This 20th Anniversary Symposium, which also honored the American Association for Cancer Research in its 100th Anniversary, was held in Trento, Italy, on June 11 to 13, 2008, and was co-chaired by Enrico Mihich, David Livingston (Dana-Farber Cancer institute, Boston, MA), and Marco Pierotti (Istituto Nazionale Tumori, Milan, Italy). Discussions were made on molecular biology of cancer cells, the role of tumor microenvironment in tumor progression, genetic and epigenetic determinants of cancer cell biology, proliferation controls, novel target therapeutics, and signal transduction mechanisms. The identification of new sites of potential intervention was a major focus of this symposium.

Carlo Croce (Ohio State University Comprehensive Cancer Center, Columbus, OH) outlined the discovery of microRNAs, the mapping of microRNA genes to chromosomal regions, their relationships to DNA fragile sites, their function in human cancer initiation and progression, and their expression in malignant versus normal cells [miR15 and miR16 were discovered at a chromosome 13 region deleted in chronic lymphocytic leukemia (CLL)]. In transgenic mice expressing a specific human oncogene, TCL1, a B-cell leukemia develops resembling aggressive CLL. Depending on cell context, microRNAs can have suppressor or oncogene-like functions. The specific patterns of microRNA expression in normal tissue can help to identify the unknown primary site of origin of a metastasis. MicroRNAs can be specifically deregulated in transgenic mice, which can lead to delineation of their role in B-cell leukemias, lymphomas, and other types of cancer. Many genes that are important for stress responses, cell survival, proliferation, and differentiation are regulated by microRNAs. Certain cytokines like IFNβ affect the expression of certain microRNAs. Differential expression of microRNA genes can be affected by their location in cancer-associated genomic regions, epigenetic mechanisms, and alterations in the microRNA processing machinery.

Carol Prives (Columbia University, New York, NY) discussed how p53 selects specific promoters of genes controlling certain key cell processes like cell cycle and death. The nuclear export factor hCAS/Cse1L, a p53 coregulator and selectivity factor, associates with some p53 target gene promoters in a p53-independent manner and cooperates with p53 to down-regulate histone H3 trimethylation. In H1299 cells, caspase-2 activation is initiated by wild-type p53 or mutant p53Q22/S23. Although p53Q22/S23 is defective in activating most p53 target genes, it induces proapoptotic gene expression, including PIDD. PIDD is required for p53-mediated apoptosis in this system. Several forms of DNA damage require nucleolar disruption to activate p53. A number of ribosomal proteins (L5, L11, L23, and S7) can bind to Mdm2 and reduce its ubiquitination of p53 in a cell type–specific manner. Mdm2, but not its homologue MdmX, is an E3 ubiquitin ligase. MdmX, however, cooperates with Mdm2 to regulate p53.

Pier Paolo Pandolfi (Harvard Medical School, Boston, MA) discussed his studies on nucleophosmin (NPM), a multifunctional protein implicated in cancer pathogenesis through involvement in chromosomal translocations and its frequent mutation in leukemia. Transfection of NPM leads to the development of cancer. NPM is involved in shuttling between the nucleus and cytoplasm. In acute promyelocytic leukemia, after PML and PLZF, NPM is the third partner of retinoic acid receptor (RAR)-α. Depending on dose, NPM functions as an oncogene or a tumor suppressor gene. NPM heterozygosity accelerates Myc-induced lymphomagenesis and leads to myeloid leukemia, T-cell lymphomas, B-cell lymphomas, and hepatocellular carcinoma. Lack of NPM leads to features similar to human myelodysplastic syndrome. With NPM homozygosity, tumor suppression functions are seen. NPM is an example of the tumorigenic consequences of subtle variation in gene expression and aberrant protein trafficking.

