[Clinicopathological features of BAP1 mutated clear cell renal cell carcinoma].

Objective: To investigate the clinicopathological characteristics, immunophenotypes, molecular features, and differential diagnosis of BAP1 mutated clear cell renal cell carcinoma (CCRCC) for better understanding this entity. Methods: Clinical data, histological morphology, immunophenotypes and molecular characteristics of 18 BAP1 mutated CCRCC cases diagnosed at the Department of Pathology, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China from January 2020 to December 2022 were analyzed. The patients were followed up. Results: There were 17 males and 1 female patients, aged from 39 to 72 years, with an average age of 56.3 years. Sixteen patients with primary CCRCC were followed up for an average of 24 months, 7 patients had metastases occurred from 4 to 22 months postoperatively. Thirteen of the 16 patients were alive at the time of the last follow-up while 3 patients died 12, 15, and 20 months after the surgery, respectively. One patient underwent retroperitoneal mass resection, but had lung metastasis 32 months after surgery. One case received cervical tumor resection and died at 22 months after the surgery. Characteristic CCRCC regions were identified in 11 of the 18 cases. The tumor cells were arranged in papillary, alveolar, and large nest patterns. Abundant lymphoid tissue, necrosis, and psammoma bodies were seen. Tumor cells showed abundant eosinophilic cytoplasm, and sometimes exhibited rhabdoid differentiation. Round eosinophilic globules were located in the cytoplasm and extracellular matrix. There were 9 cases with WHO/International Society of Urological Pathology grade 3, and 9 cases with grade 4. PAX8 (18/18), carbonic anhydrase 9 (CA9, 16/18), CD10 (18/18), and vimentin (18/18) were positive in the vast majority of tumors.TFE3 was expressed in 5 cases, with strong expression in only 1 case. Eighteen cases were all positive for P504s. Twelve cases harbored a BAP1 mutation combined with von Hippel-Lindau (VHL) mutation, and 2 cases had mutations in BAP1, VHL and PBRM1 simultaneously. SETD2 mutation was not found in any of the cases. Conclusions: BAP1 mutated CCRCC contained papillary, alveolar, and large nest patterns, eosinophilic cytoplasm, high-grade nucleoli, and collagen globules, with P504s positivity. In practical work, when encountering CCRCC containing these features, pathologists should consider the possibility of BAP1 mutations and conduct related molecular tests.

Single-nuclei sequencing of uterine serous carcinoma reveals racial differences in immune signaling.

Significant racial disparities exist between Black and White patients with uterine serous carcinoma (USC). While the reasons for these disparities are unclear, several studies have demonstrated significantly different rates of driver mutations between racial groups, including TP53. However, limited research has investigated the transcriptional differences of tumors or the composition of the tumor microenvironment (TME) between these groups. Here, we report the single-nuclei RNA-sequencing profiles of primary USC tumors from diverse racial backgrounds. We find that there are significant differences between the tumors of Black and White patients. Tumors from Black patients exhibited higher expression of specific genes associated with aggressiveness, such as PAX8, and axon guidance and synaptic signaling pathways. We also demonstrated that T cell populations are reduced in the tumor tissue compared to matched benign, while anti-inflammatory macrophage populations are retained within the TME. Furthermore, we investigated the connection between PAX8 overexpression and immunosuppression in USC through regulation of several cytokines and chemokines. Notably, we show that PAX8 activity can influence macrophage gene expression and protein secretion. These studies provide a detailed understanding of the USC transcriptome and TME, and identify differences in tumor biology from patients of different racial backgrounds.

METTL3 regulates thyroid cancer differentiation and chemosensitivity by modulating PAX8.

