Tumor Microenvironment-Associated Pericyte Populations May Impact Therapeutic Response in Thyroid Cancer.

Thyroid cancer is the most common endocrine malignancy, and aggressive radioactive iodine refractory thyroid carcinomas still lack an effective treatment. A deeper understanding of tumor heterogeneity and microenvironment will be critical to establishing new therapeutic approaches. One of the important influencing factors of tumor heterogeneity is the diversity of cells in the tumor microenvironment. Among these are pericytes, which play an important role in blood vessel stability and angiogenesis, as well as tumor growth and metastasis. Pericytes also have stem cell-like properties and are a heterogeneous cell population, and their lineage, which has been challenging to define, may impact tumor resistance at different tumor stages. Pericytes are also important stroma cell types in the angiogenic microenvironment which express tyrosine-kinase (TK) pathways (e.g., PDGFR-ß). Although TK inhibitors (TKI) and BRAFV600E inhibitors are currently used in the clinic for thyroid cancer, their efficacy is not durable and drug resistance often develops. Characterizing the range of distinct pericyte populations and distinguishing them from other perivascular cell types may enable the identification of their specific functions in the thyroid carcinoma vasculature. This remains an essential step in developing new therapeutic strategies. Also, assessing whether thyroid tumors hold immature and/or mature vasculature with pericyte populations coverage may be key to predicting tumor response to either targeted or anti-angiogenesis therapies. It is also critical to apply different markers in order to identify pericyte populations and characterize their cell lineage. This chapter provides an overview of pericyte ontogenesis and the lineages of diverse cell populations. We also discuss the role(s) and targeting of pericytes in thyroid carcinoma, as well as their potential impact on precision targeted therapies and drug resistance.

Role of Regulatory Non-Coding RNAs in Aggressive Thyroid Cancer: Prospective Applications of Neural Network Analysis.

Thyroid cancer (TC) is the most common endocrine malignancy. Most TCs have a favorable prognosis, whereas anaplastic thyroid carcinoma (ATC) is a lethal form of cancer. Different genetic and epigenetic alterations have been identified in aggressive forms of TC such as ATC. Non-coding RNAs (ncRNAs) represent functional regulatory molecules that control chromatin reprogramming, including transcriptional and post-transcriptional mechanisms. Intriguingly, they also play an important role as coordinators of complex gene regulatory networks (GRNs) in cancer. GRN analysis can model molecular regulation in different species. Neural networks are robust computing systems for learning and modeling the dynamics or dependencies between genes, and are used for the reconstruction of large data sets. Canonical network motifs are coordinated by ncRNAs through gene production from each transcript as well as through the generation of a single transcript that gives rise to multiple functional products by post-transcriptional modifications. In non-canonical network motifs, ncRNAs interact through binding to proteins and/or protein complexes and regulate their functions. This article overviews the potential role of ncRNAs GRNs in TC. It also suggests prospective applications of deep neural network analysis to predict ncRNA molecular language for early detection and to determine the prognosis of TC. Validation of these analyses may help in the design of more effective and precise targeted therapies against aggressive TC.

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-kappaBeta 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.

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

Although B-Raf(V600E) 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-Raf(V600E) gene set signature associated with tumor progression in PTCs. An independent cohort of B-Raf(V600E)-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-Raf(V600E) 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-Raf(V600E) cells in which either B-Raf(V600E) 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-Raf(V600E), caused a significant tumor growth delay and decreased distant metastases, without evidence of toxicity. In conclusion, B-Raf(V600E) plays an important role in PTC progression through genes (i.e., TSP-1) important in tumor invasion and metastasis. Testing of a patient's thyroid cancer for B-Raf(V600E) will yield important information about potential tumor aggressiveness and also allow for future use of targeted therapies with selective B-Raf(V600E) inhibitors, such as PLX4720.

The Tyrosine Kinase Inhibitor Lenvatinib Inhibits Anaplastic Thyroid Carcinoma Growth by Targeting Pericytes in the Tumor Microenvironment.

