Circular RNA circLIFR suppresses papillary thyroid cancer progression by modulating the miR-429/TIMP2 axis.

PURPOSE: Circular RNAs (circRNAs) are increasingly recognized for their important roles in various cancers, including papillary thyroid cancer (PTC). The specific mechanisms by which the circLIF receptor subunit alpha (circLIFR, hsa_circ_0072309) influences PTC progression remain largely unknown. METHODS: In our study, CircLIFR, miR-429, and TIMP2 levels were assessed using reverse transcription-quantitative PCR. The roles of circLIFR and miR-429 in PTC cells were determined using Cell Counting Kit-8, colony formation, wound healing, and Transwell assays. Western blotting was utilized to examine the levels of TIMP2. The direct interaction between circLIFR, TIMP2, and miR-429 was confirmed using dual-luciferase reporter, RNA immunoprecipitation, and fluorescence in situ hybridization assays. RESULTS: In PTC tissues and cells, a decrease in circLIFR and TIMP2 levels, accompanied by an increase in miR-429 levels, was observed. Overexpression of circLIFR or downregulation of miR-429 effectively suppressed the proliferation and migration of PTC cells. Conversely, the knockdown of circLIFR or overexpression of miR-429 had the opposite effect. Furthermore, circLIFR overexpression suppressed tumor growth in vivo. Mechanistically, circLIFR modulated TIMP2 expression by serving as a sponge for miR-429. Rescue experiments indicated that the antitumor effect of circLIFR could be reversed by miR-429. CONCLUSION: This study confirmed circLIFR as a novel tumor suppressor delayed PTC progression through the miR-429/TIMP2 axis. These findings suggested that circLIFR held promise as a potential therapeutic target for PTC.

B7-H3 and ICAM-1 are potentially therapeutic targets for thyroid carcinoma.

Although most differentiated thyroid carcinoma has a clinically favorable prognosis, some of specific types of thyroid cancer (such as anaplastic thyroid carcinoma and advanced papillary thyroid carcinoma) show fatal outcomes and require novel treatments. Immunotherapy is a promising avenue for the treatment of advanced thyroid carcinoma. B7-H3 (B7 homolog 3 protein) and ICAM-1 (intercellular adhesion molecule 1), as two important immune checkpoints (ICPs), is becoming hopeful target spots for immunotherapy. A growing amount of evidence has suggested that B7-H3 and ICAM-1 are upregulated in papillary thyroid carcinoma. However, their expression level in specific types of thyroid cancer remains largely unclear. In the present study, we explored the expression level of B7-H3 and ICAM-1 in different types of thyroid carcinoma. In the groups of the TCGA cohort, both B7-H3 and ICAM-1 mRNA were highly expressed in thyroid carcinoma. Furthermore, the patients with Stage2, 61-80y, Follicular thyroid papillary carcinoma and N0 had lower B7-H3 and ICAM-1 mRNA expression. In the groups of our cohort, PTCs and ATCs showed frequently moderate to strong expression of B7-H3 and ICAM-1 protein expression. The significant relevance of B7-H3 staining score with ICAM-1 staining score was observed in TCGA database and our cohort, which might open avenues for the combination therapy in advanced thyroid cancer.

[Bioinformatic analysis of CCND2 expression in papillary thyroid carcinoma and its impact on immune infiltration].

