Metastasis Research Society–American Association for Cancer Research Joint Conference on Metastasis

Introduction

By analogy, the study of metastasis is like a group of blind people studying an elephant. Each describes the pachyderm based on the part (s)he touches, but none comprehends the whole elephant because his/her exposure is limited. Likewise, the complexity of metastasis can only be appreciated when one either steps back from a specialized area and examines the landscape or communicates with other researchers studying other aspects of the process. Biology provides only correlative mechanistic insight, whereas molecular biology and biochemistry are meaningless when disconnected from the biology. Knowledge from all disciplines is of limited value if it cannot be translated into clinical practice. So, nearly 500 scientists and clinicians gathered in Vancouver, British Columbia, for a jointly sponsored conference on cancer metastasis from August 3 to 7, 2008.

The metastasis field has reached an important crossroads. The conference was less focused upon reflection of past progress than outlining directions for future research and translation of new findings into clinical practice. The meeting did not generate formal recommendations; rather, it focused upon emerging common themes.

Joán Massagué (Memorial Sloan Kettering Cancer Center, New York, NY), keynote speaker, presented data highlighting how cancer development and progression are regulated by three classes of genes—initiating (cancer-causing), progression, and virulence (aggressiveness; ref. 1). His deconstruction of the metastatic process reinforced how metastasis is not equivalent to tumorigenicity. Moreover, he showed how altered expression of these genes is affected by environmental signals to which cancer cells are exposed.

Dan Welch (University of Alabama-Birmingham, Birmingham, AL), recipient of the 2008 Metastasis Research Society's Paget-Ewing Award, then followed with a lecture entitled “The ‘M’ Word,” which offered an historical overview of major findings in metastasis research in alliterative terms. The mobilization of cells virtually mocks those trying to treat cancer. But manipulable models have allowed discovery of microenvironmental influences on metastasis-regulatory genes. Recent findings have introduced the possibility of managing metastases until cure is possible (2).

The two opening lectures summarized overarching themes. First, metastasis is no longer an incomprehensible black box. Mechanistic insights are providing targets for therapeutic intervention. But, there is still urgent need for more and improved models. Second, context is critical. Tumor cell–tumor cell and tumor-stromal interactions control cellular behaviors involved throughout the metastatic cascade. Third, cures will require input from experts in multiple disciplines, identification of common patterns used by other organisms and cells, and dialog with oncologists.

Lost and Found in Translation

In recent years, several metastasis promoting and suppressing genes have been identified (3, 4). Rik Thompson (St. Vincent's Institute, Fitzroy, Australia) developed an epithelial-to-mesenchymal transition (EMT; ref. 5) classifier signature that is up-regulated in invasive human breast tumors. Interestingly, a proportion of cells also shared markers with breast cancer stem cells, suggesting that expression of EMT-associated proteins may confer a survival advantage.

microRNA (miR) is an abundant class of small nonprotein-coding RNA that control gene and protein expression (6, 7). miR can be tumor suppressing, oncogenic (8), prometastatic (9, 10), or antimetastatic (11). Doug Hurst (University of Alabama-Birmingham, Birmingham, AL) and Dan Welch reported that transduction of miR-146 (which is up-regulated by the BRMS1 metastasis suppressor; ref. 12) into MDA-MB-231 cells inhibited migration and invasion in vitro as well as metastasis in vivo. They further showed that BRMS1 down-regulated known metastasis-promoting miR and up-regulated other metastasis-suppressive miR. Dawn Cochrane (University of Colorado at Denver, Aurora, CO) showed that miR-200c repressed ZEB1, with concomitant restoration of E-cadherin expression, suggesting that miR-200c may be involved in EMT. The clinical promise of miR was discussed by Eric Marcusson (Regulus Therapeutics, Carlsbad, CA), who reported on strategies to target three proinvasive, potentially prometastatic miR (miR-21, miR-10b, and miR-122) in glioma, breast cancer, and liver carcinoma, respectively.

