Publications

BACKGROUND: The c-Myc oncoprotein is activated in the majority of colorectal cancers (CRCs), whereas the TGF-β pathway is frequently affected by loss-of-function mutations, for example in SMAD2/3/4 genes. The canonical model places Myc downstream of inhibitory TGF-β signaling. However, we previously demonstrated that Myc also inhibits TGF-β signaling through the miR-17 92 microRNA cluster, raising the question about functional relationships between these two pathways.

METHODS: We engineered a series of genetically complex murine and human CRC cell lines in which Myc and TGF-β activities could be manipulated simultaneously. This was achieved through retroviral expression of the Myc-estrogen receptor fusion protein and through Smad4 short hairpin RNA knockdown. Cell lines thus modified were injected subcutaneously in immunocompromised mice, and the resultant tumors (n = 5-10 per treatment group) were analyzed for overall growth and neovascularization. Additionally, the distribution of MYC and TGF-β pathway mutations was analyzed in previously profiled human CRC samples.

RESULTS: In kras-mutated/trp53-deleted murine colonocytes, either Myc activation or TGF-β inactivation increased tumor sizes and microvascular densities approximately 1.5- to 2.5-fold, chiefly through downregulation of thrombospondin-1 and related type I repeat-containing proteins. Combining Myc activation with TGF-β inactivation did not further accelerate tumorigenesis. This redundancy and the negative effect of TGF-β signaling on angiogenesis were also demonstrated using xenografts of human CRC cell lines. Furthermore, the analysis of the Cancer Genome Atlas data revealed that in CRC without microsatellite instability, overexpression of Myc and inactivation of Smads (including acquired mutations in SMAD2) are mutually exclusive, with odds ratio less than 0.1.

CONCLUSIONS: In human CRC, gain-of-function alterations in Myc and loss-of-function alterations in TGF-β exhibit a masking epistatic interaction and are functionally redundant.

Myopericytoma (MPC) is a rare tumor with perivascular proliferation of pluripotent stem-cell-like pericytes. Although indolent, MPC may be locally aggressive with recurrent disease. The pathogenesis and diagnostic biomarkers of MPC are poorly understood. We discovered that 15% of benign MPCs (thyroid, skin; 3 of 20 samples) harbored BRAF(WT/V600E); 33.3% (1 of 3 samples) of BRAF(WT/V600E)-MPCs were multifocal/infiltrative/recurrent. Patient-MPC and primary MPC cells harbored BRAF(WT/V600E), were clonal and expressed pericytic-differentiation biomarkers crucial for its microenvironment. BRAF(WT/V600E)-positive thyroid MPC primary cells triggered in vitro (8.8-fold increase) and in vivo (3.6-fold increase) angiogenesis. Anti-BRAF(V600E) therapy with vemurafenib disrupted angiogenic and metabolic properties ( 3-fold decrease) with down-regulation ( 2.2-fold decrease) of some extracellular-matrix (ECM) factors and ECM-associated long non-coding RNA (LincRNA) expression, with no effects in BRAF(WT)-pericytes. Vemurafenib also inhibited ( 3-fold decrease) cell viability in vitro and in BRAF(WT/V600E)-positive thyroid MPC patient-derived xenograft (PDX) mice (n = 5 mice per group). We established the first BRAF(WT/V600E)-dependent thyroid MPC cell culture. Our findings identify BRAF(WT/V600E) as a novel genetic aberration in MPC pathogenesis and MPC-associated biomarkers and imply that anti-BRAF(V600E) agents may be useful adjuvant therapy in BRAF(WT/V600E)-MPC patients. Patients with BRAF(WT/V600E)-MPC should be closely followed because of the risk for multifocality/recurrence.

BACKGROUND AND RATIONALE: Anaplastic thyroid cancer (ATC) is characterized by pleomorphic cells, has a poor prognosis, is highly devastating disease, and is not curable. No reliable biomarkers of metastatic potential, helpful for early diagnosis of ATC and therapeutic response have been found yet. Thrombospondin-1 (TSP-1) plays a fundamental role in cancer progression by regulating cell stromal cross-talk in the tumor microenvironment.

GOALS: Our goal was to understand whether TSP-1 could affect protein levels of its integrin receptors (e.g., ITGα3, α6, and β1) and cell morphology in BRAF(V600E)-ATC cells in vitro and in vivo.

EXPERIMENTAL DESIGN: Anaplastic thyroid cancer-derived cell cultures and western blotting were used to assess integrin protein expression upon TSP-1 silencing. Immunohistochemistry was performed on orthotopic primary human ATC and metastatic ATC in lung tissue to compare TSP-1 and integrin protein expression levels.

RESULTS: TSP-1 knock-down down-regulates ITGα3, α6, and β1 in BRAF(V600E)-human ATC cells. BRAF(V600E)-ATC cells with TSP-1 knock-down were rounded compared to control cells, which displayed a spread morphology. TSP-1 knock-down also reduced TSP-1, ITGα3, α6, and β1 protein expression levels in vivo in the ATC microenvironment, which is enriched in stromal and inflammatory cells.

