Sandra Ryeom

An oncogene–tumor suppressor cascade drives metastatic prostate cancer by coordinately activating Ras and nuclear factor-κB

Metastasis is responsible for the majority of prostate cancer–related deaths; however, little is known about the molecular mechanisms that underlie this process. Here we identify an oncogene–tumor suppressor cascade that promotes prostate cancer growth and metastasis by coordinately activating the small GTPase Ras and nuclear factor-κB (NF-κB). Specifically, we show that loss of the Ras GTPase-activating protein (RasGAP) gene DAB2IP induces metastatic prostate cancer in an orthotopic mouse tumor model. Notably, DAB2IP functions as a signaling scaffold that coordinately regulates Ras and NF-κB through distinct domains to promote tumor growth and metastasis, respectively. DAB2IP is suppressed in human prostate cancer, where its expression inversely correlates with tumor grade and predicts prognosis. Moreover, we report that epigenetic silencing of DAB2IP is a key mechanism by which the polycomb-group protein histone-lysine N-methyltransferase EZH2 activates Ras and NF-κB and triggers metastasis. These studies define the mechanism by which two major pathways can be simultaneously activated in metastatic prostate cancer and establish EZH2 as a driver of metastasis.

Down's syndrome suppression of tumour growth and the role of the calcineurin inhibitor DSCR1

The incidence of many cancer types is significantly reduced in individuals with Down’s syndrome, and it is thought that this broad cancer protection is conferred by the increased expression of one or more of the 231 supernumerary genes on the extra copy of chromosome 21. One such gene is Down’s syndrome candidate region-1 (DSCR1, also known as RCAN1), which encodes a protein that suppresses vascular endothelial growth factor (VEGF)-mediated angiogenic signalling by the calcineurin pathway. Here we show that DSCR1 is increased in Down’s syndrome tissues and in a mouse model of Down’s syndrome. Furthermore, we show that the modest increase in expression afforded by a single extra transgenic copy of Dscr1 is sufficient to confer significant suppression of tumour growth in mice, and that such resistance is a consequence of a deficit in tumour angiogenesis arising from suppression of the calcineurin pathway. We also provide evidence that attenuation of calcineurin activity by DSCR1, together with another chromosome 21 gene Dyrk1a, may be sufficient to markedly diminish angiogenesis. These data provide a mechanism for the reduced cancer incidence in Down’s syndrome and identify the calcineurin signalling pathway, and its regulators DSCR1 and DYRK1A, as potential therapeutic targets in cancers arising in all individuals.

CD4+ T Cells Contribute to the Remodeling of the Microenvironment Required for Sustained Tumor Regression upon Oncogene Inactivation

Oncogene addiction is thought to occur cell autonomously. Immune effectors are implicated in the initiation and restraint of tumorigenesis, but their role in oncogene inactivation-mediated tumor regression is unclear. Here, we show that an intact immune system, specifically CD4(+) T cells, is required for the induction of cellular senescence, shutdown of angiogenesis, and chemokine expression resulting in sustained tumor regression upon inactivation of the MYC or BCR-ABL oncogenes in mouse models of T cell acute lymphoblastic lymphoma and pro-B cell leukemia, respectively. Moreover, immune effectors knocked out for thrombospondins failed to induce sustained tumor regression. Hence, CD4(+) T cells are required for the remodeling of the tumor microenvironment through the expression of chemokines, such as thrombospondins, in order to elicit oncogene addiction.

TORC1 Is Essential for NF1-Associated Malignancies

Inactivating mutations in NF1 underlie the prevalent familial cancer syndrome neurofibromatosis type 1 [1]. The NF1-encoded protein is a Ras GTPase-activating protein (RasGAP) [2]. Accordingly, Ras is aberrantly activated in NF1-deficient tumors; however, it is unknown which effector pathways critically function in tumor development. Here we provide in vivo evidence that TORC1/mTOR activity is essential for tumorigenesis. Specifically, we show that the mTOR inhibitor rapamycin potently suppresses the growth of aggressive NF1-associated malignancies in a genetically engineered murine model. However, in these tumors rapamycin does not function via mechanisms generally assumed to mediate tumor suppression, including inhibition of HIF-1alpha and indirect suppression of AKT, but does suppress the mTOR target Cyclin D1 [3]. These results demonstrate that mTOR inhibitors may be an effective targeted therapy for this commonly untreatable malignancy. Moreover, they indicate that mTOR inhibitors do not suppress all tumor types via the same mechanism, suggesting that current biomarkers that rely on HIF-1alpha suppression may not be informative for all cancers. Finally, our results reveal important differences between the effects of mTOR inhibition on the microvasculature in genetically engineered versus xenograft models and indicate that the former may be required for effective preclinical screening with this class of inhibitors.

