SWI/SNF regulates a transcriptional program that induces senescence to prevent liver cancer.

Oncogene-induced senescence (OIS) is a potent tumor suppressor mechanism. To identify senescence regulators relevant to cancer, we screened an shRNA library targeting genes deleted in hepatocellular carcinoma (HCC). Here, we describe how knockdown of the SWI/SNF component ARID1B prevents OIS and cooperates with RAS to induce liver tumors. ARID1B controls p16INK4a and p21CIP1a transcription but also regulates DNA damage, oxidative stress, and p53 induction, suggesting that SWI/SNF uses additional mechanisms to regulate senescence. To systematically identify SWI/SNF targets regulating senescence, we carried out a focused shRNA screen. We discovered several new senescence regulators, including ENTPD7, an enzyme that hydrolyses nucleotides. ENTPD7 affects oxidative stress, DNA damage, and senescence. Importantly, expression of ENTPD7 or inhibition of nucleotide synthesis in ARID1B-depleted cells results in re-establishment of senescence. Our results identify novel mechanisms by which epigenetic regulators can affect tumor progression and suggest that prosenescence therapies could be employed against SWI/SNF-mutated cancers.

A MYC-aurora kinase A protein complex represents an actionable drug target in p53-altered liver cancer.

MYC oncoproteins are involved in the genesis and maintenance of the majority of human tumors but are considered undruggable. By using a direct in vivo shRNA screen, we show that liver cancer cells that have mutations in the gene encoding the tumor suppressor protein p53 (Trp53 in mice and TP53 in humans) and that are driven by the oncoprotein NRAS become addicted to MYC stabilization via a mechanism mediated by aurora kinase A (AURKA). This MYC stabilization enables the tumor cells to overcome a latent G2/M cell cycle arrest that is mediated by AURKA and the tumor suppressor protein p19(ARF). MYC directly binds to AURKA, and inhibition of this protein-protein interaction by conformation-changing AURKA inhibitors results in subsequent MYC degradation and cell death. These conformation-changing AURKA inhibitors, with one of them currently being tested in early clinical trials, suppressed tumor growth and prolonged survival in mice bearing Trp53-deficient, NRAS-driven MYC-expressing hepatocellular carcinomas (HCCs). TP53-mutated human HCCs revealed increased AURKA expression and a positive correlation between AURKA and MYC expression. In xenograft models, mice bearing TP53-mutated or TP53-deleted human HCCs were hypersensitive to treatment with conformation-changing AURKA inhibitors, thus suggesting a therapeutic strategy for this subgroup of human HCCs.

In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer.

In solid tumors, resistance to therapy inevitably develops upon treatment with cytotoxic drugs or molecularly targeted therapies. Here, we describe a system that enables pooled shRNA screening directly in mouse hepatocellular carcinomas (HCC) in vivo to identify genes likely to be involved in therapy resistance. Using a focused shRNA library targeting genes located within focal genomic amplifications of human HCC, we screened for genes whose inhibition increased the therapeutic efficacy of the multikinase inhibitor sorafenib. Both shRNA-mediated and pharmacological silencing of Mapk14 (p38α) were found to sensitize mouse HCC to sorafenib therapy and prolong survival by abrogating Mapk14-dependent activation of Mek-Erk and Atf2 signaling. Elevated Mapk14-Atf2 signaling predicted poor response to sorafenib therapy in human HCC, and sorafenib resistance of p-Mapk14-expressing HCC cells could be reverted by silencing Mapk14. Our results suggest that a combination of sorafenib and Mapk14 blockade is a promising approach to overcoming therapy resistance of human HCC.

A Direct in vivo RNAi screen identifies MKK4 as a key regulator of liver regeneration.