Some miR act by inhibiting protein translation (6, 13). Coupled with emerging data that metastasis regulatory genes can regulate miR and eukaryotic translation initiation factor 4E (eIF4E), the rate-limiting protein in mRNA translation, Nahum Sonenberg (McGill University, Montreal, Canada) reported that overexpression of eIF4E enhanced its ability to unwind mRNA with high order secondary structures, resulting in increased translation of messages (14). Jeremy Graff and colleagues (Eli Lilly & Co., Indianapolis, IN) sought to exploit this finding as a potential therapeutic. Using eIF4E-specific antisense oligonucleotides, Graff and colleagues (15, 16) found significantly reduced tumor growth of human tumor xenografts with no significant host toxicity, although the oligonucleotides could also target murine eIF4E. Clinical trials have already begun.

Jong-Heun Lee and Pat Steeg (National Cancer Institute, Bethesda, MD) supported potential roles of mRNA processing and translation in metastasis control via differential regulation of two genes, Edg2 and Gemin5 (17, 18), by the metastasis suppressor Nm23-H1. The link is that Gemin5 is involved in ribonucleoprotein complex assembly (19).

Tumor Cells in Isolation Do Not Make Metastases

Metastatic cell behavior is determined by the context(s) in which cells find themselves. Tumor-tumor and tumor-stromal crosstalk, whether local or long distance, is increasingly recognized as key to controlling invasion and metastasis (20). Unfortunately, myriad cell types and molecules along with synchronous and asynchronous signaling complicates microenvironmental studies. Nonetheless, some common themes are emerging in the signaling realm.

Bruce Zetter (Children's Hospital, Boston MA) showed that high levels of antizyme—which negatively regulates the secretion of the polyamine, spermine—correlated with prostate cancer aggressiveness and inhibited prostate cancer cell growth. Polyamines are ubiquitous signaling molecules and play important roles in chromatin structure (14). Previous studies have shown that inhibition of polyamine synthesis can alter cancer metastasis (21–23).

Marsha Rosner (University of Chicago, Chicago, IL) detailed how Raf kinase inhibitor protein, a metastasis suppressor (24), acts as a tumor suppressor by regulating the spindle checkpoint and enhances expression of other tumor suppressor molecules such as the miR, Let-7. Her studies highlighted how miR expression may be context-dependent.

Invasion has long been recognized as a necessary step in metastasis, occurring through a variety of means. Erik Sahai (Cancer Research UK, London, England) showed, using elegant intravital microscopy (25), that cancer cell motility is diverse, with low motility in cohesive/more differentiated areas and high motility in less-adhesive/less-differentiated areas. Motility of single cells was dependent on nuclear Smad2 activation by transforming growth factor (TGF)β1. Because TGFβ family molecules are implicated in EMT and are differentially expressed in various tissues (26), cellular location determines relative motility and mechanisms of/for tumor cell movement.

Although proteases, including the matrix metalloproteinases (MMP), are known to contribute to invasion through matrix barriers, relatively little attention has been paid to quantifying enzyme activity in vivo. Lynn Matrisian and colleagues (Vanderbilt University, Nashville, TN) used quenched beacons that fluoresce upon selective processing by MMPs, to address this issue. Although molecules to study in vivo activation are still being perfected, the current generation clearly shows tumor cell and stromal cell derivation of activated MMPs (27). In addition, proteases contribute to metastasis by releasing and activating matrix-bound growth factors, such as TGFβ.

Tensional forces of the matrix on tumor cells change as desmoplasia occurs or as tumor cells degrade matrices. Valerie Weaver (University of California, San Francisco, CA) provided evidence that matrix rigidity changes with concomitant alterations in signaling (28, 29). In collaboration with Janine Erler (Institute of Cancer Research, London, England), Weaver and colleagues showed that tumor-derived lysyl oxidase, which promotes collagen cross-linking, also prepares the “soil” for future seeding of bone marrow-derived cells (BMDC) and other cancer cells.

