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PDF file - 141K, RET-PTC triggers CD44-ICD and CD44-CTF production

PDF file- 142K, The RET-PTC-RAS-BRAF cascade triggers CD44 cleavage

PDF file - 102K, PKA and MEK inhibition blunts CD44-ICD mediated CREB phosphorylation

PDF file - 175K, CD44-ICD-CREB complex in human PTC cell lines

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PDF file - 78K, CD44-ICD expression in human thyroid cancer cell lines

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Cancer is an abnormal state of cells where they undergo uncontrolled proliferation and produce aggressive malignancies that cause millions of deaths every year. With the new understanding of the molecular mechanism(s) of disease progression, our knowledge about the disease is snowballing, leading to the evolution of many new therapeutic regimes and their successive trials. In the past few decades, various combinations of therapies have been proposed and are presently employed in the treatment of diverse cancers. Targeted drug therapy, immunotherapy, and personalized medicines are now largely being employed, which were not common a few years back. The field of cancer discoveries and therapeutics are evolving fast as cancer type-specific biomarkers are progressively being identified and several types of cancers are nowadays undergoing systematic therapies, extending patients’ disease-free survival thereafter. Although growing evidence shows that a systematic and targeted approach could be the future of cancer medicine, chemotherapy remains a largely opted therapeutic option despite its known side effects on the patient’s physical and psychological health. Chemotherapeutic agents/pharmaceuticals served a great purpose over the past few decades and have remained the frontline choice for advanced-stage malignancies where surgery and/or radiation therapy cannot be prescribed due to specific reasons. The present report succinctly reviews the existing and contemporary advancements in chemotherapy and assesses the status of the enrolled drugs/pharmaceuticals; it also comprehensively discusses the emerging role of specific/targeted therapeutic strategies that are presently being employed to achieve better clinical success/survival rate in cancer patients., All the authors are highly grateful and acknowledge to the authority of the respective departments and institutions for their support in carrying out this research. The authors also express their sincere gratitude to the unknown referee for critically reviewing the manuscript and suggesting useful changes. This research was funded by "Agencia Canaria de Investigación, Innovación y Sociedad de la Información (ACIISI) del Gobierno de Canarias” (No. ProID2020010134), and o´Caja Canarias (Project No. 2019SP43).

The expression level of the tumor suppressor p53 is controlled by the E3 ubiquitin ligase MDM2 with a regulatory feedback loop, which allows p53 to upregulate its inhibitor MDM2. In this manuscript we demonstrated that p90RSK binds and phosphorylates MDM2 on serine 166 both in vitro and in vivo by kinase assay, immunoblot, and co-immunoprecipitation assay; this phosphorylation increases the stability of MDM2 which in turn binds p53, ubiquitinating it and promoting its degradation by proteasome. A pharmacological inhibitor of p90RSK, BI-D1870, decreases MDM2 phosphorylation, and restores p53 function, which in turn transcriptionally increases the expression of cell cycle inhibitor p21 and of pro-apoptotic protein Bax and downregulates the anti-apoptotic protein Bcl-2, causing a block of cell proliferation, measured by a BrdU assay and growth curve, and promoting apoptosis, measured by a TUNEL assay. Finally, an immunohistochemistry evaluation of primary thyroid tumors, in which p90RSK is very active, confirms MDM2 stabilization mediated by p90RSK phosphorylation.