Background: Anaplastic thyroid carcinoma (ATC) is a rapidly fatal cancer with a median survival of a few months. Enhanced therapeutic options for durable management of ATC will rely on an understanding of genetics and the role of the tumor microenvironment. The prognosis for patients with ATC has not improved despite more detailed scrutiny of underlying tumor genetics. Pericytes in the microenvironment play a key evasive role for thyroid carcinoma (TC) cells. Lenvatinib improves outcomes in patients with radioiodine-refractory well-differentiated TC. In addition to the unclear role of pericytes in ATC, the effect and mechanism of lenvatinib efficacy on ATC have not been sufficiently elucidated. Design: We assessed pericyte enrichment in ATC. We determined the effect of lenvatinib on ATC cell growth cocultured with pericytes and in a xenograft mouse model from human BRAFWT/V600E-ATC-derived cells coimplanted with pericytes. Results: ATC samples were significantly enriched in pericytes compared with normal thyroid samples. BRAFWT/V600E-ATC-derived cells were resistant to lenvatinib treatment shown by a lack of suppression of MAPK and Akt pathways. Moreover, lenvatinib increased CD47 protein (thrombospondin-1 [TSP-1] receptor) levels over time vs. vehicle. TSP-1 levels were downregulated upon lenvatinib at late vs. early time points. Critically, ATC cells, when cocultured with pericytes, showed increased sensitivity to this therapy and ultimately decreased number of cells. The coimplantation in vivo of ATC cells with pericytes increased ATC growth and did not downregulate TSP-1 in the microenvironment in vivo. Conclusions and Implications: Pericytes are enriched in ATC samples. Lenvatinib showed inhibitory effects on BRAFWT/V600E-ATC cells in the presence of pericytes. The presence of pericytes could be crucial for effective lenvatinib treatment in patients with ATC. Degree of pericyte abundance may be an attractive prognostic marker in assessing pharmacotherapeutic options. Effective durable management of ATC will rely on an understanding not only of genetics but also on the role of the tumor microenvironment.

Case Report of CCDC149-ALK Fusion: A Novel Genetic Alteration and a Clinically Relevant Target in Metastatic Papillary Thyroid Carcinoma.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer, usually with an indolent course. ALK fusions are rare in PTC but may give rise to a more aggressive behavior. We report a novel ALK fusion, CCDC149-ALK, not previously described in PTC, detected by next-generation sequencing in a 30-year-old woman with progressive widely metastatic radioiodine-refractory (RAIR) disease to lung, muscle, and brain. The patient was started on alectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor. Within eight weeks, her palpable disease had completely regressed, and the serum thyroglobulin decreased dramatically. Restaging imaging demonstrated an objective partial response. Our case highlights the role of ALK fusions in thyroid cancer and highlights its clinical significance in PTC. We recommend deep mutational sequencing in BRAFV600E-negative RAIR PTC to identify targetable genetic alterations, including gene fusions, that may result in dramatic therapeutic benefits.

Targeting BRAFV600E with PLX4720 displays potent antimigratory and anti-invasive activity in preclinical models of human thyroid cancer.

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.

Lenvatinib Targets PDGFR-ß Pericytes and Inhibits Synergy With Thyroid Carcinoma Cells: Novel Translational Insights.

