Anaplastic Thyroid Carcinoma Cell Lines Research Articles

C5AR1-induced TLR1/2 pathway activation drives proliferation and metastasis in anaplastic thyroid cancer.

This study aimed to elucidate the role and mechanisms of Complement C5a receptor 1 (C5AR1) in driving the malignant progression of anaplastic thyroid carcinoma (ATC).C5AR1 expression was assessed in ATC tissues and cell lines. Functional assays evaluated the effects of C5AR1 knockdown on the malignant features of ATC cells. The interaction between C5AR1 and miR-335-5p was confirmed using a luciferase reporter assay and Fluorescence in situ hybridization, and the impact of C5AR1 knockdown on the Toll-like receptor (TLR) 1/2 signaling pathway was examined. In vivo studies evaluated the effects of C5AR1 modulation on tumor growth and metastasis.C5AR1 levels were elevated in ATC tumor samples and associated with poor survival in ATC patients. C5AR1 knockdown impeded ATC cell proliferation, migration, and invasion in vitro. MiR-335-5p was identified as an upstream regulator of C5AR1, which negatively modulates C5AR1 expression. C5AR1 knockdown diminished TLR1, TLR2, and myeloid differentiation primary response 88 (MyD88) levels, while C5AR1 overexpression activated this pathway. Blocking TLR1/2 signaling abrogated the oncogenic effects of C5AR1 overexpression. C5AR1 silencing inhibited tumor growth and lung metastasis of ATC cells in nude mice.C5AR1 contributes to ATC tumorigenesis and metastasis by activating the TLR1/2 pathway, and is negatively regulated by miR-335-5p. Targeting the miR-335-5p/C5AR1/TLR1/2 axis represents a potential therapeutic strategy for ATC.

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.

Honokiol Suppresses Cell Proliferation and Tumor Migration through ROS in Human Anaplastic Thyroid Cancer Cells.

Honokiol is a natural polyphenolic compound extracted from Magnolia officinali, which is commonly used material in Chinese herbal medicine, has a variety of biological functions, including anti-tumor, anti-oxidant, anti-inflammation, anti-microbial and anti-allergy. Although honokiol has numerous beneficial effects on human diseases, the underlying mechanisms of tumor metastasis are still unclear. Previously, we reported that honokiol suppresses thyroid cancer cell proliferation with cytotoxicity through cell cycle arrest, apoptosis, and dysregulation of intracellular hemostasis. Herein, we hypothesized that the antioxidant effect of honokiol might play a critical role in thyroid cancer cell proliferation and migration. The cell viability assays, cellular reactive oxygen species (ROS) activity, cell migration, and immunoblotting were performed after cells were treated with honokiol. Based on this hypothesis, we first demonstrated that honokiol suppresses cell proliferation in two human anaplastic thyroid carcinoma (ATC) cell lines, KMH-2 and ASH-3, within a dosage- and time-dependent manner by cell counting kit-8 (CCK-8) assay. Next, we examined that honokiol induced ROS activation and could be suppressed by pre-treated with an antioxidant agent, N-acetyl-l-cysteine (NAC). Furthermore, the honokiol suppressed cell proliferation can be rescued by pre-treated with NAC. Finally, we demonstrated that honokiol inhibited ATC cell migration by modulating epithelial-mesenchymal transition (EMT)-related markers by Western blotting. Taken together, we provided the potential mechanism for treating ATC cells with honokiol, which significantly suppresses tumor proliferation and inhibits tumor metastasis in vitro through reactive oxygen species (ROS) induction.

