Publications

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.

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.

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 TGFβ1, 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 TGFβ1 expression evoke gene-regulatory networks and pathways (TGFβ 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/TGFβ1 axis reduces tumor cell growth and overcomes drug resistance.

CONCLUSIONS: Pericytes shield BRAFV600E-PTC cells from targeted therapy via TSP-1 and TGFβ1, suggesting this axis as a new therapeutic target for overcoming resistance to BRAFV600E and TK inhibitors.

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.

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.

Cachexia is the result of complex metabolic alterations which cause morbidity and mortality in patients with advanced cancers including undifferentiated (anaplastic) thyroid carcinoma (ATC). ATC is a lethal disease with limited therapeutic options and unclear etiology for cachexia. We hypothesize that the BRAF(V600E) oncoprotein triggers microvascular endothelial cell tubule formation (in vitro angiogenesis) by means of factors which play a crucial role in angiogenic switch, inflammation/immune response and cachexia. We use human ATC cells and applied multiplex ELISA assay to screen for and measure angiogenic/cachectic and pro-inflammatory factors in the ATC-derived secretome. We find that vemurafenib anti-BRAF(V600E) therapy significantly reduces secreted VEGFA, VEGFC and IL6 protein levels compared to vehicle-treated ATC cells. As a result, the secretome from vemurafenib-treated ATC cells inhibits microvascular endothelial cell-related in vitro angiogenesis. Furthermore, ATC clinical samples express VEGFA, VEGFC and IL6 proteins. Our results suggest that angiogenic/cachectic and pro-inflammatory/immune response factors could play a crucial role in BRAF(V600E)-positive human ATC aggressiveness. Understanding the extent to which microenvironment-associated angiogenic factors participate in cachexia and cancer metabolism in advanced thyroid cancers will reveal new biomarkers and foster novel therapeutic approaches.

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.

Evaluation for malignancy of the adrenal cortex, adrenal cortical carcinoma (ACC), is a challenge in surgical pathology due to its relative rarity and histologic overlap with its benign counterpart, adrenocortical adenoma (ACA). We characterized a cohort of human ACC and ACA, including a molecular screen, with a goal of identifying potential diagnostic adjuncts. Thirty-six cases of ACC underwent histologic and clinical review. In the 31 ACC cases with available material and a cohort of 10 ACA cases, a multiplex nucleotide amplification molecular screen from formalin-fixed, paraffin-embedded tissue was peformed. ACCs demonstrated a wide variety of clinical and histologic characteristics with overall poor but unpredictable survival for subjects with ACC. By mutational screen, 12/31 (38.7%) carcinomas harbored CTNNB1 mutations, 1 with an additional TP53 mutation; 1 case each had isolated APC and TP53 mutations; 16 were wild-type for all tested loci; and 1 case demonstrated repeated assay failures. Two of the 10 ACA (20%) demonstrated CTNNB1 mutations by mutational screen, with no additional mutations. Immunohistochemistry for beta-catenin was performed and compared with the results of the molecular screen. Strong nuclear beta-catenin immunopositivity corresponded to the presence of CTNNB1 mutation by genotyping in 10 of 12 cases (83% sensitivity); the mismatched case(s) demonstrated strong membranous staining by immunohistochemistry. Seventeen of the 18 cases without CTNNB1 mutation showed membranous staining or did not stain (94% specificity); the mismatched case demonstrated scattered (<10%) positive nuclei. Both mutations in ACA were corroborated with immunohistochemistry for beta-catenin. No histomorphologic parameter appeared dominant in lesions with a particular mutational status. Based on these results, mutational status of CTNNB1 in adrenal cortical neoplasms can be predicted with reasonable accuracy by immunohistochemical cellular localization. Nuclear localization of beta-catenin by immunostain may be helpful in analysis of select lesions of the adrenal cortex whose biological behavior is uncertain from clinical and histologic information; a larger cohort is required to test this hypothesis.