Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice.

Up until recently, methods for generating floxed mice either conventionally or by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (CRISPR-associated protein 9) editing have been technically challenging, expensive and error-prone, or time-consuming. To circumvent these issues, several labs have started successfully using a small artificial intron to conditionally knockout (KO) a gene of interest in mice. However, many other labs are having difficulty getting the technique to work. The key problem appears to be either a failure in achieving correct splicing after the introduction of the artificial intron into the gene or, just as crucial, insufficient functional KO of the gene's protein after Cre-induced removal of the intron's branchpoint. Presented here is a guide on how to choose an appropriate exon and where to place the recombinase-regulated artificial intron (rAI) in that exon to prevent disrupting normal gene splicing while maximizing mRNA degradation after recombinase treatment. The reasoning behind each step in the guide is also discussed. Following these recommendations should increase the success rate of this easy, new, and alternative technique for producing tissue-specific KO mice.

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology.

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Undifferentiated spermatogonia regulate Cyp26b1 expression through NOTCH signaling and drive germ cell differentiation.

Cytochrome P450 family 26 subfamily B member 1 (CYP26B1) regulates the concentration of all-trans retinoic acid (RA) and plays a key role in germ cell differentiation by controlling local distribution of RA. The mechanisms regulating Cyp26b1 expression in postnatal Sertoli cells, the main components of the stem cell niche, are so far unknown. During gonad development, expression of Cyp26b1 is maintained by Steroidogenic Factor 1 (SF-1) and Sex-Determining Region Y Box-9 (SOX9), which ensure that RA is degraded and germ cell differentiation is blocked. Here, we show that the NOTCH target Hairy/Enhancer-of-Split Related with YRPW Motif 1 (HEY1), a transcriptional repressor, regulates germ cell differentiation via direct binding to the Cyp26b1 promoter and thus inhibits its expression in Sertoli cells. Further, using in vivo germ cell ablation, we demonstrate that undifferentiated type A spermatogonia are the cells that activate NOTCH signaling in Sertoli cells through their expression of the NOTCH ligand JAGGED-1 (JAG1) at stage VIII of the seminiferous epithelium cycle, therefore mediating germ cell differentiation by a ligand concentration-dependent process. These data therefore provide more insights into the mechanisms of germ cell differentiation after birth and potentially explain the spatiotemporal RA pulses driving the transition between undifferentiated to differentiating spermatogonia.-Parekh, P. A., Garcia, T. X., Waheeb, R., Jain, V., Gandhi, P., Meistrich, M. L., Shetty, G., Hofmann, M.-C. Undifferentiated spermatogonia regulate Cyp26b1 expression through NOTCH signaling and drive germ cell differentiation.

Regulation of GDNF expression in Sertoli cells.

Sertoli cells regulate male germ cell proliferation and differentiation and are a critical component of the spermatogonial stem cell (SSC) niche, where homeostasis is maintained by the interplay of several signaling pathways and growth factors. These factors are secreted by Sertoli cells located within the seminiferous epithelium, and by interstitial cells residing between the seminiferous tubules. Sertoli cells and peritubular myoid cells produce glial cell line-derived neurotrophic factor (GDNF), which binds to the RET/GFRA1 receptor complex at the surface of undifferentiated spermatogonia. GDNF is known for its ability to drive SSC self-renewal and proliferation of their direct cell progeny. Even though the effects of GDNF are well studied, our understanding of the regulation its expression is still limited. The purpose of this review is to discuss how GDNF expression in Sertoli cells is modulated within the niche, and how these mechanisms impact germ cell homeostasis.

RBPJ in mouse Sertoli cells is required for proper regulation of the testis stem cell niche.

