A common functional consequence of tumor-derived mutations within c-MYC.

The relevance of changes to the coding sequence of the c-MYC oncogene to malignancy is controversial. Overexpression of a pristine form of MYC is observed in many cancers and is sufficient to drive tumorigenesis in most contexts. Yet missense changes to MYC are found in ~50% of Burkitt's lymphomas, aggregate within an amino-terminal degron important for proteasomal destruction of MYC, and where examined profoundly enhance the tumorigenic properties of MYC in vitro and in vivo. Much of the controversy surrounding these mutants stems from the limited number of mutations that have been evaluated and their clustering within a single region of the MYC protein; the highly-conserved Myc box I (MbI) element. Here, by analysis of extant genomic data sets, we identify a previously unrecognized hotspot for tumor-associated MYC mutations, located in a conserved central portion of the protein. We show that, despite their distal location in MYC, mutations in this region precisely phenocopy those in MbI in terms of stability, in vitro transformation, growth-promoting properties, in vivo tumorigenesis and ability to escape p53-dependent tumor surveillance mechanisms. The striking parallels between the behavior of tumor-derived mutations in disparate regions of the MYC protein reveals that a common molecular process is disrupted by these mutations, implying an active role for these mutations in tumorigenesis and suggesting that different therapeutic strategies may be needed for treatment of lymphomas expressing wild type versus mutant forms of MYC protein.

The Polycomb complex PRC2 supports aberrant self-renewal in a mouse model of MLL-AF9;Nras(G12D) acute myeloid leukemia.

The Trithorax and Polycomb groups of chromatin regulators are critical for cell-lineage specification during normal development; functions that often become deregulated during tumorigenesis. As an example, oncogenic fusions of the Trithorax-related protein mixed lineage leukemia (MLL) can initiate aggressive leukemias by altering the transcriptional circuitry governing hematopoietic cell differentiation, a process that requires multiple epigenetic pathways to implement. Here we used shRNA screening to identify chromatin regulators uniquely required in a mouse model of MLL-fusion acute myeloid leukemia, which revealed a role for the Polycomb repressive complex 2 (PRC2) in maintenance of this disease. shRNA-mediated suppression of PRC2 subunits Eed, Suz12 or Ezh1/Ezh2 led to proliferation arrest and differentiation of leukemia cells, with a minimal impact on growth of several non-transformed hematopoietic cell lines. The requirement for PRC2 in leukemia is partly because of its role in direct transcriptional repression of genes that limit the self-renewal potential of hematopoietic cells, including Cdkn2a. In addition to implicating a role for PRC2 in the pathogenesis of MLL-fusion leukemia, our results suggest, more generally, that Trithorax and Polycomb group proteins can cooperate with one another to maintain aberrant lineage programs in cancer.

Granule exocytosis mediates immune surveillance of senescent cells.

Senescence is a stable cell cycle arrest program that contributes to tumor suppression, organismal aging and certain wound healing responses. During liver fibrosis, for example, hepatic stellate cells initially proliferate and secrete extracellular matrix components that produce fibrosis; however, these cells eventually senesce and are cleared by immune cells, including natural killer (NK) cells. Here, we examine how NK cells target senescent cells and assess the impact of this process on liver fibrosis. We show that granule exocytosis, but not death-receptor-mediated apoptosis, is required for NK-cell-mediated killing of senescent cells. This pathway bias is due to upregulation of the decoy death receptor, Dcr2, an established senescence marker that attenuates NK-mediated cell death. Accordingly, mice with defects in granule exocytosis accumulate senescent stellate cells and display more liver fibrosis in response to a fibrogenic agent. Our results thus provide new insights into the immune surveillance of senescent cells and reveal how granule exocytosis has a protective role against liver fibrosis.

AXL receptor kinase is a mediator of YAP-dependent oncogenic functions in hepatocellular carcinoma.

Yes-associated protein (YAP) is a downstream effector of the Hippo signaling pathway, which controls organ expansion and tissue development. We have recently defined the tumorigenic potential and clinical significance of the YAP1 oncogene in human hepatocellular carcinoma (HCC). The present study aims to define the tumorigenic properties of YAP in HCC and elucidate the related downstream signaling mechanism. In a gain-of-function study, we demonstrated that ectopic increased expression of YAP in the immortalized non-tumorigenic hepatocyte cell line MIHA confers tumorigenic and metastatic potentials, as evidenced by (1) enhanced aptitudes in cell viability, anchorage-independent growth, migration and invasion; (2) tumor formation in a xenograft mouse model; and (3) induction of HCC biomarker α-fetoprotein and activation of mitogen-activated protein kinase. Furthermore, we have identified AXL, a receptor tyrosine kinase, as a key downstream target that drives YAP-dependent oncogenic functions. RNAi-mediated knockdown of AXL expression decreased the ability of YAP-expressing MIHA cells and of the primary HCC cell line to proliferate and invade. These results indicate that AXL is a mediator of YAP-dependent oncogenic activities and implicates it as a potential therapeutic target for HCC.

