United States

The androgen receptor (AR) plays a crucial role in the development and maintenance of the male phenotype, as shown in patients with androgen insensitivity syndrome (AIS). The AR is a nuclear receptor that needs to homodimerize to execute its role as transcription factor. Dimerization can occur through three different modes: via the DNA-binding domain (DBD), via the ligand-binding domain and via an interaction between the LBD and the aminoterminal domain. Dimerization via the DBD is very well known and occurs through the D-box located in the second zinc finger. DBD dimerization is generally accepted for all steroid receptors, but some of them show that D-box mutations do not inactivate them completely. However, for the AR, no monomeric transactivation capacity was described yet. To study the role of D-box independent activity of the AR, we introduced a double point mutation in the D-box that is predicted to disrupt DBD dimerization (ARDmon). These two point mutations were found in separate patients suffering from partial AIS. In vitro studies with this mutant suggest that the remaining activity depends on the androgen response element under investigation. We show that ligand binding was not affected by the D-box mutation. Furthermore, BioID assays indicated no changes in coregulatory interactions. When introducing the corresponding mutation in mice, this led to a phenotype that is intermediate between global ARKO and wild type (WT) males. Based on their anogenital distance (AGD) and the absence of nipples at early age, the ARDmon/Y mice were identified as males. Follow-up of their AGD and body weight, however, revealed clear differences with both WT males and females. Furthermore, hypospadias was observed in the ARDmon/Y mice. Testes were smaller in size compared to the WT males and first analysis showed almost no expression of well-known AR regulated genes. Reminiscent structures of the epididymis were found.
In summary, the ARDmon mouse model shows an intermediate AIS phenotype. Via orchidectomy in combination with androgen replacement therapy, we will now investigate remaining androgen responses in the ARDmon/Y mice and elucidate response elements on which the AR can act in a D-box independent way. Additionally, we generated the first rodent PAIS model with a genetic cause of hypospadias.

ENDO 2022

Disrupting DNA-binding Domain Dimerization In The Androgen Receptor In Mice Results In a Partial Androgen Insensitivity Model

**Program**
ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

This year’s education program features:
more than 100 in-person and virtual sessions with another 70+ in-person only sessions;
six plenary sessions including plenaries on Envisioning Future Therapies for Type 1 Diabetes and Addressing Challenges in Fertility and Reproduction;
over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

**What Programming Is Included in My Virtual Meeting Registration?**
As you build your schedule during the conference, you can select the sessions that are tagged with 'Livestreamed', so you can view virtually. These sessions are being livestreamed, so they are available in real time to our virtual audience. In addition, our in-person and livestreamed sessions will be recorded, so you can access them on demand after 72 hours. We will have over 200 hours available on demand for Standard and Premium pass registrants.

Product theaters and the Communications and Career Center will also include hybrid content delivery.

**Plenaries**
Spanning the spectrum of science, clinical care, and social implications, an outstanding slate of world-renowned speakers will showcase the most cutting-edge advances on research and medicine.

**Symposia**
Our ENDO 2022 lineup of sessions offers a unique opportunity to dive into topics from nearly every major therapeutic area of endocrinology and hormone science. Listen, learn, and be inspired by world-class faculty who are at the forefront of scientific discovery and high-quality health care.

**Basic Science Pathways**

These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
Diabetes and Metabolism
Neuroendocrinology
Nuclear Receptors and Signaling
Reproductive Endocrinology

