Katie E. Hebron

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.

Macrophage deficiency of Akt2 reduces atherosclerosis in Ldlr null mice

Macrophages play crucial roles in the formation of atherosclerotic lesions. Akt, a serine/threonine protein kinase B, is vital for cell proliferation, migration, and survival. Macrophages express three Akt isoforms, Akt1, Akt2, and Akt3, but the roles of Akt1 and Akt2 in atherosclerosis in vivo remain unclear. To dissect the impact of macrophage Akt1 and Akt2 on early atherosclerosis, we generated mice with hematopoietic deficiency of Akt1 or Akt2. After 8 weeks on Western diet, Ldlr−/− mice reconstituted with Akt1−/− fetal liver cells (Akt1−/−→Ldlr−/−) had similar atherosclerotic lesion areas compared with control mice transplanted with WT cells (WT→Ldlr−/−). In contrast, Akt2−/−→Ldlr−/− mice had dramatically reduced atherosclerotic lesions compared with WT→Ldlr−/− mice of both genders. Similarly, in the setting of advanced atherosclerotic lesions, Akt2−/−→Ldlr−/− mice had smaller aortic lesions compared with WT→Ldlr−/− and Akt1−/−→Ldlr−/− mice. Importantly, Akt2−/−→Ldlr−/− mice had reduced numbers of proinflammatory blood monocytes expressing Ly-6Chi and chemokine C-C motif receptor 2. Peritoneal macrophages isolated from Akt2−/− mice were skewed toward an M2 phenotype and showed decreased expression of proinflammatory genes and reduced cell migration. Our data demonstrate that loss of Akt2 suppresses the ability of macrophages to undergo M1 polarization reducing both early and advanced atherosclerosis.

CD318 is a ligand for CD6

Significance The CD6 T cell surface glycoprotein regulates T cell activation, and CD6 is a risk gene for autoimmune diseases including multiple sclerosis (MS). Moreover, recent work indicates that CD6 is an attractive target for the development of new therapeutic approaches to autoimmune diseases such as MS. The known ligand of CD6 is CD166 (also termed ALCAM), but CD6–CD166 interactions neither explain CD6-dependent interactions with stromal cell lineages that are critical in organ-targeted autoimmune diseases nor account for effects of CD6-targeted therapeutics in autoimmune diseases. This report definitively establishes CD318 as a second ligand of CD6 and provides evidence for the importance of CD6–CD318 interactions in autoimmune diseases that affect the central nervous system and the synovial lining of joints. It has been proposed that CD6, an important regulator of T cells, functions by interacting with its currently identified ligand, CD166, but studies performed during the treatment of autoimmune conditions suggest that the CD6–CD166 interaction might not account for important functions of CD6 in autoimmune diseases. The antigen recognized by mAb 3A11 has been proposed as a new CD6 ligand distinct from CD166, yet the identity of it is hitherto unknown. We have identified this CD6 ligand as CD318, a cell surface protein previously found to be present on various epithelial cells and many tumor cells. We found that, like CD6 knockout (KO) mice, CD318 KO mice are also protected in experimental autoimmune encephalomyelitis. In humans, we found that CD318 is highly expressed in synovial tissues and participates in CD6-dependent adhesion of T cells to synovial fibroblasts. In addition, soluble CD318 is chemoattractive to T cells and levels of soluble CD318 are selectively and significantly elevated in the synovial fluid from patients with rheumatoid arthritis and juvenile inflammatory arthritis. These results establish CD318 as a ligand of CD6 and a potential target for the diagnosis and treatment of autoimmune diseases such as multiple sclerosis and inflammatory arthritis.

TRIzol and Alu qPCR-based quantification of metastatic seeding within the skeleton.

Current methods for detecting disseminated tumor cells in the skeleton are limited by expense and technical complexity. We describe a simple and inexpensive method to quantify, with single cell sensitivity, human metastatic cancer in the mouse skeleton, concurrently with host gene expression, using TRIzol-based DNA/RNA extraction and Alu sequence qPCR amplification. This approach enables precise quantification of tumor cells and corresponding host gene expression during metastatic colonization in xenograft models.

