Thyroid Tumor Cells Research Articles

Estrogen-related receptor alpha promotes thyroid tumor cell survival via a tumor subtype-specific regulation of target gene networks.

Mortalin (encoded by HSPA9) is a mitochondrial chaperone often overexpressed in cancer through as-yet-unknown mechanisms. By searching different RNA-sequencing datasets, we found that ESRRA is a transcription factor highly correlated with HSPA9 in thyroid cancer, especially in follicular, but not C cell-originated, tumors. Consistent with this correlation, ESRRA depletion decreased mortalin expression only in follicular thyroid tumor cells. Further, ESRRA expression and activity were relatively high in thyroid tumors with oncocytic characteristics, wherein ESRRA and mortalin exhibited relatively high functional overlap. Mechanistically, ESRRA directly regulated HSPA9 transcription through a novel ESRRA-responsive element located upstream of the HSPA9 promoter. Physiologically, ESRRA depletion suppressed thyroid tumor cell survival via caspase-dependent apoptosis, which ectopic mortalin expression substantially abrogated. ESRRA depletion also effectively suppressed tumor growth and mortalin expression in the xenografts of oncocytic or ESRRA-overexpressing human thyroid tumor cells in mice. Notably, our Bioinformatics analyses of patient data revealed two ESRRA target gene clusters that contrast oncocytic-like and anaplastic features of follicular thyroid tumors. These findings suggest that ESRRA is a tumor-specific regulator of mortalin expression, the ESRRA-mortalin axis has higher significance in tumors with oncocytic characteristics, and ESRRA target gene networks can refine molecular classification of thyroid cancer.

DsRNAi-mediated silencing of PIAS2beta specifically kills anaplastic carcinomas by mitotic catastrophe

The E3 SUMO ligase PIAS2 is expressed at high levels in differentiated papillary thyroid carcinomas but at low levels in anaplastic thyroid carcinomas (ATC), an undifferentiated cancer with high mortality. We show here that depletion of the PIAS2 beta isoform with a transcribed double-stranded RNA–directed RNA interference (PIAS2b-dsRNAi) specifically inhibits growth of ATC cell lines and patient primary cultures in vitro and of orthotopic patient-derived xenografts (oPDX) in vivo. Critically, PIAS2b-dsRNAi does not affect growth of normal or non-anaplastic thyroid tumor cultures (differentiated carcinoma, benign lesions) or cell lines. PIAS2b-dsRNAi also has an anti-cancer effect on other anaplastic human cancers (pancreas, lung, and gastric). Mechanistically, PIAS2b is required for proper mitotic spindle and centrosome assembly, and it is a dosage-sensitive protein in ATC. PIAS2b depletion promotes mitotic catastrophe at prophase. High-throughput proteomics reveals the proteasome (PSMC5) and spindle cytoskeleton (TUBB3) to be direct targets of PIAS2b SUMOylation at mitotic initiation. These results identify PIAS2b-dsRNAi as a promising therapy for ATC and other aggressive anaplastic carcinomas.

Investigation of dextran-coated magnetic nanoparticles encapsulated by medication and modified with folate for targeted drug delivery: Invitro and docking studies

In this study, we synthesized dextran (DEX)-coated iron oxide (Fe3O4) and encapsulated it by the anticancer drug cisplatin (CP). This nano-composite functionalized with folic acid (FA) molecules for targeted delivery. These nanoparticles (NPs) were then used as nanocarriers for the targeted administration of CP for treating thyroid tumor cancer. The Fe3O4 NPs were synthesized using a chemical technique. The development of the Fe3O4@DEX-CP-FA nanosystem was achieved using a reverse microemulsion technique. The morphological features of the NPs were characterized using several methods, including dynamic light scattering, atomic force microscopy, vibrating sample magnetometry, high-resolution transmission electron microscopy, and field emission scanning electron microscopy. The docking study has verified that nanoparticles have anti-proliferative effects in thyroid cancer by attaching strongly to molecular targets. The MTT test evaluated the effects of a curative Fe3O4@DEX-CP-FA effect on cancerous and non-cancerous cell lines. The results obtained from the formulation of Fe3O4@DEX-CP-FA revealed the presence of spherical particles with an average size of 107.3 nm, a zeta potential of −28.7 mV, and a polydispersity value of 0.055 mV. Depending on the dose, the nanosystem exhibited cytotoxic effects on thyroid tumor (TT) and normal human thyroid (NHT) cell lines. The study findings indicate that Fe3O4@DEX-CP-FA demonstrated sustained antitumor efficacy while not revealing a harmful impact on normal cells.