Transgenic, inducible RNAi in megakaryocytes and platelets in mice.

BACKGROUND: RNA interference (RNAi) is a powerful tool for suppressing gene function. The tetracycline (tet)-regulated expression system has recently been adapted to allow inducible RNAi in mice, however its efficiency in a particular cell type in vivo depends on a transgenic tet transactivator expression pattern and is often highly variable. OBJECTIVE: We aimed to establish a transgenic strategy that allows efficient and inducible gene knockdown in particular hematopoietic lineages in mice. METHODS AND RESULTS: Using a tet-regulated reporter gene strategy, we found that transgenic mice expressing the rtTA (tet-on) transactivator under control of the cytomegalovirus (CMV) promoter (CMV-rtTA mice) display inducible reporter gene expression with unusual and near-complete efficiency in megakaryocytes and platelets. To test whether the CMV-rtTA transgene can drive inducible and efficient gene knockdown within this lineage, we generated a novel mouse strain harboring a tet-regulated short hairpin RNA (shRNA) targeting Bcl-x(L) , a pro-survival Bcl-2 family member known to be essential for maintaining platelet survival. Doxycycline treatment of adult mice carrying both transgenes induces shRNA expression, depletes Bcl-x(L) in megakaryocytes and triggers severe thrombocytopenia, whereas doxycycline withdrawal shuts off shRNA expression, normalizes Bcl-x(L) levels and restores platelet numbers. These effects are akin to those observed with drugs that target Bcl-x(L) , clearly demonstrating that this transgenic system allows efficient and inducible inhibition of genes in megakaryocytes and platelets. CONCLUSIONS: We have established a novel transgenic strategy for inducible gene knockdown in megakaryocytes and platelets that will be useful for characterizing genes involved in platelet production and function in adult mice.

Implications of cellular senescence in tissue damage response, tumor suppression, and stem cell biology.

Cellular senescence is characterized by an irreversible cell cycle arrest that, when bypassed by mutation, contributes to cellular immortalization. Activated oncogenes induce a hyperproliferative response, which might be one of the senescence cues. We have found that expression of such an oncogene, Akt, causes senescence in primary mouse hepatoblasts in vitro. Additionally, AKT-driven tumors undergo senescence in vivo following p53 reactivation and show signs of differentiation. In another in vivo system, i.e., liver fibrosis, hyperproliferative signaling through AKT might be a driving force of the senescence in activated hepatic stellate cells. Senescent cells up-regulate and secrete molecules that, on the one hand, can reinforce the arrest and, on the other hand, can signal to an innate immune system to clear the senescent cells. The mechanisms governing senescence and immortalization are overlapping with those regulating self-renewal and differentiation. These respective control mechanisms, or their disregulation, are involved in multiple pathological conditions including fibrosis, wound healing, and cancer. Understanding extracellular cues that regulate these processes may enable new therapies for these conditions.

Analysis of the apoptotic and therapeutic activities of histone deacetylase inhibitors by using a mouse model of B cell lymphoma.

Histone deacetylase inhibitors (HDACi) can elicit a range of biological responses that affect tumor growth and survival, including inhibition of cell cycle progression, induction of tumor cell-selective apoptosis, suppression of angiogenesis, and modulation of immune responses, and show promising activity against hematological malignancies in clinical trials. Using the Emu-myc model of B cell lymphoma, we screened tumors with defined genetic alterations in apoptotic pathways for therapeutic responsiveness to the HDACi vorinostat. We demonstrated a direct correlation between induction of tumor cell apoptosis in vivo and therapeutic efficacy. Vorinostat did not require p53 activity or a functional death receptor pathway to kill Emu-myc lymphomas and mediate a therapeutic response but depended on activation of the intrinsic apoptotic pathway with the proapoptotic BH3-only proteins Bid and Bim playing an important role. Our studies provide important information regarding the mechanisms of action of HDACi that have broad implications regarding stratification of patients receiving HDACi therapy alone or in combination with other anticancer agents.

p400 function is required for the adenovirus E1A-mediated suppression of EGFR and tumour cell killing.

