Day 1: Wednesday, September 23

Keynote Lectures

New Dimensions in Cell Migration and Matrix Interactions

Kenneth M Yamada, MD, PhD¹, Andrew D Doyle, PhD¹, Tomohiro Onodera, MD, PhD¹, Takayoshi Sakai, DDS, PhD^1,2, Jeff Chi-feng Hsu, PhD1 , Vira V Artym, PhD^1,3 , and Kazue Matsumoto^1
1Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland; ²Depertment of Oral-Facial Disorders, Osaka University Graduate School of Dentistry, Osaka, Japan; ³Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington DC

Cells migrate, tumors invade, and tissues remodel in three-dimensional (3D) environments. However, most mechanistic studies in cell biology are conducted using traditional tissue culture on flat, two-dimensional (2D) substrates. Recent studies from a number of laboratories show that many fundamental biological properties of cells are governed by dimensionality and other physical properties of the local environment. Cells interacting with extracellular matrices that are 2D versus 3D differ in cell morphology, signaling, modes of migration, and requirements for cell-surface integrins and proteases. Another notable feature of 3D matrices in vivo is that many are fibrillar rather than flat or uniform in texture. Interestingly, "1D" fibrillar lines on a flat surface can mimic many of the properties of a cell-derived 3D fibrillar matrix in terms of cell morphology, migration mode, and even Golgi and centrosome orientation. The requirement for proteases in tumor cell invasion can also differ depending on the type of matrix. These studies emphasize the importance of choosing one's model system carefully, because mechanistic conclusions can differ significantly depending upon dimensionality and other physical properties. In fact, for some studies, a 1D system can provide more accurate physiological insights into 3D biology than regular 2D tissue culture.

The spatial arrangement and dynamics of 3D cell and tissue movements are particularly important in tissue remodeling processes involved in embryonic development, cancer, and tissue engineering. 3D culture and direct imaging of cells undergoing tissue remodeling in organ formation have revealed roles for integrins, fibronectin, and cell motility. Organ morphogenesis, as well as 3D in vitro reconstitution of the initial steps of this process starting from single cells, involves cell-surface interactions dependent on integrins, E-cadherin, and dramatic epithelial cell movements. The mechanisms that lead from undifferentiated, amorphous collections of cells to a complex branched structure in organ development involve cell-matrix interaction, novel regulatory molecules such as Cleftin, and coordinated effects on cell-cell and cell-matrix adhesion systems associated with transient epithelial cell motility. These applications of 3D model systems, combined with live-tissue imaging, should continue to provide new insights into the mechanisms of cell migration, invasion, and tissue remodeling.

Rho GTPase Signaling in Migration and Morphogenesis

Alan Hall¹, Joanne Durgan¹, Aron Jaffe^1,2 and Noriko Kaji¹. ¹Memorial Sloan-Kettering Cancer Center, Cell Biology Program, 1275 York Avenue, New York, NY 10065. ²Current address: Novartis Institutes for Biomedical Research, Inc., 250 Massachusetts Avenue, Cambridge, MA

Rho family GTPases control signal transduction pathways that regulate the assembly and spatial organization of the actin cytoskeleton and associated cell-matrix and cell-cell adhesion complexes. Rho regulates contractile actin:myosin filament and focal adhesion assembly, Rac regulates the formation of membrane protrusions (lamellipodia), while Cdc42 triggers filopodial extensions at the cell periphery. In addition, Rho GTPases promote a diverse set of other cellular activities, including changes in gene transcription, the establishment of cell polarity, cell cycle progression, the organization and dynamics of the microtubule cytoskeleton and the activation of a variety of enzymes including PI 3-kinase and NADPH oxidases. The actin and microtubule cytoskeletons play a central role in driving many of the dynamic aspects of cell behavior and our particular focus is to determine the molecular mechanisms by which Rho GTPases regulate cell migration and cell morphogenesis. The disruption of normal tissue architecture and the appearance of inappropriate migratory activity are two defining characteristics associated with cancer progression towards an invasive and metastatic phenotype. Our long-term goal is to identify the mechanisms that drive these changes.

