Carmelo Nucera

The landscape of somatic copy-number alteration across human cancers

A powerful way to discover key genes with causal roles in oncogenesis is to identify genomic regions that undergo frequent alteration in human cancers. Here we present high-resolution analyses of somatic copy-number alterations (SCNAs) from 3,131 cancer specimens, belonging largely to 26 histological types. We identify 158 regions of focal SCNA that are altered at significant frequency across several cancer types, of which 122 cannot be explained by the presence of a known cancer target gene located within these regions. Several gene families are enriched among these regions of focal SCNA, including the BCL2 family of apoptosis regulators and the NF-κΒ pathway. We show that cancer cells containing amplifications surrounding the MCL1 and BCL2L1 anti-apoptotic genes depend on the expression of these genes for survival. Finally, we demonstrate that a large majority of SCNAs identified in individual cancer types are present in several cancer types.

BRAF(V600E) mutation and the biology of papillary thyroid cancer

BRAF((V600E)) mutation is the most frequent genetic alteration in papillary thyroid carcinomas (PTCs) that are 80-90% of all thyroid cancers. We evaluated the relationship between BRAF((V600E)) and tumor, host, and environmental factors in PTCs from all geographical areas of Sicily. By PCR, BRAF((V600E)) was investigated in a series of 323 PTCs diagnosed in 2002-2005. The correlation between clinicopathological tumor, host, and environmental characteristics and the presence of BRAF((V600E)) were evaluated by both univariate and multivariate analyses. BRAF((V600E)) was found in 38.6% PTCs, with a 52% frequency in the classical PTCs and 26.4% in the tall cell variant. Univariate analysis indicated that BRAF((V600E)) was associated with greater tumor size (P=0.0048), extra-thyroid invasion (P<0.0001), and cervical lymph nodal metastases (P=0.0001). Multivariate logistic regression analysis confirmed that BRAF((V600E)) was an independent predictor of extra-thyroid invasion (P=0.0001) and cervical lymph nodal metastasis (P=0.0005). The association between BRAF((V600E)) and extra-thyroid invasion was also found in micro-PTCs (P=0.006). In 60 classical PTCs, BRAF((V600E)) was positively correlated with matrix metalloproteinase-9 expression (P=0.0047), suggesting a possible mechanism for BRAF((V600E)) effect on PTC invasiveness. No association was found between BRAF((V600E)) and patient age, gender, or iodine intake. In contrast, a strong association was found with residency in Eastern Sicily (P<0.0001 compared with Western Sicily). These results indicate that BRAF((V600E)) mutation is a marker of aggressive disease in both micro- and macro-PTCs. Moreover, for the first time, a possible link between BRAF((V600E)) mutation and environmental carcinogens is suggested.

B-Raf(V600E) and thrombospondin-1 promote thyroid cancer progression.

Although B-RafV600E is the most common somatic mutation in papillary thyroid carcinoma (PTC), how it induces tumor aggressiveness is not fully understood. Using gene set enrichment analysis and in vitro and in vivo functional studies, we identified and validated a B-RafV600E gene set signature associated with tumor progression in PTCs. An independent cohort of B-RafV600E-positive PTCs showed significantly higher expression levels of many extracellular matrix genes compared with controls. We performed extensive in vitro and in vivo validations on thrombospondin-1 (TSP-1), because it has been previously shown to be important in the regulation of tumor angiogenesis and metastasis and is present in abundance in tumor stroma. Knockdown of B-RafV600E resulted in TSP-1 down-regulation and a reduction of adhesion and migration/invasion of human thyroid cancer cells. Knockdown of TSP-1 resulted in a similar phenotype. B-RafV600E cells in which either B-RafV600E or TSP-1 were knocked down were implanted orthotopically into the thyroids of immunocompromised mice, resulting in significant reduction in tumor size and fewer pulmonary metastases from the primary carcinoma as compared with the control cells. Treatment of orthotopic thyroid tumors, initiated 1 week after tumor cell implantation with PLX4720, an orally available selective inhibitor of B-RafV600E, caused a significant tumor growth delay and decreased distant metastases, without evidence of toxicity. In conclusion, B-RafV600E plays an important role in PTC progression through genes (i.e., TSP-1) important in tumor invasion and metastasis. Testing of a patients thyroid cancer for B-RafV600E will yield important information about potential tumor aggressiveness and also allow for future use of targeted therapies with selective B-RafV600E inhibitors, such as PLX4720.