We have recently shown that E1A protein of human adenovirus downregulates epidermal growth factor receptor (EGFR) expression and induces apoptosis in head and neck (HNSCC) and lung cancer cells independently of their p53 status. E1A has five isoforms of which the major ones E1A12S and E1A13S regulate transcription of cellular genes by binding to transcriptional modulators such as pRB, CtBP, p300 and p400. In this study, we have identified E1A12S isoform to have the highest effect on EGFR suppression and induction of apoptosis in HNSCC cells. Similar to Ad5, E1A12S from human adenovirus types 2, 3, 9 and 12 suppressed EGFR, whereas E1A12S of adenovirus types 4 and 40 had no effect on EGFR expression. Using deletion mutants of E1A12S we have shown that interaction of E1A with p400, but not p300 or pRB, is required for EGFR suppression and apoptosis. Inhibition of p400 by short hairpin RNA confirmed that HNSCC cells with reduced p400 expression were less sensitive to E1A-induced suppression of EGFR and apoptosis. p300 function was shown to be dispensable, as cells expressing E1A mutants that are unable to bind p300, or p300 knockout cells, remained sensitive to E1A-induced apoptosis. In summary, this study identifies p400 as an important mediator of E1A-induced downregulation of EGFR and apoptosis.

Targeting survivin via PI3K but not c-akt/PKB by anticancer drugs in immature neutrophils.

Myelosuppression is the most common unwanted side effect associated with the administration of anticancer drugs, and infections remain a common cause of death in chemotherapy-treated patients. Several mechanisms of the cytotoxicity of these drugs have been proposed and may synergistically operate in a given cell. Survivin expression has been associated with cancer, but recent reports suggest that this molecule is also expressed in several immature and mature hematopoietic cells. Here, we provide evidence that treatment of immature neutrophils with anticancer drugs reduced endogenous survivin levels causing apoptosis. The anticancer drugs did not directly target survivin, instead they blocked the activity of phosphatidylinositol-3-OH kinase, which regulated survivin expression and apoptosis in these cells. Strikingly, and in contrast to other cells, this pathway did not involve the serine/threonine kinase c-akt/PKB. Moreover, in combination with anticancer drug therapy, rapamycin did not induce increased myelosuppression in an experimental lymphoma mouse model. These data suggest that drugs that block either c-akt/PKB or signaling molecules located distal to c-akt/PKB may preferentially induce apoptosis of cancer cells as they exhibit no cytotoxicity for immature neutrophils.

Generation and analysis of genetically defined liver carcinomas derived from bipotential liver progenitors.

Hepatocellular carcinoma is a chemoresistant cancer and a leading cause of cancer mortality; however, the molecular mechanisms responsible for the aggressive nature of this disease are poorly understood. In this study, we developed a new liver cancer mouse model that is based on the ex vivo genetic manipulation of embryonic liver progenitor cells (hepatoblasts). After retroviral gene transfer of oncogenes or short hairpin RNAs targeting tumor suppressor genes, genetically altered liver progenitor cells are seeded into the liver of otherwise normal recipient mice. We show that histopathology of the engineered liver carcinomas reveals features of the human disease. Furthermore, representational oligonucleotide microarray analysis (ROMA) of murine liver tumors initiated by two defined genetic hits revealed spontaneously acquired genetic alterations that are characteristic for human hepatocellular carcinoma. This model provides a powerful platform for applications like cancer gene discovery or high-throughput preclinical drug testing.

Bcl-2 mediates chemoresistance in matched pairs of primary E(mu)-myc lymphomas in vivo.

