Sarcomas: genetics, signalling, and cellular origins. Part 2: TET-independent fusion proteins and receptor tyrosine kinase mutations.

Although the mechanisms that underlie sarcoma development are still poorly understood, the identification of non-random chromosomal translocations and receptor tyrosine kinase mutations associated with defined sarcoma types has provided new insight into the pathogenesis of these tumours. In Part 1 of the review (J Pathol 2007;213:4-20), we addressed sarcomas that express fusion genes containing TET gene family products. Part 2 of the review summarizes our current understanding of the implications of fusion genes that do not contain TET family members in sarcoma development, as well as that of specific mutations in genes encoding receptor tyrosine kinases (RTKs). The final section will serve as a summary of both reviews and will attempt to provide a synthesis of some of the emerging principles of sarcomagenesis.

Sarcomas: genetics, signalling, and cellular origins. Part 1: The fellowship of TET.

Sarcomas comprise some of the most aggressive solid tumours that, for the most part, respond poorly to chemo- and radiation therapy and are associated with a sombre prognosis when surgical removal cannot be performed or is incomplete. Partly because of their lower frequency, sarcomas have not been studied as intensively as carcinomas and haematopoietic malignancies, and the molecular mechanisms that underlie their pathogenesis are only beginning to be understood. Even more enigmatic is the identity of the primary cells from which these tumours originate. Over the past 25 years, however, several non-random chromosomal translocations have been found to be associated with defined sarcomas. Each of these translocations generates a fusion gene believed to be directly related to the pathogenesis of the sarcoma in which it is expressed. The corresponding fusion proteins provide a unique tool not only to study the process of sarcoma development, but also to identify cells that are permissive for their putative oncogenic properties. This is the first of two reviews that cover the mechanisms whereby specific fusion/mutant gene products participate in sarcoma development and the cellular context that may provide the necessary permissiveness for their expression and oncogenicity. Part 1 of the review focuses on sarcomas that express fusion genes containing TET gene family products, including EWSR1, TLS/FUS, and TAFII68. Part 2 (J Pathol 2007; DOI: 10.1002/path.2008) summarizes our current understanding of the genetic and cellular origins of sarcomas expressing fusion genes exclusive of TET family members; it also covers soft tissue malignancies harbouring specific mutations in RTK-encoding genes, the prototype of which are gastrointestinal stromal tumours (GIST).

Exogenous mesenchymal stem cells localize to the kidney by means of CD44 following acute tubular injury.

Mesenchymal stem cells (MSC) were recently shown to migrate to injured tissues when transplanted systemically. The mechanisms underlying the migration and homing of these cells is, however, unclear. In this study, we examine the role of CD44 and its major ligand, hyaluronic acid, in the trafficking of intravenously injected MSC in the glycerol-induced mouse model of acute renal failure (ARF). In vitro, hyaluronic acid promoted a dose-dependent migration of the stem cells that was inhibited by an anti-CD44 blocking monoclonal antibody. In vivo, stem cells injected into mice with ARF migrated to the injured kidney where hyaluronic acid expression was increased. Their presence correlated with morphological and functional recovery. Renal localization of the MSC was blocked by pre-incubation with the CD44 blocking antibody or by soluble hyaluronic acid. Stem cells derived from CD44 knockout mice did not localize to the injured kidney and did not accelerate morphological or functional recovery. Reconstitution by transfection of CD44 knockout stem cells with cDNA encoding wild-type CD44, but not a loss of function CD44 unable to bind hyaluronic acid, restored in vitro migration and in vivo localization of the cells to injured kidneys. We suggest that CD44 and hyaluronic acid interactions recruit exogenous MSC to injured renal tissue and enhance renal regeneration.

Inhibition of the hyaluronan-CD44 interaction by merlin contributes to the tumor-suppressor activity of merlin.