David Lyden and Selena Granitto (Weill Cornell Medical College, New York, NY) presented data indicating that mobilization of VEGFR1/2⁺ BMDC to distant sites predispose the latter to metastasis development (30). This so-called “premetastatic niche” includes retained BMDC, restructured matrix, and altered growth factor milieu. Interestingly, they also found that LYVE-1⁺ BMDC surrounding the invasive edges of tumors. VEGFR1 colocalized with LYVE-1–expressing lymph vessels, suggesting involvement of progenitor cells in lymphangiogenesis. Using intravital lymphangiography, Dai Fukumura (Harvard Medical School, Boston, MA) elaborated on published results that VEGF-C–mediated cancer cell dissemination through lymphatic vessels to lymph nodes (31) could be inhibited by an anti-VEGFR3 antibody, AZD2171 (tyrosine kinase inhibitor), or through inhibition of nitric oxide synthase(s). Involvement of hematopoietic and mesenchymal stem cells was further supported by findings from Makoto Taketo's (Kyoto University, Kyoto, Japan) laboratory. Immature myeloid cells (CD34⁺ CCR1⁺) accumulate at invasive fronts, tracking tumor cells expressing the chemokine, CCL9 (32). Taken together, BMDC seem to both prime the premetastatic niche, promote local invasion, and regulate tumor vascularity.

Taking advantage of neural stem cells’ ability to home to sites of brain injury, Brunhilde Felding-Habermann (Scripps Research Institute, La Jolla, CA) proposed a Trojan horse approach to treat brain metastases. The delivery system could overcome restricted drug bioavailability due to the blood-brain barrier. However, Pat Steeg and Diane Palmieri (National Cancer Institute, Bethesda, MD) presented data from their collaborator, Quentin Smith (Texas Tech University, Lubbock, TX), showing unexpected findings that the blood-brain barrier is not as restrictive as originally believed. These same investigators are addressing the increasing problem of brain metastasis associated with breast cancer patients receiving trastuzumab.⁸ Using an updated model of brain metastasis, efficacy of the orally available tyrosine kinase inhibitor of EGFR and HER2, Lapatinib (33), or a broad-spectrum histone deacetylase inhibitor, vorinostat, was shown. However, due to the heterogeneity of individual brain metastases, it is likely that a “toolbox” of drugs will be necessary for more complete treatment.

Translating Laboratory Findings to the Clinic and Vice Versa

With the emphasis on targeting invasion during the 1980s and 1990s, MMP inhibitors rapidly proceeded into clinical trials. Failure of MMP inhibitors forced re-evaluation of which steps in metastasis are valid targets and how to assess better the efficacy in clinical trials. A discussion of translating basic research to the clinic was led by Pat Steeg and George Sledge (Indiana University Cancer Center, Indianapolis, IN). Several key questions were raised in Dr. Sledge's presentation on how to design an antimetastatic clinical trial: What is (are) a reasonable targets? Are we targeting the untreatable? Will we eradicate metastases or delay progression? Which tumor types are most amenable to cost-effective clinical trials? How will antimetastatic therapies be used in an adjuvant setting?

Despite the fact that many of these questions remain incompletely answered, preclinical studies targeting metastasis were reported (some reviewed in ref. 34). Saburo Sone (University of Tokushima, Tokushima, Japan) used a small molecule multikinase inhibitor, E7080, to inhibit mesothelioma. Charles Hart (Threshold Pharmaceuticals, Redwood City, CA) provided data for a hypoxia-activated prodrug, TH-302, which cross-links DNA when activated. Anil Bagri and colleagues (Genentech, South San Francisco, CA) developed functional blocking antibodies against neuropilin 1 and 2 to be used against vascular remodeling and lymphangiogenesis, respectively (35). Dan Theodorescu (University of Virginia, Charlottesville, VA) explained how the study of the metastasis suppressor, Rho GDP dissociation inhibitor 2 (36), has been useful for understanding translation of known drugs to the clinic.