Day 3: Friday, September 25

Session III: Cancer

Cell Biology and Cancer, In Translation

Ira Mellman, PhD, Genentech, Inc., South San Francisco, CA

Cancer remains one of or most daunting health problems, and one of our most challenging scientific problems. Although decades of study have yielded a wealth of information concerning fundamental mechanisms, there has been only modest success at translating this information into effective therapies. There are many reasons for this, cancer’s inherent complexity and heterogeneity topping the list. Fortunately, we are beginning to see the introduction of a new generation of therapeutics based on our accumulated understanding. Yet using these agents effectively will require a comparably deep understanding of how they actually work to affect human cancer at the level of the patient, and at the level of cancer cell biology. Such an effort requires no “translation”, as it is directed precisely at understanding the mechanism of action of cancer therapy in human cancer.

Receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR, Her1) and Her2, are clinically validated targets in many important carcinomas, where either mutation or overexpression of wild type receptor can contribute to oncogenesis. One therapeutic approach has been to generate inhibitory monoclonal antibodies, with two such agents (Erbitux® and Vectabix®, neither a Genentech product) currently in use in cancers bearing alterations in EGFR. EGFR signaling is preceded by dimerization of the receptor, an event triggered by a conformational change in the receptor’s extracellular domain due to ligand binding. The alteration exposes a “dimerization arm” from domain II, although recent studies have suggested that dimerization reflects the ligand-dependent interaction of a membrane proximal region of the receptor’s cytoplasmic domain. In either event, dimerization results in the receptor’s activation by autophosphorylation. We have now provided a precise picture of how EGFR activation occurs by developing a quantum dot-based approach to tracking single molecules in living cells. By studying time-dependent alterations in receptor diffusivity, we find that EGFR undergoes spontaneous and reversible dimerization, even in the absence of ligand. Although dimer formation was stabilized entirely by the receptor’s dimerization arm, dimerization did not lead to signaling. It did, however, prime the receptors for activation by enhancing ligand binding affinity and decreasing the lag time exhibited between ligand binding and phosphorylation or adapter recruitment. Remarkably, receptor dimers were polarized on the plasma membrane, with enrichment at peripheral sites high in actin. Thus, signal transduction itself was polarized, exhibiting another level of control exhibited by mitogenic ligands over cellular responses. We are currently using these approaches to understand how therapeutic antibodies to EGFR, as well as other RTKs, act to exhibit their effects.

The Cell Biology of PTEN/ AKT Regulation in Cancer

Muhan Chen¹, Jernej Murn¹, Christopher Pratt¹, Martha Zeeman¹, Audrey O’Neill², Charles L. Sawyers^3,5, William Gerald³, Carlos CordonCardo⁴, Alexandra C. Newton², Brett S. Carver³, and Lloyd C. Trotman^1
1Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, NY
²University of California San Diego, CA
³ Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY
⁴ Department of Pathology, Columbia University, New York, NY
⁵ Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY

The PTEN tumor suppressor is among the most frequently lost or mutated genes of human cancer. By reverting PI-3 Kinase activity, PTEN inhibits AKT kinase activation and thus antagonizes cell survival and proliferation. Through modeling of partial and complete Pten-loss a non-canonical dose-outcome relationship has emerged for this tumor suppressor in a variety of solid tumors as well as in primary cells: While partial Pten-loss initiates disease in prostate, its complete inactivation paradoxically blocks tumorigenesis through p53-dependent cellular senescence arrest, which thus constitutes a cell-intrinsic tumor defense mechanism. To circumvent this arrest, cancer cells use all facets of gene deregulation including Pten-degradation to proliferate in haploinsufficiency until the p53-response is lost. While these findings underscore the role of Pten regulation in cancer, we have recently uncovered an equally critical role for Akt signal termination, as Pp2a-mediated Akt dephosphorylation in the nucleus is essential for prevention of colon and prostate cancer after partial loss of Pten.