Abstract 5082: Novel TSHR CAR T therapy for targeting oncocytic carcinoma

Abstract Oncocytic carcinoma (Hurthle cell carcinoma) has the highest incidence of metastasis among the differentiated thyroid cancers. Metastatic disease is reported at the initial diagnosis in 10-20% of patients and 34% overall. Thyroid-stimulating hormone receptor (TSHR) expression is limited to the thyroid gland. It is abundantly expressed on thyroid tumor cells, making TSHR a compelling target for advanced thyroid cancer diagnostics and therapeutics. We developed a potent anticancer strategy for treating oncocytic carcinoma using a novel thyroid hormone stimulating receptor (TSHR)- targeted (chimeric antigen receptor) CAR T cell therapy. TSHR CAR T was generated by transducing T cells derived from healthy donors. In vitro effector functions (cytotoxicity, proliferation, degranulation) were tested in TSHR+ tumor models, and subsequent in vivo studies using NSG mice were performed to assess anti-tumor activity efficacy. We observed strong anti-tumor activity against metastatic oncocytic carcinoma cell line XCT.UC1 using the TSHR CAR T therapy in vivo. Re-challenging treated mice with XCT.UC1 cells reactivated TSHR CART cells with enhanced proliferation and complete suppression of tumor growth of the newly injected tumor cells. Our work represents a promising strategy to improve treatment outcomes of patients with metastatic oncocytic carcinoma that may provide a durable response. Citation Format: Justyna J. Gleba, Aylin Alasonyalilar-Demirer, Matthew Pawlush, Claudia Manriquez-Roman, Elizabeth L. Siegler, Robert C. Smallridge, Abba Zubair, Han W. Tun, Saad S. Kenderian, John A. Copland. Novel TSHR CAR T therapy for targeting oncocytic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5082.

Autophagy sustains mitochondrial respiration and determines resistance to BRAFV600E inhibition in thyroid carcinoma cells

ABSTRACT BRAFV600E is the most prevalent mutation in thyroid cancer and correlates with poor prognosis and therapy resistance. Although selective inhibitors of BRAFV600E have been developed, more advanced tumors such as anaplastic thyroid carcinomas show a poor response in clinical trials. Therefore, the study of alternative survival mechanisms is needed. Since metabolic changes have been related to malignant progression, in this work we explore metabolic dependencies of thyroid tumor cells to exploit them therapeutically. Our results show that respiration of thyroid carcinoma cells is highly dependent on fatty acid oxidation and, in turn, fatty acid mitochondrial availability is regulated through macroautophagy/autophagy. Furthermore, we show that both lysosomal inhibition and the knockout of the essential autophagy gene, ATG7, lead to enhanced lipolysis; although this effect is not essential for survival of thyroid carcinoma cells. We also demonstrate that following inhibition of either autophagy or fatty acid oxidation, thyroid tumor cells compensate oxidative phosphorylation deficiency with an increase in glycolysis. In contrast to lipolysis induction, upon autophagy inhibition, glycolytic boost in autophagy-deficient cells is essential for survival and, importantly, correlates with a higher sensitivity to the BRAFV600E selective inhibitor, vemurafenib. In agreement, downregulation of the glycolytic pathway results in enhanced mitochondrial respiration and vemurafenib resistance. Our work provides new insights into the role of autophagy in thyroid cancer metabolism and supports mitochondrial targeting in combination with vemurafenib to eliminate BRAFV600E-positive thyroid carcinoma cells. Abbreviations: AMP: adenosine monophosphate; ATC: anaplastic thyroid carcinoma; ATG: autophagy related; ATP: adenosine triphosphate; BRAF: B-Raf proto-oncogene, serine/threonine kinase; Cas9: CRISPR-associated protein; CREB: cAMP responsive element binding protein; CRISPR: clustered regularly interspaced short palindromic repeats; 2DG: 2-deoxyglucose; FA: fatty acid; FAO: fatty acid oxidation; FASN: fatty acid synthase; FCCP: trifluoromethoxy carbonyl cyanide phenylhydrazone; LAMP1: lysosomal associated membrane protein 1; LIPE/HSL: lipase E, hormone sensitive type; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OCR: oxygen consumption rate; OXPHOS: oxidative phosphorylation; PRKA/PKA: protein kinase cAMP-activated; PTC: papillary thyroid carcinoma; SREBF1/SREBP1: sterol regulatory element binding transcription factor 1.

