Abcc4, ATP-binding cassette subfamily C member 4; CCK-8, Cell Counting Kit-8; FPKM, fragments per kilobase of transcript per million mapped reads; OS, overall survival; PTC, p53mut/+;Tsc1fl/fl;Alb-Cre; siRNA, small interfering RNA; TCGA, The Cancer Genome Atlas.
      Next, we examined Abcc4 protein expression by IHC in a cohort of 106 paired human HCC and adjacent non-cancerous liver tissue samples obtained from Southwest Hospital, Chongqing, China. We found that Abcc4 expression was increased in 61.32% (65/106) of the HCC samples compared with adjacent normal tissues (Figs. 5C and S6D). Kaplan-Meier analysis demonstrated that increased Abcc4 expression was positively correlated with poor survival in patients with HCC (p = 0.0008; Fig. 5D). We next examined the relationships between Abcc4 protein expression and clinicopathological parameters. We found that Abcc4 expression was positively associated with the largest tumour size (unpaired Student's t test, p = 0.019), tumour differentiation, TNM stage, and vascular thrombosis (Fig. 5E–H, Table 1), indicating that Abcc4 expression correlated with HCC disease progression. To extend this work, we analysed the associations between overall survival and various risk factors in 106 HCC tissue samples. Univariate and multivariate analyses demonstrated that Abcc4 was an independent risk factor for poor survival in patients with HCC that could be used prognostically. The results showed that Abcc4 and vascular thrombosis status were strongly associated with overall survival in patients with HCC, suggesting that Abcc4 expression is an independent predictor of poor overall survival in patients with HCC (overall survival: hazard ratio 1.887; 95% CI 1.23–3.466; p = 0.006; Table S3).
      Table 1Relationships between Abcc4 and clinicopathologic characteristics in patients with HCC.
      CharacteristicsPatients

      n (%)
      Abcc4 expression levelp value
      LowHigh
      Sex
       Male96 (90.6)38580.554
       Female10 (9.4)37
      Age (years)
       ≤6082 (77.4)31510.733
       >6024 (22.6)1014
      TNM stage
       I32 (30.2)20120.001
      p ≤0.05 was considered statistically significant.
       II10 (9.4)46
       III47 (44.3)1631
       IV17 (16.1)116
      Lymph node metastasis
       Negative101 (95.3)41600.069
       Positive5 (4.7)05
      Tumour size (cm)
       <518 (17)1170.032
      p ≤0.05 was considered statistically significant.
       ≥588 (83)3058
      Pathological grading
       Well7 (6.6)520.011
      p ≤0.05 was considered statistically significant.
       Moderate71 (67)3140
       Poor28 (26.4)523
      Vascular thrombosis
       Negative70 (66)32380.038
      p ≤0.05 was considered statistically significant.
       Positive36 (34)927
      Recurrence
       Negative27 (25.5)13140.242
       Positive79 (74.5)2851
      Intrahepatic metastasis
       Negative71 (67)31400.134
       Positive35 (33)1025
      Abcc4, ATP-binding cassette subfamily C member 4.
      Statistical analyses were carried out using the Pearson χ2 test.
      p ≤0.05 was considered statistically significant.
      To further validate the function of Abcc4 in HCC, we examined cell proliferation and migration in 2 different HCC cell lines, including the p53 mutated HCC cell line Huh7 and primary mouse PTC cells. Silencing of Abcc4 significantly reduced Abcc4 protein expression and inhibited cell proliferation (Fig. 5I, J). The transwell assay indicated that silencing of Abcc4 dramatically inhibited cell migration in Huh7 cells (Fig. 5K).

      mTOR inhibition significantly blocks hepatocarcinogenesis triggered by p53 (haplo)insufficiency and Tsc1 insufficiency

