Differential diagnosis of thyroid cancer and benign nodules is still one of the most challenging issues in the field of endocrinology. To overcome overdiagnosis of papillary thyroid carcinomas (PTC) and the consecutive overtreatment of multinodular diseases, the search for easily accessible, sensitive and accurate biomarkers is critical. Several micro-RNAs (miRNAs) freely circulating in peripheral blood or enclosed in extracellular vesicles (EVs) have been proposed as potential biomarkers from non-invasive liquid biopsies. However, protocols are rarely comparable and conflicting data exist in the literature. In this work, we aimed to assess the diagnostic value of six micro-RNAs by comparing their expression in thyroid tissue to their abundance in bulk plasma and in plasma-EVs, before and after thyroid surgery. Plasma-EVs were isolated using a sequential density- and size-based fractionation, followed by in-depth characterization, confirming EV purity. Micro-RNA levels were measured by RT-qPCR in thyroid tissue, plasma and plasma-EVs. Among the six candidates, only miR-146b-5p and miR-21a-5p displayed a significant differential abundance in purified plasma-derived EVs from patients with PTC and benign disease. However, no difference could be demonstrated in bulk plasma through our cohort of patients. Overall, our work supports the use of a well-defined protocol of plasma-EV miRNAs purification for biomarker discovery, rather than the use of freely circulating miRNAs in bulk plasma. Our work also demonstrates that standardized pre-analytical and analytical procedures as well as optimized EV-miRNAs detection methods are essential.
Ophélie Delcorte, Julie Craps, Siam Mahibullah, Catherine Spourquet, Ludovic D’Auria, Patrick Van Der Smissen, Chantal Dessy, Etienne Marbaix, Michel Mourad, and Christophe E Pierreux
Feng Xu, Yali Ling, Jingjing Yuan, Qin Zeng, Lusha Li, Dexing Dai, Xuedi Xia, Ruoman Sun, Ran Zhang, and Zhongjian Xie
Differentiated thyroid carcinoma (DTC) is the most common endocrine malignancy and highly expresses the receptor for 1,25-dihydroxyvitamin D (1,25(OH)2D). However, it is unclear whether 1,25(OH)2D regulates DTC proliferation and differentiation. Here, we found that 1,25(OH)2D3 inhibited proliferation but not differentiation of the DTC cells. Notably, CYP27B1was elevated in DTC cells and 25-hydroxyvitamin D3 (25(OH)D3) reduced DTC cell proliferation. Knockdown of VDR did not affect the anti-proliferative effects of 1,25(OH)2D3. However, knockdown of CCAAT enhancer-binding protein β (C/EBPβ)abolished 1,25(OH)2D3-suppressed DTC cell proliferation. In addition, 1,25(OH)2D3 induced phosphorylation and translocation of C/EBPβto the nucleus from the cytoplasm. However, inhibition of p38 mitogen-activated protein kinases (MAPK) abrogated 1,25(OH)2D3-induced phosphorylation and nuclear translocation of C/EBPβas well as 1,25(OH)2D3-suppressed DTC cell proliferation. Knockdown of C/EBPβreduced the expression of Notch3. Knockdown of Notch3 blocked 1,25(OH)2D3-suppressed DTC cell proliferation. In the DTC cell-derived xenograft SCID mouse, knockdown of C/EBPβmarkedly increased tumor growth and proliferation and decreased apoptosis. In DTC patients, C/EBPβwas predominantly located in the cytoplasm of DTC cells in the tumor tissue when compared with adjacent non-cancerous tissue in which C/EBPβis located in the nucleus. In conclusion, C/EBPβstimulated Notch3signaling via the p38 MAPK-dependent pathway mediates the inhibitory effect of 1,25(OH)2D on DTC cell proliferation.