Mechanisms of vemurafenib-induced anti-tumor effects in ATC FRO cells

BackgroundAnaplastic Thyroid Carcinoma (ATC) is a rare and deadly malignant tumor in humans. It is prone to developing resistance to radiotherapy and chemotherapy. Molecular targeted therapy offers a novel way to treat ATC. The BRAF mutation is closely associated with many cancers, including thyroid carcinoma. Vemurafenib, a small-molecule inhibitor, is specifically designed to target the mutant serine/threonine kinase BRAF. The objective of this study is to elucidate the regulatory mechanisms underlying the effects of vemurafenib on human anaplastic thyroid carcinoma cell line FRO and to assess its potential therapeutic role. MethodsThe effects of vemurafenib on the proliferation of FRO cells were assessed by the CCK-8 method and Colony-forming assay. Transwell chambers and scratch tests were employed to examine the impact of vemurafenib on the invasion and migration of FRO cells. Apoptosis and cycle distribution of FRO cells were analyzed by tunel assay and flow cytometry. The effects of vemurafenib on the expression of BRAF-activated non-protein coding RNA (BANCR), Bax, Bcl2, and E-cadherin were evaluated by qRT-PCR. Furthermore, the effects of vemurafenib on the expression of phosphoinositol-3-kinase (PI3K)/phosphoinositol-3-kinase (AKT) pathway-related proteins, BRAF, CyclinD1, Bcl-2, Bax, and E-cadherin proteins in FRO cells were investigated through the western-blot method. All experiments were conducted in three replicates. ResultsVemurafenib was observed to inhibit proliferation and induce apoptosis in a dose- and time-dependent manner (P < 0.05). The formation of FRO cell colonies, as well as migration and invasion, all showed a dose-dependent reduction (P < 0.05). Flow cytometric analysis indicated G0/G1 cell cycle arrest (P < 0.05). QRT-PCR revealed that vemurafenib could suppress the expression of BANCR and Bcl2 while increasing the expression of Bax and E-cadherin in a dose-dependent manner (P < 0.05). The protein expression levels of Bax and E-cadherin were up-regulated significantly, and the expression levels of BRAF, CyclinD1, Bcl-2, p-PI3K, p-AKT, and p-mTOR were markedly down-regulated with increasing concentrations of vemurafenib (P < 0.05). ConclusionsThe proliferation and metastasis of FRO cells can be suppressed by vemurafenib through the silencing of BRAF and BANCR expression, inhibition of PI3K/AKT signaling pathway activation, induction of apoptosis, and cell cycle arrest.

Evaluation of the Therapeutic Effects of Harmine on Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid carcinoma (ATC) is an extremely difficult disease to tackle, with an overall patient survival of only a few months. The currently used therapeutic drugs, such as kinase inhibitors or immune checkpoint inhibitors, can prolong patient survival but fail to eradicate the tumor. In addition, the onset of drug resistance and adverse side-effects over time drastically reduce the chances of treatment. We recently showed that Twist1, a transcription factor involved in the epithelial mesenchymal transition (EMT), was strongly upregulated in ATC, and we wondered whether it might represent a therapeutic target in ATC patients. To investigate this hypothesis, the effects of harmine, a β-carboline alkaloid shown to induce degradation of the Twist1 protein and to possess antitumoral activity in different cancer types, were evaluated on two ATC-derived cell lines, BHT-101 and CAL-62. The results obtained demonstrated that, in both cell lines, harmine reduced the level of Twist1 protein and reverted the EMT, as suggested by the augmentation of E-cadherin and decrease in fibronectin expression. The drug also inhibited cell proliferation and migration in a dose-dependent manner and significantly reduced the anchorage-independent growth of both ATC cell lines. Harmine was also capable of inducing apoptosis in BHT-101 cells, but not in CAL-62 ones. Finally, the activation of PI3K/Akt signaling, but not that of the MAPK, was drastically reduced in treated cells. Overall, these in vitro data suggest that harmine could represent a new therapeutic option for ATC treatment.