CONTEXT: Pericyte populations abundantly express tyrosine kinases (eg, platelet-derived growth factor receptor-ß [PDGFR-ß]) and impact therapeutic response. Lenvatinib is a clinically available tyrosine kinase inhibitor that also targets PDGFR-ß. Duration of therapeutic response was shorter in patients with greater disease burden and metastasis. Patients may develop drug resistance and tumor progression. OBJECTIVES: Develop a gene signature of pericyte abundance to assess with tumor aggressiveness and determine both the response of thyroid-derived pericytes to lenvatinib and their synergies with thyroid carcinoma-derived cells. DESIGN: Using a new gene signature, we estimated the relative abundance of pericytes in papillary thyroid carcinoma (PTC) and normal thyroid (NT) TCGA samples. We also cocultured CD90+;PAX8- thyroid-derived pericytes and BRAFWT/V600E-PTC-derived cells to determine effects of coculture on paracrine communications and lenvatinib response. RESULTS: Pericyte abundance is significantly higher in BRAFV600E-PTC with hTERT mutations and copy number alterations compared with NT or BRAFWT-PTC samples, even when data are corrected for clinical-pathologic confounders. We have identified upregulated pathways important for tumor survival, immunomodulation, RNA transcription, cell-cycle regulation, and cholesterol metabolism. Pericyte growth is significantly increased by platelet-derived growth factor-BB, which activates phospho(p)-PDGFR-ß, pERK1/2, and pAKT. Lenvatinib strongly inhibits pericyte viability by down-regulating MAPK, pAKT, and p-p70S6-kinase downstream PDGFR-ß. Critically, lenvatinib significantly induces higher BRAFWT/V600E-PTC cell death when cocultured with pericytes, as a result of pericyte targeting via PDGFR-ß. CONCLUSIONS: This is the first thyroid-specific model of lenvatinib therapeutic efficacy against pericyte viability, which disadvantages BRAFWT/V600E-PTC growth. Assessing pericyte abundance in patients with PTC could be essential to selection rationales for appropriate targeted therapy with lenvatinib.

Fine-Tuning Lipid Metabolism by Targeting Mitochondria-Associated Acetyl-CoA-Carboxylase 2 in BRAFV600E Papillary Thyroid Carcinoma.

Background: BRAFV600E acts as an ATP-dependent cytosolic kinase. BRAFV600E inhibitors are widely available, but resistance to them is widely reported in the clinic. Lipid metabolism (fatty acids) is fundamental for energy and to control cell stress. Whether and how BRAFV600E impacts lipid metabolism regulation in papillary thyroid carcinoma (PTC) is still unknown. Acetyl-CoA carboxylase (ACC) is a rate-limiting enzyme for de novo lipid synthesis and inhibition of fatty acid oxidation (FAO). ACC1 and ACC2 genes encode distinct isoforms of ACC. The aim of our study was to determine the relationship between BRAFV600E and ACC in PTC. Methods: We performed RNA-seq and DNA copy number analyses in PTC and normal thyroid (NT) in The Cancer Genome Atlas samples. Validations were performed by using assays on PTC-derived cell lines of differing BRAF status and a xenograft mouse model derived from a heterozygous BRAFWT/V600E PTC-derived cell line with knockdown (sh) of ACC1 or ACC2. Results:ACC2 mRNA expression was significantly downregulated in BRAFV600E-PTC vs. BRAFWT-PTC or NT clinical samples. ACC2 protein levels were downregulated in BRAFV600E-PTC cell lines vs. the BRAFWT/WT PTC cell line. Vemurafenib increased ACC2 (and to a lesser extent ACC1) mRNA levels in PTC-derived cell lines in a BRAFV600E allelic dose-dependent manner. BRAFV600E inhibition increased de novo lipid synthesis rates, and decreased FAO due to oxygen consumption rate (OCR), and extracellular acidification rate (ECAR), after addition of palmitate. Only shACC2 significantly increased OCR rates due to FAO, while it decreased ECAR in BRAFV600E PTC-derived cells vs. controls. BRAFV600E inhibition synergized with shACC2 to increase intracellular reactive oxygen species production, leading to increased cell proliferation and, ultimately, vemurafenib resistance. Mice implanted with a BRAFWT/V600E PTC-derived cell line with shACC2 showed significantly increased tumor growth after vemurafenib treatment, while vehicle-treated controls, or shGFP control cells treated with vemurafenib showed stable tumor growth. Conclusions: These findings suggest a potential link between BRAFV600E and lipid metabolism regulation in PTC. BRAFV600E downregulates ACC2 levels, which deregulates de novo lipid synthesis, FAO due to OCR, and ECAR rates. ShACC2 may contribute to vemurafenib resistance and increased tumor growth. ACC2 rescue may represent a novel molecular strategy for overcoming resistance to BRAFV600E inhibitors in refractory PTC.