Yeon-Sook Choi, Hyemi Kwon, Mi-Hyeon You, Tae Yong Kim, Won Bae Kim, Young Kee Shong, Min Ji Jeon, and Won Gu Kim
Dabrafenib is a BRAF kinase inhibitor approved for treatment of BRAF-mutated anaplastic thyroid carcinoma (ATC) in combination with trametinib. Erlotinib is a tyrosine kinase inhibitor of EGF receptor (EGFR). We evaluated effects of dabrafenib and erlotinib combination treatment on ATC cells in vitro and in vivo. Cell proliferation, colony formation, apoptosis, and migration of ATC cells harboring a BRAF mutation (BHT101, 8505C, and SW1736) were evaluated after treatment with dabrafenib in combination with erlotinib or trametinib. The changes in activation of mitogen extracellular kinase (MEK) and extracellular signal-related kinase (ERK) signaling were also evaluated by Western blot analysis. Effects of these combinations were also evaluated using an in vivo xenograft model. First, we detected EGFR activation in dabrafenib-resistant SW1736 cells using a phospho-receptor tyrosine kinase array. A dabrafenib and erlotinib combination synergistically inhibited cell proliferation, colony formation, and migration, with an induction of apoptotic cell death in all three ATC cells, compared with dabrafenib or erlotinib alone. This synergistic effect was comparable with a dabrafenib and trametinib combination. The dabrafenib and erlotinib combination effectively inhibited phosphorylated (p)-MEK, p-ERK, and p-EGFR expressions compared with dabrafenib or erlotinib alone, while the dabrafenib and trametinib combination only inhibited p-MEK and p-ERK expressions. The dabrafenib with erlotinib or trametinib combinations also significantly suppressed tumor growth and induced apoptosis in a BHT101 xenograft model. The dabrafenib and erlotinib combination could be a potential novel treatment regimen to overcome drug resistance to dabrafenib alone in patients with BRAF-mutated ATC.
Matthew H Kulke, Fang-Shu Ou, Donna Niedzwiecki, Lucas Huebner, Pamela Kunz, Hagen F Kennecke, Edward M Wolin, Jennifer A Chan, Eileen M O’Reilly, Jeffrey A Meyerhardt, and Alan Venook
Treatment with the MTOR inhibitor everolimus improves progression-free survival (PFS) in pancreatic neuroendocrine tumors (pNETs), but it is not known if the addition of a VEGF pathway inhibitor to an MTOR inhibitor enhances antitumor activity. We performed a randomized phase II study evaluating everolimus with or without bevacizumab in patients with advanced pNETs. One hundred and fifty patients were randomized to receive everolimus 10 mg daily with or without bevacizumab 10 mg/kg i.v. every 2 weeks. Patients also received standard dose of octreotide in both arms. The primary endpoint was PFS, based on local investigator review. Treatment with the combination of everolimus and bevacizumab resulted in improved progression-free survival compared to everolimus (16.7 months compared to 14.0 months; one-sided stratified log-rank P = 0.1028; hazard ratio (HR) 0.80 (95% CI 0.56–1.13)), meeting the predefined primary endpoint. Confirmed tumor responses were observed in 31% (95% CI 20%, 41%) of patients receiving combination therapy, as compared to only 12% (95% CI 5%, 19%) of patients receiving treatment with everolimus (P = 0.0053). Median overall survival duration was similar in the everolimus and combination arm (42.5 and 42.1 months, respectively). Treatment-related toxicities were more common in the combination arm. In summary, treatment with everolimus and bevacizumab led to superior PFS and higher response rates compared to everolimus in patients with advanced pNETs. Although the higher rate of treatment-related adverse events may limit the use of this combination, our results support the continued evaluation of VEGF pathway inhibitors in pNETs.
Milagros Peña-Zanoni, Erika Yanil Faraoni, Alejandra Abeledo-Machado, Pablo Anibal Perez, Carla Agustina Marcial López, María Andrea Camilletti, Silvina Gutierrez, Susana B Rulli, and Graciela Diaz-Torga
Among pituitary adenomas, prolactinomas are the most frequently diagnosed (about 50%). Dopamine agonists are generally effective in the treatment of prolactinomas. However, a subset of about 25% of patients does not respond to these agents. The management of drug-resistant prolactinomas remains a challenge for endocrinologists and new inhibitory treatments are needed. Pituitary activins inhibit lactotroph function. Its expression and action were found reduced in animal models of lactotroph hyperplasia (female mice overexpressing the B subunit of the human chorionic gonadotrophin and female mice knockout for dopamine receptor type 2). In these models, an oophorectomy avoids prolactinoma development. Hormonal replacement with oestradiol and/or progesterone is not enough to reach the tumor size observed in transgenic females. We postulated that the loss of gonadal inhibins after an oophorectomy contributes to prevent hyperplasia development. Here, we demonstrated that an oophorectomy at 2 months age recovers the following in adulthood: (i) pituitary activin expression, (ii) activin receptor expression specifically in lactotroph population, (iii) activin biological activity in lactotrophs with a concomitant reduction of Pit-1 expression. To summarize, when an oophorectomy is performed, inhibins are lost and the inhibitory action of pituitary activins on lactotroph population is recovered, helping to prevent lactotroph hyperplasia development. These results emphasize the importance of the inhibitory action of activins on lactotroph function, positioning activins as a good therapeutic target for the treatment of resistant prolactinomas.
