Trenis D. Palmer

Targeting tumor cell motility to prevent metastasis.

Mortality and morbidity in patients with solid tumors invariably result from the disruption of normal biological function caused by disseminating tumor cells. Tumor cell migration is under intense investigation as the underlying cause of cancer metastasis. The need for tumor cell motility in the progression of metastasis has been established experimentally and is supported empirically by basic and clinical research implicating a large collection of migration-related genes. However, there are few clinical interventions designed to specifically target the motility of tumor cells and adjuvant therapy to specifically prevent cancer cell dissemination is severely limited. In an attempt to define motility targets suitable for treating metastasis, we have parsed the molecular determinants of tumor cell motility into five underlying principles including cell autonomous ability, soluble communication, cell-cell adhesion, cell-matrix adhesion, and integrating these determinants of migration on molecular scaffolds. The current challenge is to implement meaningful and sustainable inhibition of metastasis by developing clinically viable disruption of molecular targets that control these fundamental capabilities.

Lack of transforming growth factor-β signaling promotes collective cancer cell invasion through tumor-stromal crosstalk

IntroductionTransforming growth factor beta (TGF-β) has a dual role during tumor progression, initially as a suppressor and then as a promoter. Epithelial TGF-β signaling regulates fibroblast recruitment and activation. Concurrently, TGF-β signaling in stromal fibroblasts suppresses tumorigenesis in adjacent epithelia, while its ablation potentiates tumor formation. Much is known about the contribution of TGF-β signaling to tumorigenesis, yet the role of TGF-β in epithelial-stromal migration during tumor progression is poorly understood. We hypothesize that TGF-β is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion.MethodsFluorescently labeled murine mammary carcinoma cells, isolated from either MMTV-PyVmT transforming growth factor-beta receptor II knockout (TβRII KO) or TβRIIfl/fl control mice, were combined with mammary fibroblasts and xenografted onto the chicken embryo chorioallantoic membrane. These combinatorial xenografts were used as a model to study epithelial-stromal crosstalk. Intravital imaging of migration was monitored ex ovo, and metastasis was investigated in ovo. Epithelial RNA from in ovo tumors was isolated by laser capture microdissection and analyzed to identify gene expression changes in response to TGF-β signaling loss.ResultsIntravital microscopy of xenografts revealed that mammary fibroblasts promoted two migratory phenotypes dependent on epithelial TGF-β signaling: single cell/strand migration or collective migration. At epithelial-stromal boundaries, single cell/strand migration of TβRIIfl/fl carcinoma cells was characterized by expression of α-smooth muscle actin and vimentin, while collective migration of TβRII KO carcinoma cells was identified by E-cadherin+/p120+/β-catenin+ clusters. TβRII KO tumors also exhibited a twofold greater metastasis than TβRIIfl/fl tumors, attributed to enhanced extravasation ability. In TβRII KO tumor epithelium compared with TβRIIfl/fl epithelium, Igfbp4 and Tspan13 expression was upregulated while Col1α2, Bmp7, Gng11, Vcan, Tmeff1, and Dsc2 expression was downregulated. Immunoblotting and quantitative PCR analyses on cultured cells validated these targets and correlated Tmeff1 expression with disease progression of TGF-β-insensitive mammary cancer.ConclusionFibroblast-stimulated carcinoma cells utilize TGF-β signaling to drive single cell/strand migration but migrate collectively in the absence of TGF-β signaling. These migration patterns involve the signaling regulation of several epithelial-to-mesenchymal transition pathways. Our findings concerning TGF-β signaling in epithelial-stromal interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment.