Stem cells are influenced by their surrounding microenvironment, or niche. In the testis, Sertoli cells are the key niche cells directing the population size and differentiation fate of spermatogonial stem cells (SSCs). Failure to properly regulate SSCs leads to infertility or germ cell hyperplasia. Several Sertoli cell-expressed genes, such as Gdnf and Cyp26b1, have been identified as being indispensable for the proper maintenance of SSCs in their niche, but the pathways that modulate their expression have not been identified. Although we have recently found that constitutively activating NOTCH signaling in Sertoli cells leads to premature differentiation of all prospermatogonia and sterility, suggesting that there is a crucial role for this pathway in the testis stem cell niche, a true physiological function of NOTCH signaling in Sertoli cells has not been demonstrated. To this end, we conditionally ablated recombination signal binding protein for immunoglobulin kappa J region (Rbpj), a crucial mediator of NOTCH signaling, in Sertoli cells using Amh-cre. Rbpj knockout mice had: significantly increased testis sizes; increased expression of niche factors, such as Gdnf and Cyp26b1; significant increases in the number of pre- and post-meiotic germ cells, including SSCs; and, in a significant proportion of mice, testicular failure and atrophy with tubule lithiasis, possibly due to these unsustainable increases in the number of germ cells. We also identified germ cells as the NOTCH ligand-expressing cells. We conclude that NOTCH signaling in Sertoli cells is required for proper regulation of the testis stem cell niche and is a potential feedback mechanism, based on germ cell input, that governs the expression of factors that control SSC proliferation and differentiation.

Thyroid C-Cell Biology and Oncogenic Transformation.

The thyroid parafollicular cell, or commonly named "C-cell," functions in serum calcium homeostasis. Elevations in serum calcium trigger release of calcitonin from the C-cell, which in turn functions to inhibit absorption of calcium by the intestine, resorption of bone by the osteoclast, and reabsorption of calcium by renal tubular cells. Oncogenic transformation of the thyroid C-cell is thought to progress through a hyperplastic process prior to malignancy with increasing levels of serum calcitonin serving as a biomarker for tumor burden. The discovery that multiple endocrine neoplasia type 2 is caused by activating mutations of the RET gene serves to highlight the RET-RAS-MAPK signaling pathway in both initiation and progression of medullary thyroid carcinoma (MTC). Thyroid C-cells are known to express RET at high levels relative to most cell types; therefore, aberrant activation of this receptor is targeted primarily to the C-cell, providing one possible cause of tissue-specific oncogenesis. The role of RET signaling in normal C-cell function is unknown though calcitonin gene transcription appears to be sensitive to RET activation. Beyond RET, the modeling of oncogenesis in animals and screening of human tumors for candidate gene mutations have uncovered mutation of RAS family members and inactivation of Rb1 regulatory pathway as potential mediators of C-cell transformation. A growing understanding of how RET interacts with these pathways, both in normal C-cell function and during oncogenic transformation, will help in the development of novel molecular-targeted therapies.

Constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte exit from quiescence.

Notch signaling components have long been detected in Sertoli and germ cells in the developing and mature testis. However, the role of this pathway in testis development and spermatogenesis remains unknown. Using reporter mice expressing green fluorescent protein following Notch receptor activation, we found that Notch signaling was active in Sertoli cells at various fetal, neonatal, and adult stages. Since Notch signaling specifies stem cell fate in many developing and mature organ systems, we hypothesized that maintenance and differentiation of gonocytes and/or spermatogonial stem cells would be modulated through this pathway in Sertoli cells. To this end, we generated mutant mice constitutively expressing the active, intracellular domain of NOTCH1 (NICD1) in Sertoli cells. We found that mutant Sertoli cells were morphologically normal before and after birth, but presented a number of functional changes that drastically affected gonocyte numbers and physiology. We observed aberrant exit of gonocytes from mitotic arrest, migration toward cord periphery, and premature differentiation before birth. These events, presumably unsupported by the cellular microenvironment, were followed by gonocyte apoptosis and near complete disappearance of the gonocytes by day 2 after birth. Molecular analysis demonstrated that these effects are correlated with a dysregulation of Sertoli-expressed genes that are required for germ cell maintenance, such as Cyp26b1 and Gdnf. Taken together, our results demonstrate that Notch signaling is active in Sertoli cells throughout development and that proper regulation of Notch signaling in Sertoli cells is required for the maintenance of gonocytes in an undifferentiated state during fetal development.

Stem cells and nanomaterials.

Because of their ability to self-renew and differentiate into many cell types, stem cells offer the potential to be used for tissue regeneration and engineering. Much progress has recently been made in our understanding of the biology of stem cells and our ability to manipulate their proliferation and differentiation to obtain functional tissues. Similarly, nanomaterials have been recently developed that will accelerate discovery of mechanisms driving stem cell fate and their utilization in medicine. Nanoparticles have been developed that allow the labeling and tracking of stem cells and their differentiated phenotype within an organism. Nanosurfaces are engineered that mimic the extracellular matrix to which stem cells adhere and migrate. Scaffolds made of functionalized nanofibers can now be used to grow stem cells and regenerate damaged tissues and organs. However, the small scale of nanomaterials induces changes in their chemical and physical properties that might modify their interactions with cells and tissues, and render them toxic to stem cells. Therefore a thorough understanding of stem cell-nanomaterial interactions is still necessary not only to accelerate the success of medical treatments but also to ensure the safety of the tools provided by these novel technologies.