Background Improvement in genetic characterization of Colon Cancer (CC) patients is required to propose new potential targets, since surgical resection coupled to chemotherapy, presents several limits such as cancer recurrence and drug resistance. Targeted therapies have more efficacy and less toxicity than standard treatments. One of the most relevant cancer-specific actionable targets are receptor tyrosine kinases (RTKs) whose role in CC need to be better investigated. Methods We have analysed 37 CC patients using the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP). We have confirmed the somatic nature of RET variants through Sanger sequencing and assessed RET activation status and protein expression by immunofluorescence and western-blot analyses. We have used RET mutant expression vectors to evaluate the effect of selected mutations in HEK293 cells by performing proliferation, migration and clonogenic assays. Results Among the 409 cancer-related genes included in the CCP we have focused on the RTKs. Overall, we have observed 101 different potentially damaging variants distributed across 31 RTK genes in 28 patients. The most frequently mutated RTKs were FLT4, ROS1, EPH7, ERBB2, EGFR, RET, FGFR3 and FGFR4. In particular, we have identified 4 different somatic variants in 10% of CC patients in RET proto-oncogene. Among them, we have demonstrated that the G533C variant was able to activate RET by promoting dimer formation and enhancing Y1062 phosphorylation. Moreover, we have demonstrated that RET G533C variant was able to stimulate anchorage-dependent proliferation, migration and clonogenic cell survival. Notably, the effects induced by the RET G533C variant were abolished by vandetanib. Conclusions The discovery of pathogenic variants across RTK genes in 75% of the CC patients under analysis, suggests a previously underestimated role for RTKs in CC development. The identification of a gain-of-function RET mutation in CC highlights the potential use of RET in targeted therapy. Electronic supplementary material The online version of this article (10.1186/s13046-018-0746-y) contains supplementary material, which is available to authorized users.

RET receptor tyrosine kinase (RTK) acts as an ontogenetic driver in several human malignancies, including papillary and medullary thyroid carcinoma, lung adenocarcinoma, colorectal carcinoma, Spitzoid neoplasms, salivary gland carcinoma and chronic myeloproliferative disorders, secondary to as diverse genetic lesions as point-mutations, small insertions/deletions, and gene fusions. In other neoplasms, including breast and pancreatic adenocarcinoma, RET over-expression is up-regulated. Thus, small molecule compounds with RET tyrosine kinase inhibitory activity (TKIs) are being investigated for the targeted treatment of these malignancies. Multi-targeted TKIs with the RET inhibitory enzymatic activity of IC50 in the nanomolar range have entered clinical practice, registered for the treatment of medullary thyroid cancer (vandetanib, cabozantinib), radioiodine refractory non medullary thyroid cancer (lenvatinib, sorafenib) or cancers of other sites (sunitinib, ponatinib, regorafenib). This review summarizes mechanisms of RET oncogenic activity and properties of new TKIs that, at the preclinical stage, have demonstrated promising anti-RET activity.

CD44 is a marker of cancer stem-like cells and epithelial–mesenchymal transition that is overexpressed in many cancer types, including thyroid carcinoma. At extracellular and intramembranous domains, CD44 undergoes sequential metalloprotease- and γ-secretase–mediated proteolytic cleavage, releasing the intracellular protein fragment CD44-ICD, which translocates to the nucleus and activates gene transcription. Here, we show that CD44-ICD binds to the transcription factor CREB, increasing S133 phosphorylation and CREB-mediated gene transcription. CD44-ICD enhanced CREB recruitment to the cyclin D1 promoter, promoting cyclin D1 transcription and cell proliferation. Thyroid carcinoma cells harboring activated RET/PTC, RAS, or BRAF oncogenes exhibited CD44 cleavage and CD44-ICD accumulation. Chemical blockade of RET/PTC, BRAF, metalloprotease, or γ-secretase were each sufficient to blunt CD44 processing. Furthermore, thyroid cancer cell proliferation was obstructed by RNA interference–mediated knockdown of CD44 or inhibition of γ-secretase and adoptive CD44-ICD overexpression rescued cell proliferation. Together, these findings reveal a CD44-CREB signaling pathway that is needed to sustain cancer cell proliferation, potentially offering new molecular targets for therapeutic intervention in thyroid carcinoma. Cancer Res; 72(6); 1449–58. ©2012 AACR.