ALCAM/CD166 Is a TGF-β–Responsive Marker and Functional Regulator of Prostate Cancer Metastasis to Bone

The dissemination of prostate cancer to bone is a common, incurable aspect of advanced disease. Prevention and treatment of this terminal phase of prostate cancer requires improved molecular understanding of the process as well as markers indicative of molecular progression. Through biochemical analyses and loss-of-function in vivo studies, we demonstrate that the cell adhesion molecule, activated leukocyte cell adhesion molecule (ALCAM), is actively shed from metastatic prostate cancer cells by the sheddase ADAM17 in response to TGF-β. Not only is this posttranslational modification of ALCAM a marker of prostate cancer progression, the molecule is also required for effective metastasis to bone. Biochemical analysis of prostate cancer cell lines reveals that ALCAM expression and shedding is elevated in response to TGF-β signaling. Both in vitro and in vivo shedding is mediated by ADAM17. Longitudinal analysis of circulating ALCAM in tumor-bearing mice revealed that shedding of tumor, but not host-derived ALCAM is elevated during growth of the cancer. Gene-specific knockdown of ALCAM in bone-metastatic PC3 cells greatly diminished both skeletal dissemination and tumor growth in bone. The reduced growth of ALCAM knockdown cells corresponded to an increase in apoptosis (caspase-3) and decreased proliferation (Ki67). Together, these data demonstrate that the ALCAM is both a functional regulator as well as marker of prostate cancer progression.

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Integrin-Free Tetraspanin CD151 Can Inhibit Tumor Cell Motility upon Clustering and Is a Clinical Indicator of Prostate Cancer Progression

Normal physiology relies on the organization of transmembrane proteins by molecular scaffolds, such as tetraspanins. Oncogenesis frequently involves changes in their organization or expression. The tetraspanin CD151 is thought to contribute to cancer progression through direct interaction with the laminin-binding integrins α3β1 and α6β1. However, this interaction cannot explain the ability of CD151 to control migration in the absence of these integrins or on non-laminin substrates. We demonstrate that CD151 can regulate tumor cell migration without direct integrin binding and that integrin-free CD151 (CD151(free)) correlates clinically with tumor progression and metastasis. Clustering CD151(free) through its integrin-binding domain promotes accumulation in areas of cell-cell contact, leading to enhanced adhesion and inhibition of tumor cell motility in vitro and in vivo. CD151(free) clustering is a strong regulator of motility even in the absence of α3 expression but requires PKCα, suggesting that CD151 can control migration independent of its integrin associations. The histologic detection of CD151(free) in prostate cancer correlates with poor patient outcome. When CD151(free) is present, patients are more likely to recur after radical prostatectomy and progression to metastatic disease is accelerated. Multivariable analysis identifies CD151(free) as an independent predictor of survival. Moreover, the detection of CD151(free) can stratify survival among patients with elevated prostate-specific antigen levels. Cumulatively, these studies demonstrate that a subpopulation of CD151 exists on the surface of tumor cells that can regulate migration independent of its integrin partner. The clinical correlation of CD151(free) with prostate cancer progression suggests that it may contribute to the disease and predict cancer progression.

Abstract B65: Proteolytic shedding of the cell adhesion molecule ALCAM generates a spatial and context-dependent heterogeneity important for metastasis.

Heterogeneity in the tumor microenvironment is dictated in part by context-dependent changes in cell-cell interactions. Subpopulations of tumor cells can responding to local micro-environmental changes in a manner that distinguishes them biochemically as well as behaviorally from the rest of the tumor. We identified such a phenomenon in the proteolytic shedding of the cell adhesion molecule ALCAM (Activated Leukocyte Cell Adhesion Molecule). ALCAM controls cell adhesion in a variety of cell types including T-cells, epithelial cells, neurons and endothelial cell. Although ALCAM mRNA is detected in all epithelial tissues and the tumors they give rise to, the detection of cell-surface ALCAM is highly variable, being strong in some regions of the tumor while completely absent in adjacent tumor cells. Through molecular analysis of cell-surface ALCAM we determined that this adhesion molecule is shed from the surface by ADAM17 in response to cytokines from the local milieu. Although ALCAM shedding is common in cancer, the loss of ALCAM greatly diminishes metastasis, thereby suggesting that ALCAM remains necessary for tumor progression. Indeed, ALCAM shedding supports a dynamic regulation of cell adhesion which, in turn, supports tumor cell dissemination. Considering that the proteolytic processing of ALCAM is functionally important in tumor progression, the detection of ALCAM shedding should be a molecular indicator of disease progression. Indeed, through analysis of retrospective patient cohorts, we demonstrated ALCAM shedding to be a prognostic indicator of disease progression in cancers of the colon, kidney and bladder. Our observations demonstrate that spatial and context-dependent heterogeneity can be accomplished through post-translational proteolysis of cell adhesion molecules. This process enables tumor cells to respond dynamically to the local milieu and disseminate to a more hospitable environment. Identifying this proteolysis in cancer patients through the detection of ALCAM shedding can predict metastatic progression and long-term patient outcome. Citation Format: Amanda G. Hansen, Katie Hebron, Trenis D. Palmer, Shanna Arnold, Andries Zijlstra. Proteolytic shedding of the cell adhesion molecule ALCAM generates a spatial and context-dependent heterogeneity important for metastasis.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B65. doi:10.1158/1538-7445.CHTME14-B65

