Meeting Report

Twentieth Annual Pezcoller Symposium: of Cancer—20 Years of Progress Punctuatedby the Pezcoller Symposia Enrico Mihich

Roswell Park Cancer Institute, Buffalo, New York wild-type or mutant p53Q22/S23. Although p53Q22/S23 is defective Introduction in activating most p53 target genes, it induces proapoptotic gene This 20th Anniversary Symposium, which also honored the expression, including PIDD. PIDD is required for p53-mediated American Association for Cancer Research in its 100th Anniversary, in this system. Several forms of DNA damage require was held in Trento, Italy, on June 11 to 13, 2008, and was co-chaired nucleolar disruption to activate p53. A number of ribosomal by Enrico Mihich, David Livingston (Dana-Farber Cancer institute, proteins (L5, L11, L23, and S7) can bind to and reduce its Boston, MA), and Marco Pierotti (Istituto Nazionale Tumori, Milan, ubiquitination of p53 in a cell type–specific manner. Mdm2, but not Italy). Discussions were made on molecular biology of cancer cells, its homologue MdmX, is an E3 ubiquitin ligase. MdmX, however, the role of tumor microenvironment in tumor progression, genetic cooperates with Mdm2 to regulate p53. and epigenetic determinants of cancer cell biology, proliferation Pier Paolo Pandolfi (Harvard Medical School, Boston, MA) controls, novel target therapeutics, and signal transduction discussed his studies on nucleophosmin (NPM), a multifunctional mechanisms. The identification of new sites of potential interven- protein implicated in cancer pathogenesis through involvement in tion was a major focus of this symposium. chromosomal translocations and its frequent in leukemia. Transfection of NPM leads to the development of Report cancer. NPM is involved in shuttling between the nucleus and Carlo Croce (Ohio State University Comprehensive Cancer cytoplasm. In acute promyelocytic leukemia, after PML and PLZF, a Center, Columbus, OH) outlined the discovery of microRNAs, NPM is the third partner of retinoic acid receptor (RAR)- . NPM the mapping of microRNA genes to chromosomal regions, their Depending on dose, functions as an or a tumor NPM relationships to DNA fragile sites, their function in human suppressor gene. heterozygosity accelerates Myc-induced cancer initiation and progression, and their expression in lymphomagenesis and leads to myeloid leukemia, T-cell lympho- malignant versus normal cells [miR15 and miR16 were discovered mas, B-cell lymphomas, and hepatocellular carcinoma. Lack of NPM at a chromosome 13 region deleted in chronic lymphocytic leads to features similar to human myelodysplastic NPM leukemia (CLL)]. In transgenic mice expressing a specific human syndrome. With homozygosity, tumor suppression functions NPM oncogene, TCL1, a B-cell leukemia develops resembling aggressive are seen. is an example of the tumorigenic consequences of CLL. Depending on cell context, microRNAs can have suppressor subtle variation in gene expression and aberrant protein or oncogene-like functions. The specific patterns of microRNA trafficking. expression in normal tissue can help to identify the unknown Zena Werb (University of California, San Francisco, CA) used primary site of origin of a metastasis. MicroRNAs can be four-color, long-term, fluorescent imaging in mice to investigate specifically deregulated in transgenic mice, which can lead to leukocyte migration in different mammary tumor microenviron- delineation of their role in B-cell leukemias, lymphomas, and ments. T-cell migration was highest along blood vessels; myeloid other types of cancer. Many genes that are important for stress and dendritic cells were most motile at tumor-stroma borders. In responses, cell survival, proliferation, and differentiation are human breast cancer, high GATA3 tumor levels imply a good regulated by microRNAs. Certain cytokines like IFNh affect the prognosis, and low GATA3 the opposite. In mice, there is no expression of certain microRNAs. Differential expression of dissemination of GATA3-negative cells transfected with GATA3. microRNA genes can be affected by their location in cancer- Inflammatory cell increases were accompanied by tumor associated genomic regions, epigenetic mechanisms, and alter- progression. Deeper within tumors, little migration of macro- ations in the microRNA processing machinery. phages and other cells was observed. Newly recruited myeloid Carol Prives (Columbia University, New York, NY) discussed how cells extravasated and