Katharina Wang, Ina Schütze, Sebastian Gulde, Nicole Bechmann, Susan Richter, Jana Helm, Michael Lauseker, Julian Maurer, Astrid Reul, Gerald Spoettl, Barbara Klink, Doreen William, Thomas Knösel, Juliane Friemel, Michel Bihl, Achim Weber, Maria Fankhauser, Laura Schober, Diana Vetter, Martina Broglie Däppen, Christian G Ziegler, Martin Ullrich, Jens Pietzsch, Stefan R Bornstein, Christian Lottspeich, Matthias Kroiss, Martin Fassnacht, Vera Ursula Julia Wenter, Roland Ladurner, Constanze Hantel, Martin Reincke, Graeme Eisenhofer, Ashley B Grossman, Karel Pacak, Felix Beuschlein, Christoph J Auernhammer, Natalia S Pellegata, and Svenja Nölting
Aggressive pheochromocytomas and paragangliomas (PPGLs) are difficult to treat, and molecular targeting is being increasingly considered, but with variable results. This study investigates established and novel molecular-targeted drugs and chemotherapeutic agents for the treatment of PPGLs in human primary cultures and murine cell line spheroids. In PPGLs from 33 patients, including 7 metastatic PPGLs, we identified germline or somatic driver mutations in 79% of cases, allowing us to assess potential differences in drug responsivity between pseudohypoxia-associated cluster 1-related (n = 10) and kinase signaling-associated cluster 2-related (n = 14) PPGL primary cultures. Single anti-cancer drugs were either more effective in cluster 1 (cabozantinib, selpercatinib, and 5-FU) or similarly effective in both clusters (everolimus, sunitinib, alpelisib, trametinib, niraparib, entinostat, gemcitabine, AR-A014418, and high-dose zoledronic acid). High-dose estrogen and low-dose zoledronic acid were the only single substances more effective in cluster 2. Neither cluster 1- nor cluster 2-related patient primary cultures responded to HIF-2a inhibitors, temozolomide, dabrafenib, or octreotide. We showed particular efficacy of targeted combination treatments (cabozantinib/everolimus, alpelisib/everolimus, alpelisib/trametinib) in both clusters, with higher efficacy of some targeted combinations in cluster 2 and overall synergistic effects (cabozantinib/everolimus, alpelisib/trametinib) or synergistic effects in cluster 2 (alpelisib/everolimus). Cabozantinib/everolimus combination therapy, gemcitabine, and high-dose zoledronic acid appear to be promising treatment options with particularly high efficacy in SDHB-mutant and metastatic tumors. In conclusion, only minor differences regarding drug responsivity were found between cluster 1 and cluster 2: some single anti-cancer drugs were more effective in cluster 1 and some targeted combination treatments were more effective in cluster 2.
Sophie Moog, Betty Salgues, Yasmine Braik-Djellas, Thomas Viel, Daniel Balvay, Gwennhael Autret, Estelle Robidel, Anne-Paule Gimenez-Roqueplo, Bertrand Tavitian, Charlotte Lussey-Lepoutre, and Judith Favier
Therapies for metastatic SDHB-dependent pheochromocytoma and paraganglioma (PPGL) are limited and poorly efficient. New targeted therapies and identification of early non-invasive biomarkers of response are thus urgently needed for these patients. We characterized an in vivo allograft model of spontaneously immortalized murine chromaffin cells (imCC) with inactivation of the Sdhb gene by dynamic contrast-enhanced MRI (DCE-MRI) and 18FDG-PET. We evaluated the response to several therapies: IACS-010759 (mitochondrial respiratory chain complex I inhibitor), sunitinib (tyrosine kinase inhibitor with anti-angiogenic activity), talazoparib (poly ADP ribose polymerase (PARP) inhibitor) combined or not to temozolomide (alkylating agent), pharmacological inhibitors of HIF2a (PT2385 and PT2977 (belzutifan)) and molecular inactivation of HIF2a (imCC Sdhb−/− shHIF2a). Multimodal imaging was performed, including magnetic resonance spectroscopy (1H-MRS) to monitor the level of succinate in vivo. The allografted model of Sdhb−/− imCC reflected SDHB-deficient tumors, with increased angiogenesis and a particular avidity for 18FDG. After 14 days of treatment, IACS-010759, sunitinib and talazoparib at high doses allowed a significant reduction of the tumor volumes. In contrast to the tumor growth inhibition observed in Sdhb−/− shHIF2a imCC tumors, pharmacological inhibitors of HIF2a (PT2385 and belzutifan) showed no antitumor action in this model, alone or in combination with sunitinib. 1H-MRS, but not DCE-MRI, enabled the monitoring response to sunitinib, which was the best treatment in this study, promoting a decrease in succinate levels detected in vivo. This study paves the way for new therapeutic options and reveals a potential new early biomarker of response to treatment in SDHB-dependent PPGL.