OBJECTIVE: To investigate cyclin D2 (CCND2) expression in papillary thyroid carcinoma (PTC) and its association with the clinicopathological features. METHODS: The public databases TCGA, TIMER 2.0 and UALCAN were used to explore CCND2 expression level in PTC and adjacent tissues, and its diagnostic value for PTC was analyzed using ROC curves. GO enrichment analysis of CCND2-related differentially expressed genes (DEGs) in PTC was performed, and tumor immune infiltration of CCND2 in thyroid cancer was analyzed using TIMER database and CIBERSORT data source. RT-qPCR and Western blot were used to detect CCND2 expression in normal human thyroid cell line Nthy-ori-3-1 and human PTC cell lines TPC-1 and BCPAP. CCND2 expression was also detected in clinical specimens of PTC and adjacent tissues by immunohistochemistry, and its correlation with clinicopathological features of the patients were analyzed. RESULTS: Informatic analysis revealed significantly higher CCND2 mRNA expression in thyroid cancer than in the adjacent tissues (P < 0.001) in close correlation with tumor stage, gender, age, pathological subtype, and lymph node involvement (P < 0.05). ROC curve analysis showed that at the cutoff value of 4.983, the diagnostic sensitivity, specificity, and accuracy of CCND2 expression for PTC was 83.6%, 94.9%, and 78.5%, respectively. CCND2 expression was positively correlated with B cells, CD4+ T cells, and macrophages (P < 0.001) and negatively with CD8+ T cells (P < 0.01), and also correlated with memory B-cell infiltration, CD4+ T-cell memory activation, M2 macrophages, resting mast cells, and mast cell activation (P < 0.05). RT-qPCR, Western blot and immunohistochemistry showed significantly higher CCND2 expression in the PTC cells than in Nthy-ori-3-1 cells (P < 0.01) and also in clinical PTC tissues than in the adjacent tissues (P < 0.05) in correlation with tumor size, lymph node metastasis and TNM stage (P < 0.05). CONCLUSION: CCND2 overexpression is closely correlated with tumor progression and immune cell infiltration in PTC patients..

Pathomics and single-cell analysis of papillary thyroid carcinoma reveal the pro-metastatic influence of cancer-associated fibroblasts.

BACKGROUND: Papillary thyroid carcinoma (PTC) is globally prevalent and associated with an increased risk of lymph node metastasis (LNM). The role of cancer-associated fibroblasts (CAFs) in PTC remains unclear. METHODS: We collected postoperative pathological hematoxylin-eosin (HE) slides from 984 included patients with PTC to analyze the density of CAF infiltration at the invasive front of the tumor using QuPath software. The relationship between CAF density and LNM was assessed. Single-cell RNA sequencing (scRNA-seq) data from GSE193581 and GSE184362 datasets were integrated to analyze CAF infiltration in PTC. A comprehensive suite of in vitro experiments, encompassing EdU labeling, wound scratch assays, Transwell assays, and flow cytometry, were conducted to elucidate the regulatory role of CD36+CAF in two PTC cell lines, TPC1 and K1. RESULTS: A significant correlation was observed between high fibrosis density at the invasive front of the tumor and LNM. Analysis of scRNA-seq data revealed metastasis-associated myoCAFs with robust intercellular interactions. A diagnostic model based on metastasis-associated myoCAF genes was established and refined through deep learning methods. CD36 positive expression in CAFs can significantly promote the proliferation, migration, and invasion abilities of PTC cells, while inhibiting the apoptosis of PTC cells. CONCLUSION: This study addresses the significant issue of LNM risk in PTC. Analysis of postoperative HE pathological slides from a substantial patient cohort reveals a notable association between high fibrosis density at the invasive front of the tumor and LNM. Integration of scRNA-seq data comprehensively analyzes CAF infiltration in PTC, identifying metastasis-associated myoCAFs with strong intercellular interactions. In vitro experimental results indicate that CD36 positive expression in CAFs plays a promoting role in the progression of PTC. Overall, these findings provide crucial insights into the function of CAF subset in PTC metastasis.

Therapeutic targeting of TNIK in papillary thyroid carcinoma: a novel approach for tumor growth suppression.