Targeting BRAFV600E with PLX4720 Displays Potent Antimigratory and Anti-invasive Activity in Preclinical Models of Human Thyroid Cancer

The role of the B-RafV600E mutation in aggressive thyroid cancers is examined.

BRAF(V600E) and microenvironment in thyroid cancer: a functional link to drive cancer progression.

Papillary thyroid cancer (PTC) rates continue to increase in the United States and Europe, and, although most patients do well, some recur and die of their disease. Patients with PTC harboring the BRAF(V600E) mutation seem to display a more aggressive clinical behavior, but little is known about the role of this mutation in crucial processes in the tumor microenvironment, such as tumor adhesion, migration, invasion, and metastasis. The extracellular matrix (ECM) microenvironment is not merely a structural scaffold for the cellular elements of the epithelial and stromal microenvironment, but it also elicits a profound influence on cell behavior affecting viability, proliferation, adhesion, and motility. The effects of BRAF(V600E) on cell surface receptors (i.e., integrins) and ECM noncellular components [i.e., thrombospondin-1 (TSP-1) and fibronectin (FN)] seem to trigger different pathologic biological processes in a cell context-dependent manner. This review focuses on the recent progress in understanding the role of BRAF(V600E) in the regulation of some ECM noncellular components and trans-membrane receptors of the microenvironment in PTC in order to design novel targeted therapies directed at the BRAF(V600E) multifaceted signaling cascades. Some of these targeted therapeutics, such as ATP-competitive BRAF(V600E) inhibitors (i.e., orally bioavailable PLX4720 and PLX4032 compounds), are already under investigation.

SCF β-TRCP suppresses angiogenesis and thyroid cancer cell migration by promoting ubiquitination and destruction of VEGF receptor 2

The E3 ubiquitin ligase β-TRCP, acting in concert with casein kinase I, drives ubiquitination and degradation of VEGFR2, and renders human papillary thyroid cancer cells resistant to the VEGFR2 inhibitor sorafenib.

FOXA1 Is a Potential Oncogene in Anaplastic Thyroid Carcinoma

Purpose: FOXA1 is a mammalian endodermal transcription factor belonging to the human forkhead box gene family that plays a role in certain tumor types. Here, we investigated the potential role of FOXA1 in human thyroid carcinomas. Experimental Design: We examined the level of FOXA1 expression and gene copy number by immunohistochemistry and fluorescence in situ hybridization, respectively, in a cohort of benign and malignant thyroid tumors. In addition, we examined the role of FOXA1 in the proliferation of an undifferentiated thyroid carcinoma cell line by short hairpin RNA-mediated silencing. Results: We show that FOXA1 is overexpressed in human anaplastic thyroid carcinomas (ATC). In addition, we identify FOXA1 DNA copy number gain within the 14q21.1 locus in both an ATC cell line and human ATC cases. Silencing of FOXA1 in an ATC cell line causes G1 growth arrest and reduction of cell proliferation. Moreover, we observe a potential link between FOXA1 and the cell cycle machinery by identifying p27kip1 up-regulation on FOXA1 silencing. Conclusions:FOXA1 is overexpressed in aggressive thyroid cancers and involved in cell cycle progression in an ATC cell line. Therefore, FOXA1 may be an important oncogene in thyroid tumorigenesis and a potential new therapeutic target for the treatment of anaplastic thyroid cancers.