Mutation or loss of expression of merlin is responsible for neurofibromatosis type 2 (NF2), which is characterized by the development of schwannomas and other tumors of the nervous system. Like the ERM (ezrin-radixin-moesin) proteins, merlin interacts with CD44, a cell-surface receptor for hyaluronan (HA) that promotes tumorigenesis. However, the relationship between merlin and CD44 and the mechanism by which merlin exerts its tumor-suppressor function have not been elucidated. In the present study, we show that increased expression of wild-type merlin in Tr6BC1 schwannoma cells inhibits HA binding to CD44. Furthermore, we demonstrate that the residues required for this inhibitory effect and the interaction between CD44 and merlin lie within the first 50 amino acids of merlin. Overexpression of merlin inhibited subcutaneous growth of Tr6BC1 cells in immunocompromised Rag1 mice. In contrast, knocking down expression of endogenous merlin promoted tumor cell growth, as did overexpression of a merlin deletion mutant (merlinDel-1) that lacks the first 50 amino acids but not of other NH(2)-terminal deletion mutants. Together, our results demonstrate that inhibition of the CD44-HA interaction contributes to the tumor-suppressor function of merlin, and they suggest that merlin inhibits tumor growth, at least in part, by negatively regulating CD44 function.

A gene expression signature that distinguishes desmoid tumours from nodular fasciitis.

Nodular fasciitis (NF) is a rapidly growing cellular mass composed of fibroblasts/myofibroblasts, usually localized in subcutaneous tissues, that typically undergoes fibrosis and almost never recurs. Desmoid tumours (DTs) are rare forms of fibroblastic/myofibroblastic growth that arise in deep soft tissues, display a propensity for local infiltration and recurrence, but fail to metastasize. Given that both entities are primarily fibroblastic/myofibroblastic lesions with overlapping histological features, their gene expression profiles were compared to identify differentially expressed genes that may provide not only potential diagnostic markers, but also clues as to the pathogenesis of each disorder. Differentially expressed transcripts (89 clones displaying increased expression in DTs and 246 clones displaying increased expression in NF) included genes encoding several receptor and non-receptor tyrosine kinases (EPHB3, PTPRF, GNAZ, SYK, LYN, EPHA4, BIRC3), transcription factors (TWIST1, PITX2, EYA2, OAS1, MITF, TCF20), and members of the Wnt signalling pathway (AXIN2, WISP1, SFRP). Remarkably, almost one-quarter of the differentially expressed genes encode proteins associated with inflammation and tissue remodelling, including members of the interferon (IFN), tumour necrosis factor (TNF), and transforming growth factor beta (TGF-beta) signalling pathways as well as metalloproteinases (MMP1, 9, 13, 23), urokinase plasminogen activator (PLAU), and cathepsins. The observations provide the first comparative molecular characterization of desmoid tumours and nodular fasciitis and suggest that selected tyrosine kinases, transcription factors, and members of the Wnt, TGF-beta, IFN, and TNF signalling pathways may be implicated in influencing and distinguishing their fate.

Matrix metalloproteinase-7-mediated cleavage of Fas ligand protects tumor cells from chemotherapeutic drug cytotoxicity.

Recent evidence suggests that one mechanism whereby cytotoxic drugs, such as doxorubicin, kill tumors is the induction or up-regulation of Fas ligand (FasL) expression on the tumor cell surface. The ensuing engagement of Fas by FasL on adjacent cells leads to apoptosis. However, despite cytotoxic drug-induced FasL expression, Fas-sensitive tumors frequently resist chemotherapy, suggesting that they may possess a mechanism that prevents or inactivates Fas-FasL interactions. In the present work, we addressed the involvement of the FasL/Fas signaling pathway in doxorubicin-induced apoptosis and the ability of matrix metalloproteinases (MMPs) to proteolytically cleave FasL in tumor cells. Doxorubicin-induced apoptosis was inhibited by expression of soluble Fas or incubation of the tumor cells with MMP-7 but not with MMP-2 or MMP-9. Resistance to doxorubicin was also induced by expression in the tumor cells of constitutively active MMP-7 but not of a catalytically inactive mutant. Conversely, inhibition of MMP-7 expression in tumor cells by transfection of MMP-7 cDNA in antisense orientation resulted in sensitization to doxorubicin. MMP-7 efficiently cleaved recombinant FasL in vitro and reduced cell surface FasL expression. Our observations provide evidence that one mechanism whereby MMP-7 may promote tumor survival and resistance to doxorubicin is by cleaving FasL and reducing its effectiveness in triggering Fas-mediated apoptosis.