**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

technical paper

Introduction: The pituitary gland controls several mechanisms as metabolism, growth, and reproduction, in response to hypothalamic stimuli. The adequate temporal/ spatial expression of transcription factors is mandatory for a normal pituitary development. PROP1 transcription factor is widely known as a key pituitary regulator. Pathogenic variants in the PROP1 gene are the main familial causes of hypopituitarism accounting for about 54% of the cases. Aim: To characterize the expression of genes involved in pituitary cells determination and specification during pituitary high demand period such as sexual maturation compared to adulthood in the Ames mice. Methods: Weight and naso-anal length were measured. Pituitary glands were collected from 5 wild type (WT) and 5 prop1 mutant (Mut) animals with 30 (P30), 40, (P40), and 60 (P60) days after birth. RT-qPCR was performed to analyze the pituitary transcription factors Sox2, Sox3, Hesx1, Otx2, and genes codifying the hormones GH, TSH, LH/ FSH, CGA, and PRL by SYBR® Green (QIAGEN, Valencia, CA). They were normalized by endogenous genes and performed in triplicate. The target genes relative quantification was performed using the mutant related to its age paired wild type as a calibrator and the results were expressed as fold change. Immunofluorescence of SOX2 (ab97959, Abcam, UK) (1: 1600) and pituitary hormones GH, LH, and FSH (1: 250) were done in pituitaries at P60 and its aged paired control, stained with propidium iodide (1: 1000) for nucleus and the secondary antibody Alexa FluorÒ488 (ab150077, Abcam, UK) (1: 1000). Results: All mutant mice presented decreased weight and naso-anal length at the three analyzed periods. The expression of the pituitary stem/ progenitor cell marker Sox2 was increased at P30 and P60 and decreased at P40. Sox3 was increased at P30 and decreased at P40 and P60. Hesx1 was increased at P30 and P60 and decreased at P40. Otx2 was increased at all periods. At P30 the genes codifying pituitary hormones was decreased, except for Tsh and Fsh. At P40 it was observed increased expression of the genes codifying Gh, Lh, Fsh and Prl, while Tsh and Cga expression was reduced. At P60 all pituitary hormones codifying genes presented decreased expression. The immunofluorescence at P60 showed increased expression of SOX2, similar expression of FSH, and decreased expression of GH and LH related to their age paired WT. Conclusion: Despite the absence of PROP1, Hesx1 is downregulated at P40. Pituitary cell subtype differentiation and hormonal production is observed at transcriptional and protein levels, showing that pituitary cells can differentiate and produce hormones in the absence of Prop1. The recovery of pituitary capacity to differentiate in mutant suggests the involvement of another factor in the pituitary cell differentiation pathway that could compensate the lack of Prop1.

PROP1 is not essential for pituitary cells terminal differentiation

The Musashi RNA-binding protein functions as a gatekeeper of cell maturation and maintains stem cell plasticity by regulating the translation of target mRNAs. The adult anterior pituitary tissue expresses a high level of Musashi and also demonstatres a high level of cell plasticity, indicating a Musashi-dependent function in maintaining endocrine homeostasis in the anterior pituitary. Towards understanding the mechanism(s) by which Musashi functions to control cell plasticity, we have identified co-associated proteins necessary for Musashi function. The two Musashi isoforms (Musashi1 and Musashi2) are conserved across species and are structurally characterized by two N-terminal RNA-recognition motifs (RRMs, that associate with specific sequences in the 3' untranslated region of mRNAs targets) and a disordered C-terminus. Musashi uses these domains to selectively repress, or to activate, mRNA translation in a manner specific to target mRNA and cell context. Both Musashi isoforms contain two C-terminal regulatory serine residues that require phosphorylation to enable mRNA translational activation. However, the Musashi proteins do not have inherent mRNA translation function and utilize co-associated protein complexes to mediate mRNA translation. We have previously identified Musashi1 binding protein partners through mass spectrometry. Here, we have identified the protein interactions that are required for Musashi1-dependent translational activation. Knockdown of target protein levels in Xenopus laevis oocytes indicated that the PABP4, CELF2, LSM14A/B, ELAVL1, ELAVL2, ELAVL4, and PUM1 proteins are required for Musashi target mRNA translational activation. We further determined that the effect of LSM14A/B dual knockdown was in fact specific to LSM14B alone. LSM14B is an RNA-binding protein that is required for RNA granule formation, RNA transcript metabolism, and mRNA translation. Studies in mouse oocytes have shown that LSM14B is required to maintain transcript levels of the Musashi1 target Cyclin B1 (Ccnb1) mRNA prior to mRNA translation. In the adult murine pituitary, LSM14B is ubiquitously expressed in both hormone-producing cells and in stem cells as indicated through single cell RNA sequencing. These findings indicate that LSM14B and Musashi1 may function cooperatively to regulate pituitary hormone production and cell differentiation in response to changes in physiological demand.