Drug-induced inhibition of HMGA and EZH2 activity as a possible therapy for anaplastic thyroid carcinoma

ABSTRACT Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and lethal neoplasms in humans, and just limited progresses have been made to extend patient survival and decrease ATC-associated mortality. Thus, the identification of novel therapeutic strategies for treating ATC is needed. Recently, our group has identified two proteins with oncogenic activity, namely HMGA1 and EZH2, with pivotal roles in ATC cancer progression. Therefore, we tested the ability of trabectedin, a HMGA1-targeting drug, and GSK126, an inhibitor of EZH2 enzymatic activity, to impair cell viability of four ATC-derived cell lines. In the present study, we first confirmed the overexpression of HMGA1 and EZH2 in all ATC-derived cell lines and tissues compared to the normal primary thyroid cells and tissues. Then, treatment of the ATC cell lines with trabectedin and GSK126 resulted in a drastic induction of apoptotic cell death, which increased when the ATC cell lines were treated with a combination of both drugs. Conversely, normal primary human thyroid cells did not show any significant reduction in their viability when exposed to the same drugs. Noteworthy, both drugs induced the deregulation of EZH2- and HMGA1-controlled genes. Altogether, these findings propose the combination of trabectedin and GSK126 as possible novel strategy for ATC therapy.

LncRNA H19 Influences Cellular Activities via the miR-454-3p/BHLHE40 Axis in Anaplastic Thyroid Carcinoma.

Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy threatening patients' life quality. Our previous study has demonstrated that inhibition of long non-coding RNA H19 (lncRNA h19; H19) blocks ATC growth and metastasis. The current study aimed to further explore the potential mechanism of H19 in ATC. Expression of H19, miR-454-3p, and BHLHE40 mRNA was measured using RT-qPCR in tissue samples and cell lines. The dual-luciferase reporter assay and Pearson correlation analysis were used to explore the interaction among H19, miR-454-3p, and BHLHE40. The biological process of proliferation, migration, and invasion was determined using loss- or gain-function CCK-8 and Transwell assays. Western blot assay was used to evaluate the changes in protein levels. H19 was elevated in ATC tissues and cell lines. Based on online prediction database results, miR-454-3p might be a target of H19, and BHLHE40 might be a direct target of miR-454-3p. miR-454-3p expression was decreased in ATC and had a negative interaction with H19. BHLHE40 mRNA expression was increased and has a negative correlation with miR-454-3p and a positive correlation with H19. Downregulation of miR-454-3p and upregulation of BHLHE40 could reverse the decreased cellular activities caused by si-H19. Moreover, the silence of H19 modulates BHLHE40 to affect the PI3K/AKT protein levels and apoptotic-related protein levels. The current study provided a potential detailed mechanism of H19 in ATC, and lncRNA H19-miR-454-3p-BHLHE40 interaction may be a new experimental basis for prognosis and targeted therapy for ATC patients.

SPRY4 as a Potential Mediator of the Anti-Tumoral Role of Macrophages in Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid carcinoma (ATC) is the most lethal subtype of thyroid cancer, with high invasive and metastatic potential, not responding to conventional treatments. Its aggressiveness may be influenced by macrophages, which are abundant cells in the tumor microenvironment. To investigate the role of macrophages in ATC aggressiveness, indirect co-cultures were established between ATC cell lines and THP-1-derived macrophages. Macrophages significantly increased both the migration and invasion of T235 cells (p < 0.01; p < 0.01), contrasting with a decrease in C3948 (p < 0.001; p < 0.05), with mild effects in T238 migration (p < 0.01) and C643 invasion (p < 0.05). Flow cytometry showed upregulation of CD80 (pro-inflammatory, anti-tumoral) and downregulation of CD163 (anti-inflammatory, pro-tumoral) in macrophages from co-culture with T235 (p < 0.05) and C3948 (p < 0.05), respectively. Accordingly, we found an upregulation of secreted pro-inflammatory mediators (e.g., GM-CSF, IL-1α; p < 0.05) in C3948-macrophage co-cultures. Proteomic analysis showed the upregulation of SPRY4, an inhibitor of the MAPK pathway, in C3948 cells from co-culture. SPRY4 silencing promoted cancer cell invasion, reverting the reduced invasion of C3948 caused by macrophages. Our findings support that macrophages play a role in ATC cell aggressiveness. SPRY4 is a possible modulator of macrophage-ATC cell communication, with a tumor suppressor role relevant for therapeutic purposes.

Antitumor Activity of Berberine by Activating Autophagy and Apoptosis in CAL-62 and BHT-101 Anaplastic Thyroid Carcinoma Cell Lines.