A Novel Orthotopic Mouse Model of Human Anaplastic Thyroid Carcinoma

BACKGROUND Orthotopic mouse models of human cancer represent an important in vivo tool for drug testing and validation. Most of the human thyroid carcinoma cell lines used in orthotopic or subcutaneous models are likely of melanoma and colon cancer. Here, we report and characterize a novel orthotopic model of human thyroid carcinoma using a unique thyroid cancer cell line. METHODS We used the cell line 8505c, originated from a thyroid tumor histologically characterized by anaplastic carcinoma cell features. We injected 8505c cells engineered using a green fluorescent protein-positive lentiviral vector orthotopically into the thyroid of severe combined immunodeficient mice. RESULTS Orthotopic implantation with the 8505c cells produced thyroid tumors after 5 weeks, showing large neck masses, with histopathologic features of a high-grade neoplasm (anaplasia, necrosis, high mitotic and proliferative indexes, p53 positivity, extrathyroidal invasion, lymph node and distant metastases) and immunoprofile of follicular thyroid cell origin with positivity for thyroid transcription factor-1 and PAX8, and for cytokeratins. CONCLUSIONS Here we describe a novel orthotopic thyroid carcinoma model using 8505c cells. This model can prove to be a reliable and useful tool to investigate in vivo biological mechanisms determining thyroid cancer aggressiveness, and to test novel therapeutics for the treatment of refractory or advanced thyroid cancers.

Late Intervention with anti-BRAFV600E Therapy Induces Tumor Regression in an Orthotopic Mouse Model of Human Anaplastic Thyroid Cancer

Human anaplastic thyroid cancer (ATC) is a lethal disease with an advanced clinical presentation and median survival of 3 months. The BRAF(V600E) oncoprotein is a potent transforming factor that causes human thyroid cancer cell progression in vitro and in vivo; therefore, we sought to target this oncoprotein in a late intervention model of ATC in vivo. We used the human ATC cell line 8505c, which harbors the BRAF(V600E) and TP53(R248G) mutations. Immunocompromised mice were randomized to receive the selective anti-BRAF(V600E) inhibitor, PLX4720, or vehicle by oral gavage 28 d after tumor implantation, 1 wk before all animals typically die due to widespread metastatic lung disease and neck compressive symptoms in this model. Mice were euthanized weekly to evaluate tumor volume and metastases. Control mice showed progressive tumor growth and lung metastases by 35 d after tumor implantation. At that time, all control mice had large tumors, were cachectic, and were euthanized due to their tumor-related weight loss. PLX4720-treated mice, however, showed a significant decrease in tumor volume and lung metastases in addition to a reversal of tumor-related weight loss. Mouse survival was extended to 49 d in PLX4720-treated animals. PLX4720 treatment inhibited cell cycle progression from 28 d to 49 d in vivo. PLX4720 induces striking tumor regression and reversal of cachexia in an in vivo model of advanced thyroid cancer that harbors the BRAF(V600E) mutation.

Personalized Therapy in Patients With Anaplastic Thyroid Cancer: Targeting Genetic and Epigenetic Alterations

CONTEXT Anaplastic thyroid cancer (ATC) is the most lethal of all thyroid cancers and one of the most aggressive human carcinomas. In the search for effective treatment options, research toward targeted, personalized therapies is proving to be a path with great potential. As we gain a deeper understanding of the genetic (eg, BRAF(V600E), PIK3CA, TP53, hTERT mutations, etc) and epigenetic (eg, histone methylation, histone de-acetylation, microRNA regulatory circuits, etc) alterations driving ATC, we are able to find targets when developing novel therapies to improve the lives of patients. Beyond development, we can look into the effectiveness of already approved targeted therapies (eg, anti-BRAF(V600E) selective inhibitors, tyrosine kinase inhibitors, histone deacetylase inhibitors, inhibitors of DNA methylation, etc) to potentially test in ATC after learning the molecular mechanisms that aid in tumor progression. DESIGN We performed a literature analysis in Medline through the PubMed web site for studies published between 2003 and 2014 using the following main keywords: anaplastic thyroid cancer, genetic and epigenetic alterations. OBJECTIVE Here, we outlined the common pathways that are altered in ATC, including the BRAF(V600E)/ERK1/2-MEK1/2 and PI3K-AKT pathways. We then examined the current research looking into personalized, potential targeted therapies in ATC, mentioning those that have been tentatively advanced into clinical trials and those with the potential to reach that stage. We also reviewed side effects of the current and potential targeted therapies used in patients with advanced thyroid cancer. CONCLUSIONS DNA and RNA next-generation sequencing analysis will be fundamental to unraveling a precise medicine and therapy in patients with ATC. Indeed, given the deep biological heterogeneity/complexity and high histological grade of this malignancy and its tumor microenvironment, personalized therapeutic approaches possibly based on the use of combinatorial targeted therapy will provide a rational approach when finding the optimal way to improve treatments for patients with ATC.