One of the limiting factors to advancement of metastasis research has been the paucity of clinical material. Quite simply, metastatic tissue (especially matched with primary tumor material from the same patient) has not been routinely collected. Saraswati Sukumar (Johns Hopkins University, Baltimore, MD) reported on a rapid autopsy program that has been useful for comparing metastases with the primary tumor at the molecular level (37). She further showed that metastases are heterogeneous, suggesting that any particular targeted therapy will not be effective against all metastases.

Marie-France Poupon (Institut Curie, Paris, France) has generated at least 30 new xenograft models of various subtypes of human breast cancer after direct s.c. implantation of the tumor tissue into immunocompromised mice (38). Xenografts could be passaged in mice without loss of tumor architecture and are being used to determine responses to existing and new chemotherapies.

New Paradigms: Looking at Metastasis through New Lenses

To understand metastasis at the cellular and molecular levels, new models, novel methods, and new views are necessary. In Meet-the-Expert sessions, Diane Palmieri, Pnina Brodt (McGill University Health Centre, Montréal, QC, Canada) and Steven Gallinger (University of Toronto, Toronto, ON, Canada), and Andrea Mastro (The Pennsylvania State University, University Park, PA), and Evan Keller (University of Michigan, Ann Arbor, MI) discussed the need for better models to study brain metastases, liver metastases, and bone metastases, respectively. Mastro and Keller described their work with breast and prostate cancer metastasis to bone, respectively. Venkatesh Krishnan (The Pennsylvania State University, University Park, PA) presented a new in vitro model of breast cancer colonization of bone in a novel three-dimensional bioreactor in which bone cells could be grown for extended times (39). Metastatic MDA-MB-231 breast carcinoma cells not only penetrated the bone-like material but invaded in a manner similar to that observed in biopsies.

Melody Swartz (Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland) discussed the active role of the lymphatic system and autocrine chemokine gradients during invasion and metastasis (40, 41). Elena Deryugina (Scripps Research Institute, La Jolla, CA) reported an antiapoptotic role for CUB domain containing protein-1/subtractive immunization metastasis-associated 135-kDa protein, a molecule that was discovered using a screen for metastasis-blocking monoclonal antibodies (42, 43).

Sometimes it is important to step away to see big things more clearly. In an “out of the box” session, Carrie Rinker-Schaeffer (University of Chicago, Chicago, IL) considered the concept of bacterial quorum sensing (societal interactions) and how those principles could be applied to the study of metastasis (recently reviewed in ref. 44). Michael Federle (Princeton University, Princeton, NJ; ref. 45) and Matthew Parsek (University of Washington School of Medicine, Seattle, WA; refs. 46, 47) presented how bacterial societies need cell-cell interactions as well as appropriate microenvironments and/or autoinducers to act as a colony, analogous to colonization of tissues by metastatic cells. The New Paradigms session introduced several different approaches and ideas as well as reminding everyone to consider new ways to collect data and evaluate old data.

Signaling: Information Flows Both Ways

For tumor cells to successfully metastasize, they must both interact with, and respond to, their environment. Harold Moses (Vanderbilt University, Nashville, TN) used a murine knockout of TGFβ type II receptor to show how TGFβ regulates the tumor microenvironment and tumor cell behavior (48, 49). Similarly, Yan Xu (Indiana University, Indianapolis, IN) showed metastasis suppression and promotion functions of ovarian cancer G protein coupled receptor-1 (50), depending on the presence or absence of macrophages, respectively. Susan Bellis (University of Alabama-Birmingham, Birmingham, AL) presented data showing the importance of the glycosylation status of the β1 integrin, which is selectively sialylated by ST6Gal-1 sialyltransferase, leading to enhanced adhesion and migration (51, 52). Robin Anderson (Peter MacCallum Cancer Centre, Melbourne, Australia), using a syngeneic mouse model of breast cancer, showed that 4T1 cell expression of βthree integrin promoted metastasis to bone and lung without affecting primary tumor growth. In each case, the modifications affected how metastatic tumor cells communicate with the local environment.