To advance our understanding of PI 3-Kinase signal termination and the role of its cellular localization in cancer, we are studying the second class of known phosphatases of AKT, namely the PHLPP genes. To this end, we follow prostate tumorigenesis in cohorts of Phlpp knockout mice, which have a normal or hyperactive PI 3-K pathway by virtue of heterozygosity for Pten. In this presentation I will focus on the consequences of this compound inactivation at the cellular and organismal levels and present their relevance to human prostate cancer.

Role of Shelterin in Cancer and Aging

Maria A. Blasco, Telomeres and Telomerase group, Spanish National Cancer Centre (CNIO), Madrid 28029, Spain

Shelterin is a highly conserved chromosome end-capping complex, which encompasses TRF1, TRF2, TPP1, POT1, TIN2 and RAP1. Although extensive cell culture studies suggest that shelterin is essential for telomere regulation, its role in telomere biology and disease in the context of the organism remained elusive due to lack of viable loss-of-function mouse models. I will report on mice and cells conditionally deleted for TRF1 and TPP1. Absence of TRF1 and TPP1 results in perinatal mortality, severe skin atrophy and hyperpigmentation, and widespread epithelial dysplasia, which are associated to induction of telomere-instigated DNA damage, activation of the p53/p21 and p16 pathways, and cell cycle arrest in vivo. Abrogation of the p53 and RB pathways in Trf1 and Tpp1-deleted mouse embryonic fibroblasts (MEF) bypasses senescence but results in widespread chromosomal instability concomitant with activation of the ATM/ATR DNA damage signalling pathways and phosphorylation of the downstream kinases Chk1 and Chk2. In vivo, p53 deficiency ameliorates mouse survival and leads to development of malignant squamous cell carcinomas in the skin of Trf1 and Tpp1 deficient mice, demonstrating a tumor suppressor activity of shelterin components. Finally, we show that the levels of some shelterin components decrease during physiological organismal aging. Together, these results show that dysfunction of telomere-associated proteins is sufficient to produce severe telomeric damage and loss of telomere capping in the absence of telomere shortening, resulting into premature tissue degeneration, acquisition of chromosomal aberrations and neoplastic lesions.

Cell Polarity Pathways as Regulators or Morphogenesis and Tumorigenesis

Senthil Muthuswamy, PhD, Ontario Cancer Institute, University of Toronto, Canada
Cold Spring Harbor Laboratory, NY

In mammals, the role polarity proteins play during tumorigenesis is not well understood. I will discuss our findings that demonstrate that Par6 polarity proteins function as downstream targets of oncogenes like ErbB2 and that disruption of a polarity protein Scribble cooperates with oncogenes during initiation of tumorigenesis. I will also discuss our recent findings on the role played by the polarity protein Scribble in regulating asymmetric cell division and epithelial differentiation of mammary progenitors. These findings identify polarity proteins as novel regulators of differentiation and tumorigenesis.

Invadopodia, Specialized Cell Structures for Cancer Invasion

Kevin M. Branch¹, Nelson R. Alexander¹, Aron Parekh¹, Scott A. Guelcher², Alissa M. Weaver¹
Departments of Cancer Biology¹ and Chemical Engineering², Vanderbilt University, Nashville, TN

Cells sense both physical and chemical signals from the external environment. Tissue density has been associated with enhanced risk of cancer formation and invasiveness; however the underlying basis is unclear. We investigated the effect of rigid and crosslinked extracellular matrix (ECM) substrates on the formation and function of invadopodia, specialized subcellular structures that degrade ECM. We find that invasive cancer cells are highly responsive to the physical microenvironment, with mechanotransduction signaling promoting invasiveness of cells. These data suggest a potential mechanism, via rigidity-induced invadopodia activity, for the reported correlation of tissue density with cancer aggressiveness.