Anaplastic thyroid carcinoma (ATC) is the most lethal thyroid carcinoma. Doxorubicin (DOX) is the only drug approved for anaplastic thyroid cancer treatment, but its clinical use is restricted due to irreversible tissue toxicity. Berberine (BER), an isoquinoline alkaloid extracted from Coptidis Rhizoma, has been proposed to have antitumor activity in many cancers. However, the underlying mechanisms by which BER regulates apoptosis and autophagy in ATC remain unclear. Thus, the present study aimed to assess the therapeutic effect of BER in human ATC cell lines CAL-62 and BHT-101 as well as the underlying mechanisms. In addition, we assessed the antitumor effects of a combination of BER and DOX in ATC cells. The cell viability of CAL-62 and BTH-101 with treatment of BER for different hours was measured by CCK-8 assay, and cell apoptosis was assessed by clone formation assay and flow cytometric analysis. The protein levels of apoptosis protein, autophagy-related proteins and PI3K/AKT/mTORpathway were determined Using Western blot. Autophagy in cells was observed with GFP-LC3 plasmid using confocal fluorescent microscopy. Flow cytometry was used to detect intracellular ROS. The present results showed that BER significantly inhibited cell growth and induced apoptosis in ATC cells. BER treatment also significantly upregulated the expression of LC3B-II and increased the number of GFP-LC3 puncta in ATC cells. Inhibition of autophagy by 3-methyladenine (3-MA) suppressed BER-induced autophagic cell death. Moreover, BER induced the generation of reactive oxygen species (ROS). Mechanistically, we demonstrated that BER regulated the autophagy and apoptosis of human ATC cells through the PI3K/AKT/mTOR pathways. Furthermore, BER and DOX cooperated to promote apoptosis and autophagy in ATC cells. Taken together, the present findings indicated that BER induces apoptosis and autophagic cell death by activating ROS and regulating the PI3K/AKT/mTOR signaling pathway.

ASPM promotes migration and invasion of anaplastic thyroid carcinoma by stabilizing KIF11.

Abnormal spindle-like microcephaly-associated (ASPM) protein is crucial to the mitotic spindle function during cell replication and tumor progression in multiple tumor types. However, the effect of ASPM in anaplastic thyroid carcinoma (ATC) has not yet been understood. The present study is to elucidate the function of ASPM in the migration and invasion of ATC. ASPM expression is incrementally upregulated in ATC tissues and cell lines. Knockout (KO) of ASPM pronouncedly attenuates the migration and invasion of ATC cells. ASPM KO significantly reduces the transcript levels of Vimentin, N-cadherin, and Snail and increases E-cadherin and Occludin, thereby inhibiting epithelial-to-mesenchymal transition (EMT). Mechanistically, ASPM regulates the movement of ATC cells by inhibiting the ubiquitin degradation of KIF11 and thus stabilizing it via direct binding to it. Moreover, xenograft tumors in nude mice proved that KO of ASPM could ameliorate tumorigenesis and tumor growth accompanied by a decreased protein expression of KIF11 and an inhibition of EMT. In conclusion, ASPM is a potentially useful therapeutic target for ATC. Our results also reveal a novel mechanism by which ASPM inhibits the ubiquitin process in KIF11.

Validation of EZH2 Inhibitor Efficiency in Anaplastic Thyroid Carcinoma Cell Lines.

