Luis Miguel Alonso-Colmenar (Microscopy and Cytometry Research Center; Faculty of Medicine; Complutense University of Madrid; Madrid, Spain) served as Guest Editor for this issue's special focus series on Eph/Ephrin in cancer.
β1 integrin: An emerging player in the modulation of tumorigenesis and response to therapy
Historically, a hallmark of tumorigenesis was the ability to grow in an anchorage-independent manner. Hence, tumors were thought to proliferate and survive independently of integrin attachment to the substratum. However, recent data suggest that integrins regulate not only tumor cell proliferation, survival and migration, but may also influence their response to anti-cancer agents. Interestingly, these influences are largely masked by growth of tumor cells in the standard, yet artificial, environment of 2D cell culture, but are readily apparent under 3D in vitro culture conditions and in tumor growth in vivo. We, and others, have recently demonstrated that the β1 integrin subunit controls the growth and invasion of prostate tumor cells in 3D culture conditions. Recently, the importance of integrins has also been demonstrated using tissue specific conditional knockout strategies in transgenic mouse tumor models, where they control primary tumor growth and dictate the site of metastatic spread. Furthermore, integrin-extracellular matrix interactions may modulate the response of tumors to standard chemotherapy agents or radiation. Taken together, these results highlight the important role of integrins in regulating tumor growth and metastasis; however, point out that the evaluation of their contribution to these processes requires appropriate contextual modeling.
Small heat shock proteins in cellular adhesion and migration: Evidence from Plasmodium genetics
Cellular locomotion and adhesion critically depend on regulated turnover of filamentous actin. Biochemical data from diverse model systems support a role for the family of small heat shock proteins (HSPBs) in microfilament regulation. The small chaperones could either act directly, through competition with the motor myosin, or indirectly, through modulation of actin depolymerizing factor/cofilin activity. However, a direct link between HSPBs and actin-based cellular motility remained to be established. In a recent experimental genetics study, we provided evidence for regulation of Plasmodium motility by HSPB6/Hsp20. The infectious forms of malaria parasites, termed sporozoites, display fast and continuous substrate-dependent motility, which is largely driven by turnover of actin microfilaments. Sporozoite gliding locomotion is essential to avoid destruction by host defense mechanisms and to ultimately reach a hepatocyte, the target cell, where to transform and replicate. Genetic ablation of Plasmodium HSP20 dramatically changed sporozoite speed and substrate adhesion, resulting in impaired natural malaria transmission. In this article, we discuss the function of Hsp20 in this fast-moving unicellular protozoan and implications for the roles of HSPBs in adhesion and migration of eukaryotic cells.
Repulsive guidance molecule A (RGMa): A molecule for all seasons
RGMa (repulsive guidance molecule a) was the first identified molecule that possessed the necessary functional activity to repulse and steer growth cones to their target in the brain. By binding to its neogenin receptor, RGMa caused the collapse of growth cones and encouraged axons to grow along specific trajectories in vitro. Although originally characterized in 1990, RGMa was not conclusively shown to mediate axon guidance in vivo for another 12 years. Loss-of-function analysis in mice revealed that RGMa may play a more important role in neural tube morphogenesis. RGMa has now emerged as a molecule with pleiotropic roles involving cell adhesion, cell migration, cell polarity and cell differentiation which together strongly influence early morphogenetic events as well as immune responses. RGMa can be regarded as a molecule for all seasons.
Altered patterns of filopodia production in CHO cells heterologously expressing zebra finch CB1 cannabinoid receptors
Recent findings indicate that cannabinoid-altered vocal development involves elevated densities of dendritic spines in a subset of brain regions involved in zebra finch song learning and production suggesting that cannabinoid receptor activation may regulate cell structure. Here we report that activation of zebra finch CB1 receptors (zfCB1, delivered by a lentivector to CHO cells) produces dose-dependent biphasic effects on the mean length of filopodia expressed: Low agonist concentrations (3 nM WIN55212-2) increase lengths while higher concentrations reduce them. In contrast, treatment of zfCB1-expressing cells with the antagonist/inverse agonist SR141716A causes increases in both mean filopodia length and number at 30 and 100 nM. These results demonstrate that CB1 receptor activation can differentially influence filiopodia elongation depending on dose, and demonstrate that manipulation of cannabinoid receptor activity is capable of modulating cell morphology.
Eph/ephrin signaling in cancer: Intricate, puzzling and ... fascinating!
The Eph receptor tyrosine kinases family and their membrane bound ligands, the ephrins, represents a complex signaling network of cell communication for cell sorting during tissue patterning in development and in the normal physiology and homeostasis of adult tissues. This molecular family has adapted to evolving tissue complexity in multicellular organisms through the emergence of more members and complex mechanisms of expression and signaling that result in the fine-tuning of cell positioning. Since their initial identification from an erythropoietin producing hepatocellular (Eph) carcinoma cell line in 1987, Eph/ephrin signaling has been a matter of intensive investigation for their plausible role in cancer. Similarly to their context dependent modus operandi in normal tissues, Eph/ephrin signaling in cancer is an intricate and puzzling network of events that tumors “manage” to their benefit in multiple aspects like cell adhesion to substrate, migration, invasion or growth.
