Membrane type 1 matrix metalloproteinase regulates anaplastic thyroid carcinoma cell growth and invasion into the collagen matrix.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive cancer types; however, the molecular mechanism contributing to the aggressive characteristics remain unclear. Membrane type 1 matrix metalloproteinase (MT1-MMP) plays an important role in cancer invasion and has been associated with a poor prognosis in various malignant neoplasms. In this study, we investigated the relationship between MT1-MMP expression and the proliferation and invasion of ATC cells, along with the association with clinicopathologic factors in patients with ATC. Suppression of MT1-MMP reduced the proliferation and invasion of ATC cells, and suppressed ERK activity, indicating a role in cancer cell proliferation in collagen matrix culture conditions. The expression of MT1-MMP was detected in 29 of 34 (85.3%) surgical specimens from ATC patients. In addition, the expression of MT1-MMP in the tumor lesion was higher than that of normal and stromal tissues. Collectively, these results suggest that elevated MT1-MMP expression plays a role in the pathogenesis of ATC, which may promote its aggressive characteristics such as proliferation and invasion, highlighting a potential new therapeutic target.

Potentiated anti-tumor effects of BETi by MEKi in anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) is an aggressive malignancy with limited treatment options. We explored novel treatment modalities by targeting epigenetic modifications using inhibitors of BET (e.g. BRD4) activity. We evaluated the efficacy in the treatment of ATC of a novel BET inhibitor, PLX51107 (PLX), currently in clinical trials for other solid tumors and hematologic malignancies, alone or combined with a MEK inhibitor, PD0325901(PD). To elucidate the effects of these inhibitors on growth of ATC, we treated ATC cells derived from patient tumors (THJ-11T and THJ-16T cells) and mouse xenograft tumors with inhibitors. We found PLX and PD inhibitors singly inhibited proliferation of both human ATC cells lines, but together exhibited stronger inhibition of proliferation. In mouse xenografts, the combination treatment almost totally blocked growth in xenograft tumors derived from both ATC cells. PD effectively attenuated MEK-ERK signaling, which was further enhanced by PLX in the combined treatment in cultured cells and tumors. Importantly, the combination of PLX and PD acted synergistically to suppress MYC transcription to increase p27 in decreasing tumor cell proliferation. PLX and PD cooperated to upregulate pro-apoptotic proteins to promote apoptosis. These two inhibitors converged to reduce the binding of BRD4 to the MYC promoter to suppress the MYC expression. These findings indicate that combined treatment of BET and MEK-ERK inhibitors was more effective to treat ATC than single targeted treatment. Synergistic suppression of MYC transcription via collaborative actions on chromatin modifications suggested that targeting epigenetic modifications could provide novel treatment opportunities for ATC.

New therapies for anaplastic thyroid cancer.

Anaplastic thyroid cancer is a rare but extremely aggressive type of thyroid cancer. Treatment typically consists of surgery, external beam radiotherapy and cytotoxic chemotherapy. However, available literature suggests only modest survival benefit for cytotoxic chemotherapy. Recent advances have suggested the combination of BRAF and MEK inhibition may have a profound and durable effect on patients with BRAFV600E-mutated anaplastic thyroid cancer, with a response rate of 69%. Other systemic treatments, including immunotherapies, have also shown promising but more limited results. Many clinical trials assessing the efficacy of kinase inhibitors and immunotherapies are currently ongoing.

Cystathione ß-Synthase Is Increased in Thyroid Malignancies.

BACKGROUND: Cystathione ß-synthase (CBS) catalyzes the conversion of homocysteine and cysteine to hydrogen sulfide (H2S) and cystathione, via the trans-sulfuration pathway. CBS protein expression levels are increased in several different human malignancies, with increased protein expression correlating with parameters such as tumor stage, anaplasia, metastases, and chemotherapy resistance. MATERIALS AND METHODS: This study employed tissue microarrays to examine CBS expression in benign thyroid tissue, thyroid oncocytomas, thyroid follicular adenomas, and in follicular, papillary, anaplastic, and medullary thyroid carcinomas. RESULTS: CBS expression was increased in all thyroid carcinomas types compared to benign thyroid tissue, but not in thyroid follicular adenomas or oncocytomas. A similar pattern was observed for nicotinamide phosphoribosyltransferase (NAMPT) tissue microarray analysis comparing thyroid adenomas and follicular carcinomas. CONCLUSION: For the first time, we showed that an H2S-syntheszing enzyme plays a role in thyroid malignancies. Additionally, our data suggest that CBS and NAMPT immunohistochemistry may be useful in differentiating follicular adenomas from follicular carcinomas.