Cell-laden hydrogels in integrated microfluidic devices for long-term cell culture and tubulogenesis assays.

A hydrogel biochip combining microfluidic mixing and orthogonal supplementation strategies is developed and validated to allow facile generation of libraries of optically transparent 3D culture microenvironments. Live, on-chip tracing of embryonic stem cell differentiation and endothelial cell tubulogenesis confirms that the platform can be used to both create communities of discrete 3D microenvironments as well as to locally monitor subsequent divergent responses at both single cell and multi-cell scales.

Review article: new treatments for advanced differentiated thyroid cancers and potential mechanisms of drug resistance.

The treatment of advanced, radioiodine refractory, differentiated thyroid cancers (RR-DTCs) has undergone major advancements in the last decade, causing a paradigm shift in the management and prognosis of these patients. Better understanding of the molecular drivers of tumorigenesis and access to next generation sequencing of tumors have led to the development and Food and Drug Administration (FDA)-approval of numerous targeted therapies for RR-DTCs, including antiangiogenic multikinase inhibitors, and more recently, fusion-specific kinase inhibitors such as RET inhibitors and NTRK inhibitors. BRAF + MEK inhibitors have also been approved for BRAF-mutated solid tumors and are routinely used in RR-DTCs in many centers. However, none of the currently available treatments are curative, and most patients will ultimately show progression. Current research efforts are therefore focused on identifying resistance mechanisms to tyrosine kinase inhibitors and ways to overcome them. Various novel treatment strategies are under investigation, including immunotherapy, redifferentiation therapy, and second-generation kinase inhibitors. In this review, we will discuss currently available drugs for advanced RR-DTCs, potential mechanisms of drug resistance and future therapeutic avenues.

Fibronectin Contributes to a BRAF Inhibitor-driven Invasive Phenotype in Thyroid Cancer through EGR1, Which Can Be Blocked by Inhibition of ERK1/2.

Mutations in BRAF are common in advanced papillary and anaplastic thyroid cancer (PTC and ATC). However, patients with BRAF-mutant PTC currently lack therapies targeting this pathway. Despite the approved combination of BRAF and MEK1/2 inhibition for patients with BRAF-mutant ATC, these patients often progress. Thus, we screened a panel of BRAF-mutant thyroid cancer cell lines to identify new therapeutic strategies. We showed that thyroid cancer cells resistant to BRAF inhibition (BRAFi) exhibit an increase in invasion and a proinvasive secretome in response to BRAFi. Using reverse-phase protein array (RPPA), we identified a nearly 2-fold increase in expression of the extracellular matrix protein, fibronectin, in response to BRAFi treatment, and a corresponding 1.8- to 3.0-fold increase in fibronectin secretion. Accordingly, the addition of exogenous fibronectin phenocopied the BRAFi-induced increase in invasion while depletion of fibronectin in resistant cells resulted in loss of increased invasion. We further showed that BRAFi-induced invasion can be blocked by inhibition of ERK1/2. In a BRAFi-resistant patient-derived xenograft model, we found that dual inhibition of BRAF and ERK1/2 slowed tumor growth and decreased circulating fibronectin. Using RNA sequencing, we identified EGR1 as a top downregulated gene in response to combined BRAF/ERK1/2 inhibition, and we further showed that EGR1 is necessary for a BRAFi-induced increase in invasion and for induction of fibronectin in response to BRAFi. IMPLICATIONS: Together, these data show that increased invasion represents a new mechanism of resistance to BRAF inhibition in thyroid cancer that can be targeted with an ERK1/2 inhibitor.

BCL2L11 Induction Mediates Sensitivity to Src and MEK1/2 Inhibition in Thyroid Cancer.