Selpercatinib combination with the mitochondria-targeted antioxidant MitoQ effectively suppresses RET–mutant thyroid cancer

Genetic alternation of REarranged during Transfection (RET) that leads to constitutive RET activation is a crucial etiological factor for thyroid cancer. RET is known to regulate mitochondrial processes, although the underlying molecular mechanisms remain unclear. We previously showed that the multi-kinase inhibitors vandetanib and cabozantinib increase the mitochondrial membrane potential (Δψm) in RET-mutated thyroid tumor cells and that this effect can be exploited to increase mitochondrial enrichment of Δψm-sensitive agents in the tumor cells. In this study, we hypothesized that the RET-selective inhibitor, selpercatinib, can increase Δψm and, subsequently, tumor cell uptake of the mitochondria-targeted ubiquinone (MitoQ) to the level to break the mitochondrial homeostasis and induce lethal responses in RET-mutated thyroid tumor cells. We show that selpercatinib significantly increased Δψm, and its combination with MitoQ synergistically suppressed RET-mutated human thyroid tumor cells, which we validated using RET-targeted genetic approaches. Selpercatinib and MitoQ, in combination, also suppressed CCDC6-RET fusion cell line xenografts in mice and prolonged animal survival more effectively than single treatments of each agent. Moreover, we treated two patients with CCDC6-RET or RETM918T thyroid cancer, who could not take selpercatinib at regular doses due to adverse effects, with a dose-reduced selpercatinib and MitoQ combination. In response to this combination therapy, both patients showed tumor reduction. The quality of life of one patient significantly improved over a year until the tumor relapsed. This combination of selpercatinib with MitoQ may have therapeutic potential for patients with RET-mutated tumors and intolerant to regular selpercatinib doses.

Cancer-associated fibroblast-derived periostin promotes papillary thyroid tumor growth through integrin-FAK-STAT3 signaling.

Background: Periostin (POSTN) is a critical extracellular matrix protein in various tumor microenvironments. However, the function of POSTN in thyroid cancer progression remains largely unknown. Methods: Postn and Rag1 knock-out mice and orthotopic mouse models were used to determine the role of POSTN on papillary thyroid tumor progression. Immunofluorescence, cell co-culture, fluorescence in situ hybridization, chromatin immunoprecipitation assay, recombinant protein and inhibitor treatment were performed to explore the underlying mechanisms of POSTN-promoted papillary thyroid tumor growth. Results: POSTN is up-regulated in papillary thyroid tumors and negatively correlates with the overall survival of patients with thyroid cancer. Cancer-associated fibroblast (CAF)-derived POSTN promotes papillary thyroid tumor growth in vivo and in vitro. POSTN deficiency in CAFs significantly impairs CAF-promoted papillary thyroid tumor growth. POSTN promotes papillary thyroid tumor cell proliferation and IL-4 expression through integrin-FAK-STAT3 signaling. In turn, tumor cell-derived IL-4 induces the activation of CAFs and stimulates POSTN expression by activating STAT6. We reveal the crucial role of CAF-derived POSTN and tumor cell-derived IL-4 in driving the development of papillary thyroid tumors through the POSTN-integrin-FAK-STAT3-IL-4 pathway in tumor cells and IL-4-STAT6-POSTN signaling in CAFs. Conclusion: Our findings underscore the significance of POSTN and IL-4 as critical molecular mediators in the dynamic interplay between CAFs and tumor cells, ultimately supporting the growth of papillary thyroid tumors.

GLP-1 receptor agonist liraglutide inhibits the proliferation and migration of thyroid cancer cells.

This research was done to find out how liraglutide affected the growth and movement of two human thyroid cancer cell lines overexpressing GLP-1 receptor (migration of medullary thyroid cancer TT/GLP-1R and papillary thyroid carcinoma TCP-1/GLP-1R). Flow cytometer and cAMP assays were used to identify the expression and activation of GLP-1R in two stable cell lines. Counting Kit-8 for Cells was applied to examine the proliferative ability of cells in two stable cell lines after treatment with different concentrations of liraglutide at indicated time points. To track the capacity for cell migration, the Transwell test was utilized. We found that liralutide-activated GLP-1R could significantly reduce the growth and metastasis of two kinds of thyroid tumor cells, and the inhibitory effect was dose- and time- dependent. The phosphorylatios of Akt, S6K1, and 70SK declined after receiving liraglutide therapy In our previous studies, we found that the GLP-1 receptor agonist Liraglutide inhibited the proliferation and migration of thyroid cancer cells through the PI3K/Akt/mTOR pathway. This finding provides a theoretical basis for the treatment of diabetes mellitus complicated with medullary thyroid cancer, and is relatively safe.

