Inhibiting ERK dimerization ameliorates BRAF-driven anaplastic thyroid cancer.

BACKGROUND: RAS-to-ERK signaling is crucial for the onset and progression of advanced thyroid carcinoma, and blocking ERK dimerization provides a therapeutic benefit in several human carcinomas. Here we analyzed the effects of DEL-22379, a relatively specific ERK dimerization inhibitor, on the activation of the RAS-to-ERK signaling cascade and on tumor-related processes in vitro and in vivo. METHODS: We used a panel of four human anaplastic thyroid carcinoma (ATC) cell lines harboring BRAF or RAS mutations to analyze ERK dynamics and tumor-specific characteristics. We also assessed the impact of DEL-22379 on the transcriptional landscape of ATC cell lines using RNA-sequencing and evaluated its therapeutic efficacy in an orthotopic mouse model of ATC. RESULTS: DEL-22379 impaired upstream ERK activation in BRAF- but not RAS-mutant cells. Cell viability and metastasis-related processes were attenuated by DEL-22379 treatment, but mostly in BRAF-mutant cells, whereas in vivo tumor growth and dissemination were strongly reduced for BRAF-mutant cells and mildly reduced for RAS-mutant cells. Transcriptomics analyses indicated that DEL-22379 modulated the transcriptional landscape of BRAF- and RAS-mutant cells in opposite directions. CONCLUSIONS: Our findings establish that BRAF- and RAS-mutant thyroid cells respond differentially to DEL-22379, which cannot be explained by the previously described mechanism of action of the inhibitor. Nonetheless, DEL-22379 demonstrated significant anti-tumor effects against BRAF-mutant cells in vivo with an apparent lack of toxicity, making it an interesting candidate for the development of combinatorial treatments. Our data underscore the differences elicited by the specific driver mutation for thyroid cancer onset and progression, which should be considered for experimental and clinical approaches.

Gbetagamma dimers released in response to thyrotropin activate phosphoinositide 3-kinase and regulate gene expression in thyroid cells.

Signaling by TSH through its receptor leads to the dissociation of trimeric G proteins into Galpha and Gbetagamma. Galphas activates adenylyl cyclase, which increases cAMP levels that induce several effects in the thyroid cell, including transcription of the sodium-iodide symporter (NIS) gene through a mechanism involving Pax8 binding to the NIS promoter. Much less is known about the function of Gbetagamma in thyroid differentiation, and therefore we studied their role in TSH signaling. Gbetagamma overexpression inhibits NIS promoter activation and reduces NIS protein accumulation in response to TSH and forskolin. Conversely, inhibition of Gbetagamma-dependent pathways increases NIS promoter activity elicited by TSH but does not modify forskolin-induced activation. Gbetagamma dimers are being released from the Gs subfamily of proteins, because cholera toxin mimics the effects elicited by TSH, whereas pertussis toxin has no effect on NIS promoter activity. We also found that TSH stimulates Akt phosphorylation in a phosphoinositide 3-kinase (PI3K)-dependent and cAMP-independent manner. This is mediated by Gbetagamma, because its overexpression or specific sequestration, respectively, increased or reduced phosphorylated Akt levels upon TSH stimulation. Gbetagamma sequestration increases NIS protein levels induced by TSH and Pax8 binding to the NIS promoter, which is also increased by PI3K inhibition. This is, at least in part, caused by Gbetagamma-mediated Pax8 exclusion from the nucleus that is attenuated when PI3K activity is blocked. These data unequivocally demonstrate that Gbetagamma released by TSH action stimulate PI3K, inhibiting NIS gene expression in a cAMP-independent manner due to a decrease in Pax8 binding to the NIS promoter.

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice.

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

Regulators of the RAS-ERK pathway as therapeutic targets in thyroid cancer.

Thyroid cancer is mostly an ERK-driven carcinoma, as up to 70% of thyroid carcinomas are caused by mutations that activate the RAS/ERK mitogenic signaling pathway. The incidence of thyroid cancer has been steadily increasing for the last four decades; yet, there is still no effective treatment for advanced thyroid carcinomas. Current research efforts are focused on impairing ERK signaling with small-molecule inhibitors, mainly at the level of BRAF and MEK. However, despite initial promising results in animal models, the clinical success of these inhibitors has been limited by the emergence of tumor resistance and relapse. The RAS/ERK pathway is an extremely complex signaling cascade with multiple points of control, offering many potential therapeutic targets: from the modulatory proteins regulating the activation state of RAS proteins to the scaffolding proteins of the pathway that provide spatial specificity to the signals, and finally, the negative feedbacks and phosphatases responsible for inactivating the pathway. The aim of this review is to give an overview of the biology of RAS/ERK regulators in human cancer highlighting relevant information on thyroid cancer and future areas of research.