Background: Thyroid cancer (TC) is a common endocrine cancer with a favourable prognosis. However, poor patient prognosis due to TC dedifferentiation is becoming an urgent challenge. Recently, methyltransferase-like 3 (METTL3)-mediated N6 -methyladenosine (m6A) modification has been demonstrated to play an important role in the occurrence and progression of various cancers and a tumour suppressor role in TC. However, the mechanism of METTL3 in TC remains unclear. Methods: The correlation between METTL3 and prognosis in TC patients was evaluated by immunohistochemistry. Mettl3fl/flBrafV600ETPO-cre TC mouse models and RNA-seq were used to investigate the underlying molecular mechanism, which was further validated by in vitro experiments. The target gene of METTL3 was identified, and the complete m6A modification process was described. The phenomenon of low expression of METTL3 in TC was explained by identifying miRNAs that regulate METTL3. Results: We observed that METTL3 expression was negatively associated with tumour progression and poor prognosis in TC. Mechanistically, silencing METTL3 promoted the progression and dedifferentiation of papillary thyroid carcinoma (PTC) both in vivo and in vitro. Moreover, overexpressing METTL3 promoted the sensitivity of PTC and anaplastic thyroid cancer (ATC) cells to chemotherapeutic drugs and iodine-131 (131I) administration. Overall, the METTL3/PAX8/YTHDC1 axis has been revealed to play a pivotal role in repressing tumour occurrence, and is antagonized by miR-493-5p.

Premature Classification of Early-stage Endometrioid Ovarian Carcinoma With Mesonephric-like Differentiation as Mesonephric-like Adenocarcinoma.

Ovarian mesonephric-like adenocarcinoma (MLA) is a rare tumor with potential origins in endometriosis and Müllerian-type epithelial tumors. The morphologic patterns of MLA overlap with those of endometrioid ovarian carcinoma (EnOC). We speculated that a subset of MLAs would be classified as EnOCs. In this study, we attempted to identify MLAs from malignant endometrioid tumors. Given that the study patients with MLAs had both endometrioid-like and mesonephric-like morphologies, we defined mesonephric-like differentiation (MLD) as an endometrioid tumor with focal or diffuse MLA morphology and immunophenotype. Twelve patients exhibited mesonephric-like morphologic patterns. Immunohistochemistry analysis for CD10, TTF-1, estrogen receptor (ER), GATA3, calretinin, and PAX8 expression was done using whole-section slides. Two patients without the MLA immunophenotype were excluded. Ten patients with EnOCs with MLD (8.3%) were identified from a cohort of 121 patients with malignant endometrioid tumors. All 10 patients were positive for TTF-1 and/or GATA3. Most patients were ER-negative. Morphologically, MLD was associated with papillary thyroid carcinoma-like nuclei, flattened cells, tubular, nested, reticular, or glomeruloid architecture, and infiltrative growth. All 10 patients had pre-existing endometriosis and/or adenofibromas. Among the EnOCs with MLD, 5 had coexisting components such as EnOC grade 1 [(G1), cases 4, 7, and 9], mucinous borderline tumor (case 1), and dedifferentiated carcinoma (case 10), with distinct borders between EnOC with MLD and the other components. Nine of the 10 MLA patients (90%) harbored KRAS hotspot mutations. In addition, 4 patients harboring other components shared common KRAS hotspot mutations. No significant prognostic differences were observed between patients with and without MLD. Based on our findings, we suggest that EnOC with MLD, especially in the early stages and without high-grade components, should be considered a subtype of EnOC. Overtreatment should be avoided in such patients, particularly in the early stages. In this study, as the characteristics between EnOC with MLD and MLA were not distinguishable, we considered both conditions to be on the same spectrum. EnOCs with MLD exhibit the MLA phenotype during disease progression and are prematurely classified as MLA. Nevertheless, more patients with EnOC who have MLD/MLA are required for a more robust comparison between conventional EnOC according to staging and grading.