Inhibiting 6-phosphogluconate dehydrogenase reverses doxorubicin resistance in anaplastic thyroid cancer via inhibiting NADPH-dependent metabolic reprogramming.

Anaplastic thyroid carcinoma (ATC) is the most aggressive type of thyroid malignancies and resistant to chemotherapy. Little is known on the underlying mechanisms of ATC resistance to chemotherapy. In our work, we identified that 6-phosphogluconate dehydrogenase (6PGD) is critically involved in the development of ATC resistance to doxorubicin. We found that 6PGD mRNA, protein and enzyme activity levels are significantly upregulated in ATC cells during the prolonged exposure to doxorubicin in a time-dependent manner. 6PGD inhibition by genetic and pharmacological approaches significantly inhibits growth and survival of ATC cells that are highly resistant to doxorubicin. Consistently, 6PGD inhibition also sensitizes ATC cells to doxorubicin treatment. Of note, we observed the decreased level of NADPH, NADH and enzymatic activity of sirtuin-1 in response to 6PGD inhibition in doxorubicin-resistant ATC cells. Lactate level was also reduced by 6PGD inhibition. All these indicate that 6PGD inhibition disrupts metabolic reprogramming in doxorubicin-resistant ATC cells. Our work demonstrates 6PGD activation-mediated resistance in response to doxorubicin and provides an alternative therapeutic strategy to overcome resistance to chemotherapy for ATC treatment. Our findings also highlight the importance of metabolic reprogramming in ATC chemoresistance.

Dual effects for lovastatin in anaplastic thyroid cancer: the pivotal effect of transketolase (TKT) on lovastatin and tumor proliferation.

This study tested the hypothesis that the effects of lovastatin on anaplastic thyroid cancer cell growth are mediated by upregulation of transketolase (TKT) expression. The effects of lovastatin on TKT protein levels in ARO cells were determined using western blot and proteomic analyses. After treatment with lovastatin and oxythiamine, the in vitro and in vivo growth of ARO cells was determined using 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assays and tumor xenografts in nude mice. TKT protein expression in the ARO tumors was assessed using immunohistochemistry analysis. Proteomic analysis revealed that 25 µM lovastatin upregulated TKT expression. Co-treatment of ARO cells with 1 µM lovastatin + 1 µM oxythiamine increased TKT protein expression compared with control levels; however, no differences were observed with 10 µM lovastatin + 1 µM oxythiamine. Furthermore, treatment with either oxythiamine or lovastatin alone reduced ARO tumor expression of TKT, as well as decreased ARO cell proliferation in vitro and tumor growth in vivo. However, mice treated with both lovastatin and oxythiamine at the same time had tumor volumes similar to that of the untreated control group. We conclude that either lovastatin or oxythiamine reduced ARO cell growth; however, the combination of these drugs resulted in antagonism of ARO tumor growth.

Silencing of hTERT blocks growth and migration of anaplastic thyroid cancer cells.

Mutations in the hTERT promoter responsible for constitutive telomerase activity are the most frequent genetic alteration detected in anaplastic thyroid cancer (ATC), and proposed as diagnostic and prognostic biomarker in these tumours. In this study we analyzed hTERT expression in a series of human ATCs and investigated the effects of small-interfering RNA-mediated silencing of hTERT on viability and migration and invasive properties of three human ATC cell lines. Expression of hTERT mRNA resulted increased in 8/10 ATCs compared to normal thyroid tissues. Silencing of hTERT in CAL-62, 8505C and SW1736 cells did not modify telomere length but determined a significant decrease (about 50%) of cell proliferation in all cell lines and a great reduction (about 50%) of migration and invasion capacity. These finding demonstrate that hTERT may be considered as a molecular target for ATC treatment.

Aldehyde dehydrogenase activity plays no functional role in stem cell-like properties in anaplastic thyroid cancer cell lines.