Context: The oncogenic BRAFV600E mutation results in an active structural conformation characterized by greatly elevated ERK activity. However, additional cellular effects caused by subcellular action of BRAFV600E remain to be identified. Objective: To explore these effects, differences in the subcellular localization of wild-type and mutant BRAF in thyroid cancer were investigated. Results: A significant proportion of endogenous and exogenous BRAFV600E, but not wild-type BRAF, was detected in the mitochondrial fraction, similar to other BRAF mutants including BRAFV600D, BRAFV600K, BRAFV600R, and BRAFG469A, which showed elevated kinase activity and mitochondrial localization. Induced expression of BRAFV600E suppressed the apoptotic responses against staurosporine and TNFα/cycloheximide. Interestingly, the mitochondrial localization and antiapoptotic activities of BRAFV600E were unaffected by sorafenib and U0126 suppression of MAPK kinase (MEK) and ERK activities. Similarly, although the RAF inhibitor sorafenib effectively inhibited MEK/ERK activation, it did not block the mitochondrial localization of BRAFV600E. In addition, inducible expression of BRAFV600E increased the glucose uptake rate and decreased O2 consumption, suggesting that BRAFV600E reduces mitochondrial oxidative phosphorylation, a signature feature of cancer cells. Again, these metabolic alterations resulted by BRAFV600E expression were not affected by the treatment of thyroid cells by sorafenib. Therefore, RAF and MEK inhibitors are unable to block the antiapoptotic activity of BRAFV600E or correct the high glucose uptake rate and glycolytic activity and suppressed mitochondrial oxidative phosphorylation induced by BRAFV600E. Conclusions: The mitochondrial localization observed in oncogenic BRAF mutants might be related to their altered responses to apoptotic stimuli and characteristic metabolic phenotypes found in thyroid cancer. The inability of MEK and RAF inhibitors, U0126 and sorafenib, respectively, to block the mitochondrial localization of BRAFV600E has additional therapeutic implications for BRAFV600E-positive thyroid cancers.

BRAF gene mutations have been associated with human cancers. Among the naturally occurring mutations, two that involve amino acids of the conserved DFG motif in the activation loop (D594V and G596R), appear to be inactivating. Aim of this study was to analyze the molecular mechanisms involved in the loss of function of B-Raf inactivating mutation G596R. Furthermore, the ability of the B-Raf DFG motif mutants to generate heterodimers with C-Raf and the possible functional consequences of the B-Raf/C-Raf heterodimer formation was examined. Wet molecular experiments in HEK293T cells demonstrate that B-RafG596R is a kinase-impaired mutant. Molecular dynamics simulations show that the loss of function of B-RafG596R depends on a restraining effect of Arg596 on the catalytic residue Asp594, which results in the loss of the appropriate spatial localization and/or conformation of the latter necessary for anchoring ATP to the enzyme. Exploration of B-Raf/C-Raf heterodimer formation indicates the occurrence of functioning heterodimers in the case of all the DFG B-Raf mutants, independently from the expected differences in spatial conformation of the activation loop, although the transforming activity of the mutants appear negligible. In conclusion, this study delivers novel information on the functional properties of the B-Raf DFG motif inactivating mutants and on the mechanisms driving B-Raf/C-Raf heterodimerization and consequent C-Raf transactivation.

RET/papillary thyroid carcinoma (RET/PTC) oncoproteins result from the in-frame fusion of the RET receptor tyrosine kinase domain with protein dimerization motifs encoded by heterologous genes. Here, we show that RET/PTC stimulates the β-catenin pathway. By stimulating PI3K/AKT and Ras/extracellular signal–regulated kinase (ERK), RET/PTC promotes glycogen synthase kinase 3β (GSK3β) phosphorylation, thereby reducing GSK3β-mediated NH2-terminal β-catenin (Ser33/Ser37/Thr41) phosphorylation. In addition, RET/PTC physically interacts with β-catenin and increases its phosphotyrosine content. The increased free pool of S/T(nonphospho)/Y(phospho)β-catenin is stabilized as a result of the reduced binding affinity for the Axin/GSK3β complex and activates the transcription factor T-cell factor/lymphoid enhancer factor. Moreover, through the ERK pathway, RET/PTC stimulates cyclic AMP–responsive element binding protein (CREB) phosphorylation and promotes the formation of a β-catenin-CREB-CREB-binding protein/p300 transcriptional complex. Transcriptional complexes containing β-catenin are recruited to the cyclin D1 promoter and a cyclin D1 gene promoter reporter is active in RET/PTC–expressing cells. Silencing of β-catenin by small interfering RNA inhibits proliferation of RET/PTC–transformed PC Cl3 thyrocytes, whereas a constitutively active form of β-catenin stimulates autonomous proliferation of thyroid cells. Thus, multiple signaling events downstream from RET/PTC converge on β-catenin to stimulate cell proliferation. [Cancer Res 2009;69(5):1867–76]