CONCLUSION: TSP-1 silencing causes changes in ITG levels and ATC cell morphology. The assessment of TSP-1 and ITG levels might contribute to earlier metastatic potential of BRAF(V600E)-positive aggressive thyroid cancers, and allow improved patient selection for clinical trials.

Angiogenesis plays an important role in cancer and in many other human diseases. Vascular endothelial growth factor-A (VEGF-A), the best known angiogenic factor, was originally discovered as a potent vascular permeability factor (VPF), suggesting that other vascular permeabilizing agents, such as histamine and serotonin, might also have angiogenic activity. We recently demonstrated that, like VEGF-A, histamine and serotonin up-regulate the orphan nuclear receptor and transcription factor TR3 (mouse homolog Nur77) and that TR3/Nur77 is essential for their vascular permeabilizing activities. We now report that histamine and serotonin are also angiogenic factors that, at low micromolar concentrations, induce endothelial cell proliferation, migration and tube formation in vitro, and angiogenesis in vivo. All of these responses are mediated through specific histamine and serotonin receptors, are independent of VEGF-A, and are directly dependent on TR3/Nur77. Initially, the angiogenic response closely resembled that induced by VEGF-A, with generation of "mother" vessels. However, after  10 days, mother vessels began to regress as histamine and serotonin, unlike VEGF-A, up-regulated the potent angiogenesis inhibitor thrombospondin-1, thereby triggering a negative feedback loop. Thus, histamine and serotonin induce an angiogenic response that fits the time scale of acute inflammation.

Thrombospondins TSP-1 and TSP-2 are potent endogenous inhibitors of angiogenesis. They inhibit angiogenesis through direct effects on endothelial cell migration, proliferation, survival, and apoptosis and by antagonizing the activity of VEGF. Several of the membrane receptor systems and signal transduction molecules that mediate the effects of TSP-1 and TSP-2 have been elucidated. TSP-1 and TSP-2 exert their direct effects through CD36, CD47, and integrins. Recent data indicate that CD36 and β1 integrins collaborate to transmit the signals that are initiated by TSP-1 and TSP-2. Furthermore, these receptors appear to associate with VEGFR2 to form a platform for the integration of positive and negative signals for angiogenesis. Cross talk between pro- and antiangiogenic signal transduction pathways may enable TSP-1 and TSP-2 to inhibit angiogenesis by antagonizing survival pathways while also activating apoptotic pathways. CD36 and CD47 are both involved in the suppression of nitric oxide (NO). Advances in understanding of the molecular regulation of angiogenesis by TSP have paved the way for innovations in experimental treatment of cancers and will likely continue to offer vast avenues for discovery in other disease processes as well.

Thrombospondin (Thbs) proteins are induced in sites of tissue damage or active remodeling. The endoplasmic reticulum (ER) stress response is also prominently induced with disease where it regulates protein production and resolution of misfolded proteins. Here we describe a function for Thbs as ER-resident effectors of an adaptive ER stress response. Thbs4 cardiac-specific transgenic mice were protected from myocardial injury, whereas Thbs4(-/-) mice were sensitized to cardiac maladaptation. Thbs induction produced a unique profile of adaptive ER stress response factors and expansion of the ER and downstream vesicles. Thbs bind the ER lumenal domain of activating transcription factor 6α (Atf6α) to promote its nuclear shuttling. Thbs4(-/-) mice showed blunted activation of Atf6α and other ER stress-response factors with injury, and Thbs4-mediated protection was lost upon Atf6α deletion. Hence, Thbs can function inside the cell during disease remodeling to augment ER function and protect through a mechanism involving regulation of Atf6α.

The incidence of human papillary thyroid cancer (PTC) is increasing and an aggressive subtype of this disease is resistant to treatment with vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor. VEGFR2 promotes angiogenesis by triggering endothelial cell proliferation and migration. However, the molecular mechanisms governing VEGFR2 stability in vivo remain unknown. Additionally, whether VEGFR2 influences PTC cell migration is not clear. We show that the ubiquitin E3 ligase SCF(β-TRCP) promotes ubiquitination and destruction of VEGFR2 in a casein kinase I (CKI)-dependent manner. β-TRCP knockdown or CKI inhibition causes accumulation of VEGFR2, resulting in increased activity of signaling pathways downstream of VEGFR2. β-TRCP-depleted endothelial cells exhibit enhanced migration and angiogenesis in vitro. Furthermore, β-TRCP knockdown increased angiogenesis and vessel branching in zebrafish. Importantly, we found an inverse correlation between β-TRCP protein levels and angiogenesis in PTC. We also show that β-TRCP inhibits cell migration and decreases sensitivity to the VEGFR2 inhibitor sorafenib in poorly differentiated PTC cells. These results provide a new biomarker that may aid a rational use of tyrosine kinase inhibitors to treat refractory PTC.