The prognosis of anaplastic thyroid carcinoma (ATC) is poor, and there is currently no established treatment to improve its outcome. We previously reported that enhancer of zeste homolog 2 (EZH2) was highly expressed in ATC, and may be a therapeutic target; however, the effects of EZH2 on ATC growth currently remain unknown. We investigated the effects of an EZH2 inhibitor (DZNep) on four ATC cell lines (8305C, KTA1, TTA1 and TTA2). We performed a gene panel analysis of all ATC cell lines to identify differences in DZNep sensitivity between the cell lines. To investigate the effects of DZNep on the recovery of differentiation, we assessed changes in thyroid differentiation markers (TDMs) before and after the DZNep treatment using PCR. EZH2 was expressed in all ATC cell lines. The cell-reducing effects of DZNep were detected in all ATC cell lines, and were the strongest in KTA1 cells followed by TTA2 cells. The TTA1 and 8305C cell lines, which showed weak cell-reducing effects, had TP53 mutations. No changes in TDMs were observed in any ATC cell line. DZNep, an EZH2 inhibitor, exerted suppressive effects on the growth of ATC cell lines and has potential as a therapeutic strategy; however, its effects may be attenuated in ATC with TP53 mutations.

MiR-210 Expression Is Strongly Hypoxia-Induced in Anaplastic Thyroid Cancer Cell Lines and Is Associated with Extracellular Vesicles and Argonaute-2.

Hypoxia, or low oxygen tension, is frequently found in highly proliferative solid tumors such as anaplastic thyroid carcinoma (ATC) and is believed to promote resistance to chemotherapy and radiation. Identifying hypoxic cells for targeted therapy may thus be an effective approach to treating aggressive cancers. Here, we explore the potential of the well-known hypoxia-responsive microRNA (miRNA) miR-210-3p as a cellular and extracellular biological marker of hypoxia. We compare miRNA expression across several ATC and papillary thyroid cancer (PTC) cell lines. In the ATC cell line SW1736, miR-210-3p expression levels indicate hypoxia during exposure to low oxygen conditions (2% O2). Furthermore, when released by SW1736 cells into the extracellular space, miR-210-3p is associated with RNA carriers such as extracellular vesicles (EVs) and Argonaute-2 (AGO2), making it a potential extracellular marker for hypoxia.

BI-847325, a selective dual MEK and Aurora kinases inhibitor, reduces aggressive behavior of anaplastic thyroid carcinoma on an in vitro three-dimensional culture.

Anaplastic thyroid carcinoma (ATC) is the most aggressive subtype of thyroid cancer. In this study, we used a three-dimensional in vitro system to evaluate the effect of a dual MEK/Aurora kinase inhibitor, BI-847325 anticancer drug, on several cellular and molecular processes involved in cancer progression. Human ATC cell lines, C643 and SW1736, were grown in alginate hydrogel and treated with IC50 values of BI-847325. The effect of BI-847325 on inhibition of kinases function of MEK1/2 and Aurora kinase B (AURKB) was evaluated via Western blot analysis of phospho-ERK1/2 and phospho-Histone H3 levels. Sodium/iodide symporter (NIS) and thyroglobulin (Tg), as two thyroid-specific differentiation markers, were measured by qRT-PCR as well as flow cytometry and immunoradiometric assay. Apoptosis was assessed by Annexin V/PI flow cytometry and BIM, NFκB1, and NFκB2 expressions. Cell cycle distribution and proliferation were determined via P16, AURKA, and AURKB expressions as well as PI and CFSE flow cytometry assays. Multidrug resistance was evaluated by examining the expression of MDR1 and MRP1. Angiogenesis and invasion were investigated by VEGF expression and F-actin labeling with Alexa Fluor 549 Phalloidin. Western blot results showed that BI-847325 inhibits MEK1/2 and AURKB functions by decreasing phospho-ERK1/2 and phospho-Histone H3 levels. BI-847325 induced thyroid differentiation markers and apoptosis in ATC cell lines. Inversely, BI-847325 intervention decreased multidrug resistance, cell cycle progression, proliferation, angiogenesis, and invasion at the molecular and/or cellular levels. The results of the present study suggest that BI-857,325 might be an effective multi-targeted anticancer drug for ATC treatment.

Nuclear-targeted EGF receptor enhances proliferation and migration of human anaplastic thyroid cancer cells.