Zena Werb (University of California, San Francisco, CA) used four-color, long-term, fluorescent imaging in mice to investigate leukocyte migration in different mammary tumor microenvironments. T-cell migration was highest along blood vessels; myeloid and dendritic cells were most motile at tumor-stroma borders. In human breast cancer, high GATA3 tumor levels imply a good prognosis, and low GATA3 the opposite. In mice, there is no dissemination of GATA3-negative cells transfected with GATA3. Inflammatory cell increases were accompanied by tumor progression. Deeper within tumors, little migration of macrophages and other cells was observed. Newly recruited myeloid cells extravasated and infiltrated the tissue. Dextran-ingesting, low-migratory myeloid cells were activated (M2) macrophages. Myeloid cell infiltration and migration were increased in mice challenged with fluorescently labeled necrotic tumor debris. Once the first cell made contact with the debris, other cells changed direction within minutes and migrated toward it. Thus, leukocyte behavior is conditioned by the nature of the tumor microenvironment.

Mina Bissell (Lawrence Berkeley National Laboratory, Berkeley, CA) described two sets of results that support the dynamic reciprocity between ECM signaling, the cytoskeleton, chromatin organization, and the dominance of tissue structure in mammary function and dysfunction. Signaling for functional differentiation depends on both prolactin and laminin. Prolactin transiently activates STAT5, but laminin organizes chromatin and allows stable STAT5 activation, required for β casein expression. Ductal branching as a model for controlled invasion depends on the geometric shape of the collagen substrata (micropatterns) together with the inhibitory diffusion of transforming growth factor β (TGFβ). Thus, normal or malignant behaviors depend on molecular signals intertwined with selected mechanical influences.

Jacques Pouyssegur (Institute of Signaling, Developmental Biology and Cancer Research, Nice, France) discussed tumor progression in the face of hypoxia and acidic microenvironments. Oxygen sensing is a central control mechanism of vasculogenesis and energy metabolism. Hypoxia-inducible factor (HIF) and some HIF-induced markers play major roles in tumor resistance to nutrient-depleted and acidic microenvironments. Two HIF-induced “BH3-only” proteins trigger tumor cell survival by inducing autophagy. Tumor cells express two HIF-dependent, membrane-bound carbonic anhydrases, acidifying the extracellular milieu and ensuring a more alkaline intracellular pH favoring maintenance of ATP levels and survival in hostile acidic tumor microenvironments. Targeting HIF-regulated factors that control intracellular pH could have enhanced drug-induced tumor regression.

Napoleone Ferrara (Genentech, Inc., San Francisco, CA) discussed vascular endothelial growth factor (VEGF)-A, involved in both the physiologic and pathologic growth of blood vessels. A humanized anti–VEGF-A monoclonal antibody (bevacizumab) blocks the function of all isoforms of VEGF and has clinical benefit. Vascular growth might be regulated by myeloid cells. Responses to anti-VEGF are reduced by co-injecting Gr1+ myeloid cells from animals carrying VEGF-independent tumors. Tumor implantation, as well as granulocyte colony-stimulating factor (G-CSF), increases Bv8 protein in CD11b+GRI+ bone marrow cells. Bv8 stimulates hematopoiesis and bone marrow cell mobility. Combinations of anti-VEGF plus anti-Bv8 inhibit several tumor xenografts. Anti-Bv8 reduces tumor angiogenesis and growth. Dr. Ferrara hypothesized that secreted Bv8 protein is a mediator of myeloid cell–dependent tumor angiogenesis and may contribute to refractoriness to anti-VEGF therapy.