      Considering that mTOR activation is involved in p53 loss, we investigated the therapeutic effect of the mTOR inhibitor rapamycin on hepatocarcinogenesis triggered by p53 (haplo)insufficiency and Tsc1 insufficiency. PTC mice (n = 14) and TC mice (n = 12) received i.p. injections of a vehicle or rapamycin every other day for 6 weeks beginning 8 weeks after injection of DEN/CCl4. As shown in Fig. 6A and B, rapamycin significantly reduced the tumour number and largest tumour size in both the PTC and TC mice compared with their counterparts in the vehicle treatment groups. In addition, we found that PTC mice developed many more tumours and the largest tumours compared with TC mice, whereas no significant difference was observed between these 2 groups after rapamycin treatment, indicating a greater susceptibility of PTC than TC mice to the mTOR inhibitor rapamycin. To confirm the inactivation of mTOR after rapamycin treatment, staining for phosphorylated proteins was performed. The PTC and TC tumours were p-mTORSer2448-, p-S6Ser235/236-, and p-4EBP1Thr37/46-positive in the vehicle treatment groups, while these signals were dramatically reduced in the rapamycin treatment groups (Fig. 6C). In line with this finding, the Abcc4 signal was significantly reduced in the rapamycin treatment group compared with the vehicle group in both PTC and TC tumours (Fig. 6C). To extend this work, the effect of rapamycin on 2 distinct primary liver cancer cell lines isolated from different tumours was examined. Cell proliferation was faster in PTC than in TC cells, both of which were susceptible to the anticancer effect of rapamycin treatment (Fig. 6D, E). To further validate this finding, PTC primary cells and Huh7 cells were treated with rapamycin or sapanisertib (a new selective inhibitor of mTORC1/2).
      • Moore K.N.
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      Phase I study of the investigational oral mTORC1/2 inhibitor sapanisertib (TAK-228): tolerability and food effects of a milled formulation in patients with advanced solid tumours.
      The data showed that the expression of Abcc4 was significantly reduced by rapamycin and sapanisertib, leading to inhibition of cell proliferation and indicating that Abcc4 is the downstream target of mTOR (Figs. 6F and S7A, B). Taken together, these results indicate that inhibition of mTOR activation represents a potential therapeutic strategy for p53 (haplo)insufficiency and Tsc1 loss-driven hepatocarcinogenesis.
      Figure thumbnail gr6
      Fig. 6mTOR inhibitor significantly blocks hepatocarcinogenesis triggered by p53 (haplo)insufficiency and Tsc1 insufficiency.
      (A) Representative tumorigenesis images of TC and PTC mice that received or did not receive rapamycin (4 mg/kg. i.p.) treatment are shown. (B) Tumour number and largest tumour size were analysed in TC and PTC mice after vehicle or rapamycin treatment. (C) Representative consecutive IHC images of p-mTORSer2448, p-4EBP1Thr37/46, p-S6Ser235/236, and Abcc4 staining are shown. (D and E) Representative proliferation curves for TC and PTC primary cells treated with or without rapamycin (100 nM) for 24 or 48 h are shown. These 2 distinct cell lines of the indicated genotypes were isolated from different tumours. (F) Cell proliferation of PTC primary cells was analysed using the CCK-8 assay after rapamycin (100 nM) or sapanisertib (1.4 μM) treatment for different time intervals. Images were obtained at 10× or 40× magnification; scale bar: 25 or 100 μm. Two-way ANOVA with Bonferroni's multiple comparisons test was used in (B) and (C). One-way ANOVA with Tukey's multiple comparisons test was used in (D–F). ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. Abcc4, ATP-binding cassette subfamily C member 4; CCK-8, Cell Counting Kit-8; IHC, immunohistochemistry; mTOR, mammalian target of rapamycin; n.s., no significant; PTC, p53mut/+;Tsc1fl/fl;Alb-Cre; Tsc1, tuberous sclerosis complex 1.