Epidermal growth factor (EGF) has various important physiological functions, which it exerts by binding to the epidermal growth factor receptor (EGFR). Reports show that EGF expression is strongly correlated with the occurrence and development of many types of tumour. To date, however, the relationship between EGF/EGFR and the occurrence and development of thyroid carcinoma remains unclear. In the current study, we investigated this phenomenon using human anaplastic thyroid carcinoma cell lines (SUN-80). The results indicated that EGF triggered the EGFR-mediated intracellular signalling pathway, including signal transducers and activators of transcription 1/3/5 (STAT1/3/5) and protein kinase B (AKT) in a time- and dose-dependent manner. In addition, results from EGF-induced EGFR internalization and co-localization analyses showed that clathrin, Rab5/7, and EEA1 play critical roles in the intracellular trafficking of EGF/EGFR. Interestingly, EGF triggered EGFR translocation into the nucleus, while nuclear-localized EGFR affected cell cycle distribution, thereby significantly promoting the ration of S phase. Overall, these findings indicated that nuclear EGFR exerts biological activity and physiological functions, including changing cell cycle, which in turn promotes proliferation and migration of SUN-80 cells. These findings lay a foundation for further explorations seeking to understand the biological effects of the EGF/EGFR system on the occurrence and development of thyroid cancer.

Inhibiting ERK dimerization ameliorates BRAF-driven anaplastic thyroid cancer

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

A-Kinase Interacting Protein 1 Knockdown Restores Chemosensitivity via Inactivating PI3K/AKT and β-Catenin Pathways in Anaplastic Thyroid Carcinoma.

BackgroundA-kinase interacting protein 1 (AKIP1) promotes tumor progression and chemoresistance in several malignancies; meanwhile, it is related to higher tumor size and recurrence risk of papillary thyroid carcinoma, while the role of AKIP1 in anaplastic thyroid carcinoma (ATC) is unclear. The aim of this study is to explore the effect of AKIP1 knockdown on cell malignant behaviors and doxorubicin resistance in ATC.MethodsAKIP1 knockdown was conducted in ATC cell lines (8505C and CAL-62 cells) by siRNA; then, cell viability, apoptosis, invasion, PI3K/AKT and β-catenin pathways, and doxorubicin sensitivity were detected. Subsequently, doxorubicin-resistant 8505C cells (8505C/Dox) were established. Additionally, AKIP1 was modified in 8505C and 8505C/Dox cells that underwent doxorubicin treatment by siRNA or overexpression plasmid, followed by cellular function and pathway detection.ResultsAKIP1 was elevated in FRO, 8505C, CAL-62, and KHM-5M cells compared to control cells (all p < 0.05). Subsequently, AKIP1 knockdown elevated apoptosis, inhibited viability and invasion, and inactivated PI3K/AKT and β-catenin pathways in 8505C and CAL-62 cells (all p < 0.05). AKIP1 knockdown decreased relative cell viability in doxorubicin-treated 8505C and CAL-62 cells; then, AKIP1 was elevated in 8505C/Dox cells compared to 8505C cells (all p < 0.05). Furthermore, AKIP1 knockdown restored doxorubicin sensitivity (reflected by decreased cell viability and invasion, and increased apoptosis), but inactivated PI3K/AKT and β-catenin pathways in doxorubicin-treated 8505C/Dox cells. However, AKIP1 overexpression presented an opposite effect on these functions and pathways in doxorubicin-treated 8505C cells.ConclusionAKIP1 knockdown decreases cell survival and invasion while promoting sensitivity to doxorubicin via inactivating PI3K/AKT and β-catenin pathways in ATC.

HSA-MnO2-131I Combined Imaging and Treatment of Anaplastic Thyroid Carcinoma.