infiltrated the tissue. Dextran-ingesting, p53 selects specific promoters of genes controlling certain key cell low-migratory myeloid cells were activated (M2) macrophages. processes like cell cycle and death. The nuclear export factor Myeloid cell infiltration and migration were increased in mice hCAS/Cse1L, a p53 coregulator and selectivity factor, associates challenged with fluorescently labeled necrotic tumor debris. Once with some p53 target gene promoters in a p53-independent the first cell made contact with the debris, other cells changed manner and cooperates with p53 to down-regulate histone H3 direction within minutes and migrated toward it. Thus, leukocyte trimethylation. In H1299 cells, caspase-2 activation is initiated by behavior is conditioned by the nature of the tumor microenvi- ronment. Mina Bissell (Lawrence Berkeley National Laboratory, Berkeley, CA) described two sets of results that support the dynamic Requests for reprints: Enrico Mihich, Roswell Park Cancer Institute, Elm & reciprocity between ECM signaling, the cytoskeleton, chromatin Carlton Streets, Buffalo, NY 14263. Phone: 716-845-8226; Fax: 716-845-3351; E-mail: organization, and the dominance of tissue structure in mammary [email protected]. I2009 American Association for Cancer Research. function and dysfunction. Signaling for functional differentiation doi:10.1158/0008-5472.CAN-08-3155 depends on both prolactin and laminin. Prolactin transiently www.aacrjournals.org 407 Cancer Res 2009; 69: (2). January 15, 2009

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2009 American Association for Cancer Research. Cancer Research activates STAT5, but laminin organizes chromatin and allows Abnormal genetic steps underlying the most common forms of stable STAT5 activation, required for h casein expression. Ductal human cancer might not emerge without the effects of major branching as a model for controlled invasion depends on the epigenetic alterations involving important gene sets. These effects geometric shape of the collagen substrata (micropatterns) often arise during cancer initiation and deepen during progression. together with the inhibitory diffusion of transforming growth DNA hypermethylation of CpG islands in gene promoter regions factor h (TGFh). Thus, normal or malignant behaviors depend likely ‘‘locks in’’ the aberrant transcriptional repression of involved on molecular signals intertwined with selected mechanical genes. A patient’s cancer harbors several hundred such genes, many influences. of which are important for embryogenesis; the number of genes Jacques Pouyssegur (Institute of Signaling, Developmental simultaneously DNA hypermethylated in a cancer is reminiscent of Biology and Cancer Research, Nice, France) discussed tumor the number of CpG island–containing genes marked, in embryonic progression in the face of hypoxia and acidic microenvironments. stem and progenitor cells, by the silencing protein complex Oxygen sensing is a central control mechanism of vasculogenesis polycomb (PcG). Approximately f50% of the DNA hypermethy- and energy metabolism. Hypoxia-inducible factor (HIF) and some lated genes in colon cancer cells are among the f10% of HIF-induced markers play major roles in tumor resistance to embryonic cell genes marked by PcG. Methylation in stem/ nutrient-depleted and acidic microenvironments. Two HIF- progenitor cells may be essential in supporting clonal expansion induced ‘‘BH3-only’’ proteins trigger tumor cell survival by for cancer development. Evidence for this comes from mouse inducing autophagy. Tumor cells express two HIF-dependent, knockout studies of the HIC-1 gene, which is never mutated but membrane-bound carbonic anhydrases, acidifying the extracellu- often silenced by DNA hypermethylation, in which epigenetic lar milieu and ensuring a more alkaline intracellular pH favoring silencing of this gene helps foster the earliest steps in cancer maintenance of ATP levels and survival in hostile acidic tumor formation. microenvironments. Targeting HIF-regulated factors that control Federica Cavallo (Ospedale San Luigi Gonzaga, Turin, Italy) intracellular pH could have enhanced drug-induced tumor argued that antitumor vaccines can inhibit progression of early regression. neoplastic lesions. The mammary glands from BALB-neuT mice Napoleone Ferrara (Genentech, Inc., San Francisco, CA) were taken at various ages to track progression from atypical discussed vascular endothelial growth factor (VEGF)-A, involved hyperplasia to invasive cancer. Gene expression profiles were in both the physiologic and pathologic growth of blood vessels. A generated using mouse arrays including more than 32,000 genes. humanized anti–VEGF-A monoclonal antibody (bevacizumab) Oncoantigens with high homogeneous expression in human cancer blocks the function of all isoforms of VEGF and has clinical cells, and not in normal ones, were retained for evaluation as were benefit. Vascular growth might be regulated by myeloid cells. tumor microenvironment-associated oncoantigens. Peripheral tol- Responses to anti-VEGF are reduced by co-injecting Gr1+ myeloid erance to these oncoantigens can be interfered by inhibiting cells from animals carrying VEGF-independent tumors. Tumor suppressor cytokines and changing the regulatory T-cell (Treg) implantation, as well as granulocyte colony-stimulating factor function. Analysis of the expression of 169 microRNAs revealed + + (G-CSF), increases Bv8 protein in CD11b GRI bone marrow cells. differences between tumors and pregnant BALB/c mammary Bv8 stimulates hematopoiesis and bone marrow cell mobility. glands, but only slight differences between atypical hyperplasia Combinations of anti-VEGF plus anti-Bv8 inhibit several tumor and neoplastic lesions, suggesting that microRNA disregulation is a xenografts. Anti-Bv8 reduces tumor angiogenesis and growth. very early event during tumor progression. Dr. Ferrara hypothesized that secreted Bv8 protein is a mediator Karen Vousden (Beatson Institute for Cancer Research, Glasgow, of myeloid cell–dependent tumor angiogenesis and may contribute United Kingdom) stressed that understanding what regulates the to refractoriness to anti-VEGF therapy. choice of responses to p53 and how to achieve a maximum As Allan Balmain (University of California, San Francisco, CA) difference between cancer cell death and normal cell survival are indicated, multiple germ-line polymorphisms (single-nucleotide important for a tumor therapy implemented by p53 reactivation. polymorphisms) influence cancer susceptibility controlling tumor p53 induces apoptosis primarily through the activation of PUMA, multiplicity, size, or the probability of malignant progression. Each which can also induce autophagy. A novel p53 target gene, TIGAR, genetic modifier has a weak effect. Major components of cancer encodes a protein that contains similarity to the phosphatase susceptibility are due to interactions between low-penetrance domain of the bifunctional enzyme PFK-2/FBPase-2, a regulator of genetic modifiers, which provide tools for recognition of individuals glycolysis. TIGAR enhances the oxidative branch of the pentose at risk of cancer development and, ideally, for prevention strategies. phosphate pathway, conferring resistance to by Using mouse interspecies crosses, genetic loci have been identified enhancing NADPH production. This likely provides the biochem- that confer increased or decreased risk of cancer development of ical reducing function necessary to restore reduced glutathione. skin, lung, or the lymphoid system; in some cases, causative genes Ectopic expression of TIGAR protects cells from reactive oxygen and polymorphisms are known. Mus spretus was crossed with NIH/ species– and p53-induced cell death. TIGAR is a representative FVB mice and then backcrossed to the FVB strain; the expression of p53-inducible gene that contributes to the survival of cells >20,000 genes in the normal skin was measured, and susceptibility undergoing oxidative stress. to skin tumors development was evaluated. Construction of a gene Fabrizio d’Adda di Fagagna (Institute of Molecular Oncology, expression architecture in normal skin allowed identification of Italian Foundation for Cancer Research, Milan, Italy) reported on features contributing to skin tumor susceptibility in individual senescent cells. Oncogene-induced cell proliferation and transfor- mice. Bioinformatic tools have been developed to construct single- mation are restrained by senescence. He showed that oncogene- nucleotide polymorphisms and gene expression networks associat- induced senescence is triggered by DNA-damage response ed with phenotypes involved in cancer susceptibility. activation. Inactivation of DNA-damage response abrogates Stephen Baylin (Johns Hopkins Comprehensive Cancer Center, oncogene-induced senescence and promotes cell transformation. Baltimore, MD) stressed that cancer is also an epigenetic disease. Oncogene-induced senescence resulted from DNA-damage