Angiopoietin-2 is implicated in the regulation of tumor angiogenesis.

We addressed the effect of angiopoietin expression on tumor growth and metastasis. Overexpression of angiopoietin-2 (Ang-2) in Lewis lung carcinoma and TA3 mammary carcinoma cells inhibited their ability to form metastatic tumors and prolonged the survival of mice injected with the corresponding transfectants. In contrast, angiopoietin-1 (Ang-1) overexpression had no detectable effect on the ability of either tumor type to disseminate. Tumors derived from Ang-2-overexpressing cells displayed aberrant angiogenic vessels that took the form of vascular cords or aggregated vascular endothelial cells with few associated smooth muscle cells. These vascular cords or aggregates were accompanied by endothelial and tumor cell apoptosis, suggesting that an imbalance in Ang-2 expression with respect to Ang-1 and vascular endothelial growth factor may disrupt angiogenesis and tumor survival in vivo. Our observations suggest that Ang-2 may play an important role in regulating tumor angiogenesis.

Decrease in epidermal CD44 expression as a potential mechanism for abnormal hyaluronate accumulation in superficial dermis in lichen sclerosus et atrophicus.

CD44 is a polymorphic integral membrane glycoprotein that serves as the principal cell surface receptor for hyaluronate, the major component of the extracellular matrix. CD44 is abundantly found in the skin and functions as a cell adhesion molecule. In a recent study we have observed a massive dermal accumulation of hyaluronate as a result of the in vivo selective suppression of CD44 in keratinocytes in mice expressing a keratin 5 promoter-driven CD44 anti-sense transgene. As the histologic features of the dorsal skin of these transgenic mice display some similarities to those of the skin lesions of lichen sclerosus et atrophicus, we explored the nature of the material accumulated in the dermis of genital and extragenital lesions of 14 patients with lichen sclerosus et atrophicus by Alcian Blue and human CD44 receptor globulin stainings, as well as the epidermal expression of CD44 protein and mRNA by immunohistochemistry and in situ hybridization. In this study we provide evidence that hyaluronate is accumulated in the superficial dermis of lichen sclerosus et atrophicus lesions, in particular by the use of human CD44 receptor globulin staining, which binds specifically to hyaluronate. In addition we show that the protein and mRNA expression of CD44 in the epidermis of the involved lichen sclerosus et atrophicus skin from genital and extragenital areas is significantly decreased, and in some cases completely lost. In contrast, keratinocyte CD44 expression was un-altered in the skin lesions of lupus erythematosus, scleroderma and reticular erythematous mucinosis, despite the presence of a mucinous material in the dermis. These results suggest that a decrease in CD44 in the keratinocytes may be correlated with an abnormal dermal accumulation of hyaluronate in the lesions of lichen sclerosus et atrophicus, and may play a pathogenetic role in this disease. J Invest Dermatol 115:1054-1058 2000

CD44 as a receptor for colonization of the pharynx by group A Streptococcus.

The pharynx is the primary reservoir for strains of group A STREPTOCOCCUS: (GAS) associated both with pharyngitis (streptococcal sore throat) and with invasive or "flesh-eating" soft tissue infections. We now report that CD44, a hyaluronic acid-binding protein that mediates human cell-cell- and cell-extracellular matrix-binding interactions, functions as a receptor for GAS colonization of the pharynx in vivo. We found that attachment of GAS to murine epithelial keratinocytes was mediated by binding of the GAS hyaluronic acid capsular polysaccharide to CD44. In studies of transgenic mice with a selective defect in epithelial expression of CD44, GAS adherence to CD44-deficient keratinocytes in vitro was reduced compared with adherence to keratinocytes expressing normal levels of CD44. After intranasal inoculation, GAS colonized the oropharynx of wild-type mice but failed to colonize transgenic mice deficient in CD44 expression. GAS colonization of wild-type mice could be blocked by coadministration of mAb to CD44 or by pretreatment of the animals with exogenous hyaluronic acid. These results provide evidence that CD44 serves as a receptor for GAS colonization of the pharynx and support the potential efficacy of disrupting the interaction between the GAS hyaluronic acid capsule and CD44 as a novel approach to preventing pharyngeal infection.