Maternal thyroid hormones are transcriptionally active during embryo-foetal development: results from a novel transgenic mouse model.

Even though several studies highlighted the role of maternal thyroid hormones (THs) during embryo-foetal development, direct evidence of their interaction with embryonic thyroid receptors (TRs) is still lacking. We generated a transgenic mouse model ubiquitously expressing a reporter gene tracing TH action during development. We engineered a construct (TRE2×) containing two TH-responsive elements controlling the expression of the LacZ reporter gene, which encodes ß-galactosidase (ß-gal). The specificity of the TRE2× activation by TH was evaluated in NIH3T3 cells by cotransfecting TRE2× along with TRs, retinoic or oestrogen receptors in the presence of their specific ligands. TRE2× transgene was microinjected into the zygotes, implanted in pseudopregnant BDF1 (a first-generation (F1) hybrid from a cross of C57BL/6 female and a DBA/2 male) mice and transgenic mouse models were developed. ß-gal expression was assayed in tissue sections of transgenic mouse embryos at different stages of development. In vitro, TRE2× transactivation was observed only following physiological T3 stimulation, mediated exclusively by TRs. In vivo, ß-gal staining, absent until embryonic day 9.5-10.5 (E9.5-E10.5), was observed as early as E11.5-E12.5 in different primordia (i.e. central nervous system, sense organs, intestine, etc.) of the TRE2× transgenic embryos, while the foetal thyroid function (FTF) was still inactive. Immunohistochemistry for TRs essentially colocalized with ß-gal staining. No ß-gal staining was detected in embryos of hypothyroid transgenic mice. Importantly, treatment with T3 in hypothyroid TRE2× transgenic mice rescued ß-gal expression. Our results provide in vivo direct evidence that during embryonic life and before the onset of FTF, maternal THs are transcriptionally active through the action of embryonic TRs. This model may have clinical relevance and may be employed to design end-point assays for new molecules affecting THs action.

Role of B-Raf(V600E) in differentiated thyroid cancer and preclinical validation of compounds against B-Raf(V600E).

B-Raf(V600E), an oncogenic protein kinase, is the most frequent genetic alteration in papillary thyroid carcinomas (PTC). PTC represents 80-90% of all thyroid cancers and over the past five years, more than 200 manuscripts have been published about the relationship between "B-Raf(V600E) and thyroid cancer". B-Raf(V600E) genetically arises from a transversion point mutation (valine-to-glutamate substitution at amino acid residue-600, V600E) and leads to over activation of the mitogen-activated protein kinases (MAPK) signaling pathway. The MAPK pathway is essential for transmitting proliferation signals generated by cell surface receptors and cytoplasmic signaling elements to the nucleus. In many cancers, including thyroid cancer, B-Raf(V600E) appears to play a crucial role in cell proliferation, survival and de-differentiation. In thyroid cancer, the V600E mutation occurs with greater frequently in aggressive subtypes of PTC, and in individuals that present at advanced stages of disease with extra-thyroidal extension and/or lymph node metastases. B-Raf(V600E) is considered a marker of aggressive disease in both PTC (>1 cm) and micro-PTC (

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 G(1) growth arrest and reduction of cell proliferation. Moreover, we observe a potential link between FOXA1 and the cell cycle machinery by identifying p27(kip1) 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.

Corrigendum: Thrombospondin-1 Silencing Down-Regulates Integrin Expression Levels in Human Anaplastic Thyroid Cancer Cells With BRAFV600E: New Insights in the Host Tissue Adaptation and Homeostasis of Tumor Microenvironment.

[This corrects the article DOI: 10.3389/fendo.2013.00189.].

Clonal Reconstruction of Thyroid Cancer: An Essential Strategy for Preventing Resistance to Ultra-Precision Therapy.