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

Alternative splicing of ALCAM enables tunable regulation of cell-cell adhesion through differential proteolysis

While many adhesion receptors are known to influence tumor progression, the mechanisms by which they dynamically regulate cell-cell adhesion remain elusive. We previously identified Activated Leukocyte Cell Adhesion Molecule (ALCAM) as a clinically relevant driver of metastasis and hypothesized that a tunable mechanism of ectodomain shedding regulates its contribution to dissemination. To test this hypothesis, we examined an under-explored ALCAM splice variant (ALCAM-Iso2) and demonstrated that loss of the membrane-proximal region of ALCAM (exon 13) increased metastasis four-fold. Mechanistic studies identified a novel MMP14-dependent membrane distal cleavage site in ALCAM-Iso2, which mediated a ten-fold increase in shedding, thereby decreasing cellular cohesion. Importantly, the loss of cohesion is not limited to the cell capable of shedding because the released extracellular domain diminished cohesion of non-shedding cells through disruption of ALCAM-ALCAM interactions. ALCAM-Iso2-dominated expression in bladder cancer tissue, compared to normal bladder, further emphasizes that ALCAM alternative splicing may contribute to clinical disease progression. The requirement for both the loss of exon 13 and the gain of metalloprotease activity suggests that ALCAM shedding and concomitant regulation of tumor cell adhesion is a locally tunable process.

Epithelial-Mesenchymal Transition Induces Podocalyxin to Promote Extravasation via Ezrin Signaling

SUMMARY The epithelial-mesenchymal transition (EMT) endows carcinoma cells with traits needed to complete many of the steps leading to metastasis formation, but its contributions specifically to the late step of extravasation remain understudied. We find that breast cancer cells that have undergone an EMT extravasate more efficiently from blood vessels both in vitro and in vivo. Analysis of gene expression changes associated with the EMT program led to the identification of an EMTinduced cell-surface protein, podocalyxin (PODXL), as a key mediator of extravasation in mesenchymal breast and pancreatic carcinoma cells. PODXL promotes extravasation through direct interaction of its intracellular domain with the cytoskeletal linker protein ezrin. Ezrin proceeds to establish dorsal cortical polarity, enabling the transition of cancer cells from a non-polarized, rounded cell morphology to an invasive extravasation-competent shape. Hence, the EMT program can directly enhance the efficiency of extravasation and subsequent metastasis formation through a PODXL-ezrin signaling axis.

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 4958: Fluorescent barcoding offers increased dimensionality in tracking tumor cells in vitro and in vivo

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Fluorescent or luminescent imaging of tumors in animal models is a critical element in dynamic and longitudinal monitoring of tumor burden, tumor growth, and metastatic dissemination. Currently, most imaging strategies are limited to one or two different colors per animal. To improve utility of current imaging technologies, we have developed a library of fluorescent and luminescent tracking vectors designed to expand the color dimensions possible within a single model through a multi-label approach to fluorescent barcoding. The library was built using Multisite Gateway® Cloning technology, which offers a fast and simple recombination-based cloning approach that allows for efficient expression of several genes driven by one promoter on a single vector backbone. Our lentiviral-based vectors express firefly luciferase and one of seven spectrally unique fluorescent proteins covering the entire fluorescent protein color palette from blue to far-red. We have stably labeled bone-derived MDA-MB-231 cells with the vectors by viral transduction. Using these individually colored cell lines, we have demonstrated that the seven FPs are uniquely identifiable by spectral un-mixing with the Maestro Q Imaging System and by flow cytometry. The combination of luciferase and fluorescent labeling allows us to monitor tumor growth by luciferase activity and distinguish individual cell populations by their fluorescent label. We are currently verifying that the cells retain stable expression of the vectors in mouse models; preliminary results are promising. In the future, this system will be used to develop an elegant multi-label-based fluorescent barcoding strategy that will allow us to identify individual cell populations from a heterogeneous environment in vivo. By exploiting combinations of the seven fluorescent proteins, we could potentially create over 200 uniquely identifiable cells populations. This ability will not only reduce the number of animals necessary per experiment (as control and several experimental populations can be individually analyzed in a single mouse); but will also allow us to mimic the heterogeneous environment common to all human tumors. Citation Format: Katie E. Hebron, Tatiana Ketova, Shanna Arnold, H. Charles Manning, Julie Sterling, Florent Elefteriou, Andries Zijsltra. Fluorescent barcoding offers increased dimensionality in tracking tumor cells in vitro and in vivo. [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 4958. doi:10.1158/1538-7445.AM2014-4958