Clinical and molecular impact of concurrent thyroid autoimmune disease and thyroid cancer: From the bench to bedside.

The recent incorporation of immune checkpoint inhibitors targeting the PD-1 (programmed cell death receptor 1) and CTLA-4 (cytotoxic T lymphocyte antigen 4) pathways into the therapeutic armamentarium of cancer has increased the need to understand the correlation between the immune system, autoimmunity, and malignant neoplasms. Both autoimmune thyroid diseases and thyroid cancer are common clinical conditions. The molecular pathology of autoimmune thyroid diseases is characterized by the important impact of the PD-1/PD-L1 axis, an important inhibitory pathway involved in the regulation of T-cell responses. Insufficient inhibitory pathways may prone the thyroid tissue to a self-destructive immune response that leads to hypothyroidism. On the other hand, the PD-1/PD-L1 axis and other co-inhibitory pathways are the cornerstones of the immune escape mechanisms in thyroid cancer, which is a mechanism through which the immune response fails to recognize and eradicate thyroid tumor cells. This common mechanism raises the idea that thyroid autoimmunity and thyroid cancer may be opposite sides of the same coin, meaning that both conditions share similar molecular signatures. When associated with thyroid autoimmunity, thyroid cancer may have a less aggressive presentation, even though the molecular explanation of this clinical consequence is unclear. More studies are warranted to elucidate the molecular link between thyroid autoimmune disease and thyroid cancer. The prognostic impact that thyroid autoimmune disease, especially chronic lymphocytic thyroiditis, may exert on thyroid cancer raises important insights that can help physicians to better individualize the management of patients with thyroid cancer.

HHV-6A Infection of Papillary Thyroid Cancer Cells Induces Several Effects Related to Cancer Progression.

Recent studies have shown that thyrocytes are permissive to HHV-6A infection and that the virus may contribute to the pathogenesis of autoimmune thyroiditis. Thyroid autoimmune diseases increase the risk of papillary cancer, which is not surprising considering that chronic inflammation activates pathways that are also pro-oncogenic. Moreover, in this condition, cell proliferation is stimulated as an attempt to repair tissue damage caused by the inflammatory process. Interestingly, it has been reported that the well-differentiated papillary thyroid carcinoma (PTC), the less aggressive form of thyroid tumor, may progress to the more aggressive follicular thyroid carcinoma (FTC) and eventually to the anaplastic thyroid carcinoma (ATC), and that to such progression contributes the presence of an inflammatory/immune suppressive tumor microenvironment. In this study, we investigated whether papillary tumor cells (BCPAP) could be infected by human herpes virus-6A (HHV-6A), and if viral infection could induce effects related to cancer progression. We found that the virus dysregulated the expression of several microRNAs, such as miR-155, miR-9, and the miR-221/222 cluster, which are involved in different steps of carcinogenesis, and increased the secretion of pro-inflammatory cytokines, particularly IL-6, which may also sustain thyroid tumor cell growth and promote cancer progression. Genomic instability and the expression of PTEN, reported to act as an oncogene in mutp53-carrying cells such as BCPAP, also increased following HHV-6A-infection. These findings suggest that a ubiquitous herpesvirus such as HHV-6A, which displays a marked tropism for thyrocytes, could be involved in the progression of PTC towards more aggressive forms of thyroid tumor.

Telomerase Upregulation Induces Progression of Mouse BrafV600E-Driven Thyroid Cancers and Triggers Nontelomeric Effects.

Telomerase-driven cancer progression activates pathways that can be dissected and perhaps therapeutically exploited.

Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells.