Purpose Compelling evidence suggests that nanoparticles (NPs) play a crucial role in cancer therapy. NPs templated with human serum albumin (HSA) has good retention in tumors. Manganese dioxide (MnO2) has been used to enhance the effect of radiotherapy. In this study, synthesized NPs using HSA-MnO2 labeled 131I to perform both imaging and therapy for anaplastic thyroid carcinoma (ATC). Method HSA-MnO2 was synthesized via HSA using a simple biomineralization method, and then labeled with Na131I by the chloramine T method. The cytotoxicity and biosafety of HSA-MnO2 were evaluated by the MTT test. The proliferation-inhibiting effect of HSA-MnO2-131I was evaluated in papillary thyroid cancer cell lines (K1, BCPAP, and KTC) and anaplastic thyroid carcinoma cell lines (Cal62, THJ16T, and ARO). For further translational application in medicine, we established a model of transplantable subcutaneously tumors in BALB\\c-nu mice to assess the anti-tumor effect of HSA-MnO2-131I. The imaging effects of NPs were evaluated by MRI and SPECT/CT. Results The MTT test proved that the HSA-MnO2 had low toxicity. HSA-MnO2-131I significantly inhibited the proliferation of PTC and ATC cell lines. In addition, the results unveiled that HSA-MnO2-131I exhibited dual-modality MR/SPECT imaging for thyroid cancer visualization. In particular, HSA-MnO2-131I had an enhanced T1 signal in MR. Using SPECT/CT, we observed that HSA-MnO2-131I had good retention in tumor tissue, which was helpful for the diagnosis and treatment of tumor. In vivo assays indicated that the NPs led to a reduction in radioresistance in the tumor hypoxic microenvironment. Conclusion The nanomaterial had a simple synthesis method, good water solubility and biosafety, and good retention in tumor tissue. Hence, it could be used for SPECT/CT and MR dual mode imaging and therapy with radioiodine of tumor cells. The experimental results provided a feasible solution for combining radiotherapy and dual-model imaging by NPs for cancer diagnosis and treatment.

Lysyl oxidase promotes anaplastic thyroid carcinoma cell proliferation and metastasis mediated via BMP1.

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive solid tumor with no effective treatment at present. Because of the rapid growth and aggressiveness, nearly all patients die within six months after developing ATC. Hence, more research regarding novel therapeutic targets for ATC is urgently needed. Single-cell RNA sequencing data and microarray data of ATC were retrieved from the Gene Expression Omnibus (GEO) database. Cell clustering was performed using the Seurat package. Then, differential expression and functional enrichment analyses were performed. Gene set enrichment analysis (GSEA) was further used to investigate the functional enrichment of lysyl oxidase (LOX) and bone morphogenetic protein-1 (BMP1). The expression levels of LOX and BMP1 were measured using quantitative real-time PCR and Western blot. LOX and BMP1 were knocked down using si-RNAs. Cell proliferation was evaluated by the CCK-8 and clone formation assays. Cell migration and invasion were assessed by the wound healing assay and Transwell assay, respectively. LOX was upregulated at the single-cell level, as well as in ATC tissues and cell lines. LOX knockdown significantly inhibited ATC cell proliferation. Furthermore, the migration and invasion of ATC cells were remarkably inhibited after LOX inhibition. In addition, BMP1 regulated LOX expression in 8505C cells, while BMP1 overexpression restored the LOX activity blocked by the LOX inhibitor BAPN. BMP1 could also induce the cell proliferation and metastasis of ATC. LOX/BMP1 mediates the malignant progression of ATC, highlighting the potential application of LOX/BMP1 in the treatment of ATC. This study provides new insights for efficient therapeutic agents based on the LOX/BMP1 axis.

Alginate-based 3D cell culture technique to evaluate the half-maximal inhibitory concentration: an in vitro model of anticancer drug study for anaplastic thyroid carcinoma