The Role of the PAX Genes in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is a significant oncological challenge due to its heterogeneous nature and limited treatment options. The PAX developmental gene family encodes nine highly conserved transcription factors that play crucial roles in embryonic development and organogenesis, which have been implicated in the occurrence and development of RCC. This review explores the molecular landscape of RCC, with a specific focus on the role of the PAX gene family in RCC tumorigenesis and disease progression. Of the various RCC subtypes, clear cell renal cell carcinoma (ccRCC) is the most prevalent, characterized by the loss of the von Hippel-Lindau (VHL) tumor suppressor gene. Here, we review the published literature on the expression patterns and functional implications of PAX genes, particularly PAX2 and PAX8, in the three most common RCC subtypes, including ccRCC, papillary RCC (PRCC), and chromophobe RCC (ChRCC). Further, we review the interactions and potential biological mechanisms involving PAX genes and VHL loss in driving the pathogenesis of RCC, including the key signaling pathways mediated by VHL in ccRCC and associated mechanisms implicating PAX. Lastly, concurrent with our update regarding PAX gene research in RCC, we review and comment on the targeting of PAX towards the development of novel RCC therapies.

In Silico Search for Drug Candidates Targeting the PAX8-PPARγ Fusion Protein in Thyroid Cancer.

The PAX8/PPARγ rearrangement, producing the PAX8-PPARγ fusion protein (PPFP), is thought to play an essential role in the oncogenesis of thyroid follicular tumors. To identify PPFP-targeted drug candidates and establish an early standard of care for thyroid tumors, we performed ensemble-docking-based compound screening. Specifically, we investigated the pocket structure that should be adopted to search for a promising ligand compound for the PPFP; the position of the ligand-binding pocket on the PPARγ side of the PPFP is similar to that of PPARγ; however, the shape is slightly different between them due to environmental factors. We developed a method for selecting a PPFP structure with a relevant pocket and high prediction accuracy for ligand binding. This method was validated using PPARγ, whose structure and activity values are known for many compounds. Then, we performed docking calculations to the PPFP for 97 drug or drug-like compounds registered in the DrugBank database with a thiazolidine backbone, which is one of the characteristics of ligands that bind well to PPARγ. Furthermore, the binding affinities of promising ligand candidates were estimated more reliably using the molecular mechanics Poisson-Boltzmann surface area method. Thus, we propose promising drug candidates for the PPFP with a thiazolidine backbone.

Diagnostic Value of Immunostaining for Thyroid Transcription Factor 1 (TTF1) and Paired Box 8 (PAX8) in Distinguishing Pulmonary Metastases of Mesonephric and Mesonephric-like Adenocarcinomas from Primary Lung Adenocarcinomas.

BACKGROUND/AIM: Both mesonephric adenocarcinoma (MA) and mesonephric-like adenocarcinoma (MLA) express thyroid transcription factor 1 (TTF1). TTF1 is also considered a highly sensitive and specific diagnostic marker for primary lung adenocarcinoma (PLA). However, distinguishing PLA from pulmonary metastatic MA/MLA (PMM) based on the expression of TTF1 alone can be difficult. This study aimed to investigate the expression of TTF1 and paired box 8 (PAX8) and assess their value in distinguishing PMM from PLA. PATIENTS AND METHODS: We reviewed the electronic medical records and pathology slides of eight PMM cases. We conducted immunostaining for TTF1 and PAX8 in 6, 8, and 21 cases of primary MA/MLA, PMM, and PLA, respectively. RESULTS: Two patients with stage IB uterine MLA developed lung metastases at 5 and 57 months after hysterectomy. Solitary pulmonary nodules were suspected to be primary lung cancer in two patients. Compared to primary tumors, all matched PMMs exhibited reduced TTF1 immunoreactivity. In contrast, the majority of PLAs showed uniform and intense TTF1 expression. All except one PMM exhibited diffuse and strong PAX8 expression, while only one PLA showed focal and weak PAX8 expression. CONCLUSION: Immunostaining for TTF1 and PAX8 can help in distinguishing PMM from PLA in the diagnosis of pulmonary lesions detected in patients with a history of MA/MLA.

Pax protein depletion in proximal tubules triggers conserved mechanisms of resistance to acute ischemic kidney injury preventing transition to chronic kidney disease.