Functional association of the stem cell protein Musashi with LSM14B in control of mRNA translation

G protein-coupled receptors (GPCRs) are the largest protein family and amenable for pharmacological manipulation. GPCRs in the neuroendocrine and enteroendocrine systems respond to various biological cues and in turn exert crucial roles in maintaining whole body metabolic homeostasis. For example, GPCRs in the gastrointestinal tract are involved in maintaining glucose and energy homeostasis by regulating the release of gut hormones in response to luminal dietary nutrients as well as microbial metabolites. We recently identified that an orphan GPCR, Gpr17, was co-expressed in glucagon-like peptide-1 (GLP-1)-expressing EECs in human and rodent intestinal epithelium. We reported that acute genetic ablation of Gpr17 in intestinal epithelium (iKO) improved oral glucose tolerance and glucose-stimulated insulin secretion (GSIS) in female and male mice through increased secretion of incretin hormone in response to nutrient ingestion (Cell Reports, 2022). In the neuroendocrine system, we previously identified Gpr17 as the transcription target of Forkhead box protein O1 (FoxO1) in the hypothalamus and generated hypothalamic neuron specific Gpr17 knockout animals (Cell, 2012). Our results demonstrated that agouti-related peptide (AgRP) neuron Gpr17 knockout mice had increased satiety during fasting-refeeding challenge (Diabetes, 2015) and that proopiomelanocortin (POMC) neuron Gpr17 knockout mice had increased alpha-melanocyte stimulating hormone (aMSH) processing and increased firing of POMC neurons (Nutrition & Diabetes, 2019). However, how Gpr17 may regulate energy balance especially regarding leptin hormone signaling is unclear. To address this question, we used genetic knockout approach to generate Gpr17 conditional knockout in leptin receptor expressing neurons in the brain. We found Gpr17 deficiency in leptin receptor expressing neurons increased leptin sensitivity in animals and resulted in reduced food intake, which corresponded with reduced inflammatory response in the hypothalamus. Moreover, male Gpr17 whole body knockout mice have improved intraperitoneal glucose tolerance, and increased insulin sensitivity in hyperinsulinemic-euglycemic clamp studies. In conclusion, our collective body of work showed that ablation of Gpr17 signaling in the neuroendocrine and enteroendocrine systems led to improved neurohormonal regulation to maintain metabolic homeostasis, indicating functionally targeting this GPCR may lead to better therapeutic outcome of diabetes and obesity.

Endocrine mechanisms of an orphan G protein-coupled Receptor regulating metabolic homeostasis

Background: Prolactin (PRL) is responsible for the development of mammary glands and milk production. STAT1 and STAT3 are genes that express transcription factors that mediate PRL's intracellular signaling, but an association between their function and galactorrhea has not yet been identified. Here we describe three patients with somatic STAT mutations and galactorrhea.
A 27-year-old woman with hyper-IgE syndrome (Job's Syndrome) due to a STAT3 deletion mutation (p.Val463del) associated with recurrent infections reported thin, white, bilateral discharge with breast manipulation beginning in adolescence. She did not have spontaneous leakage or pain. At presentation, PRL was slightly elevated to 27.9 (ref. 2.0-25.0 mcg/L) with otherwise normal levels of anterior pituitary hormones. She was not on any drugs known to cause hyperprolactinemia. MRI showed a 5x4 mm microadenoma without any mass effect.
A 32-year-old woman also with Job's syndrome due to a heterozygous loss-of-function STAT3 mutation (p.R382Q) associated with recurrent infections and papillary thyroid cancer described unilateral, chronic, thin, white nipple discharge with light touch of her right breast for "as long as she could remember." She had a history of breast abscesses, but at presentation there was no evidence of abscess or infection. PRL was 15.1 mcg/L, and other anterior pituitary hormones were normal. MRI showed a structurally normal pituitary.
A 22-year-old woman with a STAT1 activating mutation (p.E235A) associated with recurrent infections, autoimmune hypothyroidism, T1DM, and hypogonadotropic hypogonadism requiring pubertal induction at age 12y reported bilateral galactorrhea starting at age 18y. A white, milky discharge could be expressed with light manipulation, but no discharge was produced spontaneously. PRL was 16.2 mcg/L with serial dilution and has consistently been in the reference range. Anterior pituitary evaluation was normal except for elevated TSH of 12.5 (ref. 0.27-4.2) consistent with autoimmune hypothyroidism. MRI revealed a diffusely enlarged pituitary gland (12mm in the cranio-caudal dimension) without focal hypo-enhancement.
Conclusions: It is known that PRL signaling involves the JAK/STAT pathway, and an analysis of human milk cells showed upregulated STAT3 and downregulated STAT1 during lactation (PMID 26909879). These three cases provide clinical evidence for a mechanistic connection between STAT signaling and galactorrhea and suggest that STAT3 and STAT1 function in opposite directions to regulate lactation. In our cases, increased STAT1 or decreased STAT3 activity are associated with normal-PRL, inducible galactorrhea. Beyond understanding the specific intracellular signaling pathways leading to galactorrhea, these observations may be utilized for developing therapies for those with impaired lactation.

