CDK4 phosphorylation status and rational use for combining CDK4/6 and BRAF/MEK inhibition in advanced thyroid carcinomas.

Background: CDK4/6 inhibitors (CDK4/6i) have been established as standard treatment against advanced Estrogen Receptor-positive breast cancers. These drugs are being tested against several cancers, including in combinations with other therapies. We identified the T172-phosphorylation of CDK4 as the step determining its activity, retinoblastoma protein (RB) inactivation, cell cycle commitment and sensitivity to CDK4/6i. Poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinomas, the latter considered one of the most lethal human malignancies, represent major clinical challenges. Several molecular evidence suggest that CDK4/6i could be considered for treating these advanced thyroid cancers. Methods: We analyzed by two-dimensional gel electrophoresis the CDK4 modification profile and the presence of T172-phosphorylated CDK4 in a collection of 98 fresh-frozen tissues and in 21 cell lines. A sub-cohort of samples was characterized by RNA sequencing and immunohistochemistry. Sensitivity to CDK4/6i (palbociclib and abemaciclib) was assessed by BrdU incorporation/viability assays. Treatment of cell lines with CDK4/6i and combination with BRAF/MEK inhibitors (dabrafenib/trametinib) was comprehensively evaluated by western blot, characterization of immunoprecipitated CDK4 and CDK2 complexes and clonogenic assays. Results: CDK4 phosphorylation was detected in all well-differentiated thyroid carcinomas (n=29), 19/20 PDTC, 16/23 ATC and 18/21 thyroid cancer cell lines, including 11 ATC-derived ones. Tumors and cell lines without phosphorylated CDK4 presented very high p16CDKN2A levels, which were associated with proliferative activity. Absence of CDK4 phosphorylation in cell lines was associated with CDK4/6i insensitivity. RB1 defects (the primary cause of intrinsic CDK4/6i resistance) were not found in 5/7 tumors without detectable phosphorylated CDK4. A previously developed 11-gene expression signature identified the likely unresponsive tumors, lacking CDK4 phosphorylation. In cell lines, palbociclib synergized with dabrafenib/trametinib by completely and permanently arresting proliferation. These combinations prevented resistance mechanisms induced by palbociclib, most notably Cyclin E1-CDK2 activation and a paradoxical stabilization of phosphorylated CDK4 complexes. Conclusion: Our study supports further clinical evaluation of CDK4/6i and their combination with anti-BRAF/MEK therapies as a novel effective treatment against advanced thyroid tumors. Moreover, the complementary use of our 11 genes predictor with p16/KI67 evaluation could represent a prompt tool for recognizing the intrinsically CDK4/6i insensitive patients, who are potentially better candidates to immediate chemotherapy.

Unraveling the Roles of miR-204-5p and HMGA2 in Papillary Thyroid Cancer Tumorigenesis.

Thyroid cancer is the most common endocrine malignant tumor with an increasing incidence rate. Although differentiated types of thyroid cancer generally present good clinical outcomes, some dedifferentiate into aggressive and lethal forms. However, the molecular mechanisms governing aggressiveness and dedifferentiation are still poorly understood. Aberrant expression of miRNAs is often correlated to tumor development, and miR-204-5p has previously been identified in papillary thyroid carcinoma as downregulated and associated with aggressiveness. This study aimed to explore its role in thyroid tumorigenesis. To address this, gain-of-function experiments were performed by transiently transfecting miR-204-5p in thyroid cancer cell lines. Then, the clinical relevance of our data was evaluated in vivo. We prove that this miRNA inhibits cell invasion by regulating several targets associated with an epithelial-mesenchymal transition, such as SNAI2, TGFBR2, SOX4 and HMGA2. HMGA2 expression is regulated by the MAPK pathway but not by the PI3K, IGF1R or TGFß pathways, and the inhibition of cell invasion by miR-204-5p involves direct binding and repression of HMGA2. Finally, we confirmed in vivo the relationship between miR-204-5p and HMGA2 in human PTC and a corresponding mouse model. Our data suggest that HMGA2 inhibition offers promising perspectives for thyroid cancer treatment.

Dynamic Cancer Cell Heterogeneity: Diagnostic and Therapeutic Implications.