We previously described the role of low-density lipoprotein (LDL) in aggressiveness in papillary thyroid cancer (PTC). Moreover, the MAPK signaling pathway in the presence of BRAF V600E mutation is associated with more aggressive PTC. Although the link between MAPK cascade and LDL receptor (LDLR) expression has been previously described, it is unknown whether LDL can potentiate the adverse effects of PTC through it. We aimed to investigate whether the presence of LDL might accelerate the oncogenic processes through MAPK pathway in presence or absence of BRAF V600E in two thyroid cell lines: TPC1 and BCPAP (wild-type and BRAF V600E, respectively). LDLR, PI3K-AKT and RAS/RAF/MAPK (MEK)/ERK were analyzed via Western blot; cell proliferation was measured via MTT assay, cell migration was studied through wound-healing assay and LDL uptake was analyzed by fluorometric and confocal analysis. TPC1 demonstrated a time-specific downregulation of the LDLR, while BCPAP resulted in a receptor deregulation after LDL exposition. LDL uptake was increased in BCPAP over-time, as well as cell proliferation (20% higher) in comparison to TPC1. Both cell lines differed in migration pattern with a wound closure of 83.5 ± 9.7% after LDL coculture in TPC1, while a loss in the adhesion capacity was detected in BCPAP. The siRNA knockdown of LDLR in LDL-treated BCPAP cells resulted in a p-ERK expression downregulation and cell proliferation modulation, demonstrating a link between LDLR and MAPK pathway. The modulation of BRAF-V600E using vemurafenib-impaired LDLR expression decreased cellular proliferation. Our results suggest that LDLR regulation is cell line-specific, regulating the RAS/RAF/MAPK (MEK)/ERK pathway in the LDL-signaling cascade and where BRAF V600E can play a critical role. In conclusion, targeting LDLR and this downstream signaling cascade, could be a new therapeutic strategy for PTC with more aggressive behavior, especially in those harboring BRAF V600E.

Abstract 5074: Addition of MAPK inhibitors to prime and sensitize poorly differentiated thyroid cancers as a strategy to improve TSHR-CART cell therapy antitumor activity

Abstract Thyroid cancer is the most common endocrine cancer in the US, and its incidence is rising. Most thyroid cancer deaths are attributed to treatment-refractory, metastatic tumors. Thyroid stimulating hormone receptor (TSRH) expression is largely limited to the thyroid gland and is abundantly expressed on thyroid tumor cells, making TSRH a compelling target for advanced thyroid cancer diagnostics and therapeutics. Therefore, we developed a novel TSHR-targeted chimeric antigen receptor (CAR) T cell therapy to treat aggressive thyroid cancers. TSHR-CAR constructs were cloned into a lentiviral CAR construct containing 4-1BB and CD3ζ. First, we demonstrated potent TSHR-CART antigen-specific anti-tumor activity in vitro. Then, NOD-SCID-γ-/- (NSG) mice were inoculated subcutaneously with TSHR+ tumor cells and randomized by tumor volume to treatment with TSHR-CART cells or control Untransduced T cells (UTD). Treatment with TSHR-CART cells resulted in dose-dependent antitumor activity and prolonged survival. De-differentiated anaplastic thyroid cancers (ATC) downregulate TSHR. Our TSHR immunohistochemistry results corroborated these findings and displayed minimal TSHR protein expression, precluding successful TSHR-CART treatment. We therefore sought to sensitize these tumors with MAPK inhibitors, as a strategy to upregulate TSHR expression in patients with metastatic thyroid cancer. TSHR expression was upregulated in patient-derived xenograft (PDX) ATC models after one week of daily administration of the MAPK inhibitors (p=0.0024). After confirming that MAPK inhibition does not dampen TSHR-CART effector functions, we tested sequential and combination therapy of TSHR-CART with MEK and BRAF inhibition in vivo. NSG mice were engrafted with ATC BRAF-mutant PDX tumors and randomized by tumor volume to daily oral treatment with placebo or trametinib (MEK inhibitor) plus dabrafenib (BRAF inhibitor). One week later, mice received either UTD or TSHR-CART. Mice conditioned with trametinib plus dabrafenib (p=0.0018) and subsequently treated with TSHR-CART showed superior antitumor activity. However, the improved antitumor activity in this setting was transient. We therefore tested the durability of TSHR upregulation following MEK/BRAF inhibition and demonstrated that TSHR upregulation lasts less than 48-72 hours after discontinuation. Finally, we tested the combination of TSHR CART cells with MEK/BRAF inhibitors in ATC BRAF-mutant PDX tumors. Here, combining TSHR-CART cells with MEK/BRAF inhibitors result in durable control of the tumors. Collectively, our findings indicate that MEK/BRAF inhibition of de-differentiated thyroid cancers upregulated TSHR expression and enhanced TSHR-CART antitumor activity. This work represents a viable strategy to improve outcomes of patients with aggressive, metastatic thyroid cancers. Citation Format: Claudia Manriquez Roman, Kendall J. Schick, Justyna J. Gleba, Truc N. Huynh, Elizabeth L. Siegler, James L. Miller, Aylin Alasonyalilar Demirer, Matthew L. Pawlush, Ahmet Biligili, Long K. Mai, Erin Tapper, Leo R. Sakemura, Michelle J. Cox, Carli M. Stewart, Ismail Can, Ekene J. Ogbodo, Gaofeng Cui, Georges Mer, Gloria R. Olivier, Yushi Qiu, Robert C. Smallridge, Zubair C. Abba, Han W. Tun, John A. Copland, Saad S. Kenderian. Addition of MAPK inhibitors to prime and sensitize poorly differentiated thyroid cancers as a strategy to improve TSHR-CART cell therapy antitumor activity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5074.