The liver harbors a distinct capacity for endogenous regeneration; however, liver regeneration is often impaired in disease and therefore insufficient to compensate for the loss of hepatocytes and organ function. Here we describe a functional genetic approach for the identification of gene targets that can be exploited to increase the regenerative capacity of hepatocytes. Pools of small hairpin RNAs (shRNAs) were directly and stably delivered into mouse livers to screen for genes modulating liver regeneration. Our studies identify the dual-specific kinase MKK4 as a master regulator of liver regeneration. MKK4 silencing robustly increased the regenerative capacity of hepatocytes in mouse models of liver regeneration and acute and chronic liver failure. Mechanistically, induction of MKK7 and a JNK1-dependent activation of the AP1 transcription factor ATF2 and the Ets factor ELK1 are crucial for increased regeneration of hepatocytes with MKK4 silencing.

Adaptive immunity suppresses formation and progression of diethylnitrosamine-induced liver cancer.

BACKGROUND: Hepatocellular carcinoma (HCC) is a typical inflammation-associated cancer, but may also provoke antitumour immune responses whose significance and underlying mechanisms are incompletely understood. OBJECTIVE: To characterise immune responses in the diethylnitrosamine (DEN)-liver cancer mouse model. DESIGN: Tumour development and immune cell functions upon DEN treatment were compared between C57BL/6 wild-type (WT), chemokine scavenging receptor D6-deficient, B cell- (Igh6), CD4 T cell- (MHC-II) and T-/B cell-deficient (Rag1) mice. Relevance for human HCC was tested by comparing gene array results from 139 HCC tissues. RESULTS: The induction of premalignant lesions after 24 weeks and of HCC-like tumours after 42 weeks by DEN in mice was accompanied by significant leucocyte infiltration in the liver and upregulation of distinct intrahepatic chemokines (CCL2, CCL5, CXCL9). Macrophages and CD8 (cytotoxic) T cells were most prominently enriched in tumour-bearing livers, similar to samples from human HCC. Myeloid-derived suppressor cells (MDSC) increased in extrahepatic compartments of DEN-treated mice (bone marrow, spleen). The contribution of immune cell subsets for DEN-induced hepatocarcinogenesis was functionally dissected. In D6(-/-) mice, which lack the chemokine scavenging receptor D6, hepatic macrophage infiltration was significantly increased, but tumour formation and progression did not differ from that of WT mice. In contrast, progression of hepatic tumours (numbers, diameters, tumour load) was strikingly enhanced in T-/B cell-deficient Rag1(-/-) mice upon DEN treatment. When mice deficient for B cells (Igh6(-/-), µMT) or major histocompatibility complex II were used, the data indicated that T cells prevent initial tumour formation, while B cells critically limit growth of established tumours. Accordingly, in tumour-bearing mice antibody production against liver-related model antigen was enhanced, indicating tumour-associated B cell activation. In agreement, T and B cell pathways were differentially regulated in gene array analyses from 139 human HCC tissues and significantly associated with patients' survival. CONCLUSIONS: Distinct axes of the adaptive immune system, which are also prognostic in human HCC, actively suppress DEN-induced hepatocarcinogenesis by controlling tumour formation and progression.

Senescence surveillance of pre-malignant hepatocytes limits liver cancer development.

Upon the aberrant activation of oncogenes, normal cells can enter the cellular senescence program, a state of stable cell-cycle arrest, which represents an important barrier against tumour development in vivo. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it was reported that this 'secretory phenotype' can have pro- as well as anti-tumorigenic effects. Here we show that oncogene-induced senescence occurs in otherwise normal murine hepatocytes in vivo. Pre-malignant senescent hepatocytes secrete chemo- and cytokines and are subject to immune-mediated clearance (designated as 'senescence surveillance'), which depends on an intact CD4(+) T-cell-mediated adaptive immune response. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of murine hepatocellular carcinomas (HCCs), thus showing that senescence surveillance is important for tumour suppression in vivo. In accordance with these observations, ras-specific Th1 lymphocytes could be detected in mice, in which oncogene-induced senescence had been triggered by hepatic expression of Nras(G12V). We also found that CD4(+) T cells require monocytes/macrophages to execute the clearance of senescent hepatocytes. Our study indicates that senescence surveillance represents an important extrinsic component of the senescence anti-tumour barrier, and illustrates how the cellular senescence program is involved in tumour immune surveillance by mounting specific immune responses against antigens expressed in pre-malignant senescent cells.