TAZ/WWTR1 Mediates the Pulmonary Effects of NKX2-1 Mutations in Brain-Lung-Thyroid Syndrome.

Context: Identification of a frameshift heterozygous mutation in the transcription factor NKX2-1 in a patient with brain-lung-thyroid syndrome (BLTS) and life-threatening lung emphysema. Objective: To study the genetic defect that causes this complex phenotype and dissect the molecular mechanism underlying this syndrome through functional analysis. Methods: Mutational study by DNA sequencing, generation of expression vectors, site-directed mutagenesis, protein-DNA-binding assays, luciferase reporter gene assays, confocal microscopy, coimmunoprecipitation, and bioinformatics analysis. Results: We identified a mutation [p.(Val75Glyfs*334)] in the amino-terminal domain of the NKX2-1 gene, which was functionally compared with a previously identified mutation [p.(Ala276Argfs*75)] in the carboxy-terminal domain in other patients with BLTS but without signs of respiratory distress. Both mutations showed similar protein expression profiles, subcellular localization, and deleterious effects on thyroid-, brain-, and lung-specific promoter activity. Coexpression of the coactivator TAZ/WWTR1 (transcriptional coactivator with PDZ-binding motif/WW domain-containing transcription regulator protein 1) restored the transactivation properties of p.(Ala276Argfs*75) but not p.(Val75Glyfs*334) NKX2-1 on a lung-specific promoter, although both NKX2-1 mutants could interact equally with TAZ/WWTR1. The retention of residual transcriptional activity in the carboxy-terminal mutant, which was absent in the amino-terminal mutant, allowed the functional rescue by TAZ/WWTR1. Conclusions: Our results support a mechanistic model involving TAZ/WWTR1 in the development of human congenital emphysema, suggesting that this protein could be a transcriptional modifier of the lung phenotype in BLTS.

Key signaling pathways in thyroid cancer.

Whole genome sequencing approaches have provided unprecedented insights into the genetic lesions responsible for the onset, progression and dedifferentiation of various types of thyroid carcinomas. Through these efforts, the MAPK and PI3K signaling cascades have emerged as the main activation pathways implicated in thyroid tumorigenesis. The nature of these essential pathways is highly complex, with hundreds of components, multiple points of crosstalk, different subcellular localizations and with the ability to potentially regulate many cellular processes. Small-molecule inhibitors targeting key kinases of these pathways hold great promise as novel therapeutics and several have reached clinical trials. However, while some remarkable responses have been reported, the development of resistance remains a matter of concern and limits the benefit for patients. In this review, we discuss the latest findings on the major components of the MAPK and PI3K pathways, including their mechanisms of activation in physiological and pathological contexts, their genetic alterations with respect to the different types of thyroid carcinomas and the more relevant drugs designed to block their activity.

DARPP-32 (dopamine and 3',5'-cyclic adenosine monophosphate-regulated neuronal phosphoprotein) is essential for the maintenance of thyroid differentiation.

Coordination of events leading to differentiation is mediated by the concerted action of multiple signal transduction pathways. In general, the uncoupling of mechanisms linking differentiation to cell cycle exit is a hallmark of cancer, yet the identity and regulation of molecules integrating signal transduction pathways remains largely unknown. One notable exception is DARPP-32 (dopamine and cAMP-regulated neuronal phosphoprotein, molecular mass, 32 kDa), a third messenger that integrates multiple signaling pathways in the brain. Thyroid cells represent an excellent model for understanding the coupling of signal transduction pathways leading to both proliferation and differentiation. The cooperative action of IGF-I and TSH together, but not alone, enable thyroid cells to proliferate while maintaining their differentiated state. How signaling downstream from these molecules is integrated is not known. Here we show that DARPP-32 expression is targeted by TSH and IGF-I in thyrocytes. Significantly, dedifferentiated, tumoral, or Ras-transformed thyrocytes fail to express DARPP-32 whereas short interfering RNA-mediated silencing of DARPP-32 expression in normally differentiated thyroid cells results in loss of differentiation markers such as thyroid transcription factor 1, Pax8, thyroglobulin, and the Na/I symporter. Consistently, DARPP-32 reexpression in ras-transformed cells results in reactivation of the otherwise silent thyroglobulin and thyroperoxidase promoter. Thus, DARPP-32 is critical for the maintenance of thyroid differentiation by TSH and IGF-I, and loss of DARPP-32 expression may be a characteristic of thyroid cancer. Our results also raise the possibility that DARPP-32 may play a similar role in the maintenance of differentiation of a range of other cell types.