      Discussion

      The mTOR signalling pathway is a central regulator of cell growth and survival that can be negatively regulated by adverse environmental conditions, such as nutrient limitation.
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      Sorafenib is the FDA-approved first-line drug for HCC treatment.
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      However, to improve treatment effects, patients need to be selected before rapamycin/rapalog treatment. Based on our study, HCC mice with mTOR hyperactivation are eligible for therapy with rapamycin. The application of mTOR inhibitors in these different settings will be discussed in more detail in the future.
      There are frequent mutational disruptions in multiple key and canonical components of the mTOR signalling cascade. Mutations in Tsc1 and Tsc2, which negatively control the mTOR pathway and are the most frequently mutated proteins that are likely to be the underlying cause of Tsc1/2 loss, define a subset of human HCCs with relatively aggressive tumour behaviours. The mTOR signalling cascade represents one of the most prominent therapeutic targets in HCC. In this study, we found that compared with Tsc1 deficiency, p53 (haplo)insufficiency could further promote mTOR hyperactivation, suggesting that a mutation-dependent mechanism is likely to be another major cause leading to mTOR hyperactivation in HCC. This finding is consistent with the results of previous studies showing that p53 loss promotes the development of metastatic pancreatic ductal adenocarcinoma via mTOR hyperactivation in response to Tsc1 deficiency.
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      A subset of metastatic pancreatic ductal adenocarcinomas depends quantitatively on oncogenic Kras/Mek/Erk-induced hyperactive mTOR signalling.
      Interestingly, hyperactivated mTOR stimulated cancer cells that were relatively susceptible to rapamycin treatment, as indicated by the observed strong anticancer effect of rapamycin on mTOR-hyperactivated HCC (PTC tumours) compared with general HCC (TC tumours) in vivo. Furthermore, in primary hepatic cells, compared with inhibition of mTOR alone, dual inhibition of mTOR and PI3K effectively decreased mTOR (hyper)activity and inhibited cell proliferation. Our study reveals a personalised therapeutic option for a novel molecular subset of patients with HCC with Tsc1 deficiency (mTOR activation) and p53 mutations that are susceptible to dual inhibition of mTOR and PI3K signals.
      Abcc4 seems to play a key role in p53/PTEN/Akt/mTOR signalling. Abcc4, also named multidrug resistance protein 4, is part of the C subfamily of ATP-binding cassette (ABC) transporters, which are integral membrane proteins found in all kinds of organisms. These transporters utilise energy from ATP binding and hydrolysis to transport a variety of substrates across the biological lipid bilayer. ABC transporters are separated into 7 different subfamilies, ABCA–ABCG.
      • Dean M.
      • Rzhetsky A.
      • Allikmets R.
      The human ATP-binding cassette (ABC) transporter superfamily.
      ,
      • Hardy D.
      • Bill R.M.
      • Jawhari A.
      • Rothnie A.J.
      Functional expression of multidrug resistance protein 4 MRP4/ABCC4.
      Abcc4 has thus far been reported to have close relations with breast cancer, colorectal adenoma, acute lymphoblastic leukaemia, osteosarcoma, and pancreatic ductal adenocarcinoma in terms of proliferation and metastasis.
      • Carozzo A.
      • Yaneff A.
      • Gómez N.
      • Di Siervi N.
      • Sahores A.
      • Diez F.
      • et al.
      Identification of MRP4/ABCC4 as a target for reducing the proliferation of pancreatic ductal adenocarcinoma cells by modulating the cAMP efflux.
      • He Z.
      • Hu B.
      • Tang L.
      • Zheng S.
      • Sun Y.
      • Sheng Z.
      • et al.
      The overexpression of MRP4 is related to multidrug resistance in osteosarcoma cells.
      • Hu D.
      • Li M.
      • Su J.
      • Miao K.
      • Qiu X.
      Dual-targeting of miR-124-3p and ABCC4 promotes sensitivity to adriamycin in breast cancer cells.
      • Murray J.
      • Valli E.
      • Yu D.M.T.
      • Truong A.M.
      • Gifford A.J.
      • Eden G.L.
      • et al.
      Suppression of the ATP-binding cassette transporter ABCC4 impairs neuroblastoma tumour growth and sensitises to irinotecan in vivo.
      • Pereira C.
      • Queirós S.
      • Galaghar A.
      • Sousa H.
      • Marcos-Pinto R.
      • Pimentel-Nunes P.
      • et al.
      Influence of genetic polymorphisms in prostaglandin E2 pathway (COX-2/HPGD/SLCO2A1/ABCC4) on the risk for colorectal adenoma development and recurrence after polypectomy.
      • Tanaka Y.
      However, the function of Abcc4 in HCC has been rarely reported. In this study, we found that (1) Abcc4 was significantly overexpressed in HCC tumours with p53 (haplo)deficiency and Tsc1 deficiency; (2) Abcc4 expression was also increased in p53-deficient liver tissue compared with wild-type liver tissue; (3) Abcc4 expression was highly increased in HCC tissue compared with adjacent tissue from the same patient, and increased Abcc4 expression was positively associated with poor survival in patients with HCC with mutated p53 rather than wild-type p53; and (4) Abcc4 was positively associated with cell proliferation and metastasis in HCC and primary liver cells. These findings indicated that Abcc4 played a pivotal role in p53/mTOR signalling-triggered HCC tumorigenesis and could be used to label an aggressive subset of human HCC. However, there are 3 major questions worthy of further investigation: (1) How does the oncogenic p53/PTEN/Akt-mTOR axis control the expression of Abcc4? (2) What substrates does Abcc4 transport across the biological lipid bilayer in hepatocarcinogenesis? (3) How does Abcc4 affect the proliferation, invasion, and migration of HCC tumours?
      In conclusion, our results demonstrate that (1) Tsc1 deficiency facilitates p53 (haplo)insufficiency-mediated activation of the PTEN/Akt/mTOR axis to drive HCC tumorigenesis and metastasis; (2) dual inhibition of mTOR and PI3K effectively decreases mTOR (hyper)activity; (3) the oncogenic activity of the Akt/mTOR axis relies on Abcc4, which also labels an aggressive subtype of human HCC; and (4) transgenic mouse models can be used to specify an HCC subset, and one of the related biomarkers is Abcc4.

