Anti-apoptotic BCL-2 proteins govern cellular outcome following B-RAF(V600E) inhibition and can be targeted to reduce resistance.

In theory, pharmacological inhibition of oncogenic signaling is an effective strategy to halt cellular proliferation, induce apoptosis and eliminate cancer cells. In practice, drugs (for example, PLX-4032) that inhibit oncogenes like B-RAFV600E provide relatively short-term success in patients, owing to a combination of incomplete cellular responses and the development of resistance. To define the relationship between PLX-4032-induced responses and resistance, we interrogated the contributions of anti-apoptotic BCL-2 proteins in determining the fate of B-RAFV600E-inhibited melanoma cells. Although PLX-4032 eliminated B-RAFV600E signaling leading to marked cell cycle arrest, only a fraction of cells eventually underwent apoptosis. These data proposed two hypotheses regarding B-RAFV600E inhibition: (1) only a few cells generate a pro-apoptotic signal, or (2) all the cells generate a pro-apoptotic signal but the majority silences this pathway to ensure survival. Indeed, the latter hypothesis is supported by our observations as the addition of ABT-737, an inhibitor to anti-apoptotic BCL-2 proteins, revealed massive apoptosis following PLX-4032 exposure. B-RAFV600E inhibition alone sensitized cells to the mitochondrial pathway of apoptosis characterized by the rapid accumulation of BIM on the outer mitochondrial membrane, which could be functionally revealed by ABT-737 to promote apoptosis and loss of clonogenic survival. Furthermore, PLX-4032-resistant cells demonstrated collateral resistance to conventional chemotherapy, yet could be re-sensitized to PLX-4032 by BCL-2 family inhibition in vivo and conventional chemotherapies in vitro. Our data suggest that inhibiting anti-apoptotic BCL-2 proteins will enhance primary responses to PLX-4032, along with reducing the development of resistance to both targeted and conventional therapies.

Lymphatic function, lymphangiogenesis, and cancer metastasis.

The lymphatic system serves as the primary route for the metastasis of many cancers and the extent of lymph node involvement is the most important indicator of tumor aggressiveness. Despite the apparent importance of the lymphatic vessels for tumor dissemination, it has remained unclear whether activation of lymphatic endothelial cells may affect tumor progression and metastasis and the molecular mechanisms of lymphangiogenesis are just beginning to be elucidated. This overview describes the unique structural and functional characteristics of the lymphatic vessels that render them particularly suitable for invasion by tumor cells and for their efficient transport to lymph nodes. Recent evidence indicates occurrence of tumor lymphangiogenesis and its correlation with metastasis. Molecular regulation of tumor lymphangiogenesis, its significance for tumor metastasis, and implications for cancer therapy are discussed.

Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma.

Interactions of tumor cells with lymphatic vessels are of paramount importance for tumor progression, however, the underlying molecular mechanisms are poorly understood. Whereas enlarged lymphatic vessels are frequently observed at the periphery of malignant melanomas, it has remained unclear whether intratumoral lymphangiogenesis occurs within these tumors. Here, we demonstrate the presence of intratumoral lymphatics and enlargement of lymphatic vessels at the tumor periphery in vascular endothelial growth factor (VEGF)-C-overexpressing human melanomas transplanted onto nude mice. VEGF-C expression also resulted in enhanced tumor angiogenesis, indicating a coordinated regulation of lymphangiogenesis and angiogenesis in melanoma progression. The specific biological effects of VEGF-C were critically dependent on its proteolytic processing in vivo. Furthermore, VEGF-C induced chemotaxis of macrophages in vitro and in vivo, revealing a potential function of VEGF-C as an immunomodulator. Taken together, our results identify VEGF-C as multifunctional factor involved in regulating tumor lymphangiogenesis, angiogenesis, and immune response.

Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.