Tumor Escape from Endogenous, Extracellular Matrix–Associated Angiogenesis Inhibitors by Up-Regulation of Multiple Proangiogenic Factors

Purpose: Thrombospondin-1 (Tsp1), endostatin, and tumstatin are extracellular matrix–associated proteins that inhibit angiogenesis. We examined the mechanisms by which tumor cells may bypass the antiangiogenic effects of these endogenous regulators. Experimental Design: CT26 colon and RenCa renal carcinoma cells were stably transfected with Tsp1, endostatin, or tumstatin cDNA. Subcutaneous and metastatic tumor growth in syngeneic mice was analyzed. Expression of proangiogenic factors in resulting tumors was measured by quantitative real-time PCR. The combination of Tsp1 and vascular endothelial growth factor (VEGF) receptor-2 inhibition was also examined. Results: There was significant suppression of angiogenesis in flank tumors and liver metastases formed from cells overexpressing Tsp1, endostatin, or tumstatin. However, all tumors ultimately escaped angiogenesis inhibition. The combination of all three angiogenesis inhibitors had no additive effect beyond overexpression of a single inhibitor. Using quantitative real-time PCR, we found that VEGF and platelet-derived growth factor (PDGF)-A levels were routinely up-regulated at least 5-fold in all CT26 tumors overexpressing any antiangiogenic protein, and there were variable increases in angiopoietin 2 (Ang2), basic fibroblast growth factor, and PDGF-B. In contrast, RenCa tumors, which have high baseline levels of VEGF and PDGF-B, relied on basic fibroblast growth factor, Ang1, and PDGF-A up-regulation to counteract Tsp1 overexpression. Growth of CT26 cells with Tsp1 overexpression was suppressed when anti–VEGFR-2 treatment was added. Conclusions: Cancer cells with overexpression of three different endogenous angiogenesis inhibitor eventually escape angiogenesis inhibition by up-regulation of various proangiogenic factors. Tsp1, endostatin, and tumstatin may be functionally redundant in this system. These endogenous angiogenesis inhibitors are likely best used in combination with the blockade of proangiogenic pathways or with traditional chemotherapy or radiation therapy.

Sustained regression of tumors upon MYC inactivation requires p53 or thrombospondin-1 to reverse the angiogenic switch

The targeted inactivation of oncogenes offers a rational therapeutic approach for the treatment of cancer. However, the therapeutic inactivation of a single oncogene has been associated with tumor recurrence. Therefore, it is necessary to develop strategies to override mechanisms of tumor escape from oncogene dependence. We report here that the targeted inactivation of MYC is sufficient to induce sustained regression of hematopoietic tumors in transgenic mice, except in tumors that had lost p53 function. p53 negative tumors were unable to be completely eliminated, as demonstrated by the kinetics of tumor cell elimination revealed by bioluminescence imaging. Histological examination revealed that upon MYC inactivation, the loss of p53 led to a deficiency in thrombospondin-1 (TSP-1) expression, a potent antiangiogenic protein, and the subsequent inability to shut off angiogenesis. Restoration of p53 expression in these tumors re-established TSP-1 expression. This permitted the suppression of angiogenesis and subsequent sustained tumor regression upon MYC inactivation. Similarly, the restoration of TSP-1 alone in p53 negative tumors resulted in the shut down of angiogenesis and led to sustained tumor regression upon MYC inactivation. Hence, the complete regression of tumor mass driven by inactivation of the MYC oncogene requires the p53-dependent induction of TSP-1 and the shut down of angiogenesis. Notably, overexpression of TSP-1 alone did not influence tumor growth. Therefore, the combined inactivation of oncogenes and angiogenesis may be a more clinically effective treatment of cancer. We conclude that angiogenesis is an essential component of oncogene addiction.

Analysis of hypoxia-related gene expression in sarcomas and effect of hypoxia on RNA interference of vascular endothelial cell growth factor A.

Vascular endothelial cell growth factor A (VEGF-A) and hypoxia play important roles in tumor angiogenesis. VEGF-A gene expression is up-regulated in tumors under hypoxic conditions, yet it is unclear how such up-regulation will affect the efficacy of RNA interference strategies targeting VEGF-A. Four potential short interfering RNA (siRNA) sequences for the VEGF-A gene were cloned into expression plasmids and transfected into HT1080 human fibrosarcoma cells. Stable transfection of these plasmids decreased VEGF-A mRNA levels and protein secretion by up to 99%. Our analysis of >100 hypoxia-related genes using oligonucleotide microarrays of 38 human sarcoma samples and 14 normal tissues identified distinctly different patterns of expression between sarcomas and normal tissues as assessed by hierarchical clustering analysis. Numerous hypoxia-related genes were significantly up-regulated in sarcomas including hypoxia-inducible factor 1alpha (HIF-1alpha). Exposure of wild-type HT1080 cells to 1% hypoxia resulted in HIF-1alpha up-regulation and a 74% increase in VEGF-A secretion as compared with secretion under normoxic conditions. Surprisingly, stable cell lines expressing VEGF-A siRNAs silenced VEGF-A expression equally well in hypoxia and normoxia. S.c. injection of cells with VEGF-A siRNAs into athymic nude mice led to slower-growing tumors, decreased blood vessel density, and greater apoptosis when compared with controls. Immunofluorescence analysis of tumor sections revealed areas of HIF-1alpha nuclear expression, suggesting areas of hypoxia, in both control tumors and VEGF-suppressed tumors. We conclude that hypoxia plays an important role in human sarcomas but hypoxic up-regulation of VEGF-A expression does not attenuate the efficacy of VEGF-A RNA interference.