Associations Between Signal Transducer and Activator of Transcription (STAT) Mutations and Galactorrhea

Background: Central precocious puberty (CPP) results from early reactivation of the hypothalamic-pituitary-gonadal (HPG) axis. Four monogenic causes of CPP have been described (KISS, KISS1R MKRN3 and DLK1). Rare loss-of-function mutations of DLK1, a maternally imprinted gene located on chromosome 14q32.2, were identified in families with CPP. Both inherited or de novo DLK1 mutations were described in these affected families.
Objective: To investigate genetic abnormalities in the DLK1 in a French cohort of patients with idiopathic CPP.
Patients: Genomic DNA was obtained from 122 patients (99 girls and 23 boys) with sporadic or familial CPP. Automatic sequencing of the coding regions of DLK1 gene (5 exons, using Sanger method) was performed in all cases, including 91 familial and 31 sporadic cases.
Results A pathogenic heterozygous variant in the DLK1 gene (c.372C>A p.Cys124Ter (rs749564412) was identified in a girl with sporadic CPP associated with overweight. This nonsense mutation was considered rare (gnomAD total population 1/251,082) and it was located in the third EGF-like repeat domain in the extracellular region. The affected girl had progressive breast development at 4.5 years and accelerated growth velocity. She had breast development Tanner stage 4 at 5.5 years and bone age (BA) was 6 yr. Her height was 114.5 cm and BMI 18.16 (BMI Z-score = 2.48). Basal LH and FSH was 0.6 IU/L and 5.7 IU/L, respectively. GnRH-stimulated LH and FSH peaks were 14 IU/L and 18 IU/L, respectively, with LH: FSH ratio of 0.77. Other hormonal results were normal (estradiol: 11 pg/mL; ACTH: 11 pg/mL; IGF-1: 235 ng/mL; cortisol: 77 ng/mL). Brain MRI was normal. Pelvic ultrasound revealed uterus length: 49 mm, mucosal thickness of 4 mm, ovaries 20 and 25 mm with follicles less than 10 mm. She was treated with a depot GnRH analog (triptorelin). The DLK1 variant was inherited from her asymptomatic father who had a height of 181 cm. Familial history of CPP history was denied.
Conclusion: DLK1 loss-of-function are rare genetic cause of familial or sporadic CPP. Novel genetic findings in this factor reinforce its role in the physiopathology of the premature HPG axis reactivation and precocious sexual development in humans.

A novel nonsense mutation in Delta-like non-canonical Notch ligand 1 gene (DLK1) in a French girl with central precocious puberty

Thyroid cancer will become 4th most common cancer in women by 2030. Thyroid cancer is the most prevalent endocrine malignancy of women with an age-standardized rate of 10.1/100,000 while in men it is 3.1/100,000.
The microenvironment of thyroid cancer has a high degree of immune invasion, characterized by the presence of T effector (Teff, CD4, and CD8) cells. In our previous study based on 127 human FNA thyroid samples, we reported a dominant T cell species characterized by the deficiency of CD4 and CD8 expression (CD3+CD4-CD8-) Double Negative (DN) T cells, seem to have a regulatory function towards thyroid cancer immune tolerance (1-3).
Here, in another cohort study (46 patients), FNAs of thyroids showed that DN T-cells were significantly more abundant in lymphocytic infiltrates of thyroid cancer patients (post-operatively confirmed diagnosis by an academic pathologist), and if the DN T cell population exceeded our defined threshold (>9.14%), it indicated a high likelihood of cancer presence (96.6% sensitivity, 95% CI, 0.915 to 1.000). Conversely, DN T cells below the threshold (<9.14%) provide 100% specificity in predicting benignity of thyroid nodules.  This study identifies DN T cell quantification as an accurate rule-out test. The high specificity of the test is promising since it abolishes false-positive diagnoses and unnecessary surgical procedures. Current molecular diagnostic testing, based on the identification of tumor gene mutations in FNA samples usually reduces the risk of cancer rather than guarantee the absence of cancer which leads to 70% of unnecessary thyroidectomies.
The present study proposes DN T cell quantification as a diagnostic signature for thyroid cancer. Its integration with RNA transcriptomics may provide a simplified technology for thyroid cancer screening and active surveillance.
References
1.  Imam S, Paparodis R, Sharma D, et al. 2014 Lymphocytic profiling in thyroid cancer provides clues for failure of tumor immunity. EndocrRelat Cancer 21.
2.  Imam S, Dar P, Paparodis R, et al. 2019 Nature of coexisting thyroid autoimmune disease determines success or failure of tumor immunity in thyroid cancer. J Immunother Cancer 7.
3.  Imam S, Jaume J C 2021 Stratifying risk of malignancy in indeterminate thyroid nodules and immuno-genomic markers for early detection of thyroid cancer. International Application No. PCT/US2021/012057, Publication Number WO/2021/138661

