RESUMO
Anaplastic thyroid cancer (ATC) is a rare and lethal form of thyroid cancer that requires urgent investigation of new molecular targets involved in its aggressive biology. In this context, the overactivation of Polycomb Repressive Complex 2/EZH2, which induces chromatin compaction, is frequently observed in aggressive solid tumors, making the EZH2 methyltransferase a potential target for treatment. However, the deregulation of chromatin accessibility is yet not fully investigated in thyroid cancer. In this study, EZH2 expression was modulated by CRISPR/Cas9-mediated gene editing and pharmacologically inhibited with EZH2 inhibitor EPZ6438 alone or in combination with the MAPK inhibitor U0126. The results showed that CRISPR/Cas9-induced EZH2 gene editing reduced cell growth, migration and invasion in vitro and resulted in a 90% reduction in tumor growth when EZH2-edited cells were injected into an immunocompromised mouse model. Immunohistochemistry analysis of the tumors revealed reduced tumor cell proliferation and less recruitment of cancer-associated fibroblasts in the EZH2-edited tumors compared to the control tumors. Moreover, EZH2 inhibition induced thyroid-differentiation genes' expression and mesenchymal-to-epithelial transition (MET) in ATC cells. Thus, this study shows that targeting EZH2 could be a promising neoadjuvant treatment for ATC, as it promotes antitumoral effects in vitro and in vivo and induces cell differentiation.
Assuntos
Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Animais , Camundongos , Linhagem Celular Tumoral , Proliferação de Células , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Complexo Repressor Polycomb 2/metabolismo , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , HumanosRESUMO
Thyroid cancer is the most common endocrine malignancy, and the characterization of the genetic alterations in coding-genes that drive thyroid cancer are well consolidated in MAPK signaling. In the context of non-coding RNAs, microRNAs (miRNAs) are small non-coding RNAs that, when deregulated, cooperate to promote tumorigenesis by targeting mRNAs, many of which are proto-oncogenes and tumor suppressors. In thyroid cancer, miR-146b-5p is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of miR-146b-5p results in anti-tumoral effect. Therefore, inactivating miR-146b has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the MIR146B gene in an aggressive anaplastic thyroid cancer (ATC) cell line. For that, we designed two single-guide RNAs cloned into plasmids to direct Cas9 nickase (Cas9n) to the genomic region of the pre-mir-146b structure to target miR-146b-5p and miR-146b-3p sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n-miR-146b-GuideA-puromycin and pSp-Cas9n-miR-146b-GuideB-GFP plasmids in KTC2 cells and selected the puromycin resistant + GFP positive clones (KTC2-Cl). As a result, we observed that the ATC cell line KTC2-Cl1 showed a 60% decrease in the expression of miR-146b-5p compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the MIR146B edited sequence shows a 5 nt deletion in the miR-146b-5p region and a 1 nt deletion in the miR-146b-3p region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the MIR146B miRNA gene and reduce miR-146b-5p expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Marcação de Genes , MicroRNAs , RNA Neoplásico , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Animais , Linhagem Celular , Movimento Celular/genética , Sobrevivência Celular/genética , Xenoenxertos , Humanos , Camundongos , MicroRNAs/biossíntese , MicroRNAs/genética , Transplante de Neoplasias , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/metabolismo , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologiaRESUMO
Thyroid cancer is the most common endocrine malignancy. Although most thyroid cancer patients are successfully treated and have an excellent prognosis, a percentage of these patients will develop aggressive disease and, eventually, progress to anaplastic thyroid cancer. Since most patients with this type of aggressive thyroid carcinoma will die from the disease, new treatment strategies are urgently needed. Tumor cells live in a complex and dynamic tumor microenvironment composed of different types of stromal cells. Cancer-associated fibroblasts (CAFs) are one of the most important cell components in the tumor microenvironment of most solid tumors, including thyroid cancer. CAFs originate mainly from mesenchymal cells and resident fibroblasts that are activated and reprogrammed in response to paracrine factors and cytokines produced and released by tumor cells. Upon reprogramming, which is distinguished by the expression of different marker proteins, CAFs synthesize and secret soluble factors. The secretome of CAFs directly impacts different functions of tumor cells. This bi-directional interplay between CAFs and tumor cells within the tumor microenvironment ends up fostering tumor cancer progression. CAFs are therefore key regulators of tumor progression and represent an under-explored therapeutic target in thyroid cancer.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Carcinoma Anaplásico da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Microambiente Tumoral , Fibroblastos , Humanos , Comunicação Parácrina , Células Estromais , Neoplasias da Glândula Tireoide/patologiaRESUMO