Papillary thyroid carcinoma (PTC) is a common endocrine malignancy. The pathology of PTC is far from clear. As a kinase that can be targeted, the role of TNIK in PTC has not been investigated. This study was focused on the effects and molecular mechanisms of TNIK in PTC. Both public datasets and clinical specimens were used to verify TNIK expression. The effects of TNIK were investigated in both cell lines and mice models. Transcriptome analysis was used to explore the underlying mechanism of TNIK. Immunofluorescence, wound healing, and qRT-PCR assays were used to validate the mechanism of TNIK in PTC. The therapeutic effects of TNIK inhibitor NCB-0846 were evaluated by flow cytometry, western blot, and subcutaneous xenografts mice. TNIK expression was upregulated in PTC tissues. TNIK knockdown could suppress cell proliferation and tumor growth in no matter cell models or nude mice. The transcriptome analysis, GO enrichment analysis, and GSEA analysis results indicated TNIK was highly correlated with cytoskeleton, cell motility, and Wnt pathways. The mechanistic studies demonstrated that TNIK regulated cytoskeleton remodeling and promoted cell migration. NCB-0846 significantly inhibited TNIK kinase activity, induced cell apoptosis, and activated apoptosis-related proteins in a dose-dependent manner. In addition, NCB-0846 inhibited tumor growth in tumor-bearing mice. In summary, we proposed a novel regulatory mechanism in which TNIK-mediated cytoskeleton remodeling and cell migration to regulate tumor progression in PTC. TNIK is a therapeutic target in PTC and NCB-0846 would act as a novel targeted drug for PTC therapy.

CLIP170 inhibits the metastasis and EMT of papillary thyroid cancer through the TGF-ß pathway.

Metastasis poses a significant challenge in combating tumors. Even in papillary thyroid cancer (PTC), which typically exhibits a favorable prognosis, high recurrence rates are attributed to metastasis. Cytoplasmic linker protein 170 (CLIP170) functions as a classical microtubule plus-end tracking protein (+TIP) and has shown close association with cell migration. Nevertheless, the specific impact of CLIP170 on PTC cells remains to be elucidated. Our analysis of the GEO and TCGA databases unveiled an association between CLIP170 and the progression of PTC. To explore the impact of CLIP170 on PTC cells, we conducted various assays. We evaluated its effects through CCK-8, wound healing assay, and transwell assay after knocking down CLIP170. Additionally, the influence of CLIP170 on the cellular actin structure was examined via immunofluorescence; we further investigated the molecular expressions of epithelial-mesenchymal transition (EMT) and the transforming growth factor-ß (TGF-ß) signaling pathways through Western blotting and RT-qPCR. These findings were substantiated through an in vivo nude mouse model of lung metastasis. We observed a decreased expression of CLIP170 in PTC in contrast to normal thyroid tissue. Functionally, the knockdown of CLIP170 (CLIP170KD) notably enhanced the metastatic potential and EMT of PTC cells, both in vitro and in vivo. Mechanistically, CLIP170KD triggered the activation of the TGF-ß pathway, subsequently promoting tumor cell migration, invasion, and EMT. Remarkably, the TGF-ß inhibitor LY2157299 effectively countered TGF-ß activity and significantly reversed tumor metastasis and EMT induced by CLIP170 knockdown. In summary, these findings collectively propose CLIP170 as a promising therapeutic target to mitigate metastatic tendencies in PTC.

Homogentisic acid metabolism inhibits papillary thyroid carcinoma proliferation through ROS and p21-induced cell cycle arrest.

Thyroid cancer is one of the most common primary endocrine malignancies worldwide, and papillary thyroid carcinoma (PTC) is the predominant histological type observed therein. Although PTC has been studied extensively, our understanding of the altered metabolism and metabolic profile of PTC tumors is limited. We identified that the content of metabolite homogentisic acid (HGA) in PTC tissues was lower than that in adjacent non-cancerous tissues. We evaluated the potential of HGA as a novel molecular marker in the diagnosis of PTC tumors, as well as its ability to indicate the degree of malignancy. Studies have further shown that HGA contributes to reactive oxygen species (ROS) associated oxidative stress, leading to toxicity and inhibition of proliferation. In addition, HGA caused an increase in p21 expression levels in PTC cells and induced G1 arrest. Moreover, we found that the low HGA content in PTC tumors was due to the low expression levels of tyrosine aminotransferase (TAT) and p-hydroxyphenylpyruvate hydroxylase (HPD), which catalyze the conversion of tyrosine to HGA. The low expression levels of TAT and HPD are strongly associated with a higher probability of PTC tumor invasion and metastasis. Our study demonstrates that HGA could be used to diagnose PTC and provides mechanisms linking altered HGA levels to the biological behavior of PTC tumors.