Thyroid carcinoma cells are resistant to FAS-mediated apoptosis but sensitive to tumor necrosis factor-related apoptosis-inducing ligand.

Fas (APO-1/CD95) is a transmembrane protein of the tumor necrosis factor (TNF)/nerve growth factor receptor superfamily that induces apoptosis in susceptible normal and neoplastic cells upon cross-linking by its ligand (FasL). TNF-related apoptosis-inducing ligand (TRAIL) is a more recently identified member of the TNF superfamily that has been shown to selectively kill neoplastic cells by engaging two cell-surface receptors, DR4 and DR5. Two additional TRAIL receptors (DcR1 and DcR2) do not transmit an apoptotic signal and have been proposed to confer protection from TRAIL-induced apoptosis. We addressed the expression of Fas, DR4, and DR5 in thyroid carcinoma cell lines and in 31 thyroid carcinoma specimens by Western blot analysis and immunohistochemistry, respectively, and tested the sensitivity of thyroid carcinoma cell lines to Fas- and TRAIL-induced apoptosis. Fas was found to be expressed in most thyroid carcinoma cell lines and tissue specimens. Although cross-linking of Fas did not induce apoptosis in thyroid carcinoma cell lines, Fas-mediated apoptosis did occur in the presence of the protein synthesis inhibitor cycloheximide, suggesting the presence of a short-lived inhibitor of the Fas pathway in these cells. Cross-linking of Fas failed to induce recruitment and activation of caspase 8, whereas transfection of a constitutively active caspase 8 construct effectively killed the SW579 papillary carcinoma cell line, arguing that the action of the putative inhibitor occurs upstream of caspase 8. By contrast, recombinant TRAIL induced apoptosis in 10 of 12 thyroid carcinoma cell lines tested, by activating caspase-10 at the receptor level and triggering a caspase-mediated apoptotic cascade. Resistance to TRAIL did not correlate with DcR1 or DcR2 protein expression and was overcome by protein synthesis inhibition in 50% of the resistant cell lines. One medullary carcinoma cell line was resistant to Fas-and TRAIL-induced apoptosis, even in the presence of cycloheximide, and to transfection of constitutively active caspase-8, suggesting a different regulation of the apoptotic pathway. Our observations indicate that TRAIL effectively kills carcinomas that originate from the follicular epithelium of the thyroid gland, by inducing caspase-mediated apoptosis, and may provide a potentially potent therapeutic reagent against thyroid cancer.

Perturbation of hyaluronan interactions by soluble CD44 inhibits growth of murine mammary carcinoma cells in ascites.

Hyaluronan accumulates in ascites during intraperitoneal proliferation of TA3/St murine mammary carcinoma cells and at sites of their invasion of the peritoneal wall. To determine whether hyaluronan is functionally involved in these events, ascites tumor formation was compared in mice injected intraperitoneally with stable transfectants of TA3/St cells that overexpress soluble CD44, a hyaluronan-binding protein, versus in mice injected with transfectants expressing mutated soluble CD44 that does not bind hyaluronan. The soluble CD44 transfectants temporarily grew at a reduced rate within the peritoneal cavity, then went into G(1) arrest and were subsequently cleared from the peritoneum. However, transfectants overexpressing mutant soluble CD44 that does not bind hyaluronan exhibited similar ascites accumulation, growth rates, and cell-cycle profiles in vivo to wild-type and vector-transfected TA3/St cells, all of which continued to grow until the tumors became fatal. The soluble CD44-transfected TA3/St cells also failed to attach to and form tumors in the peritoneal wall. When grown in vitro in soft agar, the soluble CD44 transfectants exhibited a dramatic reduction in colony formation compared to wild-type, vector-transfected, and mutant soluble CD44-transfected TA3/St cells. Thus, perturbation of hyaluronan interactions by soluble CD44 has a direct effect on the growth characteristics of these tumor cells, leading to inhibition of anchorage-independent growth in vitro and ascites growth in vivo.

Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis.

We have uncovered a novel functional relationship between the hyaluronan receptor CD44, the matrix metalloproteinase-9 (MMP-9) and the multifunctional cytokine TGF-beta in the control of tumor-associated tissue remodeling. CD44 provides a cell surface docking receptor for proteolytically active MMP-9 and we show here that localization of MMP-9 to cell surface is required for its ability to promote tumor invasion and angiogenesis. Our observations also indicate that MMP-9, as well as MMP-2, proteolytically cleaves latent TGF-beta, providing a novel and potentially important mechanism for TGF-beta activation. In addition, we show that MMP-9 localization to the surface of normal keratinocytes is CD44 dependent and can activate latent TGF-beta. These observations suggest that coordinated CD44, MMP-9, and TGF-beta function may provide a physiological mechanism of tissue remodeling that can be adopted by malignant cells to promote tumor growth and invasion.

Matrix metalloproteinases in tumor invasion and metastasis.

Extensive work on the mechanisms of tumor invasion and metastasis has identified matrix metalloproteinases (MMPs) as key players in the events that underlie tumor dissemination. Studies using natural and synthetic MMP inhibitors, as well as tumor cells transfected with cDNAs encoding the MMPs characterized thus far have provided compelling evidence that MMP activity can induce or enhance tumor survival, invasion and metastasis. Because of the ability of MMPs to degrade extracellular matrix (ECM) proteins, the principal mechanism whereby MMPs promote tumor development has been thought to be the proteolytic breakdown of tissue barriers to invasion and the associated facilitation of circulating tumor cell extravasation. However, recent evidence stemming from the use of novel experimental approaches indicates that MMPs do not play a major role in the process of extravasation itself. Rather, they appear to promote intravasation (the process of penetrating the circulation following invasion of blood vessels) and regulate the relationship between tumor cells and host tissue stroma subsequent to extravasation. In addition, the discoveries that a growing number of proteolytically active MMPs may localize to the cell surface in association with adhesion receptors, and that MMP substrates include latent cytokines and growth factors, provide a new conceptual framework for the mechanisms whereby MMPs influence tumor behavior.

Activation of CD40 favors the growth and vascularization of Kaposi's sarcoma.

Although CD40 is expressed by several tumor lines and is up-regulated in tumor vascular endothelium, its role in tumor biology is still unclear. In the present study, we investigated the role of CD40 in the growth and vascularization of Kaposi's sarcoma (KS). In vitro, stimulation of CD40 induced migration of KS cells and inhibited vincristine-induced apoptosis. Similarly, the CD40 engagement on endothelial cells resulted in cell contraction, migration, and prevention of serum withdrawal-apoptosis. To understand the biological relevance of CD40 in vivo, KS cells were engineered to express and release a soluble form of CD40 (KS-sCD40) able to disrupt CD40-CD154 interaction. SCID mice s.c. injected with KS-sCD40 cells developed tumors that were significantly smaller than those induced by control cells (KS-neo). In addition, KS-sCD40 tumors showed several areas of necrosis, diffuse presence of apoptotic cells, and poor vascularization. In contrast, KS-neo tumors showed few or absent areas of necrosis and apoptosis and intense vascularization. Moreover, anti-CD40 Abs stimulated neo-angiogenesis in a murine model in which s.c. implantation of Matrigel was used as a vehicle for the delivery of mediators. These observations provide demonstration that CD40 supports tumor cell survival, growth, and neo-vascularization of KS.

Expression of L-selectin ligands by transformed endothelial cells enhances T cell-mediated rejection.