Metastasis of breast cancer occurs primarily through the lymphatic system, and the extent of lymph node involvement is a key prognostic factor for the disease. Whereas the significance of angiogenesis for tumor progression has been well documented, the ability of tumor cells to induce the growth of lymphatic vessels (lymphangiogenesis) and the presence of intratumoral lymphatic vessels have been controversial. Using a novel marker for lymphatic endothelium, LYVE-1, we demonstrate here the occurrence of intratumoral lymphangiogenesis within human breast cancers after orthotopic transplantation onto nude mice. Vascular endothelial growth factor (VEGF)-C overexpression in breast cancer cells potently increased intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs. The degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases. These results establish the occurrence and biological significance of intratumoral lymphangiogenesis in breast cancer and identify VEGF-C as a molecular link between tumor lymphangiogenesis and metastasis.

Expression of vascular endothelial growth factor induces an invasive phenotype in human squamous cell carcinomas.

Inhibition of the vascular endothelial growth factor (VEGF) receptor Flk-1 has been shown to prevent invasion of experimental squamous cell carcinomas (SCC). To directly investigate the role of VEGF in tumor invasion, we stably transfected human SCC-13 cells, which are characterized by a noninvasive phenotype in vivo, with expression vectors containing murine VEGF(164) in sense (SCC/VEGF+) or antisense (SCC/VEGF-) orientation or with vector alone (SCC/vec). SCC/vec cells formed slowly growing, well-differentiated tumors with well-defined borders between tumor and stroma, after intradermal or subcutaneous injection. In contrast, SCC/VEGF+ tumors were characterized by rapid tumor growth, with small cell groups and single cells invading into the surrounding tissue, and by admixture of blood vessels and tumor cells in areas of tumor invasion. We detected an increase in tumor vessel density and size in VEGF-overexpressing tumors, resulting in a more than fourfold increase in total vascular areas. In contrast, SCC/VEGF- clones formed noninvasive, sharply circumscribed tumors with reduced vascular density. These findings demonstrate that selective VEGF overexpression was sufficient to induce tumor invasiveness, and they provide further evidence for an active role of the tumor stroma in cancer progression.

Structure, function, and molecular control of the skin lymphatic system.

The mechanisms of angiogenesis have been studied extensively over the past years. The focus, however, has been almost exclusively on blood vessels, whereas little effort has been directed toward understanding lymphangiogenesis and the role of lymphatic vessels in physiology and pathology. The lymphatic system, acting in concert with the blood vascular system, is of fundamental importance in maintaining tissue homeostasis, and disorders of the lymphatic system are common, often resulting in chronic, disabling conditions. This overview summarizes the most important aspects of the structure and function of the lymphatic system with emphasis on the skin lymphatic vasculature and the differences between blood and lymphatic vessels. Special attention has been given to the methods employed in research of the lymphatic system. Finally, we describe molecular mechanisms involved in the regulation of lymphangiogenesis. Vascular endothelial growth factor and vascular endothelial growth factor-C, expressed by distinct skin cell populations, play an important role in the molecular control of skin angiogenesis and lymphangiogenesis.

Vascular endothelial growth factor-C (VEGF-C) and its receptors KDR and flt-4 are expressed in AIDS-associated Kaposi's sarcoma.

Kaposi's sarcoma is characterized by clusters of spindle-shaped cells that are considered to be tumor cells and by prominent vasculature. Whereas spindle cells are most likely endothelial in origin, it remains controversial whether they are of lymphatic or blood vascular derivation. To test the hypothesis that the lymphangiogenesis factor vascular endothelial growth factor-C and its receptors, KDR and flt-4, are involved in the pathogenesis of Kaposi's sarcoma, we performed in situ hybridizations and immunofluorescent stainings on human immunodeficiency virus-associated Kaposi's sarcoma. Spindle-shaped tumor cells strongly expressed KDR and flt-4 mRNA. Immunofluorescent staining confirmed expression of the flt-4 receptor in Kaposi's sarcoma cells, and double labeling revealed its colocalization with the endothelial cell marker CD31. Vascular endothelial growth factor-C was strongly expressed in blood vessels associated with Kaposi's sarcoma. In vitro, human dermal microvascular endothelial cells also expressed vascular endothelial growth factor-C mRNA that was further upregulated by vascular permeability factor/vascular endothelial growth factor. Vascular endothelial growth factor-C potently stimulated the proliferation of Kaposi's sarcoma tumor cells in vitro. These results demonstrate important paracrine functions of vascular endothelial growth factor-C, produced by blood vessels, in the pathogenesis of cutaneous Kaposi's sarcoma, and suggest a lymphatic origin and/or differentiation of Kaposi's sarcoma tumor cells.

Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas.

The function of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in epithelial tumor development has remained controversial. We studied the in vitro growth characteristics and the in vivo tumor xenograft growth of the human squamous cell carcinoma cell lines A431 and SCC-13, stably transfected to overexpress human TSP-1. Overexpression of TSP-1 inhibited tumor growth of A431 xenotransplants, and completely abolished tumor formation by SCC-13 cells. TSP-1 overexpressing A431 tumors were characterized by extensive areas of necrosis and by decreased tumor vessel number and size. The effects of TSP-1 on tumor cell growth were indirect since tumor cell proliferation rates in vivo and in vitro, anchorage-dependent and -independent growth in vitro, and susceptibility to induction of apoptosis by serum withdrawal were unchanged in TSP-1 overexpressing tumor cells. However, TSP-1 overexpression up-regulated the TSP-1 receptor CD36, leading to enhanced adhesion of A431 cells to TSP-1. These findings establish TSP-1 as a potent inhibitor of angiogenesis and tumor growth in carcinomas of the skin.

Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization.

Acquisition of invasive/metastatic potential through protease expression is an essential event in tumor progression. High levels of components of the plasminogen activation system, including urokinase, but paradoxically also its inhibitor, plasminogen activator inhibitor 1 (PAI1), have been correlated with a poor prognosis for some cancers. We report here that deficient PAI1 expression in host mice prevented local invasion and tumor vascularization of transplanted malignant keratinocytes. When this PAI1 deficiency was circumvented by intravenous injection of a replication-defective adenoviral vector expressing human PAI1, invasion and associated angiogenesis were restored. This experimental evidence demonstrates that host-produced PAI is essential for cancer cell invasion and angiogenesis.

Tumorigenic conversion of immortal human keratinocytes through stromal cell activation.

The stromal microenvironment plays a crucial role in tumor development and progression. One of the most potent activators of stromal cells is the platelet-derived growth factor (PDGF). To investigate the role of PDGF in epithelial tumor development we stably transfected immortal nontumorigenic human keratinocytes with the PDGF-B cDNA. Transfected HaCaT cells overexpressed PDGF-B but remained negative for the PDGF receptors alpha and beta (mRNA). Thus, they did not exhibit autocrine growth stimulation in vitro, but proliferation of cocultured fibroblasts was enhanced and this effect was inhibited by a neutralizing antibody to PDGF-BB. After subcutaneous injection into nude mice the transfected cells maintained high PDGF expression and formed progressively enlarging, rapidly proliferating cysts, classified as benign tumors. During early tumor development (up to 2 months), PDGF-B transfectants induced marked mesenchymal cell proliferation and angiogenesis, yet this effect vanished at later stages (2-6 months) concomitantly with increased epithelial cell proliferation and enhanced tumor growth. These results demonstrate that an activated stromal environment can promote tumorigenic conversion of nontumorigenic keratinocytes by inducing sustained epithelial hyperproliferation. This effect is apparently caused by a dual action of PDGF-BB: (i) PDGF-BB can promote tumor growth by inducing angiogenesis and stroma formation, and (ii) PDGF-activated stromal cells maintain elevated keratinocyte proliferation via a paracrine mechanism. Thus, PDGF, a major factor activated in wound healing, may play an important role as an endogenous promoter in epithelial tumor formation.

Halting angiogenesis suppresses carcinoma cell invasion.

The importance of angiogenesis in malignant tumor growth has been interpreted mainly in terms of oxygen and nutrient supply. Here we demonstrate its fundamental role for tumor invasion of malignant human keratinocytes in surface transplants on nude mice. Distinct patterns of angiogenesis and vascular endothelial growth factor receptor-2 (VEGFR-2) expression allowed us to distinguish between benign and malignant cells. Functional inactivation of VEGF-R2 by a blocking antibody disrupted ongoing angiogenesis and prevented invasion of malignant cells, without reducing tumor cell proliferation. The reversion of a malignant into a benign phenotype by halting angiogenesis demonstrates a significant function of vascular endothelium for tumor invasion.