Identifying potential breath biomarkers for early diagnosis of papillary thyroid cancer based on solid-phase microextraction gas chromatography-high resolution mass spectrometry with metabolomics.

INTRODUCTION: Thyroid cancer incidence rate has increased substantially worldwide in recent years. Fine needle aspiration biopsy (FNAB) is currently the golden standard of thyroid cancer diagnosis, which however, is invasive and costly. In contrast, breath analysis is a non-invasive, safe and simple sampling method combined with a promising metabolomics approach, which is suitable for early cancer diagnosis in high volume population. OBJECTIVES: This study aims to achieve a more comprehensive and definitive exhaled breath metabolism profile in papillary thyroid cancer patients (PTCs). METHODS: We studied both end-tidal and mixed expiratory breath, solid-phase microextraction gas chromatography coupled with high resolution mass spectrometry (SPME-GC-HRMS) was used to analyze the breath samples. Multivariate combined univariate analysis was applied to identify potential breath biomarkers. RESULTS: The biomarkers identified in end-tidal and mixed expiratory breath mainly included alkanes, olefins, enols, enones, esters, aromatic compounds, and fluorine and chlorine containing organic compounds. The area under the curve (AUC) values of combined biomarkers were 0.974 (sensitivity: 96.1%, specificity: 90.2%) and 0.909 (sensitivity: 98.0%, specificity: 74.5%), respectively, for the end-tidal and mixed expiratory breath, indicating of reliability of the sampling and analysis method CONCLUSION: This work not only successfully established a standard metabolomic approach for early diagnosis of PTC, but also revealed the necessity of using both the two breath types for comprehensive analysis of the biomarkers.

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.

Targeting miR-31 represses tumourigenesis and dedifferentiation of BRAFV600E-associated thyroid carcinoma.

BACKGROUND: BRAFV600E is the most common genetic mutation in differentiated thyroid cancer (DTC) occurring in 60% of patients and drives malignant tumour cell phenotypes including proliferation, metastasis and immune-escape. BRAFV600E-mutated papillary thyroid cancer (PTC) also displays greatly reduced expression of thyroid differentiation markers, thus tendency to radioactive iodine (RAI) refractory and poor prognosis. Therefore, understanding the molecular mechanisms and main oncogenic events underlying BRAFV600E will guide future therapy development. METHODS: Bioinformatics and clinical specimen analyses, genetic manipulation of BRAFV600E-induced PTC model, functional and mechanism exploration guided with transcriptomic screening, as well as systematic rescue experiments were applied to investigate miR-31 function within BRAFV600E-induced thyroid cancer development. Besides, nanoparticles carrying miR-31 antagomirs were testified to alleviate 131I iodide therapy on PTC models. RESULTS: We identify miR-31 as a significantly increased onco-miR in BRAFV600E-associated PTC that promotes tumour progression, metastasis and RAI refractoriness via sustained Wnt/ß-catenin signalling. Mechanistically, highly activated BRAF/MAPK pathway induces miR-31 expression via c-Jun-mediated transcriptional regulation across in vitro and transgenic mouse models. MiR-31 in turn facilitates ß-catenin stabilisation via directly repressing tumour suppressors CEBPA and DACH1, which direct the expression of multiple essential Wnt/ß-catenin pathway inhibitors. Genetic functional assays showed that thyroid-specific knockout of miR-31 inhibited BRAFV600E-induced PTC progression, and strikingly, enhanced expression of sodium-iodide symporter and other thyroid differentiation markers, thus promoted 131I uptake. Nanoparticle-mediated application of anti-miR-31 antagomirs markedly elevated radio-sensitivity of BRAFV600E-induced PTC tumours to 131I therapy, and efficiently suppressed tumour progression in the pre-clinical mouse model. CONCLUSIONS: Our findings elucidate a novel BRAF/MAPK-miR-31-Wnt/ß-catenin regulatory mechanism underlying clinically BRAFV600E-associated DTC tumourigenesis and dedifferentiation, also highlight a potential adjuvant therapeutic strategy for advanced DTC.