Acute kidney injury (AKI) is a common condition that lacks effective treatments. In part, this shortcoming is due to an incomplete understanding of the genetic mechanisms that control pathogenesis and recovery. Identifying the molecular and genetic regulators unique to nephron segments that dictate vulnerability to injury and regenerative potential could lead to new therapeutic targets to treat ischemic kidney injury. Pax2 and Pax8 are homologous transcription factors with overlapping functions that are critical for kidney development and are re-activated in AKI. Here, we examined the role of Pax2 and Pax8 in recovery from ischemic AKI and found them upregulated after severe AKI and correlated with chronic injury. Surprisingly, proximal-tubule-selective deletion of Pax2 and Pax8 resulted in a less severe chronic injury phenotype. This effect was mediated by protection against the acute insult, similar to pre-conditioning. Prior to injury, Pax2 and Pax8 mutant mice develop a unique subpopulation of proximal tubule cells in the S3 segment that displayed features usually seen only in acute or chronic injury. The expression signature of these cells was strongly enriched with genes associated with other mechanisms of protection against ischemic AKI including caloric restriction, hypoxic pre-conditioning, and female sex. Thus, our results identified a novel role for Pax2 and Pax8 in mature proximal tubules that regulates critical genes and pathways involved in both the injury response and protection from ischemic AKI.

PAX8 modulates the tumor microenvironment of high grade serous ovarian cancer through changes in the secretome.

High grade serous ovarian cancer (HGSC) arises from the fimbriated end of the fallopian tube epithelium (FTE), and in some cases, the ovarian surface epithelium (OSE). PAX8 is a commonly used biomarker for HGSC and is expressed in ∼90% of HGSC. Although the OSE does not express PAX8, murine models of HGSC derived from the OSE acquire PAX8, suggesting that it is not only a marker of Müllerian origin, but also an essential part of cancer progression, potentially from both the OSE and FTE. Previously, we have shown that PAX8 loss in HGSC cells causes tumor cell death and reduces cell migration and invasion. Herein, secretome analysis was performed in PAX8 deleted cells and we identified a reduction of the extracellular matrix (ECM) components, collagen and fibronectin. Immunoblotting and immunofluorescence in PAX8 deleted HGSC cells further validated the results from the secretome analysis. PAX8 loss reduced the amount of secreted TGFbeta, a cytokine that plays a crucial role in remodelling the tumor microenvironment. Furthermore, PAX8 loss reduced the integrity of 3D spheroids and caused a reduction of ECM proteins fibronectin and collagen in 3D cultures. Due to the ubiquitous nature of PAX8 in HGSC, regardless of cell origin, and the association of its reduced expression with decreasing tumor burden, a PAX8 inhibitor could be a promising drug target against various types of HGSC. To accomplish this, we generated a murine oviductal epithelial (MOE) cell line stably expressing PAX8 promoter-luciferase. Using this cell line, we performed a screening assay with a library of FDA-approved drugs (Prestwick Library) and quantitatively assessed these compounds for their inhibition of PAX8. We identified two hits: losartan and captropril, both inhibitors of the renin-angiotensin pathway that inhibit PAX8 expression and function. Overall, this study validates PAX8 as a regulator of ECM deposition in the tumor microenvironment.

In vitro study of glyphosate effects on thyroid cells.

Glyphosate is a pesticide, which contaminates the environment and exposes workers and general population to its residues present in foods and waters. In soil, Glyphosate is degraded in metabolites, amino-methyl-phosphonic acid (AMPA) being the main one. Glyphosate is considered a potential cancerogenic and endocrine-disruptor agent, however its adverse effects on the thyroid were evaluated only in animal models and in vitro data are still lacking. Aim of this study was to investigate whether exposure to Glyphosate could exert adverse effects on thyroid cells in vitro. Two models (adherent-2D and spheroid-3D) derived from the same cell strain Fisher-rat-thyroid-cell line-5 (FRTL-5) were employed. After exposure to Glyphosate at increasing concentrations (0.0, 0.1-0.25- 0.5-1.0-2.0-10.0 mM) we evaluated cell viability by WST-1 (adherent and spheroids), results being confirmed by propidium-iodide staining (only for spheroids). Proliferation of adherent cells was assessed by crystal violet and trypan-blue assays, the increasing volume of spheroids was taken as a measure of proliferation. We also evaluated the ability of cells to form spheroids after Glyphosate exposure. We assessed changes of reactive-oxygen-species (ROS) by the cell-permeant H2DCFDA. Glyphosate-induced changes of mRNAs encoding for thyroid-related genes (TSHR, TPO, TG, NIS, TTF-1 and PAX8) were evaluated by RT-PCR. Glyphosate reduced cell viability and proliferation in both models, even if at different concentrations. Glyphosate at the highest concentration reduced the ability of FRTL-5 to form spheroids. An increased ROS production was found in both models after exposure to Glyphosate. Finally, Glyphosate increased the mRNA levels of some thyroid related genes (TSHR, TPO, TG and TTF-1) in both models, while it increased the mRNAs of PAX8 and NIS only in the adherent model. The present study supports an adverse effect of Glyphosate on cultured thyroid cells. Glyphosate reduced cell viability and proliferation and increased ROS production in thyroid cells.