The introduction of ultra-precision targeted therapy has become a significant advancement in cancer therapeutics by creating treatments with less off target effects. Specifically with papillary thyroid carcinoma (PTC), the cancer's hallmark genetic mutation BRAFV600E can be targeted with selective inhibitors, such as vemurafenib. Despite initial positive tumor responses of regression and decreased viability, both single agent or combination agent drug treatments provide a selective pressure for drug resistant evolving clones within the overall heterogeneous tumor. Also, there are evidences suggesting that sequential monotherapy is ineffective and selects for resistant and ultimately lethal tumor clones. Reconstructing both clonal and subclonal thyroid tumor heterogeneous cell clusters for somatic mutations and epigenetic profile, copy number variation, cytogenetic alterations, and non-coding RNA expression becomes increasingly critical as different clonal enrichments implicate how the tumor may respond to drug treatment and dictate its invasive, metastatic, and progressive abilities, and predict prognosis. Therefore, development of novel preclinical and clinical empirical models supported by mathematical assessment will be the tools required for estimating the parameters of clonal and subclonal evolution, and unraveling the dormant vs. non-dormant state of thyroid cancer. In sum, novel experimental models performing the reconstruction both pre- and post-drug treatment of the thyroid tumor will enhance our understanding of clonal and sub-clonal reconstruction and tumor evolution exposed to treatments during ultra-precision targeted therapies. This approach will improve drug development strategies in thyroid oncology and identification of disease-specific biomarkers.

Coding Molecular Determinants of Thyroid Cancer Development and Progression.

Thyroid cancer is the most common endocrine malignancy. Its incidence and mortality rates have increased for patients with advanced-stage papillary thyroid cancer. The characterization of the molecular pathways essential in thyroid cancer initiation and progression has made huge progress, underlining the role of intracellular signaling to promote clonal evolution, dedifferentiation, metastasis, and drug resistance. The discovery of genetic alterations that include mutations (BRAF, hTERT), translocations, deletions (eg, 9p), and copy-number gain (eg, 1q) has provided new biological insights with clinical applications. Understanding how molecular pathways interplay is one of the key strategies to develop new therapeutic treatments and improve prognosis.

Pericytes Elicit Resistance to Vemurafenib and Sorafenib Therapy in Thyroid Carcinoma via the TSP-1/TGFß1 Axis.

PURPOSE: The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, in which pericytes are critical regulators of tyrosine-kinase (TK)-dependent signaling pathways. Although BRAFV600E and TK inhibitors are available, their efficacy as bimodal therapeutic agents in BRAFV600E-PTC is still unknown. EXPERIMENTAL DESIGN: We assessed the effects of vemurafenib (BRAFV600E inhibitor) and sorafenib (TKI) as single agents or in combination in BRAFWT/V600E-PTC and BRAFWT/WT cells using cell-autonomous, pericyte coculture, and an orthotopic mouse model. We also used BRAFWT/V600E-PTC and BRAFWT/WT-PTC clinical samples to identify differentially expressed genes fundamental to tumor microenvironment. RESULTS: Combined therapy blocks tumor cell proliferation, increases cell death, and decreases motility via BRAFV600E inhibition in thyroid tumor cells in vitro. Vemurafenib produces cytostatic effects in orthotopic tumors, whereas combined therapy (likely reflecting sorafenib activity) generates biological fluctuations with tumor inhibition alternating with tumor growth. We demonstrate that pericytes secrete TSP-1 and TGFß1, and induce the rebound of pERK1/2, pAKT and pSMAD3 levels to overcome the inhibitory effects of the targeted therapy in PTC cells. This leads to increased BRAFV600E-PTC cell survival and cell death refractoriness. We find that BRAFWT/V600E-PTC clinical samples are enriched in pericytes, and TSP1 and TGFß1 expression evoke gene-regulatory networks and pathways (TGFß signaling, metastasis, tumor growth, tumor microenvironment/ECM remodeling functions, inflammation, VEGF ligand-VEGF receptor interactions, immune modulation, etc.) in the microenvironment essential for BRAFWT/V600E-PTC cell survival. Critically, antagonism of the TSP-1/TGFß1 axis reduces tumor cell growth and overcomes drug resistance. CONCLUSIONS: Pericytes shield BRAFV600E-PTC cells from targeted therapy via TSP-1 and TGFß1, suggesting this axis as a new therapeutic target for overcoming resistance to BRAFV600E and TK inhibitors.