Decoding Cancer Immunoediting of Tumor Microenvironment Provides Immunogenomic Marker for Thyroid Cancer Screening

Progressive metastasis is the proximate cause of cancer-related mortality for the majority of patients with solid tumors. Identifying both drivers, and gatekeepers of late stage progression (metastasis progression suppressors; MPS) is crucial to improve biomarkers and treatments. We previously identified Regulator of Calcineurin 1.4 (RCAN1.4) as a MPS gene in thyroid cancer that functions in part through overexpression of the cap-n-collar transcription factor NFE2L3 (NRF3).  However, the mechanisms for RCAN1.4 loss in thyroid cancer, and whether or not this is reversible, had not be addressed. Because the RCAN 1.4 promoter contains CG rich regions reported to be hypermethyalated in other tissues, we hypothesized that RCAN 1.4 downregulation in thyroid cancer was similarly regulated. Studies were performed in both cell lines and de-identified human papillary thyroid cancer (PTC) tumor and normal tissue pairs from our Endocrine Neoplasia Repository tumor bank. Five thyroid cancer cell lines (TPC1, FTC133, BCPAP, C643, and 8505C) with different genetic drivers were selected based on low expression of RCAN 1.4. To determine if lines display methylation-regulated expression of RCAN 1.4, cells were treated with 1-10uM decitabine, a DNA methyl transferase inhibitor. In all cell lines, RCAN1.4 mRNA and protein levels increased with decitabine treatment, consistent with hypermethylation. Analysis of the literature and in silico analysis of the RCAN 1.4 gene identified CG rich regions as putative methylation targets in the proximal promoter and in intron 1. Using two different methods; 1) isolation of MseI-digestion DNA fragments using CpG MethylQuest beads followed by qPCR and 2) methylation-specific PCR (MSP) of bisulfite-modified DNA , hypermethylation of the intron 1 CG rich region was identified. There was no evidence of hypermethylation of the CG rich region of the proximal promoter. Methylation of the two regions also was assessed on 18 pairs PTC and adjacent normal tissues. Similar to the cell lines, hypermethylation was identified at intron 1 of RCAN 1.4 in PTC versus normal tissues. QRT-PCR results confirmed lower levels of RCAN1-4 mRNA in the PTC vs normal tissue. These samples also demonstrated increased NRF3 expression versus normal tissue. Taken together, these data demonstrate that RCAN 1.4 is downregulated in thyroid cancer cells and human PTCs by hypermethylation of CG rich regions in intron 1 and that this effect is reversible using demethylating agents.

The RCAN 1.4 Metastasis Progression Suppressor Gene is Hypermethylated at Intron 1 and Downregulated in Papillary Thyroid Cancer