Transcriptomic characteristics according to tumor size and SUVmax in papillary thyroid cancer patients.

The SUVmax is a measure of FDG uptake and is related with tumor aggressiveness in thyroid cancer, however, its association with molecular pathways is unclear. Here, we investigated the relationship between SUVmax and gene expression profiles in 80 papillary thyroid cancer (PTC) patients. We conducted an analysis of DEGs and enriched pathways in relation to SUVmax and tumor size. SUVmax showed a positive correlation with tumor size and correlated with glucose metabolic process. The genes that indicate thyroid differentiation, such as SLC5A5 and TPO, were negatively correlated with SUVmax. Unsupervised analysis revealed that SUVmax positively correlated with DNA replication(r = 0.29, p = 0.009), pyrimidine metabolism(r = 0.50, p < 0.0001) and purine metabolism (r = 0.42, p = 0.0001). Based on subgroups analysis, we identified that PSG5, TFF3, SOX2, SL5A5, SLC5A7, HOXD10, FER1L6, and IFNA1 genes were found to be significantly associated with tumor aggressiveness. Both high SUVmax PTMC and macro-PTC are enriched in pathways of DNA replication and cell cycle, however, gene sets for purine metabolic pathways are enriched only in high SUVmax macro-PTC but not in high SUVmax PTMC. Our findings demonstrate the molecular characteristics of high SUVmax tumor and metabolism involved in tumor growth in differentiated thyroid cancer.

Extra-nuclear TERT counteracts oxidative stress and promotes progression in papillary thyroid carcinoma.

The reactivation of TERT is associated with poor outcome in papillary thyroid cancer (PTC). Extra-telomeric functions of TERT were reported, with a protective role against oxidative stress (OS). The aim of the present study was to explore the extra-nuclear TERT localization in PTC and its role in cancer progression. TERT nuclear export under OS were analyzed in K1 PTC cell line. We investigated the role of different TERT localizations using specific TERT constructs that limit its localization to the nucleus or to the mitochondria. The effect of SRC kinase inhibitor PP2, which reduces TERT nuclear export, was investigated as well. Moreover, TERT localization was analyzed in 39 PTC tissues and correlated with the genetic profile and the level of OS, DNA damage and apoptosis in the tumors and with the clinical characteristics of the patients. We demonstrated that TERT is exported from the nucleus in response to OS induced either from H2O2 or the BRAF inhibitor PLX4720. We proved that extra-nuclear TERT reduces mitochondrial OS and induces mitochondrial fragmentation. Moreover, limiting mitochondrial TERT localization reduced proliferation, migration, AKT phosphorylation and glycolysis and increased DNA damage and p21 expression. Finally, in PTC tissues the fraction of mitochondrial/nuclear TERT resulted inversely correlated with OS and p21 expression and associated with tumor persistence. In conclusion, our data indicate that extra-nuclear TERT is involved in reducing the effect of excessive OS, thus promoting cancer cell survival. Extra-nuclear TERT may thus represent a marker of cancer progression and a possible therapeutic target in PTC.

Case report: A rare combination of aldosterone-secreting adrenocortical carcinoma and papillary thyroid carcinoma with Graves' disease.

Adrenocortical carcinoma (ACC) is a rare malignancy originating in the adrenal glands, aldosterone-producing ACC, even rarer. Papillary thyroid carcinoma (PTC), by contrast, accounts for the majority of thyroid carcinomas. We herein describe the first reported case of a female with comorbidities of aldosterone-producing ACC, PTC, and Graves' Disease(GD). The patient achieved transient clinical remission following adrenalectomy. However, three months later, aldosterone-producing ACC lung metastases emerged. Subsequently, within another three-month interval, she developed thyroid eye disease(TED). The patient died roughly one year after the adrenal operation. Exome sequencing did not reveal associations between aldosterone-producing ACC, PTC, and GD, and the underlying concurrence mechanism has yet to be elucidated. Further research of similar cases are needed to confirm potential links between the three pathologies.