Invasive follicular variant of papillary thyroid cancer harboring the NRAS mutation Q61K and presenting with bone metastasis-A case report.

INTRODUCTION: The follicular variant of papillary thyroid cancer (FVPTC) can be noninvasive or invasive. The invasive form of FVPTC commonly harbors BRAF mutations whereas RAS mutations are more often associated with noninvasive FVPTC and a favorable clinical outcome. CASE REPORT: A 47-year-old man presented with a metastasis to his right iliac bone as the initial manifestation of a 1.6cm invasive FVPTC. After total thyroidectomy, the patient underwent additional treatment, including thyroid hormone suppressive treatment to non-detectable TSH levels, repeated courses of radioiodine treatment, external beam radiation, and treatment with the tyrosine kinase inhibitor sorafenib. Despite these therapeutic efforts, the disease progressed with growth of the iliac mass and additional metastatic spread to cervical and lumbar vertebrae causing increasing pain and disability. The patient succumbed to the disease four years after presentation. Retrospective next-generation sequencing of the primary tumor using a pan-cancer targeted mutation and gene fusion panel revealed NRAS Q61K mutation and no other oncogenic alterations. DISCUSSION: The study challenges the concept that thyroid neoplasms with isolated RAS mutations are often associated with favorable clinical behavior and may be candidates for conservative management. CONCLUSION: An isolated RAS mutation in invasive FVPTC may be associated with an aggressive clinical behavior.

Vemurafenib-resistance via de novo RBM genes mutations and chromosome 5 aberrations is overcome by combined therapy with palbociclib in thyroid carcinoma with BRAFV600E.

PURPOSE: Papillary thyroid carcinoma (PTC) is the most frequent endocrine tumor. BRAFV600E represents the PTC hallmark and is targeted with selective inhibitors (e.g. vemurafenib). Although there have been promising results in clinical trials using these inhibitors, most patients develop resistance and progress. Tumor clonal diversity is proposed as one mechanism underlying drug resistance. Here we have investigated mechanisms of primary and secondary resistance to vemurafenib in BRAFWT/V600E-positive PTC patient-derived cells with P16-/- (CDKN2A-/-). EXPERIMENTAL DESIGN: Following treatment with vemurafenib, we expanded a sub-population of cells with primary resistance and characterized them genetically and cytogenetically. We have used exome sequencing, metaphase chromosome analysis, FISH and oligonucleotide SNP-microarray assays to assess clonal evolution of vemurafenib-resistant cells. Furthermore, we have validated our findings by networks and pathways analyses using PTC clinical samples. RESULTS: Vemurafenib-resistant cells grow similarly to naïve cells but are refractory to apoptosis upon treatment with vemurafenib, and accumulate in G2-M phase. We find that vemurafenib-resistant cells show amplification of chromosome 5 and de novo mutations in the RBM (RNA-binding motifs) genes family (i.e. RBMX, RBM10). RBMX knockdown in naïve-cells contributes to tetraploidization, including expansion of clones with chromosome 5 aberrations (e.g. isochromosome 5p). RBMX elicits gene regulatory networks with chromosome 5q cancer-associated genes and pathways for G2-M and DNA damage-response checkpoint regulation in BRAFWT/V600E-PTC. Importantly, combined therapy with vemurafenib plus palbociclib (inhibitor of CDK4/6, mimicking P16 functions) synergistically induces stronger apoptosis than single agents in resistant-cells and in anaplastic thyroid tumor cells harboring the heterozygous BRAFWT/V600E mutation. CONCLUSIONS: Critically, our findings suggest for the first time that targeting BRAFWT/V600E and CDK4/6 represents a novel therapeutic strategy to treat vemurafenib-resistant or vemurafenib-naïve radioiodine-refractory BRAFWT/V600E-PTC. This combined therapy could prevent selection and expansion of aggressive PTC cell sub-clones with intrinsic resistance, targeting tumor cells either with primary or secondary resistance to BRAFV600E inhibitor.

antiphospholipid antibodies syndrome associated with hyperhomocysteinemia related to MTHFR Gene C677T and A1298C heterozygous mutations in a young man with idiopathic hypoparathyroidism (DiGeorge syndrome).