Oncogenic conversion of the RET (rearranged during transfection) tyrosine kinase is associated with several cancers. A fragment-based chemical screen led to the identification of a novel RET inhibitor, Pz-1. Modeling and kinetic analysis identified Pz-1 as a type II tyrosine kinase inhibitor that is able to bind the "DFG-out" conformation of the kinase. Importantly, from a single-agent polypharmacology standpoint, Pz-1 was shown to be active on VEGFR2, which can block the blood supply required for RET-stimulated growth. In cell-based assays, 1.0 nM of Pz-1 strongly inhibited phosphorylation of all tested RET oncoproteins. At 1.0 mg kg(-1) day(-1) per os, Pz-1 abrogated the formation of tumors induced by RET-mutant fibroblasts and blocked the phosphorylation of both RET and VEGFR2 in tumor tissue. Pz-1 featured no detectable toxicity at concentrations of up to 100.0 mg kg(-1), which indicates a large therapeutic window. This study validates the effectiveness and usefulness of a medicinal chemistry/polypharmacology approach to obtain an inhibitor capable of targeting multiple oncogenic pathways.

Purpose: Calcium/calmodulin-dependent kinase II (CaMKII) is involved in the regulation of cell proliferation. Its endogenous inhibitor (hCaKIINα) is expressed in some cell types. We determined the role of CaMKII in RET-stimulated proliferation and hCaMKIINα in medullary thyroid carcinoma (MTC). Experimental Design: We analyzed the role of RET mutants on CaMKII activation in NIH3T3 and in MTC cell lines, and determined the effect of CaMKII inhibition on RET/ERK pathway and cell proliferation. Then the expression of hCaKIINα mRNA was determined by real-time PCR in primary MTC and it was correlated with some clinicopathologic parameters. Results: RET C634Y and RETM918T mutants expressed in NIH3T3 cells induced CaMKII activation. CaMKII was activated in unstimulated MTC cells carrying the same RET mutants and it was inhibited by RET inhibition. Inhibition of CaMKII in these cells induced a reduction of Raf-1, MEK, and ERK phosphorylation, cyclin D expression, and cell proliferation. hCaKIINα mRNA expression in primary MTC was very variable and did not correlate with gender and age at diagnosis. Serum calcitonin, (R2 = 0.032; P = 0.017), tumor volume (P = 0.0079), lymph node metastasis (P = 0.033), and staging (P = 0.0652) were negatively correlated with the hCaKIINα mRNA expression. Conclusions: CaMKII is activated by RET mutants and is activated at baseline in MTC cells where it mediates the oncogenic pathway leading to cell proliferation. The mRNA expression of its endogenous inhibitor hCaKIINα inversely correlates with the severity of MTC. CaMKII might represent a new target for MTC therapy and hCaKIINα is a marker of disease extension. Clin Cancer Res; 20(6); 1513–20. ©2014 AACR.

The phosphoinositides have well-defined roles in the control of cellular functions, including cytoskeleton dynamics, membrane trafficking, and cell signaling. However, the interplay among the phosphoinositides and their diffusible derivatives that originate through phospholipase A2 action (the lysophosphoinositides and glycerophosphoinositols) remains to be fully elucidated. Here we demonstrate that in PCCl3 rat thyroid cells, the intracellular levels of glycerophosphoinositol are finely modulated by ATP and norepinephrine through the P2Y metabotropic and alpha-adrenergic receptors, respectively. The enzyme involved here is phospholipase A2 IValpha (PLA2 IValpha), which in these cells specifically hydrolyzes phosphatidylinositol, forming lysophosphatidylinositol, glycerophosphoinositol, and arachidonic acid. This receptor-mediated activation of PLA2 IValpha leads to stimulation of PCCl3 cell growth. The involvement of a PLA2 IValpha-mediated pathway is demonstrated by inhibition of the increase in intracellular glycerophosphoinositol levels and cell proliferation by specific inhibitors, RNA interference, and overexpression of the dominant-negative PLA2 IValpha(1-522). Modulation of PCCl3 cell growth is not seen with inhibitors of arachidonic acid metabolism. In conclusion, these data characterize glycerophosphoinositol as a mediator of the purinergic and adrenergic regulation of PCCl3 cell proliferation, defining a novel regulatory cascade specifically involving this soluble phosphoinositide derivative and widening the involvement of the phosphoinositides in the regulation of cell function.