Naive and regulatory B-cell transcription patterns guide the increased risk of papillary thyroid carcinoma in obesity.

A growing number of studies suggest a positive association between obesity and the high incidence of papillary thyroid cancer (PTC), suggesting that the abnormal levels of adipokines associated with obesity may be a risk factor for these aggressive thyroid cancers, but the underlying regulatory mechanisms are not yet clear. We downloaded bulk RNA sequence data for subcutaneous adipose tissue (SAT) in obesity and healthy population and tumor tissues of PTC from GEO database. Through analysis of Differential Expression Genes (DEGs), Gene Set Variation Analysis (GSVA) and Weighted Correlation Network Analysis (WGCNA), we identified co-expressed genes between obesity and PTC, and their pathways were mainly enriched in the regulation of B-cells. Furthermore, through TCGA-THCA (thyroid carcinoma) cohorts analysis, we identified B-cell regulatory-related genes LEF1, TNFRSF13C, SHLD2 and SHLD3 as independent prognostic markers of PTC. Next, we explored the transcriptional regulation mechanism of the increased risk of PTC in obesity through analysis of DNA methylation CpGs data and single-cell RNA sequences (scRNA-seq) from GEO database. PTC-induced hypomethylation of the promoter region may be involved in the transcriptional regulation of these genes, while these genes were further identified in naive and regulatory B-cells of both diseases. Notably, both of the gene expressions in naive and regulatory B-cells showed high similarity in both diseases. Our data reveals the high frequency of PTC in obese populations may be explained by the comparable transcriptional patterns of naive and regulatory B-cells, and offers novel insights for the analysis of critical genes and underlying biological mechanisms for obesity and PTC.

CircPHGDH downregulation decreases papillary thyroid cancer progression through miR-122-5p/PKM2 axis.

BACKGROUND: Although papillary thyroid carcinoma (PTC) has a favorable prognosis, it could affect patient life quality and become a serious threat because of invasion and metastasis. Many investigations have suggested that circular RNAs (circRNAs) are involved in different cancer regulations. Nevertheless, circRNAs role in invasive PTC remains unclear. METHODS: In the present investigation, next-generation sequencing was applied to explore abnormal circRNA expression. The expression of circRNA phosphoglycerate dehydrogenase (circPHGDH) in PTC cell lines and tissues were examined. Then, we investigated regulatory mechanism and circPHGDH downstream targets using bioinformatics analysis and luciferase reporting analysis. Then transwell migration, Cell Counting Kit-8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used for cells migration and proliferation analysis. In vivo metastasis and tumorigenesis assays were also employed to evaluate the circPHGDH role in PTC. RESULTS: The data showcased that circPHGDH expression increased in both PTC cell lines and tissues, which suggested that circPHGDH functions in PTC progression. circPHGDH downregulation suppressed PTC invasion and proliferation in both in vivo and in vitro experiments. Bioinformatics and luciferase reporter results confirmed that both microRNA (miR)-122-5p and pyruvate kinase M2 subtype (PKM2) were downstream targets of circPHGDH. PKM2 overexpression or miR-122-5p suppression reversed PTC cell invasion and proliferation post silencing circPHGDH by restoring aerobic glycolysis. CONCLUSION: Taken together, our research found that circPHGDH downregulation reduced PTC progression via miR-122-5p/PKM2 axis regulation mediated by aerobic glycolysis.

Combined BRAF and PIM1 inhibitory therapy for papillary thyroid carcinoma based on BRAFV600E regulation of PIM1: Synergistic effect and metabolic mechanisms.