Intracellular cGMP increase is not involved in thyroid cancer cell death.

Type 5 phosphodiesterase (PDE5) inhibitors (PDE5i) lead to intracellular cyclic-guanosine monophosphate (cGMP) increase and are used for clinical treatment of erectile dysfunction. Studies found that cGMP may up/downregulate the growth of certain endocrine tumor cells, suggesting that PDE5i could impact cancer risk. We evaluated if PDE5i may modulate thyroid cancer cell growth in vitro. We used malignant (K1) and benign (Nthy-ori 3-1) thyroid cell lines, as well as the COS7 cells as a reference model. Cells were treated 0-24 h with the PDE5i vardenafil or the cGMP analog 8-br-cGMP (nM-μM range). cGMP levels and caspase 3 cleavage were evaluated by BRET, in cGMP or caspase 3 biosensor-expressing cells. Phosphorylation of the proliferation-associated extracellularly-regulated kinases 1 and 2 (ERK1/2) was evaluated by Western blotting, while nuclear fragmentation by DAPI staining. Cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Both vardenafil and 8-br-cGMP effectively induced dose-dependent cGMP BRET signals (p≤0.05) in all the cell lines. However, no differences in caspase 3 activation occurred comparing PDE5i-treated vs untreated cells, at all concentrations and time-points tested (p>0.05). These results match those obtained upon cell treatment with 8-br-cGMP, which failed in inducing caspase 3 cleavage in all the cell lines (p>0.05). Moreover, they reflect the lack of nuclear fragmentation. Interestingly, the modulation of intracellular cGMP levels with vardenafil or the analog did not impact cell viability of both malignant and benign thyroid tumor cell lines, nor the phosphorylation of ERK1/2 (p>0.05). This study demonstrates that increased cGMP levels are not linked to cell viability or death in K1 and Nthy-ori 3-1 cell lines, suggesting that PDE5i do not impact the growth of thyroid cancer cells. Since different results were previously published, further investigations are recommended to clarify the impact of PDE5i on thyroid cancer cells.

Alternation between toxic and proliferative effects of Roundup® on human thyroid cells at different concentrations.

Endocrine-disrupting and carcinogenic effects of glyphosate have long been suspected, but little is known about the effect of compounds used in real life at different concentrations, neither in normal nor in thyroid tumor cells. As cancer cells may have different sensitivities and the effect of the product containing glyphosate may be different from that produced by the active ingredient alone, including the Acceptable Occupational Exposure Level (AOEL=160µg/L) and the Acceptable Daily Intake (ADI=830µg/L) determined by ANVISA, we used two human thyroid-derived cell lines, Nthy-ori 3-1 (from normal follicular cells) and TPC-1 (from papillary carcinoma), to test 15 different concentrations of Roundup® Original DI. Trypan blue (TB), CCK-8 and BrdU assays were used to evaluate cytotoxicity, metabolic activity and proliferation with 24h and 48h exposures in technical and biological triplicates. TB showed an important toxic effect, especially after 24h of exposure, in both cell lines. The AOEL concentration caused the death of 43% and 50% of the Nthy-ori and TPC-1 cells, respectively, in 24 h, while ADI resulted in 35% and 58% of cell death. After 48h of exposure, AOEL and ADI caused a lower number of dead Nthy-ori (33% and 18%) and TPC-1 (33% and 37%) cells, respectively, suggesting that the toxic effect of the product disappears and/or both strains have repair mechanisms that protect them from longer exposures. On the other hand, the CCK-8 assay showed that small concentrations of Roundup have a proliferative effect: 6.5µg/L increased the number of both Nthy-ori and TPC-1 cells at 24h, and the BrdU assay confirmed the stimulatory effect with a 321% increase in the absorbance of Nthy-ori cells at 48h. The herbicide produced even more frequent increases in the BrdU absorbance of TPC-1 cells, mainly at 24h. We conclude that thyroid cells exposed to Roundup present a nonmonotonic dual dose–response curve. Low concentrations of the pesticide, considered acceptable, cause significant cell death but also have an important proliferative effect, especially on TPC-1 cells. This herbicide, widely used around the world, may play a role in the increased incidence rate of thyroid nodules and cancer that has been observed in recent decades.