Background: Loss of the expression of thyroid differentiation markers such as sodium iodide symporter (NIS) and, consequently, radioiodine refractoriness is observed in aggressive papillary thyroid cancer and anaplastic thyroid cancer (ATC) that may harbor the BRAFV600E mutation. Activation of the BRAFV600E oncogene in thyroid follicular cells induces the expression of the miR-17-92 cluster that comprises seven mature microRNAs (miRNAs). miRNAs are a class of endogenous small RNAs (â¼22 nt) that regulate gene expression post-transcriptionally. Indeed, miR-17-92 is overexpressed in ATC, and in silico prediction shows the potential targeting of thyroid transcription factors and tumor suppressor pathways. In this study, we aimed to investigate the role of the miR-17-92 cluster in thyroid cell differentiation and function. Methods:miR-17-92 silencing was performed using CRISPR/Cas9n-guided genomic editing of the miR-17-92 gene in the KTC2 ATC cell line, and miR-17-92 cluster or individual miRNAs were overexpressed in PCCl3 thyroid cells to evaluate the influence in thyroid cell differentiation and cell function. Results: In this study, we demonstrate that CRISPR/Cas9n gene editing of the miR-17-92 cluster results in promotion of thyroid follicular cell differentiation (NIS, thyroperoxidase, thyroglobulin, PAX8, and NKX2-1 expression) in the KTC2 ATC cell line and inhibits cell migration and proliferation by restoring transforming growth factor beta (TGF-ß) signaling pathway responsiveness. Moreover, induction of the miR-17-92 cluster in normal thyroid follicular cells strongly impairs thyroid differentiation and induces a pro-oncogenic effect by blocking TGF-ß signaling and increasing cell migration. Conclusions:miR-17-92 is a potent regulator of thyroid follicular cell differentiation, and CRISPR/Cas9n-mediated editing of the miR-17-92 gene efficiently blocks miR-17-92 expression in the KTC2 ATC cell line, resulting in improvement of thyroid differentiation. Thus, targeting miR-17-92 could provide a potential molecular approach to restoring thyroid cell differentiation and NIS expression in aggressive thyroid cancer.
Assuntos
Diferenciação Celular/fisiologia , MicroRNAs/genética , Carcinoma Anaplásico da Tireoide/genética , Células Epiteliais da Tireoide/patologia , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , MicroRNAs/metabolismo , Carcinoma Anaplásico da Tireoide/metabolismo , Carcinoma Anaplásico da Tireoide/patologia , Células Epiteliais da Tireoide/metabolismo , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologiaRESUMO
Anaplastic thyroid cancer (ATC) is one of the most aggressive human cancers. Key signal transduction pathways that regulate mitochondrial metabolism are frequently altered in ATC. Our goal was to determine the mitochondrial metabolic phenotype of ATC by studying markers of mitochondrial metabolism, specifically monocarboxylate transporter 1 (MCT1) and translocase of the outer mitochondrial membrane member 20 (TOMM20). Staining patterns of MCT1 and TOMM20 in 35 human thyroid samples (15 ATC, 12 papillary thyroid cancer [PTC], and eight non-cancerous thyroid) and nine ATC mouse orthotopic xenografts were assessed by visual and Aperio digital scoring. Staining patterns of areas involved with cancer versus areas with no evidence of cancer were evaluated independently where available. MCT1 is highly expressed in human anaplastic thyroid cancer when compared to both non-cancerous thyroid tissues and papillary thyroid cancers (P<.001 for both). TOMM20 is also highly expressed in both ATC and PTC compared to non-cancerous thyroid tissue (P<.01 for both). High MCT1 and TOMM20 expression is also found in ATC mouse xenograft tumors compared to non-cancerous thyroid tissue (P<.001). These xenograft tumors have high (13)C- pyruvate uptake. ATC has metabolic features that distinguish it from PTC and non-cancerous thyroid tissue, including high expression of MCT1 and TOMM20. PTC has low expression of MCT1 and non-cancerous thyroid tissue has low expression of both MCT1 and TOMM20. This work suggests that MCT1 blockade may specifically target ATC cells presenting an opportunity for a new drug target.