Multi-genomic analysis of 260 adrenocortical cancer patient tumors identifies novel network BIRC5-hsa-miR-335-5p-PAX8-AS1 strongly associated with poor survival.

BACKGROUND: Adrenocortical carcinoma is a rare endocrine cancer with poor overall survival. Linking survival outcomes to a common target across multiple genomic datasets incorporating microRNA-long non-coding RNA dysregulation have not been well described. We hypothesized that a multi-database analysis of microRNA-long noncoding RNA-messenger RNA regulatory networks associated with survival will identify novel biomarkers. METHODS: Significantly dysregulated genes or microRNA in adrenocortical carcinoma compared to normal adrenal was identified from sequencing data for 260 human adrenocortical carcinomas using GEO2R. The miRnet identified hub microRNA and genes and long noncoding RNA and microRNA associated with survival genes. The R2 generated Kaplan-Meier curves. The database miRTarBase linked genes associated with poor survival and dysregulated microRNA. RESULTS: Analysis of genes and microRNAs differentially regulated in >50% of datasets revealed 75 genes and 12 microRNAs were upregulated, and 167 genes and 12 microRNAs were downregulated (bonf. P < .05). Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed cell cycle, P53 signaling, arachidonic acid and innate immune response, and PI3/Akt are altered in adrenocortical carcinoma. A microRNA-target interaction network of differentially regulated microRNAs identified upregulated miRNA107, 103a-3p and 27a-3p, 16-5p, and downregulated 335-5p to have the highest degree of interaction with upregulated (ie, TPX2, CDK1, BIRC5, PRC1, CCNB1, GINS1) and downregulated (ie, RSPO3, NR2F1, TLR4, HOXA5, USP53, SLC16A9) hub genes as well as hub long noncoding RNAs XIST, NEAT1, KCNQ1OT1, and PAX8-AS1. Survival analysis revealed that the hub genes are associated with poor overall survival (P < .05) of adrenocortical carcinoma in the Cancer Genome Atlas data. CONCLUSION: A messenger RNA-microRNA-long noncoding RNA network analysis identified the BIRC5-miR335-5p-PAX8-AS1 network as one that was associated with poor overall survival in adrenocortical carcinoma, warranting further validation as a potential therapeutic target.

Significance analysis of PAX8 expression in endometrial carcinoma.

To analyze the expression and prognostic value of paired-box 8 (PAX8) expression in uterine corpus endometrial carcinoma (UCEC) by bioinformatics. The expression of PAX8 gene in UCEC was analyzed by R language and immunohistochemistry. The correlation between PAX8 expression and clinicopathological features was analyzed by R language. The prognostic factors was analyzed by univariate/multivariate regression. The survival curve of patients was analyzed by Kaplan-Meier Plotter (K-M Plotter). The diagnostic value of PAX8 in UCEC was analyzed by receiver operating characteristic curve, and the relationship between PAX8 expression and methylation was analyzed by Ualcan. The relationship between methylation and prognosis was analyzed by MethSurv database. The expression of PAX8 in cancer tissues was significantly higher than that in normal tissues. The expression of PAX8 was related to clinical stage, age, histological type, histologic grade, tumor invasion and disease-specific survival event. Univariate/multivariate regression analysis showed that clinical stage, tumor invasion, and PAX8 expression were the influence factors of overall survival (OS), while histologic grade and PAX8 expression were the influence factors of disease-specific survival, and patients with low expression had a longer OS. The area under the curve of receiver operating characteristic curve was 0.81 for PAX8 diagnosis of UCEC. PAX8 was hypomethylated in cancer tissue, and patients with hypermethylated PAX8 had a longer OS. The high expression of PAX8 induced by hypomethylation may play an important role in the occurrence and prognosis of UCEC.

