Jonathan P. Sleeman

Complex networks orchestrate epithelial–mesenchymal transitions

Epithelial–mesenchymal transition is an indispensable mechanism during morphogenesis, as without mesenchymal cells, tissues and organs will never be formed. However, epithelial-cell plasticity, coupled to the transient or permanent formation of mesenchyme, goes far beyond the problem of cell-lineage segregation. Understanding how mesenchymal cells arise from an epithelial default status will also have a strong impact in unravelling the mechanisms that control fibrosis and cancer progression.

Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells

Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Thus, HDAC inhibitors are candidate drugs for differentiation therapy of cancer. Here, we show that the well‐tolerated antiepileptic drug valproic acid is a powerful HDAC inhibitor. Valproic acid relieves HDAC‐dependent transcriptional repression and causes hyperacetylation of histones in cultured cells and in vivo. Valproic acid inhibits HDAC activity in vitro, most probably by binding to the catalytic center of HDACs. Most importantly, valproic acid induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute myeloid leukemia patients. More over, tumor growth and metastasis formation are significantly reduced in animal experiments. Therefore, valproic acid might serve as an effective drug for cancer therapy.

HYALURONATE RECEPTORS : KEY PLAYERS IN GROWTH, DIFFERENTIATION, MIGRATION AND TUMOR PROGRESSION

Hyaluronate (HA) is an abundant component of extracellular matrix that is believed to be crucial in many cellular processes, including tissue remodeling, the creation of cell-free spaces, inflammation and tumorigenesis. Although several well characterized proteins within the extracellular matrix associate with HA, it is now clear that cells can also bind and respond to HA directly, via cell-surface HA-binding proteins. The cDNAs coding for two families of such proteins, CD44 and RHAMM, have been cloned and characterized. These proteins have been implicated in a number of physiological processes, including cell migration, lymphocyte activation and tumor progression. Although many of these processes depend on an association with HA, some are apparently HA-independent, suggesting that other ligands for these receptors may be involved.

CD24 Expression Causes the Acquisition of Multiple Cellular Properties Associated with Tumor Growth and Metastasis

The glycosylphosphatidylinositol-anchored membrane protein CD24 functions as an adhesion molecule for P-selectin and L1 and plays a role in B-cell development and neurogenesis. Over the last few years, a large body of literature has also implicated CD24 expression in tumorigenesis and progression. Here, we show that ectopic CD24 expression can be sufficient to promote tumor metastasis in experimental animals. By developing a doxycycline-inducible system for the expression of CD24 in breast cancer cells, we have also analyzed the cellular properties that CD24 expression influences. We found that CD24 expression increased tumor cell proliferation. Furthermore, in addition to promoting binding to P-selectin, CD24 expression also indirectly stimulated cell adhesion to fibronectin, collagens I and IV, and laminin through the activation of alpha3beta1 and alpha4beta1 integrin activity. Moreover, CD24 expression supported rapid cell spreading and strongly induced cell motility and invasion. CD24-induced proliferation and motility were integrin independent. Together, these observations implicate CD24 in the regulation of multiple cell properties of direct relevance to tumor growth and metastasis.

Inhibition of tumour cell growth by hyperforin, a novel anticancer drug from St. John's wort that acts by induction of apoptosis

Hyperforin is a plant derived antibiotic from St. Johns wort. Here we describe a novel activity of hyperforin, namely its ability to inhibit the growth of tumour cells by induction of apoptosis. Hyperforin inhibited the growth of various human and rat tumour cell lines in vivo, with IC50 values between 3–15 μM. Treatment of tumour cells with hyperforin resulted in a dose-dependent generation of apoptotic oligonucleosomes, typical DNA-laddering and apoptosis-specific morphological changes. In MT-450 mammary carcinoma cells hyperforin increased the activity of caspase-9 and caspase-3, and hyperforin-mediated apoptosis was blocked by the broad-range caspase inhibitor zVAD.fmk. When added to MT-450 cells, hyperforin, but not paclitaxel, induced a rapid loss of the mitochondrial transmembrane potential Δψm, and subsequent morphological changes such as homogenization and vacuolization of mitochondria. Monitoring of Δψm revealed that the hyperforin-mediated mitochondrial permeability transition can not be prevented by zVAD.fmk. This indicates that mitochondrial permeabilization is a cause rather than a consequence of caspase activation. Moreover, hyperforin was capable of releasing cytochrome c from isolated mitochondria. These findings suggest that hyperforin activates a mitochondria-mediated apoptosis pathway. In vivo, hyperforin inhibited the growth of autologous MT-450 breast carcinoma in immunocompetent Wistar rats to a similar extent as the cytotoxic drug paclitaxel, without any signs of acute toxicity. Owing to the combination of significant antitumour activity, low toxicity in vivo and natural abundance of the compound, hyperforin holds the promise of being an interesting novel antineoplastic agent that deserves further laboratory and in vivo exploration.

