CD147, CD44, and the epidermal growth factor receptor (EGFR) signaling pathway cooperate to regulate breast epithelial cell invasiveness.

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777-788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer.

CD147-dependent heterogeneity in malignant and chemoresistant properties of cancer cells.

CD147 (alias emmprin or basigin), an integral plasma membrane glycoprotein and a member of the Ig superfamily, is widespread in normal tissues, but highly up-regulated in many types of malignant cancer cells. CD147 is multifunctional, with numerous binding partners. Recent studies suggest that complexes of CD147 with the hyaluronan receptor CD44 and associated transporters and receptor tyrosine kinases are enriched in the plasma membrane of cancer stem-like cells. Here, we show that subpopulations of tumor cell lines constitutively expressing high levels of cell-surface CD147 exhibit cancer stem-like cell properties; that is, they exhibit much greater invasiveness, anchorage-independent growth, spheroid formation, and drug resistance in vitro and higher tumorigenicity in vivo than those constitutively expressing low levels of cell-surface CD147. Primary CD147-rich cell subpopulations derived from mouse mammary adenocarcinomas also exhibit high levels of invasiveness and spheroid-forming capacity, whereas CD147-low cells do not. Moreover, localization at the plasma membrane of CD44, the EGF receptor, the ABCB1 and ABCG2 drug transporters, and the MCT4 monocarboxylate transporter is elevated in cells constitutively expressing high levels of cell-surface CD147. These results show that CD147 is associated with assembly of numerous pro-oncogenic proteins in the plasma membrane and may play a fundamental role in properties characteristic of cancer stem-like cells.

Regulation of invadopodia formation and activity by CD147.

A defining feature of malignant tumor progression is cellular penetration through the basement membrane and interstitial matrices that separate various cellular compartments. Accumulating evidence supports the notion that invasive cells employ specialized structures termed invadopodia to breach these structural barriers. Invadopodia are actin-based, lipid-raft-enriched membrane protrusions containing membrane-type-1 matrix metalloproteinase (MT1-MMP; also known as matrix metalloproteinase 14; MMP14) and several signaling proteins. CD147 (emmprin, basigin), an immunoglobulin superfamily protein that is associated with tumor invasion and metastasis, induces the synthesis of various matrix metalloproteinases in many systems. In this study we show that upregulation of CD147 is sufficient to induce MT1-MMP expression, invasiveness and formation of invadopodia-like structures in non-transformed, non-invasive, breast epithelial cells. We also demonstrate that CD147 and MT1-MMP are in close proximity within these invadopodia-like structures and co-fractionate in membrane compartments with the properties of lipid rafts. Moreover, manipulation of CD147 levels in invasive breast carcinoma cells causes corresponding changes in MT1-MMP expression, invasiveness and invadopodia formation and activity. These findings indicate that CD147 regulates invadopodia formation and activity, probably through assembly of MT1-MMP-containing complexes within lipid-raft domains of the invadopodia.

KSHV activation of VEGF secretion and invasion for endothelial cells is mediated through viral upregulation of emmprin-induced signal transduction.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS)-one of the most common tumors arising in the setting of immune suppression. Hallmarks of KS lesions include KSHV-infected cells of endothelial lineage and neoangiogenesis. Promigratory factors secreted in the tumor microenvironment by KSHV-infected cells promote endothelial cell (EC) migration and angiogenesis but existing therapies targeting these pathways are not widely utilized. This underscores the need for additional characterization of KSHV-host interactions relevant to EC pathogenesis to identify new therapeutic targets. We recently demonstrated that de novo infection by KSHV promotes EC invasion through upregulation of extracellular matrix metalloproteinase inducer (emmprin)-a multifunctional glycoprotein previously shown to induce tumor cell invasion and regional angiogenesis through upregulation of signal transduction and promotion of tumor-stroma interactions. This study was undertaken to determine whether EC invasion for KSHV-infected cells is induced through activation of specific signal transduction pathways and proangiogenic factors by emmprin. We found that KSHV activation of emmprin induces PI3K/Akt- and mitogen-activated protein kinase (MAPK)-dependent secretion of vascular endothelial growth factor (VEGF). Functionally, EC invasion following de novo infection is induced by emmprin-dependent PI3K/Akt and MAPK activation of VEGF. These findings support the potential utility of targeting emmprin for reducing VEGF secretion and EC migration in the KS microenvironment.