Abstract 55: Separation of tetraspanin CD151 from its integrin partner α3β1 reflects an alter migratory state and predicts prostate cancer progression

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The dysregulation of cell migration enables tumor cells to escape their tissue of origin and disseminate. Since cancer-related deaths are primarily caused by the dissemination of tumor cells, mechanisms of migration are both a target for therapy and an indicator of disease progression. The regulation of cell adhesion is widely recognized as a rate-limiting step in metastasis but how tumor cells achieve dynamic control over their adhesion receptors is poorly understood. During an analysis of prostate cancer progression we discovered that α3β1 expression is reduced and that its tetraspanin partner, CD151, is not “integrin-free”. We were able to detect integrin-free CD151 using antibodies specific to the integrin-binding domain of CD151. Dual staining of tumor tissue and normal tissue from prostate cancer patients for total CD151 and integrin-free CD151 revealed that the appearance of integrin-free CD151 corresponds with poor-patient outcome. In fact, the detection of integrin-free CD151 is an independent predictor of prostate cancer progression. Surprisingly, the clustering of integrin-free CD151 immobilizes tumor cells in vivo and prevents metastasis suggesting that the ability of CD151 to control migration does not depend on its α3β1 integrin partner. Indeed, integrin-free CD151 is now associated with non-integrin partners through which it can regulate tumor cell motility. These observations demonstrate that the appearance of integrin-free CD151 reflects the disruption of the CD151/ α3β1/laminin axis and thereby reveals an altered migratory ability in tumor cells. This has clinical as well molecular implications. Integrin-free CD151 can be used as a molecular indicator of disease progression and assist in the distinction between indolent (benign) and advanced disease (Palmer et al. 2013). In addition, the identification of new CD151 partners can provide new therapeutic targets to inhibit the motility of tumor cells that have undergone this change in migratory status. A preliminary evaluation identified a similar appearance of integrin-free CD151 in cancers derived from other tissues, suggesting that this change in molecular status is broadly applicable to most solid tumors. Palmer, et al. (2013). Integrin-free tetraspanin CD151 can inhibit tumor cell motility upon clustering and is a clinical indicator of prostate cancer progression. Cancer Research. Citation Format: Trenis Palmer, Carlos Martinez, Catalina Vasquez, Katie Hebron, Shanna Arnold, Celestial Jones-Paris, Susanne Chan, Venu Chalasani, Jose Gomez-Lemus, Andrew Williams, Joseph Chin, Giovanna Giannico, Tatiana Ketova, John Lewis, Andries Zijlstra. Separation of tetraspanin CD151 from its integrin partner α3β1 reflects an alter migratory state and predicts prostate cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 55. doi:10.1158/1538-7445.AM2014-55

Abstract 4217: Engaging CD151 inhibits cell migration and metastasis through a novel mechanism involving the cell adhesion molecule ALCAM/CD166