PAX8 in the Junction between Development and Tumorigenesis.

Normal processes of embryonic development and abnormal transformation to cancer have many parallels, and in fact many aberrant cancer cell capabilities are embryonic traits restored in a distorted, unorganized way. Some of these capabilities are cell autonomous, such as proliferation and resisting apoptosis, while others involve a complex interplay with other cells that drives significant changes in neighboring cells. The correlation between embryonic development and cancer is driven by shared proteins. Some embryonic proteins disappear after embryogenesis in adult differentiated cells and are restored in cancer, while others are retained in adult cells, acquiring new functions upon transformation to cancer. Many embryonic factors embraced by cancer cells are transcription factors; some are master regulators that play a major role in determining cell fate. The paired box (PAX) domain family of developmental transcription factors includes nine members involved in differentiation of various organs. All paired box domain proteins are involved in different cancer types carrying pro-tumorigenic or anti-tumorigenic roles. This review focuses on PAX8, a master regulator of transcription in embryonic development of the thyroid, kidney, and male and female genital tracts. We detail the role of PAX8 in each of these organ systems, describe its role during development and in the adult if known, and highlight its pro-tumorigenic role in cancers that emerge from PAX8 expressing organs.

The renal lineage factor PAX8 controls oncogenic signalling in kidney cancer.

Large-scale human genetic data1-3 have shown that cancer mutations display strong tissue-selectivity, but how this selectivity arises remains unclear. Here, using experimental models, functional genomics and analyses of patient samples, we demonstrate that the lineage transcription factor paired box 8 (PAX8) is required for oncogenic signalling by two common genetic alterations that cause clear cell renal cell carcinoma (ccRCC) in humans: the germline variant rs7948643 at 11q13.3 and somatic inactivation of the von Hippel-Lindau tumour suppressor (VHL)4-6. VHL loss, which is observed in about 90% of ccRCCs, can lead to hypoxia-inducible factor 2α (HIF2A) stabilization6,7. We show that HIF2A is preferentially recruited to PAX8-bound transcriptional enhancers, including a pro-tumorigenic cyclin D1 (CCND1) enhancer that is controlled by PAX8 and HIF2A. The ccRCC-protective allele C at rs7948643 inhibits PAX8 binding at this enhancer and downstream activation of CCND1 expression. Co-option of a PAX8-dependent physiological programme that supports the proliferation of normal renal epithelial cells is also required for MYC expression from the ccRCC metastasis-associated amplicons at 8q21.3-q24.3 (ref. 8). These results demonstrate that transcriptional lineage factors are essential for oncogenic signalling and that they mediate tissue-specific cancer risk associated with somatic and inherited genetic variants.

Oncogenic Signaling in Kidney Cancer Requires Renal Lineage Factor PAX8.

Lineage factor PAX8 is essential for downstream oncogenic signaling of ccRCC-specific driver mutations.

The transcription factor PAX8 promotes angiogenesis in ovarian cancer through interaction with SOX17.