As Allan Balmain (University of California, San Francisco, CA) indicated, multiple germ-line polymorphisms (single-nucleotide polymorphisms) influence cancer susceptibility controlling tumor multiplicity, size, or the probability of malignant progression. Each genetic modifier has a weak effect. Major components of cancer susceptibility are due to interactions between low-penetrance genetic modifiers, which provide tools for recognition of individuals at risk of cancer development and, ideally, for prevention strategies. Using mouse interspecies crosses, genetic loci have been identified that confer increased or decreased risk of cancer development of skin, lung, or the lymphoid system; in some cases, causative genes and polymorphisms are known. Mus spretus was crossed with NIH/FVB mice and then backcrossed to the FVB strain; the expression of >20,000 genes in the normal skin was measured, and susceptibility to skin tumors development was evaluated. Construction of a gene expression architecture in normal skin allowed identification of features contributing to skin tumor susceptibility in individual mice. Bioinformatic tools have been developed to construct single-nucleotide polymorphisms and gene expression networks associated with phenotypes involved in cancer susceptibility.

Stephen Baylin (Johns Hopkins Comprehensive Cancer Center, Baltimore, MD) stressed that cancer is also an epigenetic disease. Abnormal genetic steps underlying the most common forms of human cancer might not emerge without the effects of major epigenetic alterations involving important gene sets. These effects often arise during cancer initiation and deepen during progression. DNA hypermethylation of CpG islands in gene promoter regions likely “locks in” the aberrant transcriptional repression of involved genes. A patient's cancer harbors several hundred such genes, many of which are important for embryogenesis; the number of genes simultaneously DNA hypermethylated in a cancer is reminiscent of the number of CpG island–containing genes marked, in embryonic stem and progenitor cells, by the silencing protein complex polycomb (PcG). Approximately ∼50% of the DNA hypermethylated genes in colon cancer cells are among the ∼10% of embryonic cell genes marked by PcG. Methylation in stem/progenitor cells may be essential in supporting clonal expansion for cancer development. Evidence for this comes from mouse knockout studies of the HIC-1 gene, which is never mutated but often silenced by DNA hypermethylation, in which epigenetic silencing of this gene helps foster the earliest steps in cancer formation.

Federica Cavallo (Ospedale San Luigi Gonzaga, Turin, Italy) argued that antitumor vaccines can inhibit progression of early neoplastic lesions. The mammary glands from BALB-neuT mice were taken at various ages to track progression from atypical hyperplasia to invasive cancer. Gene expression profiles were generated using mouse arrays including more than 32,000 genes. Oncoantigens with high homogeneous expression in human cancer cells, and not in normal ones, were retained for evaluation as were tumor microenvironment-associated oncoantigens. Peripheral tolerance to these oncoantigens can be interfered by inhibiting suppressor cytokines and changing the regulatory T-cell (Treg) function. Analysis of the expression of 169 microRNAs revealed differences between tumors and pregnant BALB/c mammary glands, but only slight differences between atypical hyperplasia and neoplastic lesions, suggesting that microRNA disregulation is a very early event during tumor progression.

Karen Vousden (Beatson Institute for Cancer Research, Glasgow, United Kingdom) stressed that understanding what regulates the choice of responses to p53 and how to achieve a maximum difference between cancer cell death and normal cell survival are important for a tumor therapy implemented by p53 reactivation. p53 induces apoptosis primarily through the activation of PUMA, which can also induce autophagy. A novel p53 target gene, TIGAR, encodes a protein that contains similarity to the phosphatase domain of the bifunctional enzyme PFK-2/FBPase-2, a regulator of glycolysis. TIGAR enhances the oxidative branch of the pentose phosphate pathway, conferring resistance to oxidative stress by enhancing NADPH production. This likely provides the biochemical reducing function necessary to restore reduced glutathione. Ectopic expression of TIGAR protects cells from reactive oxygen species– and p53-induced cell death. TIGAR is a representative p53-inducible gene that contributes to the survival of cells undergoing oxidative stress.

Fabrizio d'Adda di Fagagna (Institute of Molecular Oncology, Italian Foundation for Cancer Research, Milan, Italy) reported on senescent cells. Oncogene-induced cell proliferation and transformation are restrained by senescence. He showed that oncogene-induced senescence is triggered by DNA-damage response activation. Inactivation of DNA-damage response abrogates oncogene-induced senescence and promotes cell transformation. Oncogene-induced senescence resulted from DNA-damage responses triggered by oncogene-induced DNA hyper-replication. In addition, he reported that senescent cells secrete inflammatory chemokines, and knockdown of interleukin (IL)-8 receptor prevents oncogene-induced senescence.