Patients with advanced thyroid cancer, including advanced papillary thyroid cancer and anaplastic thyroid cancer (ATC), have low survival rates because of the lack of efficient therapies available that can combat their aggressiveness. A total of 90% of thyroid cancers have identifiable driver mutations, which often are components of the MAPK pathway, including BRAF, RAS, and RET-fusions. In addition, Src is a non-receptor tyrosine kinase that is overexpressed and activated in thyroid cancer, which we and others have shown is a clinically relevant target. We have previously demonstrated that combined inhibition of Src with dasatinib and the MAPK pathway with trametinib synergistically inhibits growth and induces apoptosis in BRAF- and RAS-mutant thyroid cancer cells. Herein, we identified the pro-apoptotic protein BCL2L11 (BIM) as being a key mediator of sensitivity in response to combined dasatinib and trametinib treatment. Specifically, cells that are sensitive to combined dasatinib and trametinib treatment have inhibition of FAK/Src, MEK/ERK, and AKT, resulting in the dramatic upregulation of BIM, while cells that are resistant lack inhibition of AKT and have a dampened induction of BIM. Inhibition of AKT directly sensitizes resistant cells to combined dasatinib and trametinib but will not be clinically feasible. Importantly, targeting BCL-XL with the BH3-mimeitc ABT-263 is sufficient to overcome lack of BIM induction and sensitize resistant cells to combined dasatinib and trametinib treatment. This study provides evidence that combined Src and MEK1/2 inhibition is a promising therapeutic option for patients with advanced thyroid cancer and identifies BIM induction as a potential biomarker of response.

Surgery After BRAF-Directed Therapy Is Associated with Improved Survival in BRAFV600E Mutant Anaplastic Thyroid Cancer: A Single-Center Retrospective Cohort Study.

Background: The aim of this study was to describe the oncologic outcomes of patients with BRAFV600E-mutated anaplastic thyroid cancer (ATC) who had neoadjuvant BRAF-directed therapy with subsequent surgery. For context, we also reviewed patients who received BRAF-directed therapy after surgery, and those who did not have surgery after BRAF-directed therapy. Methods: This was a single-center retrospective cohort study conducted at a tertiary care cancer center in Texas from 2017 to 2021. Fifty-seven consecutive patients with BRAFV600E-mutated ATC and at least 1 month of BRAF-directed therapy were included. Primary outcomes were overall survival (OS) and progression-free survival (PFS). Results: All patients had stage IVB (35%) or IVC (65%) ATC. Approximately 70% of patients treated with BRAF-directed therapy ultimately had surgical resection of residual disease. Patients who had neoadjuvant BRAF-directed therapy followed by surgery (n = 32) had 12-month OS of 93.6% [confidence interval (CI) 84.9-100] and PFS of 84.4% [CI 71.8-96.7]. Patients who had surgery before BRAF-directed therapy (n = 12) had 12-month OS of 74.1% [CI 48.7-99.5] and PFS of 50% [CI 21.7-78.3]. Finally, patients who did not receive surgery after BRAF-directed therapy (n = 13) had 12-month OS of 38.5% [CI 12.1-64.9] and PFS of 15.4% [CI 0-35.0]. Neoadjuvant BRAF-directed therapy reduced tumor size, extent of surgery, and surgical morbidity score. Subgroup analysis suggested that any residual ATC in the surgical specimen was associated with significantly worse 12-month OS and PFS (OS = 83.3% [CI 62.6-100], PFS = 61.5% [CI 35.1-88]) compared with patients with pathologic ATC complete response (OS = 100%, PFS = 100%). Conclusions: We observed that neoadjuvant BRAF-directed therapy reduced extent of surgery and surgical morbidity. While acknowledging potential selection bias, the 12-month OS rate appeared higher in patients who had BRAF-directed therapy followed by surgery as compared with BRAF-directed therapy without surgery; yet, it was not significantly different from surgery followed by BRAF-directed therapy. PFS appeared higher in patients treated with neoadjuvant BRAF-directed therapy relative to patients in the other groups. These promising results of neoadjuvant BRAF-directed therapy followed by surgery for BRAF-mutated ATC should be confirmed in prospective clinical trials.

The spermatogonial stem cell niche in the collared peccary (Tayassu tajacu).