Closing Keynotes

Guarding the Genome: Centromeres, Aneupoidy and Tumorigenesis

Don W. Cleveland, PhD, Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA

The basic element insuring chromosome inheritance, the centromere, is not specified by DNA sequence, but by an epigenetic mark. CENP-A, a histone variant that replaces H3 only at functional centromeres, is shown to confer a unique conformational rigidity to nucleosomes, providing the basis for such an epigenetic mark. Unattached centromeres are also responsible for the mitotic checkpoint, the major cell cycle control mechanism that acts to maintain diploid chromosome content. Prevention of premature onset of anaphase requires a set of components that act at centromeres to generate a diffusible “wait anaphase” inhibitor. The mitotic checkpoint can be weakened in mice by reduction of CENP-E, a highly processive, centromere-associated kinesin required for stable capture by each centromere of the correct number of spindle microtubules. Reduction in CENP-E produces near diploid aneuploidy from missegregation of whole chromosomes. Noting a correlation of aneuploidy with tumorigenesis, nearly 100 years ago Boveri proposed aneuploidy as a cause of tumorigenesis. Boveri’s hypothesis has now been tested, revealing that whole chromosomal aneuploidy can act oncogenically, but depending on the preceding genetic damage chromosomal instability can play a previously unsuspected role in preventing tumorigenesis.

Small RNA Mediated Epigenetic Mechanism in Stem Cell Self-Renewal

Haifan Lin, PhD, Yale University, New Haven, CT

Small non-coding RNAs have emerged as key players in epigenetic regulation. Recently, a novel class of small RNAs that interact with Piwi proteins has been discovered in the mammalian and Drosophila germline. These Piwi-interacting RNAs (piRNAs) represent a distinct small RNA pathway that is widely thought to function only in the germline. In this talk, I will review our recent work with our collaborators on the epigenetic function of the Drosophila Piwi protein and its associated piRNAs in somatic cells. This work has revealed a novel epigenetic mechanism mediated by Piwi and its associated piRNAs in somatic cells that might also be applicable to the germline. Based on these results, we propose a “Piwi-piRNA guidance hypothesis” for Piwi/piRNA-mediated epigenetic programming, in which the Piwi-piRNA complex serves as a sequence-recognition machinery that recruits epigenetic effectors such as Heterochromatin Protein 1a (HP1a) to specific sites in the genome to execute epigenetic regulation.

Deconstructing Metastasis

Joan Massagué, PhD, Cancer Biology and Genetics Program, HHMI, MSKCC, New York, NY

Metastasis is a multi-stage process that selects for circulating cancer cells that can infiltrate, survive in, and colonize distant organs. The cellular origin, oncogenic alterations, tissue affinities, and circulation patterns of tumors markedly influence the sites and temporal course of metastasis. Cancer cell dissemination may be followed by a protracted period of latency before relapse in one or more organs, as in breast cancer, or by a swift colonization of multiple organs, as in lung adenocarcinoma. We have sought to incorporate this varied biology into experimental models to deconstruct metastasis. Searching for mechanisms that prime breast cancer cells for infiltration of different organs, we identified several mediators of infiltration through the non-fenestrated walls of lung and brain capillaries (EGFR ligands, COX2, TGF-induced angiopoietin-like 4), and additional mediators (sialyl transferase ST6GalNac5 and others) for infiltration though brain capillaries with blood-brain barrier. Searching for mechanisms that allow infiltrated breast cancer cells to survive as latent disease, we found that a hyperactive SRC pathway supports the responsiveness of breast cancer cells to certain survival factors in the marrow microenvironment. Searching for mechanisms that in contrast underlie the rapid metastasis of lung adenocarcinomas to brain and bone, we uncovered an involvement of WNT/TCF signaling through HOXH9 and LEF1. These striking disparities in the natural progression of different cancers bring into focus important questions about the evolution of metastatic traits, the molecular mediators involved, and the treatment opportunities to prevent metastasis.