The lymph node as a bridgehead in the metastatic dissemination of tumors.

The metastatic spread of tumors is not a random process. Distinct patterns of metastasis can be discerned which vary from tumor type to tumor type. A common pattern, particularly for carcinomas, is that regional lymph nodes are often the first organs to develop metastases. This pattern of metastasis is central to the utility of the sentinel lymphonodectomy surgical technique. However, not all tumors and tumor types metastasize first to the regional lymph nodes. The mechanisms which determine whether regional lymph nodes or other sites first develop metastases remain poorly understood. In this article I review the anatomical, cellular and molecular factors which play a role in metastatic dissemination and determine patterns of metastasis. I then explore the importance of tumor heterogeneity and the selection of metastatically competent tumor cells during systemic dissemination, and suggest that some secondary sites are more readily colonised by metastasizing cells than others. Metastases at these sites act as bridgeheads, constituting a reservoir of tumor cells which, because they have already successfully metastasized, possess many of the properties required for metastasis to further sites. These tumor cells are therefore more likely than cells in the primary tumor to acquire all of the properties required for metastasis to less favourable secondary sites. To illustrate the bridgehead concept, I argue that features of the design and function of the lymphatic system make it highly amenable to the entry of metastasizing tumor cells and the formation of lymph node metastases, and suggest that lymph node metastases form a bridgehead for further metastatic spread.

Tumor metastasis and the lymphatic vasculature.

Tumor‐associated lymphatic vessels act as a conduit by which disseminating tumor cells access regional lymph nodes and form metastases there. Lymph node metastasis is of major prognostic significance for many types of cancer, although lymph node metastases are themselves rarely life‐threatening. These observations focus our attention on understanding how tumor cells interact with the lymphatic vasculature, and why this interaction is so significant for prognosis. Tumors interact with the lymphatic vasculature in a number of ways, including vessel co‐option, chemotactic migration and invasion into lymphatic vessels and induction of lymphangiogenesis. Tumor‐induced lymphangiogenesis both locally and in regional lymph nodes has been correlatively and functionally associated with metastasis formation and poor prognosis. The investigation of the molecular regulation of lymphangiogenesis has identified ways of interfering with prolymphangiogenic signaling. Blockade of tumor‐induced lymphangiogenesis in preclinical models inhibits metastasis formation in lymph nodes and often also in other organs, suggesting that blocking the lymphatic route of dissemination might suppress metastasis formation not only in lymph nodes but also in other organs. However, randomized clinical trials that have investigated the efficacy of therapeutic removal of lymph nodes have concluded that lymph node metastases act only as indicators that primary tumors have developed metastatic potential, and do not govern the further spread of metastatic cells. To reconcile these apparently paradoxical observations we suggest a model in which tumor‐induced lymphangiogenesis and lymph node metastasis formation act as indicators that tumors are producing factors that can act systemically to promote metastasis formation in distant organs.

CD44 Acts Both as a Growth‐ and Invasiveness‐Promoting Molecule and as a Tumor‐Suppressing Cofactor

Abstract: High‐molecular‐weight splice variants of the CD44 transmembrane protein family have been implicated in tumorigenesis and metastasis formation. By contrast, in certain tumors‐for example, Burkitts lymphoma, neuroblastomas, and prostate cancer‐loss of CD44 expression seems to accompany transformation. Here we describe two modes of action of CD44 proteins. They can bind growth factors and present them to their authentic high‐affinity receptors, and thus promote proliferation and invasiveness of cells. Under these conditions the CD44 proteins recruit ERM proteins‐for example, ezrin or moesin‐to their cytoplasmic tails, thereby producing links to the cytoskeleton. This mode of action could account for the tumor‐promoting action of CD44 proteins. The second mode of action of CD44 proteins comes into play when cells reach confluent growth conditions. Under specific conditions, binding of another ligand, the ECM component hyaluronate, leads to the activation and binding to the CD44 cytoplasmic tail of the tumor suppressor protein merlin. The activation of merlin confers growth arrest, so‐called contact inhibition. This function of CD44 proteins defines them as tumor suppressors. The type of action of CD44 on a given cell will depend on the isoform pattern of CD44 expressed, on the cellular equipment with ERM protein members, on the nature of the ECM, and on yet‐unknown conditions.