BackgroundThree-dimensional (3D) cell culture methods are identified for simulating the biological microenvironment and demonstrating more similarity to in vivo circumstances. Anaplastic thyroid carcinoma (ATC) is a lethal endocrine malignancy. Despite different treatment approaches, no improvement in the survival rate of the patients has been shown. In this study, we used the 3D in vitro ATC model to investigate the cytotoxic effect of BI-847325 anticancer drug in two-dimensional (2D)- and 3D- cultured cells.MethodsHuman ATC cell lines, C643 and SW1736, were cultured in one percentage (w/v) sodium alginate. Spheroids were incubated in medium for one week. The reproducibility of the fabrication of alginate beads was evaluated. Encapsulation of the cells in alginate was examined by DAPI (4′,6-diamidino-2-phenylindole) staining. Survival of alginate-encapsulated cells was evaluated by CFSE (5,6-Carboxyfluorescein N-hydroxysuccinimidyl ester) staining. The population doubling times of C643 and SW1736 cell lines cultured in 2D monolayer as well as in 3D system were calculated. The cytotoxic effect of BI-847325 on 2D- and 3D- cultured cell lines was assessed for 24–72 h by MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] assay. Finally, the 3D culture results were compared with the 2D culture method.ResultsThe half-maximal inhibitory concentration (IC50) values of BI-847325 were higher in 3D culture compared to 2D culture. The cytotoxicity data indicated that 3D in vitro models were more resistant to chemotherapy agents.ConclusionsThe findings of this study are beneficial for developing in vitro ATC 3D models to analyze the efficacy of different chemotherapy drugs and formulations.

Abstract P120: Novel in vitro targeted combination therapies for anaplastic thyroid cancer

Abstract Purpose: To identify effective drug combination strategies targeting multiple oncogenic pathways in anaplastic thyroid cancer. Background: Treatments with BRAF and MEK inhibitors induce impressive tumor size reduction in most BRAF-mutated anaplastic thyroid carcinoma (ATC) patients. However, these drugs are ultimately ineffective due to resistance development, probably because ATC cells express multiple oncogenic pathways that can be used to bypass the treatments. Therefore, the identification of novel therapeutic strategies is still urgently needed. We had previously identified additional pathways/targets (TWIST1, MMP9, BCL2, TGFβR, and the stress kinase MAPK14/p38α) by reverse-phase protein arrays (RPPA) and microarray analysis. In this study, we report the in vitro effects of inhibitors specific for the identified targets used alone or in combination with BRAF inhibitors. Methods: We established mouse ATC cell lines (MCH2.2-Luc and PPA6-Luc) to graft into immunocompetent syngeneic animals. As a first step, these lines were treated in vitro for 72 hours with increasing concentrations of nine different inhibitors for the above targets, and the MTS colorimetric assay determined viability/IC50. We also tested the combinatorial effects of these compounds with BRAF inhibitors (vemurafenib or dabrafenib) and compared them to single agents’ effects. Also, we investigated the colony-forming ability and apoptotic response of these cell lines. Results: IC50 concentrations for nine different compounds were calculated. Both cell lines showed similar IC50 values, except that PPA6-Luc was more resistant to the BRAF inhibitor dabrafenib than MCH2.2-Luc. The MMP-9 inhibitor and the PARP inhibitor olaparib did not significantly affect the growth of these lines. Among three different MAPK14/p38α inhibitors tested, ralimetinib was the most effective (IC50 @ 15 mM). Importantly, venetoclax (BCL2 inhibitor) and harmine (TWIST1 inhibitor) effectively inhibited cell growth with an IC50 of 8 µM and 25 mM, respectively. Treatments using 0.1mM dabrafenib or 1mM vemurafenib in combination with 4 other drugs (5 mM each below the IC50) resulted in significant synergy only with ralimetinib (38% growth decrease) and harmine (37% growth decrease) compared to treatments with single agents. Colony-forming assays confirmed the growth suppression. Conclusions: Inhibiting specific targets previously identified by RPPA and microarray analysis demonstrated, for the first time, in differential ATC cell growth suppression. Notably, only a combination of specific inhibitors (dabrafenib combined with the TWIST1 or the MAPK14/p38α inhibitors) resulted in synergy, suggesting that these targeted combinations will be more effective in our preclinical ATC mouse model than the other drugs. By treating these combinations in a mouse model may contribute, in the future, to the development of clinical and therapeutic strategies for aggressive thyroid cancer. Citation Format: Muthusamy Kunnimalaiyaan, Parag Parekh, Jalyn Golden, Ying Anderson, Stephen Lai, Maria Cabanillas, Marc Zafereo, Ramona Dadu, Marie-Claude Hofmann. Novel in vitro targeted combination therapies for anaplastic thyroid cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P120.