PAX8 is a master transcription factor that is essential during embryogenesis and promotes neoplastic growth. It is expressed by the secretory cells lining the female reproductive tract, and its deletion during development results in atresia of reproductive tract organs. Nearly all ovarian carcinomas express PAX8, and its knockdown results in apoptosis of ovarian cancer cells. To explore the role of PAX8 in these tissues, we purified the PAX8 protein complex from nonmalignant fallopian tube cells and high-grade serous ovarian carcinoma cell lines. We found that PAX8 was a member of a large chromatin remodeling complex and preferentially interacted with SOX17, another developmental transcription factor. Depleting either PAX8 or SOX17 from cancer cells altered the expression of factors involved in angiogenesis and functionally disrupted tubule and capillary formation in cell culture and mouse models. PAX8 and SOX17 in ovarian cancer cells promoted the secretion of angiogenic factors by suppressing the expression of SERPINE1, which encodes a proteinase inhibitor with antiangiogenic effects. The findings reveal a non-cell-autonomous function of these transcription factors in regulating angiogenesis in ovarian cancer.

Pax8 and Nkx2-1 haploinsufficiencies differentially affect liver metabolic pathways.

Thyroid dysfunctions are associated with liver diseases ranging, in severity, from insulin resistance (IR) to hepatocellular carcinoma. The pathogenic mechanisms appear complex and are not attributable, exclusively, to the impaired thyroid hormone (TH) signalling. Using a mouse model of human congenital hypothyroidism, young double heterozygote for both NK2 homeobox 1 (Nkx2-1)- and Paired box 8 (Pax8)-null mutations (DHTP) mice, and single heterozygous Pax8+/- and Nkx2-1+/- mice, we studied the liver pathways, the endocrine and metabolic factors affected in conditions of different dysthyroidisms. Young Nkx2-1+/- females displayed a slight hyperthyroidism and, in liver, increased TH signalling (i.e. increased expression of Dio1 and Trß1) and lipogenic gene expression, with triglycerides accumulation. Hypothyroid DHTP and euthyroid Pax8+/- females shared liver and skeletal muscle IR and hepatic hypothyroidism (i.e. reduced expression of Mct8, Dio1 and TRß1), activation of AKT and increased expression of glutathione peroxidase 4. Oxidative stress and reduced mitochondrial COX activity were observed in DHTP mice only. Pax8+/- females, but, unexpectedly, not DHTP ones, displayed transcriptional activation of the hepatic (and renal) gluconeogenic pathway, hypercortisolemia, fasting hyperglycaemia and hyperinsulinemia, reduced serum ß-hydroxybutyrate, associated with hepatic AMPK activation. DHTP mice showed hypercholesterolemia and activation of mTOR. Collectively, the data indicate that heterozygote mutations of Pax8 and Nkx2-1 genes may produce multiple dysmetabolisms, even under systemic euthyroidism. Differential liver pathways and multiple hormonal axes are affected with implications for energy and nutrient homeostasis. The identified players may be specific target in the management of thyroid dysfunction-associated dysmetabolisms in terms of prevention/counteraction of IR, type 2 diabetes and related comorbidities.

Sox9 is involved in the thyroid differentiation program and is regulated by crosstalk between TSH, TGFß and thyroid transcription factors.

While the signaling pathways and transcription factors involved in the differentiation of thyroid follicular cells, both in embryonic and adult life, are increasingly well understood, the underlying mechanisms and potential crosstalk between the thyroid transcription factors Nkx2.1, Foxe1 and Pax8 and inductive signals remain unclear. Here, we focused on the transcription factor Sox9, which is expressed in Nkx2.1-positive embryonic thyroid precursor cells and is maintained from embryonic development to adulthood, but its function and control are unknown. We show that two of the main signals regulating thyroid differentiation, TSH and TGFß, modulate Sox9 expression. Specifically, TSH stimulates the cAMP/PKA pathway to transcriptionally upregulate Sox9 mRNA and protein expression, a mechanism that is mediated by the binding of CREB to a CRE site within the Sox9 promoter. Contrastingly, TGFß signals through Smad proteins to inhibit TSH-induced Sox9 transcription. Our data also reveal that Sox9 transcription is regulated by the thyroid transcription factors, particularly Pax8. Interestingly, Sox9 significantly increased the transcriptional activation of Pax8 and Foxe1 promoters and, consequently, their expression, but had no effect on Nkx2.1. Our study establishes the involvement of Sox9 in thyroid follicular cell differentiation and broadens our understanding of transcription factor regulation of thyroid function.