The oncoprotein Bcl-2 is a potent survival factor antagonizing p53-dependent and -independent apoptotic cell death. Although many anticancer agents are known to engage apoptotic pathways, the clinical impact of Bcl-2 on treatment outcome remains controversial. Since it might be difficult to assess the contribution of a single gene to treatment response in patient material due to technical considerations, we sought to address Bcl-2's role in a mouse model of primary lymphomas treated at their natural site. Driven by the E(mu)-enhancer controlled c-myc transgene, primary B cell lymphomas arise in this model by several months of age and resemble closely typical clinical and histopathological features of human non-Hodgkin lymphomas. We introduced either bcl-2 or a control construct into identical samples of freshly isolated E(mu)-myc lymphomas by retroviral gene transfer in order to obtain matched pairs of primary lymphomas differing only in their Bcl-2 status. While no Bcl-2-mediated effect was detectable in clonogenic survival assays in vitro, treatment of the genetically modified lymphoma pairs propagated in nontransgenic recipient mice revealed Bcl-2's impact on drug sensitivity in vivo. Bcl-2 efficiently blocked short- and long-term drug-mediated cell death in vivo. In a comparison of 15 matched pairs of primary lymphomas, the bcl-2 transduced sample never achieved longer remission periods than the control counterpart and most of the Bcl-2 overexpressing lymphomas failed to respond at all. We conclude that-when assessed in the physiological environmental context-MBcl-2 contributes to chemoresistance of B cell lymphomas in vivo. This model, able to test any other candidate gene, will be particularly useful to study the implications of specific mutations for drug action in vivo.

Oncogenic ras activates the ARF-p53 pathway to suppress epithelial cell transformation.

Chemically induced skin carcinomas in mice are a paradigm for epithelial neoplasia, where oncogenic ras mutations precede p53 and INK4a/ARF mutations during the progression toward malignancy. To explore the biological basis for these genetic interactions, we studied cellular responses to oncogenic ras in primary murine keratinocytes. In wild-type keratinocytes, ras induced a cell-cycle arrest that displayed some features of terminal differentiation and was accompanied by increased expression of the p19(ARF), p16(INK4a), and p53 tumor suppressors. In ARF-null keratinocytes, ras was unable to promote cell-cycle arrest, induce differentiation markers, or properly activate p53. Although oncogenic ras produced a substantial increase in both nucleolar and nucleoplasmic p19(ARF), Mdm2 did not relocalize to the nucleolus or to nuclear bodies but remained distributed throughout the nucleoplasm. This result suggests that p19(ARF) can activate p53 without overtly affecting Mdm2 subcellular localization. Nevertheless, like p53-null keratinocytes, ARF-null keratinocytes were transformed by oncogenic ras and rapidly formed carcinomas in vivo. Thus, oncogenic ras can activate the ARF-p53 program to suppress epithelial cell transformation. Disruption of this program may be important during skin carcinogenesis and the development of other carcinomas.

Inactivation of the apoptosis effector Apaf-1 in malignant melanoma.

Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy and is poorly understood at the molecular level. p53 mutations often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma. Here we show that metastatic melanomas often lose Apaf-1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis. Loss of Apaf-1 expression is accompanied by allelic loss in metastatic melanomas, but can be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (5aza2dC). Apaf-1-negative melanomas are invariably chemoresistant and are unable to execute a typical apoptotic programme in response to p53 activation. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment markedly enhances chemosensitivity and rescues the apoptotic defects associated with Apaf-1 loss. We conclude that Apaf-1 is inactivated in metastatic melanomas, which leads to defects in the execution of apoptotic cell death. Apaf-1 loss may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumour type.

Regulation of p53 by hypoxia: dissociation of transcriptional repression and apoptosis from p53-dependent transactivation.

Hypoxic stress, like DNA damage, induces p53 protein accumulation and p53-dependent apoptosis in oncogenically transformed cells. Unlike DNA damage, hypoxia does not induce p53-dependent cell cycle arrest, suggesting that p53 activity is differentially regulated by these two stresses. Here we report that hypoxia induces p53 protein accumulation, but in contrast to DNA damage, hypoxia fails to induce endogenous downstream p53 effector mRNAs and proteins. Hypoxia does not inhibit the induction of p53 target genes by ionizing radiation, indicating that p53-dependent transactivation requires a DNA damage-inducible signal that is lacking under hypoxic treatment alone. At the molecular level, DNA damage induces the interaction of p53 with the transcriptional activator p300 as well as with the transcriptional corepressor mSin3A. In contrast, hypoxia primarily induces an interaction of p53 with mSin3A, but not with p300. Pretreatment of cells with an inhibitor of histone deacetylases that relieves transcriptional repression resulted in a significant reduction of p53-dependent transrepression and hypoxia-induced apoptosis. These results led us to propose a model in which different cellular pools of p53 can modulate transcriptional activity through interactions with transcriptional coactivators or corepressors. Genotoxic stress induces both kinds of interactions, whereas stresses that lack a DNA damage component as exemplified by hypoxia primarily induce interaction with corepressors. However, inhibition of either type of interaction can result in diminished apoptotic activity.