      Abbreviations

      ABC, ATP-binding cassette; Abcc4, ATP-binding cassette subfamily C member 4; BrdU, 5-bromo-2′-deoxyuridine; DEN/CCl4, N-nitrosodiethylamine/carbon tetrachloride; IHC, immunohistochemistry; mTOR, mammalian target of rapamycin; P, p53mut/+; PI3K, phosphoinositide-3 kinase; PTC, p53mut/+;Tsc1fl/fl;Alb-Cre; Rap, rapamycin; TC, Tsc1fl/fl;Alb-Cre; TCGA, The Cancer Genome Atlas; Tsc1, tuberous sclerosis complex 1; Veh, vehicle.

      Financial support

      C-MX was supported by the Program for Young Personnel Training from Southwest Hospital ( SWH2018QNKJ-01 ), the Introduction of Special Funds for Talents from the Third Military Medical University ( Army Medical University ; 4174C6 ; 2019XQY10 ), and Natural Science Foundation of Chongqing ( cstc2019jcyj-msxmX0519 ).

      Authors' contributions

      C-MX contributed to conception and design, financial support, and administrative support. Y-DL, LF, H-QY, JZ, X-TL, X-YL, DW, G-XL, and C-MX contributed to acquisition of data. Y-DL, DH, SC, YJ, Y-JZ, and LS collected patient samples and information. Y-DL, YH, L-DZ, PB, and C-MX analysed and interpreted the data. Y-DL and C-MX performed statistical analysis. Y-DL and C-MX wrote the manuscript. All authors contributed to the final approval of the manuscript.

      Data availability statement

      The data used to support the findings of this study are included within the article.

      Conflicts of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      These results are in part based upon public data generated by TCGA Research Network: http://cancergenome.nih.gov/. We are particularly grateful to pathologist Dr. Feng Wu for extremely valuable feedback on IHC staining and for help with improving the manuscript.

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