William Kaelin (Dana-Farber Cancer Institute, Boston, MA) discussed the relationships between the VHL tumor suppressor protein (pVHL) and HIFα. pVHL is part of a ubiquitin ligase complex targeting the HIFα subunits for polyubiquitylation once they are hydroxylated by an EglN family member. HIF seems to play a prominent role in hemangioblastomas and renal cell carcinomas. EglN1 (PHD2) is the primary regulator of HIFα, whereas EglN2 and EglN3 play HIF-independent roles in the regulation of proliferation and apoptosis, respectively. A KIF1Bβ shRNA was identified in a screen for shRNAs that prevent EglN3-induced apoptosis. KIF1Bβ, a member of the kinesin family, acts downstream of EglN3; loss-of-function KIF1Bβ mutations have been identified in some pheochromocytomas and neuroblastomas. Haploinsufficiency of KIF1Bβ might be sufficient to promote tumor growth.

Pier Giuseppe Pelicci (European Institute of Oncology, Milan, Italy) discussed the biological properties of cancer stem cells. Cancer stem cells have been isolated from leukemias, breast cancer, neuroblastoma, glioblastoma, ovarin cancer, and lung cancer. Although intuitively it would seem that chemotherapy would need to target cancer stem cells to be curative, no direct evidence is as yet available to prove this hypothesis. Stem cells, like cancer stem cells, are defined by their abilities by “asymmetrical cell division” to generate more stem cells (“self-renewal”) and to produce cells that differentiate. In a lifetime, stem cells divide between 80 and 200 times. One can push stem cell proliferation by stress (e.g., during bone marrow transplantation). After bone marrow transplantation, by limiting dilution it was found that cell proliferation did not affect the number of stem cells. Increases of stem cells occur in a p21-independent manner but p21 is needed to maintain the number of functional stem cells in the presence of PML-RAR; loss of p21 prevents stem cell repopulation. p21 is increased by oncogenes expression and is increased by PAL-RAR in stem cells in a p53-independent manner. p21 is relevant and specific for leukemias and stem cell maintenance; indeed, p21 prevents the exhaustion of stem cells. In p21−/−, PML-RAR do not transplant without this being related to an increase in apoptosis or senescence. Indeed, leukemia stem cells rely on p21 to cope with DNA damage in a checkpoint manner. In conclusion, it was shown that the dependency of leukemia development on quiescent leukemia stem cells is due to transcriptional up-regulation of the cell cycle inhibitor p21 by leukemia-associated fusion proteins.

Lewis Cantley (Harvard Institutes of Medicine, Boston, MA) discussed phosphoinositide 3-kinase (PI3K) as a central enzyme in the network controlling cancer cell growth. Activating mutations in the gene encoding the p110α PI3K subunit, PIK3CA, and loss-of-function PTEN mutations are the most common PI3K-related events in solid tumors. Mouse tumors developing due to mutated forms of PIK3CA or activated AKT expression display increased expression of genes involved in glucose uptake and metabolism. A role for PI3K in angiogenesis was discussed. All embryos with class Ia PI3K genes deleted in the endothelial compartment died on day 10 to day 11 with extensive bleeding into various tissues when the embryo exhibited its first heartbeat. In mice with 70% PI3K reduction, only small microvessels were seen in B16 melanoma cells growing in Matrigel plugs, and their growth was greatly reduced. The neovasculature formed in the Matrigel plugs is more porous to small molecules indicating a defect in tight junction formation.