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL We recently demonstrated that actively promoting the formation of cell surface complexes by engaging the tetraspanin CD151 with a monoclonal antibody (mAb 1A5) inhibits metastasis by immobilizing the tumor cells. Tetraspanins influence cellular behavior via the regulation of associated partner molecules such as integrins, cell adhesion molecules, and members of the immunoglobulin superfamily. Although CD151 is able to associate with and regulate the activity of laminin-binding integrins we demonstrate that CD151 does not associate with α3α1 in the presence of mAb 1A5. Using a 2-color flow cytometric approach we mapped the domains of CD151 required for antigen recognition by mAb 1A5 and determined that 1A5 maps to the integrin 194QRD196 motif of CD151, using this information we further performed CD151 co-immunoprecipitations and demonstrated that 1A5 recognizes CD151 not associated with α3α1.To determine the identity of the protein(s) associated with the antibody-engaged CD151 and also possibly responsible for the anti-migratory, anti-metastatic phenotype we used a proteomic approach. Using tandem mass spectrometry on CD151-associated proteins co-immunoprecipitated by mAb 1A5, we detected in excess of 200 proteins. This strategy identified several putative partners of CD151. Further analysis of the CD151 associated proteins led to our discovery of the novel CD151-associated protein ALCAM/CD166, a member of the immunoglobulin superfamily. Using an RNAi-mediated knockdown approach we demonstrate that ALCAM/CD166 is required for the CD151 complex to regulate tumor cell mobility in vitro. Conversely, CD151 is required for ALCAM to control motility. Spontaneous metastasis assays using the avian chorioallantoic membrane and subcutaneous murine xenografts demonstrate thatALCAM/CD166 is indeed required for the engaged tetraspanin to inhibit tumor cell dissemination in vivo. Further mechanistic analysis of this complex revealed that the CD151/ALCAM protein complex promotes activation of the small GTPase Rap1 via activated protein kinase C. Collectively, we present data, which demonstrate that, the ALCAM-containing CD151 complex can control metastasis. This novel complex reveals how cell-cell interactions can control migration through the regulation of cell-matrix adhesions. These results also demonstrate mechanistically that tetraspanins can integrate signaling between physically distinct molecular functions and therefore behave as a molecular clutch that controls matrix adhesion in response to cell-cell interactions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4217. doi:1538-7445.AM2012-4217

Abstract 4306: Lack of TGF-β signaling induces a switch from single to collective cell migration in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Transforming growth factor-beta (TGF-β) has a dual role during tumorigenesis initially as a suppressor and then as a promoter. In stromal fibroblasts, TGF-β signaling suppresses tumorigenesis in adjacent epithelia, while its ablation potentiates tumor formation. Concurrently, epithelial TGF-β signaling regulates fibroblast recruitment and activation. Yet, the role of TGF-β in stromal-epithelial migration during tumorigenesis is unknown. We hypothesize that TGF-β is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion. We are using the chicken embryo chorioallantoic membrane (CAM) model to study this interplay by grafting both breast cancer epithelial cells, isolated from either TGF-β receptor II knockout (TβRII-deficient) or TβRII floxed (control) mice, and fibroblasts onto the CAM. Intravital microscopy revealed fibroblast stimulation of either a single cell/strand migration of control cells or collective migration of TβRII-deficient cells, both along the vasculature. Epithelial clusters at the epithelial-stromal boundaries of TβRII-deficient tumors express E-cadherin, p120, and β-catenin, while control epithelia in these regions express αSMA. TβRII-deficient tumors also exhibit a two-fold greater local CAM metastasis than control tumors. Epithelial tumor RNA isolated by laser capture microdissection was analyzed on a qPCR array. In TβRII-deficient epithelium compared to control epithelium, we found upregulation of Igfbp4 and Tspan13 and downregulation of Col1α2, Bmp7, Wnt11, Gng11, Vcan, Tmeff1, and Dsc2. Western and qPCR analyses validated these targets in vitro. For current and future studies determining the relevance of these targets to migration patterning, we achieved Wnt11 overexpression in TβRII-deficient cells, as well as knockdown of Wnt11 and Tmeff1 in control cells. Our findings about TGF-β signaling in stromal-epithelial interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4306. doi:1538-7445.AM2012-4306