Cancer Res 2009; 69: (2). January 15, 2009 408 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2009 American Association for Cancer Research. 20th Annual Pezcoller Symposium responses triggered by oncogene-induced DNA hyper-replication. The neovasculature formed in the Matrigel plugs is more porous to In addition, he reported that senescent cells secrete inflammatory small molecules indicating a defect in tight junction formation. chemokines, and knockdown of interleukin (IL)-8 receptor prevents Alberto Bardelli (Institute for Cancer Research and Treatment, oncogene-induced senescence. University of Torino Medical School, Turin, Italy) investigated the William Kaelin (Dana-Farber Cancer Institute, Boston, MA) influence of cancer on the outcome of targeted therapies. discussed the relationships between the VHL tumor suppressor Efficacy of the anti–epidermal growth factor receptor monoclonal protein (pVHL) and HIFa. pVHL is part of a ubiquitin ligase antibody cetuximab was negatively correlated with the occurrence complex targeting the HIFa subunits for polyubiquitylation once of KRAS or BRAF mutations. This allows the identification of they are hydroxylated by an EglN family member. HIF seems to play colorectal cancer patients eligible for treatment with cetuximab. a prominent role in hemangioblastomas and renal cell carcinomas. Cellular models of tumor progression have also been developed by EglN1 (PHD2) is the primary regulator of HIFa, whereas EglN2 and introducing cancer mutations in the genome of human cells using EglN3 play HIF-independent roles in the regulation of proliferation homologous recombination. Profiling targeted drugs on the and apoptosis, respectively. A KIF1Bh shRNA was identified in a mutated cells showed ‘‘oncogene addiction’’ or resistance pheno- screen for shRNAs that prevent EglN3-induced apoptosis. KIF1Bh, types. These results indicate new avenues for personalized therapies a member of the kinesin family, acts downstream of EglN3; loss-of- based on the genetic milieu of individual tumors. function KIF1Bh mutations have been identified in some William Sellers (Novartis Institute for Biomedical Research, pheochromocytomas and neuroblastomas. Haploinsufficiency of Cambridge, MA) discussed the development of new anticancer KIF1Bh might be sufficient to promote tumor growth. agents targeting the PI3K/mammalian target of rapamycin (mTOR) Pier Giuseppe Pelicci (European Institute of Oncology, Milan, pathway, which is frequently activated constitutively in human Italy) discussed the biological properties of cancer stem cells. cancers. RAD001 is a mTOR inhibitor; NVP-BEZ235 and NVP- Cancer stem cells have been isolated from leukemias, breast BGT226 are dual PI3K/mTOR inhibitors. mTOR is a regulator of cancer, neuroblastoma, glioblastoma, ovarin cancer, and lung HIF. AKT constitutive deregulation induces mTOR-dependent cancer. Although intuitively it would seem that mouse prostate intraepithelial neoplasia. RAD001 treatment led would need to target cancer stem cells to be curative, no direct to the elimination of prostate intraepithelial neoplasia. Renal evidence is as yet available to prove this hypothesis. Stem carcinoma cells lacking an intact VHL gene are dependent on HIF2 cells, like cancer stem cells, are defined by their abilities for tumorigenic potential, and recently, RAD001 has shown by ‘‘asymmetrical cell division’’ to generate more stem cells therapeutic activity in renal cell carcinoma. PI3K inhibitors have (‘‘self-renewal’’) and to produce cells that differentiate. In a entered phase I clinical trials. PIK3CA activating mutations are lifetime, stem cells divide between 80 and 200 times. One can frequent in breast and colorectal cancers. Application of the push stem cell proliferation by stress (e.g., during bone marrow imidazoquinoline NVP-BEZ235 to cells lacking an intact PTEN transplantation). After bone marrow transplantation, by limiting gene results in the dephosphorylation of AKT and AKT substrates dilution it was found that cell proliferation did not affect including GSK3h and FOXOs. TSC1-null mouse embryonic the number of stem cells. Increases of stem cells occur in a fibroblasts have constitutively phosphorylated ribosomal S6 p21-independent manner but p21 is needed to maintain the protein and lack activated AKT through a mTOR-dependent number of functional stem cells in the presence of PML-RAR; loss feedback