ß-catenin signaling is required for RAS-driven thyroid cancer through PI3K activation.

Mutations in ß-catenin are traditionally described as late events in thyroid cancer progression. However, the functional implications of ß-catenin dysregulation in the context of tumor initiating events remain unclear. The aim of this work was to investigate whether the two main oncogenic drivers in thyroid cancer, RAS and BRAF, could activate the Wnt/ß-catenin pathway. Expression of HRASV12 but not BRAFV600E in thyroid cells induced ß-catenin nuclear localization, increased ß-catenin-dependent transcriptional activity and inhibited GSK3ß. In a panel of human thyroid cancer cell lines representative of the main genetic events in thyroid cancer, ß-catenin activation was highly dependent on PI3K/AKT activity through its phosphorylation at S552, but not on MAPK. Silencing of ß-catenin expression in cell lines led to a dramatic reduction in proliferation due to an induction of senescence, which was concordant with a reduction in tumor size in nude mice. Moreover, ß-catenin silencing suppressed the expression of EMT-related genes and reduced the invasive capacity of the tumor cells. In conclusion, this work demonstrates that RAS-driven tumors induce PI3K/AKT-dependent ß-catenin activation.

FOXO1 controls thyroid cell proliferation in response to TSH and IGF-I and is involved in thyroid tumorigenesis.

TSH and insulin/IGF-I synergistically induce the proliferation of thyroid cells mainly through the cAMP and phosphatidylinositol 3-kinase (PI3K) pathways. However, the events involved in this cooperative induction remain unknown, and molecules that are potentially controlled by both TSH and IGF-I are interesting candidates as integrators of both stimuli. The finding that the PI3K pathway is frequently activated in thyroid malignancies has attracted attention to this pathway in the thyroid field. One of the targets of PI3K is Forkhead box O (FoxO)-1, a widely expressed transcription factor involved in a variety of cellular processes such as differentiation, proliferation, and apoptosis. Here we show that FoxO1 is highly expressed in differentiated rat thyroid cells and human thyroid tissue compared with human thyroid tumor-derived cells and surgically removed thyroid tumors, in which its expression is reduced. In differentiated cells, TSH/cAMP treatment decreases FoxO1 mRNA and protein levels through proteasome activation, whereas both TSH and IGF-I control FoxO1 localization by promoting a rapid exclusion from the nucleus in an Akt-dependent manner. FoxO1 can control p27(KIP1) expression in differentiated and tumor cells of the thyroid. Furthermore, FoxO1 reexpression in tumor cells promotes a decrease in their proliferation rate, whereas FoxO1 interference in differentiated cells increases their proliferation. These data point to an important role of FoxO1 in mediating the effects of TSH and IGF-I on thyroid cell proliferation and provide a link between loss of FoxO1 expression and the uncontrolled proliferation of thyroid tumor cells.

DARPP-32 is required for MAPK/ERK signaling in thyroid cells.

Modulation of MAPK signaling duration by cAMP defines its physiological output by driving cells toward proliferation or differentiation. Understanding how the kinetics of MAPK signaling are integrated with other cellular signals is a key issue in development and cancer. Here we show that dopamine and cAMP-regulated neuronal phosphoprotein, 32 kDa (DARPP-32), a protein required for thyroid cell differentiation, determines whether MAPK/ERK activation is sustained or transient. Serum, a stimulus that activates MAPK signaling and does not independently increase DARPP-32 levels results in transient activation of the MAPK pathway. By contrast, TSH + (IGF-I) activate MAPK signaling but also independently increase DARPP-32 levels. Our results are consistent with a model in which maintenance of DARPP-32 expression by TSH + IGF-I leads to sustained MAPK signaling. Moreover, the sensitivity of MAPK/ERK signaling in thyroid cells is lost when de novo DARPP-32 expression is blocked by small interfering RNA. Because both DARPP-32 levels and function as inhibitor of protein phosphatase 1, a key inhibitor of MAPK kinase activity, are governed by cAMP/protein kinase A, the results may explain why in thyroid cells cAMP signaling downstream from TSH controls the duration of MAPK pathway activity. Thus, fine-tuning of DARPP-32 levels leads to changes in the kinetics or sensitivity of MAPK/ERK signaling. Given the implications of MAPK signaling in thyroid cancer and the loss of DARPP-32 in tumor and transformed thyroid cells, DARPP-32 may represent a key therapeutic target.