Background: Integrins are cell adhesion receptors consisting of 24 transmembrane heterodimers generated from a combination of 18α integrin and 8β integrin subunits. A subset of integrins consists of receptors recognizing Arg-Gly-Asp (RGD) peptide motifs. One of the RGD-recognizing receptors is integrin αvβ3 that has been recently shown to play a role in neovascularization and progression of several cancers. Radiolabeled RGD analogs have emerged as potential imaging and therapeutic options in cancers characterized by a high expression of integrin αvβ3. Therefore, the aim of this study was to establish the expression of integrin αvβ3 in thyroid cancer (TC).
Methods: We analyzed the mRNA expression of integrin αvβ3 in 496 BRAF-like and RAS-like human TC tissue samples, including 65 paired samples of tumor vs normal tissue based on The Cancer Genome Atlas. We assessed the protein expression of integrin αvβ3 in 70 TC tissue samples and 10 normal thyroid tissues, as well as in 14 TC cell lines. BRAF-like or RAS-like tumor status was determined by BRS score based upon standard expression profiles ranging from -1 to 0 for BRAF-like cancer and 0 to 1 for RAS-like tumors. The association between BRS and αvβ3 expression was tested using the Spearman correlation coefficient (r). T-tests and paired T-tests were used to compare the continuous variables between the two groups as appropriate, and Kruskal-Wallis test was used for multiple group comparisons with an adjusted p-value of ≤ 0.05 as statistically significant.
Results: αv integrin subunit mRNA expression was significantly higher in TC than normal thyroid (log fold change 0.3, p=0.001), while the expression of the β3 subunit was similar between paired normal and malignant samples (log fold change -0.2, p=0.30).  BRAF-like tumors were characterized by a higher mRNA expression of αvβ3 integrins as documented by a moderate negative correlation between BRS and αv (r=-0.5, p<0.001) as well as β3 (r=-0.27, p<0.001). Consistently, the BRAF-like TC cell lines OCUT2 (BRS=-1), TPC1 (BRS=-0.4), K1 (BRS=-0.29), as well as Hurthle cell TC cell line XTC1 (BRS=-0.46), were characterized by the highest αvβ3 mRNA and/or protein expression. Immunostaining revealed αv expression in all malignant samples, with classic papillary TC characterized by the highest expression as compared with follicular TC (p<0.001), poorly-differentiated TC (p=0.006) and normal thyroid (p<0.001). β3 protein expression had lower intensity than αv integrin and was present in 31.8% of papillary TC, 15% of follicular TC and was not detected in poorly-differentiated TC nor normal thyroid.
Conclusions: TC is characterized by a differential expression of αvβ3 integrin, which is particularly high in the most common type of TC - BRAF-like papillary TC. The αvβ3 integrin could potentially serve as a molecular target for imaging and therapy with radiolabeled RGD analogs in TC.

Integrins as Potential Molecular Targets in Thyroid Cancer Imaging and Therapy

Thyroid cancer is the most prevalent endocrine malignancy and is estimated to have affected 52,890 individuals in the United States in the year 2020. Papillary thyroid cancer (PTC) accounts for up to 80% of all thyroid cancer diagnoses. Within PTC, BRAFV600E is the most common mutation in adults and the second most common mutation in pediatrics. While there are several validated human thyroid cancer cell lines harboring BRAF mutations, no pediatric derived cell line has been established to date, limiting generalizations in pediatric disease. Patient-derived pre-clinical models are valuable tools but are limited due to their need to be studied in vitro or in an immunocompromised host. However, in vivo mouse models recapitulate the complex interactions between tumor cells, the immune system, and components of the microenvironment, allowing more comprehensive investigation of thyroid oncogenesis and response to therapy. In this study we describe a new subcutaneous mouse model that can be used to understand differences between pediatric onset and adult onset PTC.
We have developed multiple independent, congenic murine cell lines from different stages of thyroid cancer progression. Two independent BRAFV600E-driven cell lines harboring the same genetic mutations were utilized in this study, one representing a well-differentiated tumor (WD) and one representing a poorly-differentiated tumor (PD). Despite coming from different stages of disease, both cell lines showed similar expression of the thyroid specific genes: Pax8, Ttf1, Tg, and Slc5a5, but expression of Pax8, Tg, and Slc5a5 was lower compared to WT thyrocytes. Activation of the Pi3Kinase pathway and the Akt Pathway were assessed via western blot analysis. The WD cell line had increased pERK activation compared to the PD cell line, and the PD cell line had increased pAKT compared to WD cell line.
Both cell lines were injected subcutaneously into the hind flank of Wt SJV129 mice of pediatric age (4-5 weeks) and adult age (20-22 weeks). These animals were monitored for 10 weeks post injection. While the PD cell line developed tumors at approximately the same rate and penetrance in both age groups, tumors reached end point more rapidly in the adult cohort. Adult hosts injected with the WD cell line developed tumors more rapidly than in pediatric hosts, however, tumors receded in both cohorts.
While adult and pediatric thyroid cancer share common driving mutations, there are distinct differences in disease pathogenesis between these patient populations. There are known differences in the immune system, metabolism, and other variables between pediatrics and adults. However, it is unknown what causes the difference in PTC pathogenesis between pediatric versus adult patients. We believe these new models provide a great opportunity to evaluate the role of age in PTC development and progression.