Recent studies have revealed that aldehyde dehydrogenase (ALDH) is a candidate marker for thyroid cancer stem cells, although its activity is flexible. The goal of this study is to clarify the functional significance of ALDH enzymatic activity on thyroid cancer stem cells properties in anaplastic thyroid cancer cell lines. In vitro sphere formation assay was used to judge the stemness of 4 anaplastic thyroid cancer cell lines (FRO, ACT1, 8505C, and KTC3). Two well-known ALDH inhibitors, N,N-diethylaminobenzaldehyde (DEAB) and disulfiram (DS), were first used. DEAB (50 µM) almost completely suppressed ALDH activity without affecting cell proliferation or spherogenicity. Lack of effect of ALDH suppression on spherogenicity was confirmed using shRNA for ALDH1A3, an ALDH isozyme predominantly expressed in anaplastic thyroid cancer cell lines. In contrast, an ALDH2 inhibitor DS (1 µM) inhibited spherogenicity but did not inhibit ALDH1A3 activity. Based on the recent article from another group reporting the importance of sonic hedgehog (Shh) signaling in ALDH activity and spherogenicity in thyroid cancer, the effects of the Shh inhibitor cyclopamine were also studied. Like DS, cyclopamine (1 µM) decreased spherogenicity but not ALDH activity. Finally, exogenous expression of ALDH1A3 in otherwise ALDH- TPC1 cells (a papillary thyroid cancer cell line) revealed no effect on spherogenicity. In conclusion, we here show no functional role for ALDH activity in thyroid thyroid cancer stem cells properties. That is, ALDH activity and spherogenicity are clearly dissociable. Further understanding of thyroid cancer stem cells biology in thyroid cancers remains necessary for the future development of thyroid thyroid cancer stem cells-targeted therapies.

Anaplastic lymphoma kinase L1198F and G1201E mutations identified in anaplastic thyroid cancer patients are not ligand-independent.

Activating mutations in full length anaplastic lymphoma kinase (ALK) have been reported in neuroblastoma and in anaplastic thyroid cancer. ALK-L1198F and ALK-G1201E mutations were originally identified in anaplastic thyroid cancer (ATC) and characterized as constitutively activating mutations. In this study, we employed in vitro cell culture assays together with biochemical and in vivo Drosophila analyses to characterize their sensitivity to either activation by the FAM150A (AUG-ß) and FAM150B (AUG-α) ALK ligands or inhibition by ALK inhibitors. Here we report that neither ALK-L1198F nor ALK-G1201E mutations result in ligand independent gain-of-function (GOF) activity in either in vitro biochemical analysis or the various model systems employed. ALK-L1198F is activated by the FAM150 (AUG) ligands and its ligand-dependant activity is similar to the wild type full length ALK receptor. ALK-G1201E is only very weakly activated by the FAM150 (AUG) ligands, most likely due to impaired protein stability. We conclude that neither ALK-L1198F nor ALK-G1201E displays ligand independent kinase activity, with ALK-L1198F belonging to the class of ligand dependent ALK mutations which are not constitutively active but that responds to ligand activation, while the ALK-G1201E mutation generates an unstable receptor with very low levels of kinase activity.

Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma.

Ubiquitin-specific protease 22 (USP22) aberrance has been implicated in several malignancies; however, whether USP22 plays a role in anaplastic thyroid carcinoma (ATC) remains unclear. Here, we report that USP22 expression is highly elevated in ATC tissues, which positively correlated with tumor size, extracapsular invasion, clinical stages, and poor prognosis of ATC patients. In vitro assays showed that USP22 depletion suppressed ATC cell survival and proliferation by decreasing Rb phosphorylation and cyclin D2, inactivating Akt, and simultaneously upregulating Rb; USP22 silencing restrained cell migration and invasion by inhibiting epithelial-mesenchymal transition; USP22 knockdown promoted mitochondrion- mediated and caspase-dependent apoptosis by upregulating Bax and Bid and promoting caspase-3 activation. Consistent with in vitro findings, downregulation of USP22 in ATC cells impeded tumor growth and lung metastasis in vivo. These results raise the applicability for USP22 as a useful predictor of ATC prognosis and a potential therapeutic target for ATC.