mechanism. In these cells, BEZ235 inhibits S6 phosphor- of p21 prevents stem cell repopulation. p21 is increased by ylation and activation. NVP-BEZ235 has antitumor activity in expression and is increased by PAL-RAR in stem cells xenograft models, promotes regression of PIK3CA-dependent in a p53-independent manner. p21 is relevant and specific for indigenous lung tumors in genetically engineered animals, and leukemias and stem cell maintenance; indeed, p21 prevents the has significant antiangiogenic properties. À À exhaustion of stem cells. In p21 / , PML-RAR do not transplant Stefano Piccolo (University of Padova, Padova, Italy) outlined the without this being related to an increase in apoptosis or ubiquitin-mediated control of TGFb signaling. TGFh receptors and senescence. Indeed, leukemia stem cells rely on p21 to cope Smad ubiquitination keepTGF h responsiveness under control and with DNA damage in a checkpoint manner. In conclusion, it regulate signal intensity and duration. Several E3 ubiquitin ligases was shown that the dependency of leukemia development on that influence TGFh signaling have been identified. Ubiquitination quiescent leukemia stem cells is due to transcriptional up- can also regulate target protein function, subcellular localization/ regulation of the cell cycle inhibitor p21 by leukemia-associated endocytic trafficking, protein-protein interactions, and indigenous fusion proteins. target protein activity. Smad4 ubiquitination leads to its nuclear Lewis Cantley (Harvard Institutes of Medicine, Boston, MA) exclusion. Regulative ubiquitination is reversible, as indicated by discussed phosphoinositide 3-kinase (PI3K) as a central enzyme in the existence of a whole family of ubiquitin proteases in the genome. the network controlling cancer cell growth. Activating mutations in Tadatsugu Taniguchi (University of Tokyo, Tokyo, Japan) the gene encoding the p110a PI3K subunit, PIK3CA, and loss-of- discussed IFN regulatory factors (IRF), a family of nine transcription function PTEN mutations are the most common PI3K-related factors originally identified for their IFN gene regulation, which events in solid tumors. Mouse tumors developing due to mutated have been recently found to link immune responses to invading forms of PIK3CA or activated AKT expression display increased pathogens and suppression of tumor development. IRF1 regulatory expression of genes involved in glucose uptake and metabolism. A functions include natural killer and CD4 cell differentiation. IRF1 role for PI3K in angiogenesis was discussed. All embryos with class responds to DNA damage and contributes to tumor suppression via Ia PI3K genes deleted in the endothelial compartment died on day p53 induction. IRF family members like IRF3, IRF5, and IRF7 play 10 to day 11 with extensive bleeding into various tissues when the key roles in Toll-like receptor signaling. IRF5 induces, through Toll- embryo exhibited its first heartbeat. In mice with 70% PI3K like receptor signaling, IL-6, IL-12, tumor necrosis factor a, and reduction, only small microvessels were seen in B16 melanoma cells IFNa/h. IRF5 also induces apoptosis, but not during cell cycle growing in Matrigel plugs, and their growth was greatly reduced. arrest, in response to DNA damage and has tumor suppressor www.aacrjournals.org 409 Cancer Res 2009; 69: (2). January 15, 2009

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2009 American Association for Cancer Research. Cancer Research functions through pathways that may be distinct from that of p53. toward the development of individualized, mechanism-driven IRF5 also plays a role in the Fas death receptor signaling. patient treatment. Efforts toward achieving this goal represent an important direction in cancer therapeutics. Prospects Growing knowledge of the regulation of cancer cell behavior Disclosure of Potential Conflicts of Interest and increased understanding of how cancer cells interact No potential conflicts of interest were disclosed. with the microenvironment offer new possibilities for rational therapeutic intervention. A growing repertoire of targeted therapeutics coupled with a better understanding of the in vivo Acknowledgments properties of primary cancer cells represents a starting point Received 8/20/2008; revised 10/11/2008; accepted 10/13/2008.

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Enrico Mihich

Cancer Res 2009;69:407-410.

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