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, and its incidence has increased rapidly in recent years. The BRAF inhibitor vemurafenib is effective against BRAFV600E-positive PTC; however, acquired resistance to single agent therapy frequently leads to tumor recurrence and metastasis, underscoring the need to develop tailored treatment strategies. We previously showed that the oncogenic kinase PIM1 was associated with the malignant phenotype and prognosis of PTC. In this study, we showed that sustained expression of the PIM1 protein in PTC was affected by the BRAFV600E mutation. Based on this regulatory mechanism, we tested the synergistic effects of inhibitors of BRAF (BRAFi) and PIM1 in BRAFV600E-positive PTC cell lines and xenograft tumors. LC-MS metabolomics analyses suggested that BRAFi/PIMi therapy acted by restricting the amounts of critical amino acids and nucleotides required by cancer cells as well as modulating DNA methylation. This study elucidates the role of BRAFV600E in the regulation of PIM1 in PTC and demonstrates the synergistic effect of a novel combination, BRAFi/PIMi, for the treatment of PTC. This discovery, along with the pathways that may be involved in the powerful efficacy of BRAFi/PIMi strategy from the perspective of cell metabolism, provides insight into the molecular basis of PTC progression and offers new perspectives for BRAF-resistant PTC treatment.

VDR is a potential prognostic biomarker and positively correlated with immune infiltration: a comprehensive pan-cancer analysis with experimental verification.

The vitamin D receptor (VDR) is a transcription factor that mediates a variety of biological functions of 1,25-dihydroxyvitamin D3. Although there is growing evidence of cytological and animal studies supporting the suppressive role of VDR in cancers, the conclusion is still controversial in human cancers and no systematic pan-cancer analysis of VDR is available. We explored the relationships between VDR expression and prognosis, immune infiltration, tumor microenvironment, or gene set enrichment analysis (GSEA) in 33 types of human cancers based on multiple public databases and R software. Meanwhile, the expression and role of VDR were experimentally validated in papillary thyroid cancer (PTC). VDR expression decreased in 8 types and increased in 12 types of cancer compared with normal tissues. Increased expression of VDR was associated with either good or poor prognosis in 13 cancer types. VDR expression was positively correlated with the infiltration of cancer-associated fibroblasts, macrophages, or neutrophils in 20, 12, and 10 cancer types respectively and this correlation was experimentally validated in PTC. Increased VDR expression was associated with increased percentage of stromal or immune components in tumor microenvironment (TME) in 24 cancer types. VDR positively and negatively correlated genes were enriched in immune cell function and energy metabolism pathways, respectively, in the top 9 highly lethal tumors. Additionally, VDR expression was increased in PTC and inhibited cell proliferation and migration. In conclusion, VDR is a potential prognostic biomarker and positively correlated with immune infiltration as well as stromal or immune components in TME in multiple human cancers.

Evaluation of the follicular patterned thyroid lesions based on the WHO 2022 criteria with an emphasis on the grey-zone lesions.

Follicular-patterned thyroid nodules (FPTN) are classified byWHO-2022 into benign, borderline and malignant categories. There are however, grey-zone lesions that pose a diagnostic challenge due to ambiguity in defining criteria and inter-observer variability. WHO-2022 has enumerated specific diagnostic criteria for these lesions. Accurate categorization of morphologically similar TNs is vital to reduce overtreatment of indolent lesions. In this study, we have reclassified FPTNs according to WHO-2022 criteria, emphasizing on grey-zone lesions. We studied the utility of immunohistochemistry (IHC)-CD56, HBME-1 and CK19 in distinguishing benign from malignant nodules and BRAFV600E IHC to better distinguish the (widely-invasive) encapsulated follicular variant of papillary thyroid carcinoma (FVPTC) from infiltrative FVPTC. Only those cases with dominant nodule having follicular pattern histology were included and re-evaluated for following histopathological features-focality, encapsulation, circumscription, nuclear PTC features, capsular-invasion, angio-invasion, papillae and necrosis. IHC findings for above-mentioned markers were noted. Seventy-nine cases met the inclusion criteria. Amendment of original diagnosis was done in 19 % cases. BRAFV600E IHC was positive in the two cases of infiltrative FVPTC while it was negative in all nine IE (invasive encapsulated) FVPTCs. Diffuse HBME1 was noted in most malignant nodules (61 %) while CD56 was expressed more often in benign lesions (70 %). CK19 was positive in lesions displaying nuclear PTC features (86 %). Using WHO 2022 criteria, we were able to re-classify follicular thyroid lesions with greater confidence. Appropriate IHC panel in adjunct to histology aids in categorizing challenging cases.