Several researchers emphasized the importance of the phosophoinositide pathway. Shoukat Dedhar (BC Cancer Research Centre, Vancouver, BC, Canada) used the 4T1 model to identify roles for integrin-linked kinase, which interacts with phosphatidylinositol (3,4,5) trisphosphate (53), in cancer progression. Daryll DeWald (Utah State University, Logan, UT) showed that phosphoinositide signaling was attenuated at multiple levels by BRMS1, leading to reduced metastasis. Suzanne Eccles (Institute for Cancer Research, Sutton, England) presented clinical data indicating the efficacy of inhibiting phospholipase C and phosphatidylinositol-3-OH kinase and, thus, the potential for anti-invasive and anti-metastatic therapy.

Detection of Dormant Cells: Looking for What Is Barely There

The final session, chaired by Menashe Bar-Eli (M. D. Anderson Cancer Center, Houston, TX) and Suzanne Eccles, focused on detection of disseminated cancer cells, and their potential to remain dormant or to form metastatic colonies. This session connected with data presented by Dan Welch in the opening session, who showed that reexpression of the KISS1 metastasis suppressor induces dormancy in tumor cells at ectopic, but not orthotopic, sites—i.e., metastatic cells transfected with KISS1 complete all the steps of metastasis except colonization at secondary sites (54). This property, he proposed, could be exploited to manage, even if not curing, disseminated disease. Similar observations were presented by Shin Akakura (Roswell Park Cancer Institute, Buffalo, NY), who showed that Src-suppressed protein kinase C substrate (55) suppressed macroscopic lung metastasis formation. Unfortunately, the molecular mechanisms for inducing dormancy by either molecule are not yet known.

David Tarin (Moores UCSD Cancer Center, La Jolla, CA) used microarrays to reinforce involvement of multiple genes in breast cancer metastasis (56). He reiterated the nonrandom patterns of metastatic spread of human breast cancers. Interestingly, descendants of dormant cells isolated from metastasis-free organs acquired the potential to disseminate to those same organs.

Klaus Pantel (University Medical Center Hamburg-Eppendorf, Hamburg, Germany) reported the results of large clinical studies focused on methods to enrich tumors cells from the blood or in bone marrow to identify biomarkers and tailor systemic therapy for individual patients (57). Pantel proposed how finding tumor cells in “staging areas” might be important in the future development of macroscopic lesions. Finally, Ann Chambers (London Regional Cancer Centre, London, Ontario, Canada) discussed a three-dimensional culture model that can be used to mimic the transition of metastatic cells from a quiescent to a proliferative state depending upon β1 integrin signaling to induce actin stress fiber formation (58). Dr. Chambers also showed that magnetic resonance imaging could be used to detect dormant cells within the brain by labeling them with iron nanoparticles that persist over time in nondividing cells.

Perspectives and Future Directions

In addition to the scientific advancements that reveal insights into the underlying mechanisms involved in metastasis, improvements in patient survival realized from prior research have introduced new problems. Not so long ago, diagnosis with cancer was unmentionable in polite company—cancer was often called “the ‘C’ word.” Fortunately, that stigma has decreased as clinical management of cancer has improved. However, patients with metastases are now marginalized because of “the ‘M’ word.” On a positive note, increasing proportions of patients live longer with metastatic disease. On the negative side, the social science of metastatic cancer is becoming an increasing issue. The Metastasis Research Society-American Association for Cancer Research Joint Conference on Metastasis was privileged to have advocates present who seek to prioritize metastasis research and funding. The advances in basic and clinical science suggest that the scientific and medical communities are poised to further improve survival and quality of life because of advanced understanding of the metastatic process.