CONTEXT: The RET tyrosine kinase encoding gene acts as a dominantly transforming oncogene in thyroid carcinoma and other malignancies. Ponatinib (AP24534) is an oral ATP-competitive tyrosine kinase inhibitor that is in advanced clinical experimentation in leukemia. OBJECTIVE: We tested whether ponatinib inhibited RET kinase and oncogenic activity. METHODS: Ponatinib activity was studied by an in vitro RET immunocomplex kinase assay and immunoblotting. The effects of ponatinib on proliferation of human TT, MZ-CRC-1, and TPC-1 thyroid carcinoma cells, which harbor endogenous oncogenic RET alleles, and of NIH3T3 fibroblasts transfected with oncogenic RET mutants were determined. Ponatinib activity on TT cell xenografted tumors in athymic mice was measured. RESULTS: Ponatinib inhibited immunopurified RET kinase at the IC50 of 25.8 nM (95% confidence interval [CI] = 23.15-28.77 nM). It also inhibited (IC50 = 33.9 nM; 95% CI = 26.41-43.58 nM) kinase activity of RET/V804M, a RET mutant displaying resistance to other tyrosine kinase inhibitor. Ponatinib blunted phosphorylation of point-mutant and rearranged RET-derived oncoproteins and inhibited proliferation of RET-transformed fibroblasts and RET mutant thyroid carcinoma cells. Finally, after 3 weeks of treatment with ponatinib (30 mg/kg/d), the volume of TT cell (medullary thyroid carcinoma) xenografts was reduced from 133 mm(3) to an unmeasurable size (difference = 133 mm(3), 95% CI = -83 to 349 mm(3)) (P < .001). Ponatinib-treated TT cell tumors displayed a reduction in the mitotic index, RET phosphorylation, and signaling. CONCLUSIONS: Ponatinib is a potent inhibitor of RET kinase and has promising preclinical activity in models of RET-driven medullary thyroid carcinoma.

Oncogenic conversion of the RET tyrosine kinase is a frequent feature of medullary thyroid carcinoma (MTC). ZD6474 (vandetanib) is an ATP-competitive inhibitor of RET, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptors kinases. In this study, we have studied ZD6474 mechanism of action in TT and MZ-CRC-1 human MTC cell lines, carrying cysteine 634 to tryptophan (C634W) and methionine 918 to threonine (M918T) RET mutation respectively. ZD6474 blunted MTC cell proliferation and RET, Shc and p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation. Single receptor knockdown by RNA interference showed that MTC cells depended on RET for proliferation. Adoptive expression of the ZD6474-resistant V804M RET mutant rescued proliferation of TT cells under ZD6474 treatment, showing that RET is a key ZD6474 target in these MTC cells. Upon RET inhibition, adoptive stimulation of EGFR partially rescued TT cell proliferation, MAPK signaling, and expression of cell-cycle-related genes. This suggests that simultaneous inhibition of RET and EGFR by ZD6474 may overcome the risk of MTC cells to escape from RET blockade through compensatory over-activation of EGFR.

RET/papillary thyroid carcinoma (PTC), TRK-T, or activating mutations of Ras and BRaf are frequent genetic alterations in PTC, all leading to the activation of the extracellular-regulated kinase (Erk) cascade. The aim of this study was to investigate the role of calmodulin-dependent kinase II (CaMKII) in the signal transduction leading to Erk activation in PTC cells. In normal thyroid cells, CaMKII and Erk were in the inactive form in the absence of stimulation. In primary PTC cultures and in PTC cell lines harboring the oncogenes RET/PTC-1 or BRafV600E, CaMKII was active also in the absence of any stimulation. Inhibition of calmodulin or phospholipase C (PLC) attenuated the level of CaMKII activation. Expression of recombinant RET/PTC-3, BRafV600E, or RasV12 induced CaMKII activation. Inhibition of CaMKII attenuated Erk activation and DNA synthesis in thyroid papillary carcinoma (TPC-1), a cell line harboring RET/PTC-1, suggesting that CaMKII is a component of the Erk signal cascade in this cell line. In conclusion, PTCs contain an active PLC/Ca2+/calmodulin-dependent signal inducing constitutive activation of CaMKII. This kinase is activated by BRafV600E, oncogenic Ras, and by RET/PTC. CaMKII participates to the activation of the Erk pathway by oncogenic Ras and RET/PTC and contributes to their signal output, thus modulating tumor cell proliferation.