Extracellular vesicles from thyroid cancer harbor a functional machinery involved in extracellular matrix remodeling

Extracellular vesicles (EVs) participate in cell-stroma crosstalk within the tumor microenvironment and fibroblasts (Fb) contribute to tumor promotion in thyroid cancer. However, the role of tumor-stroma derived EVs still needs to be deciphered. We hypothesized that the interaction of thyroid tumor cells with Fb would liberate EVs with a specific proteomic profile, which would have an impact on EV-functionality in thyroid tumor progression-related events. Tumor (TPC-1, 8505c) and non-tumor (NThyOri) thyroid cells were co-cultured with human Fb. EVs, obtained by ultracentrifugation of conditioned media, were characterized by nanoparticle tracking analysis and western blotting. EV-proteomic analysis was performed by mass-spectrometry, and metalloproteinases (MMPs) were studied by zymography. EV-exchange was evaluated using immunofluorescence, confocal microscopy and FACS. EVs expressed classical exosome markers, with EVs from thyroid tumor cell-Fb co-cultures showing a proteomic profile related to extracellular matrix (ECM) remodeling. Bidirectional crosstalk between Fb and TPC-1 cells produced significantly more EVs than their isolated cells, and potentiated EV-functionality. In line with this, Fb-TPC-1 derived EVs induced MMP2 activation in NThyOri supernatants, and MMP2 activity could be evidenced in Fb and TPC-1 contact-independent co-cultures. Besides, MMP2 interactors allowed us to discriminate between EVs from thyroid tumoral and non-tumoral milieus. Interestingly, Fb internalized more EVs from TPC-1 than from NThyOri producing cells. Fb and thyroid tumor cell crosstalk produces specialized EVs with an ECM remodeling proteomic profile, enabling activation of MMP2 and possibly facilitating ECM-degradation, which is potentially linked with thyroid tumor progression.

Vitamin D Reduces Thyroid Cancer Cells Migration Independently From the Modulation of CCL2 and CXCL8 Chemokines Secretion

BackgroundVitamin D3 is largely involved in the regulation of calcium homeostasis. More recently, it was demonstrated that vitamin D exerts several beneficial effects against cancer progression through several mechanisms, including the reduction of cancer cells proliferation and migration. CXCL8 and CCL2 are two chemokines secreted by thyroid tumor cells. In the thyroid tumor microenvironment, these chemokines exert several pro-tumorigenic effects including the one to increase the metastatic potential. The aim of the present study was to investigate if vitamin D could modulate both thyroid cancer cell migration and their ability to secrete CCL2 and CXCL8.MethodsTPC-1 (RET/PTC rearranged) and 8505C (BRAFV600e mutated) thyroid cancer cell lines were treated with increasing concentrations of 1,25-OH-vitamin D3 (0–1,000 nM). Cell viability was assessed by WST-1 assay, cell migration was evaluated by transwell–migration chamber system, and CCL2 and CXCL8 levels were measured in the cell culture supernatants by ELISA.ResultsVitamin D did not affect cell viability but reduced, in a dose-dependent and significant manner, thyroid cancer cell migration (ANOVAs p < 0.05 for both TPC-1 and 8505C). Vitamin D differently modulated the secretion of CCL2 and CXCL8, by significantly inhibiting the secretion of CCL2 in both thyroid cancer cell lines and inhibiting the secretion of CXCL8 only in TPC-1 (ANOVAs p < 0.05).ConclusionsVitamin D treatment of thyroid cancer cell lines reduces cell migration independently from the inhibition of the secretion of pro-tumorigenic chemokines. Future studies specifically designed at clarifying the pathways involved in the different inhibitory effects of vitamin D on CCL2 and CXCL8 in thyroid cancer cells appear worthwhile.