William Beimers, Megan Braun, Kaleb Schwinefus, Keenan Pearson, Brandon Wilbanks, and Louis James Maher
A fascinating class of familial paraganglioma (PGL) neuroendocrine tumors is driven by the loss of the tricarboxylic acid (TCA) cycle enzyme succinate dehydrogenase (SDH) resulting in succinate accumulation as an oncometabolite and other metabolic derangements. Here, we exploit a Saccharomyces cerevisiae yeast model of SDH loss where accumulating succinate, and possibly reactive oxygen species, poison a dioxygenase enzyme required for sulfur scavenging. Using this model, we performed a chemical suppression screen for compounds that relieve dioxygenase inhibition. After testing 1280 pharmaceutically active compounds, we identified meclofenoxate HCl and its hydrolysis product, dimethylaminoethanol (DMAE), as suppressors of dioxygenase intoxication in SDH-loss yeast cells. We show that DMAE acts to alter metabolism so as to normalize the succinate:2-ketoglutarate ratio, improving dioxygenase function. This study raises the possibility that oncometabolite effects might be therapeutically suppressed by drugs that rewire metabolism to reduce the flux of carbon into pathological metabolic pathways.
Lucas Leite Cunha and Laura Sterian Ward
Thyroid cancer is an excellent model for studying tumor immune microenvironment, as it often shows local signs of an immune response. The tumor immune microenvironment of thyroid cancer is the heterogeneous histological space in which tumor cells coexist with host cells. The final composition of this cellular aggregate is associated with the clinical aggressiveness characteristics of the neoplasm. High-performance multiplex technologies suggest that specific genetic signatures of the tumor immune microenvironment may provide data for the delineation of a robust prognostic model. Several proposals integrate clinic, pathologic and immunological information in an attempt to translate the knowledge gained from molecular science into a more personalized approach for the treatment strategy of patients with thyroid cancer. In addition, the tumor immune microenvironment displays multiple molecular connections between cells, revealing complex crosstalk. This interesting network generates several molecular nodes that can be used as targets for immunotherapy. In this scenario, immunotherapy emerges as a promising weapon, mainly for patients with advanced thyroid cancer, both medullary and follicular cell-derived. In fact, although most patients with thyroid cancer have an excellent prognosis with current therapies, around 30% of cases evolve in an unfavorable way, leading to the urgent need to improve immunotherapy for high-risk patients. Preclinical and early clinical investigations are providing optimistic prospects, but more studies are needed to make immunotherapy a more viable and efficient tool for years to come.
Olga Karapanou, George Simeakis, Barbara Vlassopoulou, Maria Alevizaki, and Katerina Saltiki
During the last decades, the knowledge on follicular cell-derived thyroid cancer molecular biology has led to the evolution of a number of novel therapies for these tumors, mainly tyrosine kinase inhibitors. Lenvantinib, sorafenib and recently cabozantinib have been approved for differentiated thyroid cancer (DTC), while larotrectinib and entrectinib for neurotrophic-tropomyosin receptor kinase-fusion thyroid cancer. For radioiodine (RAI) refractory DTCs ongoing research aims to identify agents that may restore RAI-avidity via redifferentiation protocols (vemurafenib or dabrafenib and trametinib) or combination treatments. These treatments are based on the tumor molecular signature. The treatment with targeted therapies has changed the therapeutic strategies and the disease prognosis, however drug resistance remains the main reason for treatment failure. Thus, the understanding of both molecular pathways implicated in tumorigenesis, and tumoral escape mechanisms, are of paramount significance for the development of new therapies for DTC. The present review focuses on the molecular landscape of DTC, the approved targeted therapies as well as the mechanisms of drug resistance. Furthermore, it points to the ongoing research and the future perspectives for the development of more efficient drugs for DTC.