Context: The V600E mutation accounts for the vast majority of thyroid carcinoma-associated BRAF mutations. Objective: The aim was to study the effects of the two BRAF V600E ATP-competitive kinase inhibitors, PLX4032 and PLX4720, in thyroid carcinoma cell lines. Experimental Design: We examined the activity of PLX4032 and PLX4720 in thyroid carcinoma cell lines harboring BRAF V600E (8505C, BCPAP, SW1736, BHT101), NRAS Q61R (HTH7), KRAS G12R (CAL62), HRAS G13R (C643), or RET/PTC1 (TPC-1) oncogenes. Normal thyrocytes (PC Cl 3) were used as control. Results: Both compounds inhibited the proliferation of BRAF mutant cell lines, but not normal thyrocytes, with a half maximal effective concentration (EC50) ranging from 78-113 nM for PLX4720 and from 29-97 nM for PLX4032. Doses equal to or higher than 500 nM were required to achieve a similar effect in BRAF wild-type cancer cells. Phosphorylation of ERK 1/2 and MAPK kinase (MEK) 1/2 decreased upon PLX4032 and PLX4720 treatment in BRAF mutant thyroid carcinoma cells but not in normal thyroid cells or in cell lines harboring mutations of RAS or RET/PTC1 rearrangements. PLX4032 and PLX4720 treatment induced a G1 block and altered expression of genes involved in the control of G1-S cell-cycle transition. 8505C cell tumor xenografts were smaller in nude mice treated with PLX4032 than in control mice. This inhibition was associated with reduction of phospho-ERK and phospho-MEK levels. Conclusions: This study provides additional evidence of the promising nature of mutant BRAF as a molecular target for thyroid carcinoma cells.

Modulation of cytosolic phospholipase A2 (PLA2) expression levels and production of its metabolites have been reported in several tumor types, indicating involvement of arachidonic acid and its derivatives in tumorigenesis. Following our demonstration that the PLA2 group IV isoform α (PLA2IVα) controls TSH-independent growth of normal thyroid (PCCl3) cells, we have investigated the mitogenic role of PLA2IVα in rat thyroid cells transformed by the RET/PTC oncogenes (PC-PTC cells). We now report that PLA2IVα acts downstream of the RET/PTC oncogenes in a novel pathway controlling RET-dependent cell proliferation. In addition, we show that PLA2IVα is in its phosphorylated/active form not only in RET/PTC-transformed cells and in cells derived from human papillary carcinomas but also in lysates from tumor tissues, thus relating constitutive activation of PLA2IVα to RET/PTC-dependent tumorigenesis. Moreover, p38 stress-activated protein kinase is the downstream effector of RET/PTC that is responsible for PLA2IVα phosphorylation and activity. In summary, our data elucidate a novel mechanism in the control of thyroid tumor cell growth that is induced by the RET/PTC oncogenes and which is distinguishable from that of other oncogenes, such as BRAF. This mechanism is mediated by PLA2IVα and should be amenable to targeted pharmacologic intervention. [Cancer Res 2007;67(24):11769–78]