Pentacyclic triterpenoids from spikes of Prunella vulgaris L. with thyroid tumour cell cytostatic bioactivities

Five new triterpenoids, including four ursane types (1–4) and one oleanane type (5), together with 15 known ursane types pentacyclic triterpenoids (6–20) were isolated from the fruit spikes of Prunella vulgaris L., a traditional Chinese herbal medicine. Their structures were elucidated based on IR, HR-ESI-MS, and NMR spectroscopic data. The SW579 cell line was used to evaluate anti-thyroid cancer activities of (1–20). The results indicated that (7–9), (16), and (19) exhibited apparent inhibitory activity with IC50 values of 25.73–71.41 μM (cisplatin as positive control, IC50 14.49 ± 0.97 μM). Network pharmacology and molecular docking were also used for the prediction of the synergistic actions and the underlying mechanisms. Accordingly, four potential targets have been characterized.

Senescent Thyrocytes, Similarly to Thyroid Tumor Cells, Elicit M2-like Macrophage Polarization In Vivo.

Simple SummarySeveral studies including ours suggest a pro-tumoral role of senescent thyrocytes in thyroid tumors. On the other hand, M2-like tumor associated macrophages infiltration increases with thyroid cancer aggressiveness. In this work, we used senescent thyrocytes and thyroid tumor cells as models of early and late tumor stages, respectively, and demonstrated their in vivo capability to recruit and polarize macrophages toward a pro-tumoral M2-like phenotype. These findings pave the way for the design of new therapeutic approaches for thyroid tumors based on the elimination or activity modulation of senescent cells and/or infiltrating macrophages.Inflammation plays a critical role in thyroid cancer onset and progression. We previously characterized the in vitro interplay between macrophages and senescent human thyrocytes and thyroid tumor-derived cell lines, modeling the early and the late thyroid tumor phases, respectively. We reported that both models are able to induce pro-tumoral M2-like macrophage polarization, through the activation of the COX2-PGE2 axis. Here, we investigated the presence of macrophage infiltrating cells in mouse xenografts derived from the above described cells models. We showed that subcutaneous injection in immunodeficient mice of both senescent human thyrocytes and thyroid tumor-derived cell lines elicits macrophage recruitment. Furthermore, considering the type of macrophage infiltrate, we observed a stronger infiltration of Arginase I positive cells (M2-like). Overall, these results demonstrate the in vivo capability of senescent and tumor thyroid cells to recruit and polarize macrophages, suggesting that the promotion of a pro-tumoral microenvironment through tumor associated macrophages may occurs in late as well as in early thyroid tumor stages, favoring tumor onset and progression.

LncRNA SNHG14 promotes the proliferation, migration, and invasion of thyroid tumour cells by regulating miR-93-5p.

Long non-coding RNAs (lncRNAs) exert vital functions in the occurrence and development of various tumours. The aim of this study was to examine the regulatory effect and underlying molecular mechanism of lncRNA small nucleolar RNA host gene 14 (SNHG14) on the proliferation, invasion and migration of thyroid tumour cells. The expression of SNHG14 in thyroid tumour cell lines was determined using qRT-PCR. CCK-8 and western blot were used to detect the effects of SNHG14 on proliferation and apoptosis of thyroid tumour cells. The effect of SNHG14 on the migration and invasion of thyroid tumour cells was analyzed using immunofluorescence, wound-healing and transwell assays. A targeting relationship between SNHG14 and miR-93-5p was determined using bioinformatics software and luciferase reporter assays. In addition, CCK-8, immunofluorescence, wound-healing and transwell assays were applied to demonstrate that SNHG14 promoted the proliferation, migration and invasion of thyroid tumour cells by targeting miR-93-5p. The biological function of SNHG14 in vivo was explored through a xenograft model and immunohistochemistry. SNHG14 was upregulated in thyroid tumour cells compared with normal cells. Downregulation of SNHG14 effectively reduced the proliferation, migration and invasion of TPC-1 cells, and induced cell apoptosis. Moreover, SNHG14 directly targeted miR-93-5p and there was a negative correlation between them. Further functional experiments illustrated that miR-93-5p overexpression dramatically reversed the promoting role of SNHG14 in proliferation, migration and invasion of TPC-1 cells. Our results demonstrated that SNHG14 promotes the proliferation, invasion and migration of thyroid tumour cells by downregulating miR-93-5p.