CONTEXT: Antiphospholipid syndrome (APS, or Hughes' syndrome) is a systemic autoimmune disorder characterized by antiphospholipid antibody positivity, which may lead to arterial and/or venous thrombosis. Hyperhomocysteinemia (HHcy), variously associated with 5,10-methylene tetrahydrofolate reductase (MTHFR) gene point mutations, is also implicated in thromboembolic events. The association of APS and HHcy has already been described but has never been reported in patients with DiGeorge syndrome (DGS), the most common contiguous-gene deletion syndrome (22q11.2) in humans, whose phenotype conversely includes bleeding disorders. DATA ACQUISITION: In this report, we present the case of a 19-yr-old patient with a past medical history of learning disability and obesity affected with idiopathic hypoparathyroidism, metabolic syndrome, and diffuse vasculitis disorders. He was referred to our endocrinology clinic for the management of severe hypocalcemia. At the time of presentation he had been taking antiepileptic drugs for 2 wk and displayed facial dysmorphism (short neck, micrognathia, a small mouth, hypoplastic nasal alae, eye hypertelorism, and low-set simple ears). DGS was suspected and confirmed by both fluorescence in situ hybridization analysis and single nucleotide polymorphism-array analysis, which revealed contiguous gene microdeletion of the chromosome 22q11.2 in the minimal DiGeorge critical region, specifically at the gene locus D22S75 (N25). CONCLUSIONS: APS, revealed by anti-beta-2-glycoprotein and anti-prothrombin antibodies positivity, and moderate HHcy related to heterozygous C677T and A1298C point mutations of the MTHFR gene were identified as a possible cause of thrombotic disorder responsible for the widespread presence of cutaneous and cerebral lesions.

Effect of the micronutrient iodine in thyroid carcinoma angiogenesis.

Iodide is a micronutrient essential for thyroid hormone production. The uptake and metabolism of iodide by thyrocytes is crucial to proper thyroid function. Iodide ions are drawn into the thyroid follicular cell via the sodium-iodide symporter (NIS) in the cell membrane and become integrated into tyrosyl residues to ultimately form thyroid hormones. We sought to learn how an abnormal concentration of iodide within thyrocyte can have significant effects on the thyroid, specifically the surrounding vascular network. Insufficient levels of iodide can lead to increased expression or activity of several pathways, including vascular endothelial growth factor (VEGF). The VEGF protein fuel vessel growth (angiogenesis) and therefore enhances the nutrients available to surrounding cells. Alternatively, normal/surplus iodide levels can have inhibitory effects on angiogenesis. Varying levels of iodide in the thyroid can influence thyroid carcinoma cell proliferation and angiogenesis via regulation of the hypoxia inducible factor-1 (HIF-1) and VEGF-dependent pathway. We have reviewed a number of studies to investigate how the effect of iodide on angiogenic and oxidative stress regulation can affect the viability of thyroid carcinoma cells. The various studies outlined give key insights to the role of iodide in thyroid follicles function and vascular growth, generally highlighting that insufficient levels of iodide stimulate pathways resulting in vascular growth, and viceversa normal/surplus iodide levels inhibit such pathways. Intriguingly, TSH and iodine levels differentially regulate the expression levels of angiogenic factors. All cells, including carcinoma cells, increase uptake of blood nutrients, meaning the vascular profile is influential to tumor growth and progression. Importantly, variation in the iodine concentrations also influence BRAFV600E-mediated oncogenic activity and might deregulate tumor proliferation. Although the mechanisms are not well eluted, iodine concentrations and metabolism might have a crucial influence on thyroid carcinoma cell viability via regulation of different molecular pathways, including angiogenesis regulatory autocrine and microenvironment-mediated signals.