Hyaluronan-oligosaccharide-induced transcription of metalloproteases

Activated dendritic epidermal Langerhans cells and metastatic tumour cells share many properties. Both cell types can invade the surrounding tissue, enter the lymphatic system and travel to regional lymph nodes. We have recently shown that fragments of the extracellular matrix component hyaluronan, which are typically produced at sites of inflammation, can activate dendritic cells. Upon activation, dendritic cells upregulate expression of matrix metalloproteases (MMPs). These observations prompted us to investigate whether exposure to hyaluronan fragments also induces MMP expression in tumour cells. Here, we report that MMP-9, MMP-13 and urokinase plasminogen activator are upregulated in murine 3LL tumour cells after exposure to mixed-size hyaluronan. Similarly upregulated MMP-9 and MMP-13 expression was observed in primary fibroblasts. By using size-fractionated hyaluronan preparations, we show that the enhanced expression of MMP-9 and MMP-13 is only induced by small hyaluronan (HA) fragments. Although our data suggest that HA-fragment-induced MMP-9 and MMP-13 expression is receptor mediated, they rule out an involvement of the hyaluronan receptors CD44, RHAMM/IHAP and TLR-4. Finally, we show that HA fragment-induced MMP-9 transcription is mediated via NF-κB. Our results suggest that the metastasis-associated HA degradation in tumours might promote invasion by inducing MMP expression.

Expression of vascular endothelial growth factor (VEGF)-C and VEGF-D, and their receptor VEGFR-3, during different stages of cervical carcinogenesis.

Cervical carcinogenesis has well‐defined stages of disease progression including three grades of pre‐invasive lesions—cervical intraepithelial neoplasia grades 1–3 (CIN 1–3)—and invasive cervical cancer. However, the biological properties of CIN lesions prone to develop invasive disease are not well defined. Recent observations suggest that early invasive disease spreads to regional lymph nodes in several tumour types and that growth factors (VEGF‐C and VEGF‐D) involved in new lymphatic vessel formation may play a crucial role in this process. The present study has assessed the expression of VEGF‐C and VEGF‐D, and their receptor VEGFR‐3, in 152 cervical lesions (33 CIN 1, 33 CIN 2, 37 CIN 3, and 49 squamous cell carcinomas) to determine whether expression of lymphangiogenic factors occurs prior to invasion. The presence of lymphatic vessels was determined using LYVE‐1 and podoplanin staining, as well as double immunostaining for LYVE‐1/CD34 and podoplanin/CD34. In situ hybridization was performed to determine VEGFR‐3 mRNA expression. A significant positive correlation was found between VEGF‐C, VEGF‐D, and VEGFR‐3 expression through the different stages of cervical carcinogenesis. Significant differences in protein expression for VEGF‐C, VEGF‐D, and VEGFR‐3 were found between CIN 1–2 and CIN 3 (p < 0.001 for all), but not between CIN 3 and cervical cancer. More than 50% of the CIN 3 lesions showed moderate to strong staining for VEGF‐C and VEGF‐D, whereas most of the early pre‐cancerous lesions (CIN 1 and 2) were negative. In cervical cancer, similar observations to those in CIN 3 were found. VEGFR‐3 mRNA expression was found in the cytoplasm of epithelial neoplastic cells and VEGFR3 protein expression was found in more than 50% of CIN 3 lesions and cervical cancers, compared with 15% in CIN 1 and 2. These findings suggest an autocrine growth stimulation pattern via VEGFR‐3. Adjacent CIN 3 was present in nine cervical cancers and displayed strong expression for VEGF‐C, VEGF‐D, and VEGFR‐3. These results suggest that in cervical carcinogenesis a switch to the lymphangiogenic phenotype may occur at the stage of CIN 3. Copyright