Spatial transcriptomics reveals prognosis-associated cellular heterogeneity in the papillary thyroid carcinoma microenvironment.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common malignant endocrine tumour, and its incidence and prevalence are increasing considerably. Cellular heterogeneity in the tumour microenvironment is important for PTC prognosis. Spatial transcriptomics is a powerful technique for cellular heterogeneity study. METHODS: In conjunction with a clinical pathologist identification method, spatial transcriptomics was employed to characterise the spatial location and RNA profiles of PTC-associated cells within the tissue sections. The spatial RNA-clinical signature genes for each cell type were extracted and applied to outlining the distribution regions of specific cells on the entire section. The cellular heterogeneity of each cell type was further revealed by ContourPlot analysis, monocle analysis, trajectory analysis, ligand-receptor analysis and Gene Ontology enrichment analysis. RESULTS: The spatial distribution region of tumour cells, typical and atypical follicular cells (FCs and AFCs) and immune cells were accurately and comprehensively identified in all five PTC tissue sections. AFCs were identified as a transitional state between FCs and tumour cells, exhibiting a higher resemblance to the latter. Three tumour foci were shared among all patients out of the 13 observed. Notably, tumour foci No. 2 displayed elevated expression levels of genes associated with lower relapse-free survival in PTC patients. We discovered key ligand-receptor interactions, including LAMB3-ITGA2, FN1-ITGA3 and FN1-SDC4, involved in the transition of PTC cells from FCs to AFCs and eventually to tumour cells. High expression of these patterns correlated with reduced relapse-free survival. In the tumour immune microenvironment, reduced interaction between myeloid-derived TGFB1 and TGFBR1 in tumour focus No. 2 contributed to tumourigenesis and increased heterogeneity. The spatial RNA-clinical analysis method developed here revealed prognosis-associated cellular heterogeneity in the PTC microenvironment. CONCLUSIONS: The occurrence of tumour foci No. 2 and three enhanced ligand-receptor interactions in the AFC area/tumour foci reduced the relapse-free survival of PTC patients, potentially leading to improved prognostic strategies and targeted therapies for PTC patients.

CircNRCAM up-regulates NRCAM to promote papillary thyroid carcinoma progression.

PURPOSE: Papillary Thyroid Carcinoma (PTC) is the most prevalent subtype of Thyroid Carcinoma (THCA), a type of malignancy in the endocrine system. According to prior studies, Neural Cell Adhesion Molecule (NRCAM) has been found to be up-regulated in PTC and stimulates the proliferation and migration of PTC cells. However, the specific mechanism of NRCAM in PTC cells is not yet fully understood. Consequently, this study aimed to investigate the underlying mechanism of NRCAM in PTC cells, the findings of which could provide new insights for the development of potential treatment targets for PTC. METHODS AND RESULTS: Bioinformatics tools were utilized and a series of experiments were conducted, including Western blot, colony formation, and dual-luciferase reporter assays. The data collected indicated that NRCAM was overexpressed in THCA tissues and PTC cells. Circular RNA NRCAM (circNRCAM) was found to be highly expressed in PTC cells and to positively regulate NRCAM expression. Through loss-of-function assays, both circNRCAM and NRCAM were shown to promote the proliferation, invasion, and migration of PTC cells. Mechanistically, this study confirmed that precursor microRNA-506 (pre-miR-506) could bind with m6A demethylase AlkB Homolog 5 (ALKBH5), leading to its m6A demethylation. It was also discovered that circNRCAM could competitively bind to ALKBH5, which restrained miR-506-3p expression and promoted NRCAM expression. CONCLUSION: In summary, circNRCAM could up-regulate NRCAM by down-regulating miR-506-3p, thereby enhancing the biological behaviors of PTC cells.