In the seminiferous epithelium, spermatogonial stem cells (SSCs) are located in a particular environment called the "niche" that is controlled by the basement membrane, key testis somatic cells, and factors originating from the vascular network. However, the role of Leydig cells (LCs) as a niche component is not yet clearly elucidated. Recent studies showed that peccaries (Tayassu tajacu) present a peculiar LC cytoarchitecture in which these cells are located around the seminiferous tubule lobes, making the peccary a unique model for investigating the SSC niche. This peculiarity allowed us to subdivide the seminiferous tubule cross-sections in three different testis parenchyma regions (tubule-tubule, tubule-interstitium, and tubule-LC contact). Our aims were to characterize the different spermatogonial cell types and to determine the location and/or distribution of the SSCs along the seminiferous tubules. Compared to differentiating spermatogonia, undifferentiated spermatogonia (A(und)) presented a noticeably higher nuclear volume (P < 0.05), allowing an accurate evaluation of their distribution. Immunostaining analysis demonstrated that approximately 93% of A(und) were GDNF receptor alpha 1 positive (GFRA1(+)), and these cells were preferentially located adjacent to the interstitial compartment without LCs (P < 0.05). The expression of colony-stimulating factor 1 was observed in LCs and peritubular myoid cells (PMCs), whereas its receptor was present in LCs and in GFRA1(+) A(und). Taken together, our findings strongly suggest that LCs, different from PMCs, might play a minor role in the SSC niche and physiology and that these steroidogenic cells are probably involved in the differentiation of A(und) toward type A(1) spermatogonia.

Sertoli Cell-Germ Cell Interactions Within the Niche: Paracrine and Juxtacrine Molecular Communications.

Male germ cell development depends on multiple biological events that combine epigenetic reprogramming, cell cycle regulation, and cell migration in a spatio-temporal manner. Sertoli cells are a crucial component of the spermatogonial stem cell niche and provide essential growth factors and chemokines to developing germ cells. This review focuses mainly on the activation of master regulators of the niche in Sertoli cells and their targets, as well as on novel molecular mechanisms underlying the regulation of growth and differentiation factors such as GDNF and retinoic acid by NOTCH signaling and other pathways.

Molecular mechanisms of resistance to kinase inhibitors and redifferentiation in thyroid cancers.

Protein kinases play critical roles in cell survival, proliferation, and motility. Their dysregulation is therefore a common feature in the pathogenesis of a number of solid tumors, including thyroid cancers. Inhibiting activated protein kinases has revolutionized thyroid cancer therapy, offering a promising strategy in treating tumors refractory to radioactive iodine treatment or cytotoxic chemotherapies. However, despite satisfactory early responses, these drugs are not curative and most patients inevitably progress due to drug resistance. This review summarizes up-to-date knowledge on various mechanisms that thyroid cancer cells develop to bypass protein kinase inhibition and outlines strategies that are being explored to overcome drug resistance. Understanding how cancer cells respond to drugs and identifying novel molecular targets for therapy still represents a major challenge for the treatment of these patients.

ETV5 regulates sertoli cell chemokines involved in mouse stem/progenitor spermatogonia maintenance.

Spermatogonial stem cells are the only stem cells in the body that transmit genetic information to offspring. Although growth factors responsible for self-renewal of these cells are known, the factors and mechanisms that attract and physically maintain these cells within their microenvironment are poorly understood. Mice with targeted disruption of Ets variant gene 5 (Etv5) show total loss of stem/progenitor spermatogonia following the first wave of spermatogenesis, resulting in a Sertoli cell-only phenotype and aspermia. Microarray analysis of primary Sertoli cells from Etv5 knockout (Etv5(-/-)) versus wild-type (WT) mice revealed significant decreases in expression of several chemokines. Chemotaxis assays demonstrated that migration of stem/progenitor spermatogonia toward Etv5(-/-) Sertoli cells was significantly decreased compared to migration toward WT Sertoli cells. Interestingly, differentiating spermatogonia, spermatocytes, and round spermatids were not chemoattracted by WT Sertoli cells, whereas stem/progenitor spermatogonia showed a high and significant chemotactic index. Rescue assays using recombinant chemokines indicated that C-C-motif ligand 9 (CCL9) facilitates Sertoli cell chemoattraction of stem/progenitor spermatogonia, which express C-C-receptor type 1 (CCR1). In addition, there is protein-DNA interaction between ETV5 and Ccl9, suggesting that ETV5 might be a direct regulator of Ccl9 expression. Taken together, our data show for the first time that Sertoli cells are chemoattractive for stem/progenitor spermatogonia, and that production of specific chemokines is regulated by ETV5. Therefore, changes in chemokine production and consequent decreases in chemoattraction by Etv5(-/-) Sertoli cells helps to explain stem/progenitor spermatogonia loss in Etv5(-/-) mice.

ERM is required for transcriptional control of the spermatogonial stem cell niche.