Recent immunohistochemical studies have suggested that L-selectin ligands may be implicated in the infiltration of tumors and rejected transplants by lymphocytes. In the present study, polyoma-middle T Ag-transformed endothelial cells (H.end), which typically form in vivo immunogenic vascular tumors resembling Kaposi's sarcoma, were engineered to express L-selectin ligands by stable transfection with a cDNA encoding alpha(1,3/4)-fucosyltransferase (H.endft). The ability of these cells to form tumors in the s.c. tissues of normal and immunocompromised mice was then compared with that of H.end cells transfected with the hygromycin-resistance vector only (H. endhygro). H.endhygro cells rapidly formed local and metastatic tumors in normal syngeneic mice, leading to death within 2-3 mo postinjection. By contrast, tumors derived from H.endft cells displayed a slower rate of growth, an absence of metastasis, and marked lymphocyte infiltration. Animals bearing these tumors survived for a significantly longer duration than animals injected with H.endhygro cells. Alternatively, H.endft and H.endhygro cells formed tumors with comparable aggressiveness in immunocompromised mice, resulting in animal death within 3 wk of injection. H.endft but not H.endhygro cells supported L-selectin-dependent adhesion and cytolytic T cell activity in vitro. Taken together, our observations indicate that the in situ expression of fucosyltransferase may significantly influence the cellular immune response in endothelioma tumors. These results may be relevant in understanding the development of vascular opportunistic tumors such as Kaposi's sarcoma.

Sialylation of the sialic acid binding lectin sialoadhesin regulates its ability to mediate cell adhesion.

The macrophage-specific cell surface receptor sialoadhesin, which is a member of the newly recognized family of sialic acid binding lectins called siglecs, binds glycoprotein and glycolipid ligands containing a2-3-linked sialic acid on the surface of several leukocyte subsets. Recently, the sialic acid binding activity of the siglec CD22 has been demonstrated to be regulated by sialylation of the CD22 receptor molecule. In the present work, we show that desialylation of in vivo macrophage sialylconjugates enhances sialoadhesin-mediated lectin activity. Herein, we show that receptor sialylation of soluble sialoadhesin inhibits its binding to Jurkat cell ligands, and that charge-dependent repulsion alone cannot explain this inhibition. Furthermore, we show that the inhibitory effect of sialic acid is partially dependent on the presence of an intact exocyclic side chain. These results, in conjunction with previous findings, suggest that sialylation of siglecs by specific glycosyltransferases may be a common mechanism by which siglec-mediated adhesion is regulated.

Localization of matrix metalloproteinase 9 to the cell surface provides a mechanism for CD44-mediated tumor invasion.

The cell surface hyaluronan receptor CD44 promotes tumor growth and metastasis by mechanisms that remain poorly understood. We show here that CD44 associates with a proteolytic form of the matrix metalloproteinase-9 (MMP-9) on the surface of mouse mammary carcinoma and human melanoma cells. CD44-associated cell surface MMP-9 promotes cell-mediated collagen IV degradation in vitro and mediates tumor cell invasion of G8 myoblast monolayers. Several distinct CD44 isoforms coprecipitate with MMP-9 and CD44/MMP-9 coclustering is observed to be dependent on the ability of CD44 to form hyaluronan-induced aggregates. Disruption of CD44/MMP-9 cluster formation, by overexpression of soluble or truncated cell surface CD44, is shown to inhibit tumor invasiveness in vivo. Our observations indicate that CD44 serves to anchor MMP-9 on the cell surface and define a mechanism for CD44-mediated tumor invasion.

Inhibition of tumor growth in vivo by hyaluronan oligomers.

One of the critical events in tumor growth and metastasis is the interaction between tumor cells and host tissue stroma, mediated by different adhesion receptor repertoires in different tumor cell types. Several lines of evidence indicate that interaction between the hyaluronan receptor CD44, expressed on tumor cells, and host tissue stromal hyaluronan can enhance growth and invasiveness of certain tumors. Disruption of CD44-hyaluronan interaction by soluble recombinant CD44 has been shown to inhibit tumor formation by lymphoma and melanoma cells transfected with CD44. Since hyaluronan is a ubiquitous glycosaminoglycan polymer from which oligosaccharides of defined size can be readily purified, we tested the ability of hyaluronan oligomers to inhibit tumor formation by subcutaneously (s.c.) injected B16F10 melanoma cells. Our results indicate that hyaluronan oligomers injected at concentrations as low as 1 mg/ml can markedly inhibit B16F10 melanoma growth, providing a potentially attractive reagent for the control of local tumor development.