Expression of angiogenic switch, cachexia and inflammation factors at the crossroad in undifferentiated thyroid carcinoma with BRAF(V600E).

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.

PARP inhibitor olaparib increases the oncolytic activity of dl922-947 in in vitro and in vivo model of anaplastic thyroid carcinoma.

PARP inhibitors are mostly effective as anticancer drugs in association with DNA damaging agents. We have previously shown that the oncolytic adenovirus dl922-947 induces extensive DNA damage, therefore we hypothesized a synergistic antitumoral effect of the PARP inhibitor olaparib in association with dl922-947. Anaplastic thyroid carcinoma was chosen as model since it is a particularly aggressive tumor and, because of its localized growth, it is suitable for intratumoral treatment with oncolytic viruses. Here, we show that dl922-947 infection induces PARP activation, and we confirm in vitro and in vivo that PARP inhibition increases dl922-947 replication and oncolytic activity. In vitro, the combination with olaparib exacerbates the appearance of cell death markers, such as Annexin V positivity, caspase 3 cleavage, cytochrome C release and propidium iodide permeability. In vivo, we also observed a better viral distribution upon PARP inhibition. Changes in CD31 levels suggest a direct effect of olaparib on tumor vascularization and on the viral distribution within the tumor mass. The observation that PARP inhibition enhances the effects of dl922-947 is highly promising not only for the treatment of anaplastic thyroid carcinoma but, in general, for the treatment of other tumors that could benefit from the use of oncolytic viruses.

miR30a inhibits LOX expression and anaplastic thyroid cancer progression.

Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies, but its genetic drivers remain little understood. In this study, we report losses in expression of the miRNA miR30a, which is downregulated in ATC compared with differentiated thyroid cancer and normal tissue. miR30a downregulation was associated with advanced differentiated thyroid cancer and higher mortality. Mechanistically, we found miR30a decreased cellular invasion and migration, epithelial-mesenchymal transition marker levels, lysyl oxidase (LOX) expression, and metastatic capacity. LOX was identified as a direct target of miR30a that was overexpressed in ATC and associated with advanced differentiated thyroid cancer and higher mortality rate. Consistent with its role in other cancers, we found that LOX inhibited cell proliferation, cellular invasion, and migration and metastasis in vitro and in vivo. Together, our findings establish a critical functional role for miR30a downregulation in mediating LOX upregulation and thyroid cancer progression, with implications for LOX targeting as a rational therapeutic strategy in ATC.

Activating BRAF and PIK3CA mutations cooperate to promote anaplastic thyroid carcinogenesis.

UNLABELLED: Thyroid malignancies are the most common type of endocrine tumors. Of the various histologic subtypes, anaplastic thyroid carcinoma (ATC) represents a subset of all cases but is responsible for a significant proportion of thyroid cancer-related mortality. Indeed, ATC is regarded as one of the more aggressive and hard to treat forms of cancer. To date, there is a paucity of relevant model systems to critically evaluate how the signature genetic abnormalities detected in human ATC contribute to disease pathogenesis. Mutational activation of the BRAF protooncogene is detected in approximately 40% of papillary thyroid carcinoma (PTC) and in 25% of ATC. Moreover, in ATC, mutated BRAF is frequently found in combination with gain-of-function mutations in the p110 catalytic subunit of PI3'-Kinase (PIK3CA) or loss-of-function alterations in either the p53 (TP53) or PTEN tumor suppressors. Using mice with conditional, thyrocyte-specific expression of BRAF(V600E), we previously developed a model of PTC. However, as in humans, BRAF(V600E)-induced mouse PTC is indolent and does not lead to rapid development of end-stage disease. Here, we use mice carrying a conditional allele of PIK3CA to demonstrate that, although mutationally activated PIK3CA(H1047R) is unable to drive transformation on its own, when combined with BRAF(V600E) in thyrocytes, this leads to development of lethal ATC in mice. Combined, these data demonstrate that the BRAF(V600E) cooperates with either PIK3CA(H1074R) or with silencing of the tumor-suppressor PTEN, to promote development of anaplastic thyroid carcinoma. IMPLICATIONS: This genetically relevant mouse model of ATC will be an invaluable platform for preclinical testing of pathway-targeted therapies for the prevention and treatment of thyroid carcinoma.