Novel Murine Models of BrafV600E driven Papillary Thyroid Cancer

Background: Thyroid cancers (TC) are divided into BRAF-like and RAS-like tumors based on their molecular signature. The mTOR (mammalian target of rapamycin) signaling pathway is commonly activated in RAS-like tumors. Oncogene-driven signaling pathways are involved in the regulation of intracellular metabolism, including glycolysis and oxidative phosphorylation (OXPHOS). There is inadequate information on the role of mTOR signaling in the regulation of TC metabolism. The purpose of this study was to determine the association between mTOR signaling and mitochondrial respiration in TC.

Methods: We conducted an in vitro study using 4 TC cell lines (2 BRAF-like, 2 RAS-like) characterized by variable activation of mTOR signaling pathway, as documented by immunoblotting to quantify a ratio of p-mTOR/mTOR. OXPHOS was measured through oxygen consumption rate (OCR) of TC cells by the Seahorse XF Cell Mito Stress Test Assay. Standard MTT assay was performed to evaluate cell proliferation. A specific mTOR inhibitor (rapamycin) as well as an mTOR activator (leucine) were used for functional studies. The association between OCR and p-mTOR/mTOR was calculated by Pearson correlation coefficient (r). The one-way Anova or Kruskal-Wallis test were used for between group comparisons, as appropriate. P-values of ≤ 0.05 were considered statistically significant.

Results: The baseline activation of mTOR signaling in TC cells correlated with baseline (r=0.75, p=0.04) and maximum OCR (r=0.91, p=0.01). In addition, the RAS-like cell lines with the highest activation of mTOR showed considerable response after inhibition of mTOR with rapamycin by inhibiting baseline OCR (FTC133: 6320 ± 1531 vs 3368 ± 651 pmol/min/mg, p=0.01; THJ29T: 4484 ± 514 vs 3067 ± 620 pmol/min/mg, p=0.047), while the TC cell lines with minimal baseline mTOR activation did not show change in mTOR phosphorylation status nor basal OCR after rapamycin treatment (TPC1: 1817 ± 201 vs 1974 ± 336 pmol/min/mg, p>0.99, BCPAP: 1051 ± 162 vs 1130 ± 228 pmol/min/mg, p>0.99). Treatment with rapamycin significantly reduced cell proliferation rates in all examined cells. On the other hand, treatment with the mTOR activator leucine increased both mTOR phosphorylation and basal OCR in TC cell lines characterized by minimal baseline mTOR activation (TPC1: 1778 ± 280 vs 2372 ± 249 pmol/min/mg, p=0.004, BCPAP: 1581 ± 603 vs 2064 ± 560 pmol/min/mg, p=0.04), but did not significantly increase OCR further in cell lines with high baseline mTOR activation and OXPHOS rate  (FTC133: 4512 ± 683 vs 5355 ± 870 pmol/min/mg, p=0.27; THJ29T: 3881 ± 880 vs 4764 ± 1273 pmol/min/mg, p=0.14).

Conclusion: mTOR signaling is associated with the regulation of mitochondrial respiration as its inhibition decreases OXPHOS rate, while its activation results in increased mitochondrial respiration. The change in mitochondrial respiration might be one of the mechanisms of the growth inhibition caused by medications targeting mTOR signaling in TC.

Disrupting DNA-binding Domain Dimerization In The Androgen Receptor In Mice Results In a Partial Androgen Insensitivity Model

Downloads

Next from ENDO 2022

PROP1 is not essential for pituitary cells terminal differentiation

Similar lecture

Zebrafish in utero glucose exposure induces lipid dysregulation that is abrogated by Acetyl CoA carboxylase or glycolysis inhibition

Stay up to date with the latest Underline news!

PRESENTATIONS

CONFERENCES

COMPANY

RESOURCES