El pdf del artículo es la versión de autor.-- et al., RET/papillary thyroid carcinoma (PTC) oncoproteins result from the in-frame fusion of the RET receptor tyrosine kinase with protein dimerization motifs encoded by heterologous genes. Here, we show that RET/PTC1 activates the Rap1 small GTPase. The activation of Rap1 was dependent on the phosphorylation of RET Tyr1062. RET/PTC1 recruited a complex containing growth factor receptor binding protein 2-associated binding protein 1 (Gab1), CrkII (v-erk sarcoma virus CT10 oncogene homologue II), and C3G (Rap guanine nucleotide exchange factor 1). By using dominant-negative and small interfering duplex (small interfering RNA) oligonucleotides, we show that RET/PTC1-mediated Rap1 activation was dependent on CrkII, C3G, and Gab1. Activation of Rap1 was involved in the RET/PTC1-mediated stimulation of the BRAF kinase and the p42/p44 mitogen-activated protein kinases. Proliferation and stress fiber formation of RET/PTC1-expressing PC C1 3 thyroid follicular cells were inhibited by the dominant-negative Rap1(N17) and by Rap1-specific GTPase-activating protein. Thus, Rap1 is a downstream effector of RET/PTC and may contribute to the transformed phenotype of RET/PTC-expressing thyrocytes. ©2007 American Association for Cancer Research., Associazione Italiana per la Ricerca sul Cancro (AIRC) and the Italian Ministero per l’Istruzione, Universitaè Ricerca Scientifica (MIUR). G. De Vita and M.D. Castellone were recipient of AIRC fellowships.

Purpose: Oncogenic conversion of BRAF occurs in ∼44% of papillary thyroid carcinomas and 24% of anaplastic thyroid carcinomas. In papillary thyroid carcinomas, this mutation is associated with an unfavorable clinicopathologic outcome. Our aim was to exploit BRAF as a potential therapeutic target for thyroid carcinoma. Experimental Design: We used RNA interference to evaluate the effect of BRAF knockdown in the human anaplastic thyroid carcinoma cell lines FRO and ARO carrying the BRAF V600E (V600EBRAF) mutation. We also exploited the effect of BAY 43-9006 [N-(3-trifluoromethyl-4-chlorophenyl)-N'-(4-(2-methylcarbamoyl pyridin-4-yl)oxyphenyl)urea], a multikinase inhibitor able to inhibit RAF family kinases in a panel of six V600EBRAF-positive thyroid carcinoma cell lines and in nude mice bearing ARO cell xenografts. Statistical tests were two sided. Results: Knockdown of BRAF by small inhibitory duplex RNA, but not control small inhibitory duplex RNA, inhibited the mitogen-activated protein kinase signaling cascade and the growth of ARO and FRO cells (P < 0.0001). These effects were mimicked by thyroid carcinoma cell treatment with BAY 43-9006 (IC50 = 0.5-1 μmol/L; P < 0.0001), whereas the compound had negligible effects in normal thyrocytes. ARO cell tumor xenografts were significantly (P < 0.0001) smaller in nude mice treated with BAY 43-9006 than in control mice. This inhibition was associated with suppression of phospho–mitogen-activated protein kinase levels. Conclusions: BRAF provides signals crucial for proliferation of thyroid carcinoma cells spontaneously harboring the V600EBRAF mutation and, therefore, BRAF suppression might have therapeutic potential in V600EBRAF-positive thyroid cancer.

RAI, also named ShcC/N-Shc, one of the members of the Shc proteins family, is a substrate of the RET receptor tyrosine kinase. Here, we show that RAI forms a protein complex with both RET/MEN 2 A and RET/PTC oncoproteins. By co-immunoprecipitation, we found that RAI associates with the Grb 2-associated binder 1 (GAB 1) adapter. This association is constitutive, but, in the presence of RET oncoproteins, both RAI and GAB 1 are tyrosine-phosphorylated, and the stoichiometry of this interaction remarkably increases. Consequently, the p 85 regulatory subunit of phosphatidylinositol-3 kinase (PI-3 K) is recruited to the complex, and its downstream effector Akt is activated. We show that human thyroid cancer cell lines derived from papillary or medullary thyroid carcinoma (PTC or MTC) carrying, respectively, RET/PTC and RET/MEN 2 A oncoproteins express RAI proteins. We also show that human PTC samples express higher levels of RAI, when compared to normal thyroid tissue. In thyroid cells expressing RET/PTC 1, ectopic expression of RAI protects cells from apoptosis; on the other hand, the silencing of endogenous RAI by small inhibitory duplex RNAs in a PTC cell line that expresses endogenous RET/PTC 1, increases the rate of spontaneous apoptosis. These data suggest that RAI is a critical substrate for RET oncoproteins in thyroid carcinomas.