Trypanosoma cruzi causes Chagas disease, which is a neglected tropical disease that produces severe pathology and mortality. The mechanisms by which the parasite invades cells are not well elucidated. We recently reported that T. cruzi up-regulates the expression of thrombospondin-1 (TSP-1) to enhance the process of cellular invasion. Here we characterize a novel TSP-1 interaction with T. cruzi that enhances cellular infection. We show that labeled TSP-1 interacts specifically with the surface of T. cruzi trypomastigotes. We used TSP-1 to pull down interacting parasite surface proteins that were identified by mass spectrometry. We also show that full length TSP-1 and the N-terminal domain of TSP-1 (NTSP) interact with T. cruzi surface calreticulin (TcCRT) and other surface proteins. Pre-exposure of recombinant NTSP or TSP-1 to T. cruzi significantly enhances cellular infection of wild type mouse embryo fibroblasts (MEF) compared to the C-terminal domain of TSP-1, E3T3C1. In addition, blocking TcCRT with antibodies significantly inhibits the enhancement of cellular infection mediated by the TcCRT-TSP-1 interaction. Taken together, our findings indicate that TSP-1 interacts with TcCRT on the surface of T. cruzi through the NTSP domain and that this interaction enhances cellular infection. Thus surface TcCRT is a virulent factor that enhances the pathogenesis of T. cruzi infection through TSP-1, which is up-regulated by the parasite.

PURPOSE: B-Raf(V600E) may play a role in the progression from papillary thyroid cancer to anaplastic thyroid cancer (ATC). We tested the effects of a highly selective B-Raf(V600E) inhibitor, PLX4720, on proliferation, migration, and invasion both in human thyroid cancer cell lines (8505c(B-RafV600E) and TPC-1(RET/PTC-1 and wild-type B-Raf)) and in primary human normal thyroid (NT) follicular cells engineered with or without B-Raf(V600E).

EXPERIMENTAL DESIGN: Large-scale genotyping analysis by mass spectrometry was performed in order to analyze >900 gene mutations. Cell proliferation and migration/invasion were performed upon PLX4720 treatment in 8505c, TPC-1, and NT cells. Orthotopic implantation of either 8505c or TPC-1 cells into the thyroid of severe combined immunodeficient mice was performed. Gene validations were performed by quantitative polymerase chain reaction and immunohistochemistry.

RESULTS: We found that PLX4720 reduced in vitro cell proliferation and migration and invasion of 8505c cells, causing early downregulation of genes involved in tumor progression. PLX4720-treated NT cells overexpressing B-Raf(V600E) (heterozygous wild-type B-Raf/B-Raf(V600E)) showed significantly lower cell proliferation, migration, and invasion. PLX4720 treatment did not block cell invasion in TPC-1 cells with wild-type B-Raf, which showed very low and delayed in vivo tumor growth. In vivo, PLX4720 treatment of 8505c orthotopic thyroid tumors inhibited tumor aggressiveness and significantly upregulated the thyroid differentiation markers thyroid transcription factor 1 and paired box gene 8.

CONCLUSIONS: Here, we have shown that PLX4720 preferentially inhibits migration and invasion of B-Raf(V600E) thyroid cancer cells and tumor aggressiveness. Normal thyroid cells were generated to be heterozygous for wild-type B-Raf/B-Raf(V600E), mimicking the condition found in most human thyroid cancers. PLX4720 was effective in reducing cell proliferation, migration, and invasion in this heterozygous model. PLX4720 therapy should be tested and considered for a phase I study for the treatment of patients with B-Raf(V600E) ATC.

Thrombospondin-1 (TSP-1) is an extracellular protein critical to normal lung homeostasis, and is reported to activate latent transforming growth factor-β (TGF-β). Because active TGF-β is causally involved in lung fibrosis after bleomycin challenge, alterations in TSP-1 may be relevant to pulmonary fibrosis. We sought to determine the effects of TSP-1 deficiency on the susceptibility to bleomycin-induced pulmonary fibrosis in a murine model. Age-matched and sex-matched C57BL/6 wild-type (WT) and TSP-1-deficient mice were treated twice weekly for 4 weeks with intraperitoneal bleomycin (0.035 U/g) or PBS, and were allowed to rest 1 week before being killed. Their lungs were inflated with PBS, fixed in formalin, paraffin-embedded, and sectioned. A certified veterinary pathologist blindly scored each slide for inflammation and fibrosis. Lungs were homogenized to obtain RNA and protein for the real-time RT-PCR analysis of connective tissue growth factor (CTGF) and collagen I, and for Western blotting to detect phospho-Smad2, or total Smad2/3, respectively. In response to bleomycin treatment, measures of fibrosis and inflammation, along with CTGF and collagen I mRNA concentrations, were increased in TSP-1-deficient mice compared with WT mice. Notably, Smad 2/3 signaling was of equal strength in WT and TSP-1 knockout mice treated with bleomycin, suggesting that TSP-1 is not required for the activation of TGF-β. These results demonstrate that TSP-1 deficiency does not protect mice from systemic bleomycin challenge, and that TSP-1 deficiency is associated with increased expression of lung collagen and CTGF.