Profiling Eph receptor expression in cells and tissues: A targeted mass spectrometry approach
The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression.
Irradiation affects cellular properties and Eph receptor expression in human melanoma cells
X-ray irradiation influences metastatic properties of tumor cells and, moreover, metastasis and cellular motility can be modified by members of the Eph receptor/ephrin family of receptor tyrosine kinases. We hypothesized that irradiation-induced changes in cellular properties relevant for metastasis in melanoma cells could be mediated by Eph receptor/ephrin signaling. In this pilot study, we analyzed one pre-metastatic (Mel-Juso) and three metastatic human melanoma (Mel-Juso-L3, A375, and A2058) cells lines and predominantly found anti-metastatic effects of X-ray irradiation with impaired cell growth, clonal growth and motility. Additionally, we observed an irradiation-induced increase in adhesion paralleled by a decrease in migration in Mel-Juso and Mel-Juso-L3 cells and, in part, also in A375 cells. We further demonstrate a decrease of EphA2 both in expression and activity at 7 d after irradiation paralleled by an upregulation of EphA3. Analyzing downstream signaling after irradiation, we detected decreased Src kinase phosphorylation, but unchanged focal adhesion kinase (FAK) phosphorylation, indicating, in part, irradiation-induced downregulation of signaling via the EphA2-Src-FAK axis in melanoma cells. However, to which extent this finding contributes to the modification of metastasis-relevant cellular properties remains to be elucidated.
Eph and ephrins in epithelial stem cell niches and cancer
The family of Eph tyrosine kinase receptors is an important part of signaling pathways involved in development, tissue homeostasis and tumorigenesis. Binding and activation of the receptors by their ligands, the ephrins, result in bidirectional signaling into both receptor and ligand expressing cells. Adult stem cell niches and tumors frequently express receptors and ligands, although their function is only beginning to be understood. Thus, Eph receptors and ephrins have become important molecules for understanding basic biological processes as well as tumorigenesis, and are promising targets for potential therapeutic intervention in human disease.
Regulation and misregulation of Eph/ephrin expression
The erythropoietin-producing hepatocellular (Eph) receptors form the largest family of receptor tyrosine kinases. Upon interaction of the Eph receptors with their ligands the ephrins, signaling cascades are initiated downstream of both receptor and ligand, a feature known as bidirectional signaling. The Eph receptors and ephrin ligands mediate important roles in embryonic development, particularly in establishing tissue organization by mediating cell adhesion or cell repulsion. In several adult tissues, at least one Eph/ephrin pair is found to play critical roles in tissue physiology and homeostasis. In recent years numerous members of this family have gained considerable attention since changes in their expression levels are a typical feature in cancer cells. Despite the fact that Eph/ephrin developmental expression profiles are well documented, little is known on transcriptional and post-transcriptional mechanisms that permits their highly specific, graded, complementary or overlapping expression patterns. Therefore understanding the transcriptional and post-transcriptional mechanisms regulating Eph/ephrin expression has far-reaching significance in biology. This review provides an overview of the mechanisms regulating Eph/ephrin expression. We highlight important emerging mechanisms of Eph/ephrin regulation or misregulation such as epigenetics and miRNAs.
The multifaceted roles of Eph/ephrin signaling in breast cancer
Eph receptors and their membrane-bound ligands are intimately involved in the control of morphogenic processes during embryonic development and adult tissue homeostasis. By their ability to orchestrate cell migration, pattern formation and tissue integrity they are also prone to be involved in carcinogenic growth. In this review we concentrate on their involvement in the normal and carcinogenic development of the breast. In this context we summarize their multi-faceted functions as tumor suppressors, tumor promoters, angiogenic inducers and regulators of stem cell homeostasis.
Bone cell interactions through Eph/ephrin: Bone modeling, remodeling and associated diseases
Bones cannot properly form or be maintained without cell-cell interactions through ephrin ligands and Eph receptors. Cell culture analysis and evaluation of genetic mouse models and human diseases reveal various ephrins and Eph functions in the skeletal system. Migration, attachment and spreading of mesenchymal stem cells are regulated by ephrinB ligands and EphB receptors. ephrinB1 loss-of-function is associated with craniofrontonasal syndrome (CFNS) in humans and mice. In bone remodeling, ephrinB2 is postulated to act as a “coupling stimulator.” In that case, bidirectional signaling between osteoclastic ephrinB2 and osteoblastic EphB4 suppresses osteoclastic bone resorption and enhances osteoblastic bone formation, facilitating the transition between these two states. Parathyroid hormone (PTH) induces ephrinB2 in osteoblasts and enhances osteoblastic bone formation. In contrast to ephrinB2, ephrinA2 acts as a “coupling inhibitor,” since ephrinA2 reverse signaling into osteoclasts enhances osteoclastogenesis and EphA2 forward signaling into osteoblasts suppresses osteoblastic bone formation and mineralization. Furthermore, ephrins and Ephs likely modulate pathological conditions such as osteoarthritis, rheumatoid arthritis, multiple myeloma and osteosarcoma. This review focuses on ephrin/Eph-mediated cell-cell interactions in bone biology.