In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade.

To identify genes involved in the transformation of thyroid follicular cells, we explored, using DNA oligonucleotide microarrays, the transcriptional response of PC Cl3 rat thyroid epithelial cells to the ectopic expression of the RET/PTC oncogenes. We found that RET/PTC was able to induce the expression of CXCR4, the receptor for the chemokine CXCL12/SDF-1alpha/beta. We observed that CXCR4 expression correlated with the transforming ability of the oncoprotein and depended on the integrity of the RET/PTC-RAS/ERK signaling pathway. We found that CXCR4 was expressed in RET/PTC-positive human thyroid cancer cell lines, but not in normal thyroid cells. Furthermore, we found CXCR4 expression in human thyroid carcinomas, but not in normal thyroid samples by immunohistochemistry. Since CXCR4 has been recently implicated in tumor proliferation, motility and invasiveness, we asked whether treatment with SDF-1alpha was able to induce a biological response in thyroid cells. We observed that SDF-1alpha induced S-phase entry and survival of thyroid cells. Invasion through a reconstituted extracellular matrix was also supported by SDF-1alpha and inhibited by a blocking antibody to CXCR4. Taken together, these results suggest that human thyroid cancers bearing RET/PTC rearrangements may use the CXCR4/SDF-1alpha receptor-ligand pathway to proliferate, survive and migrate.

RET gene rearrangements, which generate chimeric RET/ PTC oncogenes, are early events in the evolution of thyroid papillary carcinomas. Expression of RET/PTC oncogenes promotes neoplastic transformation of cultured thyroid cells and of thyroid glands in transgenic mice. Notwithstanding these oncogenic effects, we have found that the expression of two RET/PTC oncogenes (H4-RET and RFG-RET) induces apoptosis of rat thyroid PC CL 3 cells. Promotion of thyroid cell death depends on the kinase activity of RET/PTC and on the phosphorylation of a tyrosine residue (tyrosine 1062) that maps in the carboxy-terminus of the RET protein. Tyrosine 1062 is essential for RET/PTC-mediated activation of the Ras/ ERK pathway. Inhibition of Ras/ERK by a dominant negative Ras or by the MEKI inhibitor, PD98059, obstructed RET/PTC-mediated apoptosis. We also show that signals transmitted by tyrosine 1062 mediate proapoptotic events like Bcl-2 down regulation and Bax upregulation, and that adoptive overexpression of Bcl-2 overcomes RET/PTC-induced apoptosis. Thus, gene rearrangements that generate RET/PTC oncogenes subvert RET function by converting it into a chronically active kinase that is constitutively phosphorylated on tyrosine 1062. In turn, Y1062 phosphorylation transmits not only mitogenic but also proapoptotic signals to thyroid cells.

XB130, a novel adaptor protein, mediates RET/PTC chromosome rearrangement-related thyroid cancer cell proliferation and survival through phosphatidyl-inositol-3-kinase (PI3K)/Akt pathway. Recently, XB130 was found in different cancer cells in the absence of RET/PTC. To determine whether RET/PTC is required of XB130-related cancer cell proliferation and survival, WRO thyroid cancer cells (with RET/PTC mutation) and A549 lung cancer cells (without RET/PTC) were treated with XB130 siRNA, and multiple Akt down-stream signals were examined. Knocking-down of XB130 inhibited G(1)-S phase progression, and induced spontaneous apoptosis and enhanced intrinsic and extrinsic apoptotic stimulus-induced cell death. Knocking-down of XB130 reduced phosphorylation of p21Cip1/WAF1, p27Kip1, FOXO3a and GSK3β, increased p21Cip1/WAF1protein levels and cleavages of caspase-8 and-9. However, the phosphorylation of FOXO1 and the protein levels of p53 were not affected by XB130 siRNA. We also found XB130 can be phosphorylated by multiple protein tyrosine kinases. These results indicate that XB130 is a substrate of multiple protein tyrosine kinases, and it can regulate cell proliferation and survival through modulating selected down-stream signals of PI3K/Akt pathway. XB130 could be involved in growth and survival of different cancer cells.