Division of spermatogonial stem cells produces daughter cells that either maintain their stem cell identity or undergo differentiation to form mature sperm. The Sertoli cell, the only somatic cell within seminiferous tubules, provides the stem cell niche through physical support and expression of surface proteins and soluble factors. Here we show that the Ets related molecule (ERM) is expressed exclusively within Sertoli cells in the testis and is required for spermatogonial stem cell self-renewal. Mice with targeted disruption of ERM have a loss of maintenance of spermatogonial stem cell self-renewal without a block in normal spermatogenic differentiation and thus have progressive germ-cell depletion and a Sertoli-cell-only syndrome. Microarray analysis of primary Sertoli cells from ERM-deficient mice showed alterations in secreted factors known to regulate the haematopoietic stem cell niche. These results identify a new function for the Ets family transcription factors in spermatogenesis and provide an example of transcriptional control of a vertebrate stem cell niche.

Clinical Utility of Circulating Cell-Free DNA Mutations in Anaplastic Thyroid Carcinoma.

Background: Anaplastic thyroid carcinoma (ATC) is an aggressive thyroid cancer that requires a rapid diagnosis and treatment to achieve disease control. Gene mutation profiling of circulating cell-free DNA (cfDNA) in peripheral blood may help to facilitate early diagnosis and treatment selection. The relatively rapid turnaround time compared with conventional tumor mutation testing is a major advantage. The objectives of this study were to examine the concordance of ATC-related mutations detected in cfDNA with those detected in the corresponding tumor tissue, and to determine the prognostic significance of cfDNA mutations in ATC patients. Methods: The ATC patients who were diagnosed and treated at The University of Texas MD Anderson Cancer Center between January 2015 and February 2018 and who had cfDNA testing were included in this study. cfDNA was collected by blood draw and was analyzed by next-generation sequencing (NGS) using the Guardant360-73 gene platform. Results: A total of 87 patients were included in the study. The most frequently mutated genes detected in cfDNA were TP53, BRAF, and PIK3CA. In 28 treatment naive ATC patients, the concordance rate of detected mutations in TP53, BRAFV600E, and PIK3CA between cfDNA and matched tissue NGS was 82.1%, 92.9%, and 92.9%, respectively. Patients with a PIK3CA mutation detected on cfDNA had worse overall survival (OS) (p = 0.03). This association was observed across various treatment modalities, including surgery, cytotoxic chemotherapy, radiation, and BRAF inhibitor (BRAFi) therapy. With regard to treatment, BRAFi therapy significantly improved ATC OS (p = 0.003). Conclusions: cfDNA is a valuable tool to evaluate a tumor's molecular profile in ATC patients. We identified high concordance rates between the gene mutations identified via cfDNA analysis and those identified from the NGS of the corresponding tumor tissue sequencing. Identified mutations in cfDNA can potentially provide timely information to guide treatment selection and evaluate the prognosis in patients with ATC.

RAC1 Alterations Induce Acquired Dabrafenib Resistance in Association with Anaplastic Transformation in a Papillary Thyroid Cancer Patient.

BRAF-activating mutations are the most frequent driver mutations in papillary thyroid cancer (PTC). Targeted inhibitors such as dabrafenib have been used in advanced BRAF-mutated PTC; however, acquired resistance to the drug is common and little is known about other effectors that may play integral roles in this resistance. In addition, the induction of PTC dedifferentiation into highly aggressive KRAS-driven anaplastic thyroid cancer (ATC) has been reported. We detected a novel RAC1 (P34R) mutation acquired during dabrafenib treatment in a progressive metastatic lesion with ATC phenotype. To identify a potential functional link between this novel mutation and tumor dedifferentiation, we developed a cell line derived from the metastatic lesion and compared its behavior to isogenic cell lines and primary tumor samples. Our data demonstrated that RAC1 mutations induce changes in cell morphology, reorganization of F-actin almost exclusively at the cell cortex, and changes in cell adhesion properties. We also established that RAC1 amplification, with or without mutation, is sufficient to drive cell proliferation and resistance to BRAF inhibition. Further, we identified polyploidy of chromosome 7, which harbors RAC1, in both the metastatic lesion and its derived cell line. Copy number amplification and overexpression of other genes located on this chromosome, such as TWIST1, EGFR, and MET were also detected, which might also lead to dabrafenib resistance. Our study suggests that polyploidy leading to increased expression of specific genes, particularly those located on chromosome 7, should be considered when analyzing aggressive thyroid tumor samples and in further treatments.