Coexpression of EpCAM, CD44 variant isoforms and claudin-7 in anaplastic thyroid carcinoma.

BACKGROUND: Anaplastic thyroid cancer is considered to be one of the most aggressive human malignancies, and the mean survival time after diagnosis is approximately six months, regardless of treatments. This study aimed to examine how EpCAM and its related molecules are involved in the characteristics of anaplastic thyroid carcinoma. METHODOLOGY/PRINCIPAL FINDINGS: Two differentiated thyroid cancer cell lines (TPC-1 and FTC-133), and two anaplastic thyroid cancer cell lines (FRO, ACT-1) were analyzed for expression of CD44 standard isoform (CD44s), CD44 variant isoforms, and EpCAM, and human aldehyde dehydrogenase-1 (ALDH1) enzymatic activity using flow cytometry. CD44s expression was higher in TPC-1 and FTC-133 than in the FRO and ACT-1, whereas ALDH1 activities were higher in FRO and ACT-1 than in TPC-1 and FTC-133. An inverse correlation between CD44s expression and ALDH1 activity was observed in all thyroid cancer cell lines. As for the expressions of CD44 variant isoforms, ACT-1 showed higher and FRO showed moderate CD44v6 expressions, whereas either TPC-1 or FTC-133 showed negative CD44v6 expression. EpCAM expressions in FRO and ACT-1 were higher than those in TPC-1 and FTC-133, and EpCAM expressions inversely correlated with those of CD44s. A positive correlation was observed between EpCAM expression and ALDH1 activity in thyroid cancer cell lines. In the RT-PCR analysis, the expression levels of EpCAM, caludin-7 and ALDH1 in FRO and ATC-1 cells were significantly higher than those in TPC-1 and FTC-133 cells. In clinical specimens of thyroid cancers, nuclear expression of EpCAM and high expression of CD44v6 were detected significantly more frequently in anaplastic carcinomas. CONCLUSIONS/SIGNIFICANCE: Our study suggests the possibility that EpCAM, together with CD44v6 and claudin-7 as well as ALDH1, may be involved in the development of the aggressive phenotype of anaplastic thyroid carcinoma. Our findings may suggest a novel therapeutic strategy for treatment of anaplastic thyroid carcinoma.

Carboplatin synergistically triggers the efficacy of photodynamic therapy via caspase 3-, 8-, and 12-dependent pathways in human anaplastic thyroid cancer cells.

Anaplastic thyroid cancer is one of the most aggressive forms of malignancies which grow very rapidly. Several conventional methods have been applied for the treatment of anaplastic thyroid cancer, but most of them were not successful in complete recovery of the patients. Therefore, a combination of two or more conventional modalities is being applied nowadays for the treatment of this type of cancer. In this present study, the combination of photodynamic therapy (PDT) and chemotherapy has been studied in anaplastic thyroid cancer. Human anaplastic thyroid cancer cells FRO were treated with a chemotherapy drug, carboplatin (cis-diammine-1,1-cyclobutanedicarboxyl-ateplatinum II (CBDCA)), and radachlorin-mediated PDT individually and in combination. Several parameters like cytotoxicity assay by MTT, apoptosis study by annexin V and propidium iodide, cell cycle analysis by flow cytometry, confocal microscopic study, and Western blot analysis for different apoptosis-related proteins like Bax, cytochrome c, caspases 3, 9, 8, and 12, etc. were studied to check the efficacy of the combination treatment as well as to find out the mechanism of this enhanced efficacy. Results showed that both PDT and CBDCA can induce apoptosis in FRO cells. However, a synergistic efficacy was observed when the cells were treated with CBDCA and PDT in combination. Changes in mitochondrial membrane potential and an increase in reactive oxygen species generation were observed in combination treatments. The enhanced expression of different apoptotic pathway-related proteins like Bax, cytochrome c, caspase 3, caspase 8, caspase 12, etc. also confirmed the higher efficacy of combination treatment. Therefore, with this combination treatment, not only a higher efficacy can be achieved but also the effective dose of the chemotherapy drug can be reduced, and hence, the adverse side effects of the chemotherapy drugs can also be controlled.