The threshold pattern of calcineurin-dependent gene expression is altered by loss of the endogenous inhibitor calcipressin.

Calcineurin links calcium signaling to transcriptional responses in the immune, nervous and cardiovascular systems. To determine the function of the calcipressins, a family of putative calcineurin inhibitors, we assessed the calcineurin-dependent process of T cell activation in mice engineered to lack the gene encoding calcipressin 1 (Csp1). Csp1 regulated calcineurin in vivo, and genes triggered in an immune response had unique transactivation thresholds for T cell receptor stimulation. In the absence of Csp1, the apparent transactivation thresholds for all these genes were shifted because of enhanced calcineurin activity. This unbridled calcineurin activity drove Fas ligand expression, which normally requires high T cell receptor stimulation and results in the premature death of T helper type 1 cells. Thus, calcipressins modulate the pattern of calcineurin-dependent transcription, and may influence calcineurin activity beyond calcium to integrate a broad array of signals into the cellular response.

Platelet-derived thrombospondin-1 is a critical negative regulator and potential biomarker of angiogenesis

The sequential events leading to tumor progression include a switch to the angiogenic phenotype, dependent on a shift in the balance between positive and negative angiogenic regulators produced by tumor and stromal cells. Although the biologic properties of many angiogenesis regulatory proteins have been studied in detail, the mechanisms of their transport and delivery in vivo during pathologic angiogenesis are not well understood. Here, we demonstrate that expression of one of the most potent angiogenesis inhibitors, thrombospondin-1, is up-regulated in the platelets of tumor-bearing mice. We establish that this up-regulation is a consequence of both increased levels of thrombospondin-1 mRNA in megakaryocytes, as well as increased numbers of megakaryocytes in the bone marrow of tumor-bearing mice. Through the use of mouse tumor models and bone marrow transplantations, we show that platelet-derived thrombospondin-1 is a critical negative regulator during the early stages of tumor angiogenesis. Collectively, our data suggest that the production and delivery of the endogenous angiogenesis inhibitor thrombospondin-1 by platelets may be a critical host response to suppress tumor growth through inhibiting tumor angiogenesis. Further, this work implicates the use of thrombospondin-1 levels in platelets as an indicator of tumor growth and regression.

Ultrastructural immunogold localization of prostaglandin endoperoxide synthase (cyclooxygenase) to non-membrane-bound cytoplasmic lipid bodies in human lung mast cells, alveolar macrophages, type II pneumocytes, and neutrophils.

Lipid bodies are non-membrane-bound, lipid-rich cytoplasmic inclusions that occur in many mammalian cell types. Because lipid bodies are more prominent in cells associated with inflammation and are repositories of arachidonyl-phospholipids, a role for lipid bodies in the oxidative metabolism of arachidonic acid to form eicosanoids has been suggested. To evaluate further whether lipid bodies, in addition to serving as non-membranous sources of substrate arachidonate, are involved in eicosanoid formation, we used cells isolated from human lung to investigate the intracellular localization of prostaglandin endoperoxide (PGH) synthase (cyclooxygenase), the key initial, rate-limiting enzyme in the formation of prostaglandins and thromboxanes. Isolated lung cells containing a mixture of mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils from short-term cultures were fixed in suspension in a dilute aldehyde mixture, post-fixed in osmium tetroxide, stained en bloc with uranyl acetate, dehydrated in a graded series of alcohols, and embedded in Epon. A post-embedding immunogold procedure was used with a primary PGH synthase monoclonal antibody and 20-nm gold-conjugated secondary antibody to demonstrate enzyme locations. Specificity controls were also done. We found PGH synthase in lipid bodies of human lung mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils. Specific secretory and lysosomal granules and plasma membranes did not express PGH synthase. Specificity controls, including omission of the primary antibody or substitution with an irrelevant antibody, were negative. Absorption of the specific PGH synthase antibody with purified solid-phase PGH synthase resulted in a marked reduction of label in lipid bodies of all four cell types. These findings establish the presence of PGH synthase in lipid bodies of human lung mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils and, in concert with previous studies, suggest that these cytoplasmic lipid-rich organelles may be non-membrane sites of eicosanoid formation.