Alberto Bardelli (Institute for Cancer Research and Treatment, University of Torino Medical School, Turin, Italy) investigated the influence of cancer mutations on the outcome of targeted therapies. Efficacy of the anti–epidermal growth factor receptor monoclonal antibody cetuximab was negatively correlated with the occurrence of KRAS or BRAF mutations. This allows the identification of colorectal cancer patients eligible for treatment with cetuximab. Cellular models of tumor progression have also been developed by introducing cancer mutations in the genome of human cells using homologous recombination. Profiling targeted drugs on the mutated cells showed “oncogene addiction” or resistance phenotypes. These results indicate new avenues for personalized therapies based on the genetic milieu of individual tumors.

William Sellers (Novartis Institute for Biomedical Research, Cambridge, MA) discussed the development of new anticancer agents targeting the PI3K/mammalian target of rapamycin (mTOR) pathway, which is frequently activated constitutively in human cancers. RAD001 is a mTOR inhibitor; NVP-BEZ235 and NVP-BGT226 are dual PI3K/mTOR inhibitors. mTOR is a regulator of HIF. AKT constitutive deregulation induces mTOR-dependent mouse prostate intraepithelial neoplasia. RAD001 treatment led to the elimination of prostate intraepithelial neoplasia. Renal carcinoma cells lacking an intact VHL gene are dependent on HIF2 for tumorigenic potential, and recently, RAD001 has shown therapeutic activity in renal cell carcinoma. PI3K inhibitors have entered phase I clinical trials. PIK3CA activating mutations are frequent in breast and colorectal cancers. Application of the imidazoquinoline NVP-BEZ235 to cells lacking an intact PTEN gene results in the dephosphorylation of AKT and AKT substrates including GSK3β and FOXOs. TSC1-null mouse embryonic fibroblasts have constitutively phosphorylated ribosomal S6 protein and lack activated AKT through a mTOR-dependent feedback mechanism. In these cells, BEZ235 inhibits S6 phosphorylation and activation. NVP-BEZ235 has antitumor activity in xenograft models, promotes regression of PIK3CA-dependent indigenous lung tumors in genetically engineered animals, and has significant antiangiogenic properties.

Stefano Piccolo (University of Padova, Padova, Italy) outlined the ubiquitin-mediated control of TGFβ signaling. TGFβ receptors and Smad ubiquitination keep TGFβ responsiveness under control and regulate signal intensity and duration. Several E3 ubiquitin ligases that influence TGFβ signaling have been identified. Ubiquitination can also regulate target protein function, subcellular localization/endocytic trafficking, protein-protein interactions, and indigenous target protein activity. Smad4 ubiquitination leads to its nuclear exclusion. Regulative ubiquitination is reversible, as indicated by the existence of a whole family of ubiquitin proteases in the genome.

Tadatsugu Taniguchi (University of Tokyo, Tokyo, Japan) discussed IFN regulatory factors (IRF), a family of nine transcription factors originally identified for their IFN gene regulation, which have been recently found to link immune responses to invading pathogens and suppression of tumor development. IRF1 regulatory functions include natural killer and CD4 cell differentiation. IRF1 responds to DNA damage and contributes to tumor suppression via p53 induction. IRF family members like IRF3, IRF5, and IRF7 play key roles in Toll-like receptor signaling. IRF5 induces, through Toll-like receptor signaling, IL-6, IL-12, tumor necrosis factor α, and IFNα/β. IRF5 also induces apoptosis, but not during cell cycle arrest, in response to DNA damage and has tumor suppressor functions through pathways that may be distinct from that of p53. IRF5 also plays a role in the Fas death receptor signaling.

Growing knowledge of the regulation of cancer cell behavior and increased understanding of how cancer cells interact 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 properties of primary cancer cells represents a starting point toward the development of individualized, mechanism-driven patient treatment. Efforts toward achieving this goal represent an important direction in cancer therapeutics.

No potential conflicts of interest were disclosed.

©2009 American Association for Cancer Research.
2009