Inhibition of heart formation by lithium is an indirect result of the disruption of tissue organization within the embryo.

Lithium is a commonly used drug for the treatment of bipolar disorder. At high doses, lithium becomes teratogenic, which is a property that has allowed this agent to serve as a useful tool for dissecting molecular pathways that regulate embryogenesis. This study was designed to examine the impact of lithium on heart formation in the developing frog for insights into the molecular regulation of cardiac specification. Embryos were exposed to lithium at the beginning of gastrulation, which produced severe malformations of the anterior end of the embryo. Although previous reports characterized this deformity as a posteriorized phenotype, histological analysis revealed that the defects were more comprehensive, with disfigurement and disorganization of all interior tissues along the anterior-posterior axis. Emerging tissues were poorly segregated and cavity formation was decreased within the embryo. Lithium exposure also completely ablated formation of the heart and prevented myocardial cell differentiation. Despite the complete absence of cardiac tissue in lithium treated embryos, exposure to lithium did not prevent myocardial differentiation of precardiac dorsal marginal zone explants. Moreover, precardiac tissue freed from the embryo subsequent to lithium treatment at gastrulation gave rise to cardiac tissue, as demonstrated by upregulation of cardiac gene expression, display of sarcomeric proteins, and formation of a contractile phenotype. Together these data indicate that lithium's effect on the developing heart was not due to direct regulation of cardiac differentiation, but an indirect consequence of disrupted tissue organization within the embryo.

Canonical WNT signaling enhances stem cell expression in the developing heart without a corresponding inhibition of cardiogenic differentiation.

WNT signaling has been shown to influence the development of the heart. Although recent data suggested that canonical WNTs promote the emergence and expansion of cardiac progenitors in the pregastrula embryo, it has long been accepted that once gastrulation begins, canonical WNT signaling needs to be suppressed for cardiac development to proceed. Yet, this latter supposition appears to be odds with the expression of multiple canonical WNTs in the developing heart. The present study examining the effect of ectopic canonical WNT signaling on cardiogenesis in the developing frog was designed to test the hypothesis that heart formation is dependent on the inhibition of canonical WNT activity at the onset of gastrulation. Here we report that cardiac differentiation of explanted precardiac tissue from the dorsal marginal zone was not suppressed by exposure to WNT1 protein, although expression of Tbx5, Tbx20, and Nkx2.5 was selectively reduced. Pharmacological activation of WNT signaling in intact embryos using the GSK3 inhibitor SB415286 did not prevent the formation of an anatomically normal and functionally sound heart, with the only defect observed being lower levels of the cardiac transcription factor Nkx2.5. In both the explant and whole embryo studies, expression of muscle genes and proteins was unaffected by ectopic canonical WNT signaling. In contrast, canonical Wnt signaling upregulated expression of the cardiac stem cell marker c-kit and pluripotency genes Oct25 and Oct60. However, this regulatory stimulation of stem cells did not come at the expense of blocking cardiac progenitors from differentiating.

High-frequency ultrasonographic imaging of avian cardiovascular development.

The chick embryo has long been a favorite model system for morphologic and physiologic studies of the developing heart, largely because of its easy visualization and amenability to experimental manipulations. However, this advantage is diminished after 5 days of incubation, when rapidly growing chorioallantoic membranes reduce visibility of the embryo. Using high-frequency ultrasound, we show that chick embryonic cardiovascular structures can be readily visualized throughout the period of Stages 9-39. At most stages of development, a simple ex ovo culture technique provided the best imaging opportunities. We have measured cardiac and vascular structures, blood flow velocities, and calculated ventricular volumes as early as Stage 11 with values comparable to those previously obtained using video microscopy. The endocardial and myocardial layers of the pre-septated heart are readily seen as well as the acellular layer of the cardiac jelly. Ventricular inflow in the pre-septated heart is biphasic, just as in the mature heart, and is converted to a monophasic (outflow) wave by ventricular contraction. Although blood has soft-tissue density at the ultrasound resolutions and developmental stages examined, its movement allowed easy discrimination of perfused vascular structures throughout the embryo. The utility of such imaging was demonstrated by documenting changes in blood flow patterns after experimental conotruncal banding.