Though heterogeneity of cancers is recognized and has been much discussed in recent years, the concept often remains overlooked in different routine examinations. Indeed, in clinical or biological articles, reviews, and textbooks, cancers and cancer cells are generally presented as evolving distinct entities rather than as an independent heterogeneous cooperative cell population with its self-oriented biology. There are, therefore, conceptual gaps which can mislead the interpretations/diagnostic and therapeutic approaches. In this short review, we wish to summarize and discuss various aspects of this dynamic evolving heterogeneity and its biological, pathological, clinical, diagnostic, and therapeutic implications, using thyroid carcinoma as an illustrative example.

Thyroid cancer under the scope of emerging technologies.

The vast majority of thyroid cancers originate from follicular cells. We outline outstanding issues at each step along the path of cancer patient care, from prevention to post-treatment follow-up and highlight how emerging technologies will help address them in the coming years. Three directions will dominate the coming technological landscape. Genomics will reveal tumoral evolutionary history and shed light on how these cancers arise from the normal epithelium and the genomics alteration driving their progression. Transcriptomics will gain cellular and spatial resolution providing a full account of intra-tumor heterogeneity and opening a window on the microenvironment supporting thyroid tumor growth. Artificial intelligence will set morphological analysis on an objective quantitative ground laying the foundations of a systematic thyroid tumor classification system. It will also integrate into unified representations the molecular and morphological perspectives on thyroid cancer.

MiR-7-5p inhibits thyroid cell proliferation by targeting the EGFR/MAPK and IRS2/PI3K signaling pathways.

The aberrant expression of miRNAs is often correlated to tumor development. MiR-7-5p is a recently discovered downregulated miRNA in thyroid papillary carcinoma (PTC). The goal of this project was to characterize its functional role in thyroid tumorigenesis and to identify the targeted modulated pathways. MiR-7-5p overexpression following transfection in TPC1 and HT-ori3 cells decreased proliferation of the two thyroid cell lines. Analysis of global transcriptome modifications showed that miR-7-5p inhibits thyroid cell proliferation by modulating the MAPK and PI3K signaling pathways which are both necessary for normal thyroid proliferation and play central roles in PTC tumorigenesis. Several effectors of these pathways are indeed targets of miR-7-5p, among which EGFR and IRS2, two upstream activators. We confirmed the upregulation of IRS2 and EGFR in human PTC and showed the existence of a negative correlation between the decreased expression of miR-7-5p and the increased expression of IRS2 or EGFR. Our results thus support a tumor-suppressor activity of miR-7-5p. The decreased expression of miR-7-5p during PTC tumorigenesis might give the cells a proliferative advantage and delivery of miR-7-5p may represent an innovative approach for therapy.

Dissecting the Role of Thyrotropin in the DNA Damage Response in Human Thyrocytes after 131I, γ Radiation and H2O2.

BACKGROUND: The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. METHODS: Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (ß radiation), γ radiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. RESULTS: Overall, the thyrocyte responses following exposure to ß or γ radiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα q cascade and a rise in intracellular reactive oxygen species (ROS) levels. ß and γ radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to ß/γ radiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. CONCLUSIONS: TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I.

Reprogramming of Energy Metabolism: Increased Expression and Roles of Pyruvate Carboxylase in Papillary Thyroid Cancer.

Background: Energy metabolism is described to be deregulated in cancer, and the Warburg effect is considered to be a major hallmark. Recently, cellular heterogeneity in tumors and the tumor microenvironment has been recognized to play an important role in several metabolic pathways in cancer. However, its contribution to papillary thyroid cancer (PTC) development and metabolism is still poorly understood. Methods: A proteomic analysis of five PTC was performed, and the cellular distribution of several upregulated metabolic proteins was investigated in the cancerous and stromal cells of these tumors. Results: Tandem mass spectrometry analysis revealed the upregulation of many metabolism-related proteins, among them pyruvate carboxylase (PC). PC knockdown in thyroid cell lines alters their proliferative and motility capacities, and measurements of oxygen consumption rates show that this enzyme is involved in the replenishment of the tricarboxylic acid cycle. Immunostainings of several upregulated metabolic proteins show that thyroid cancer cells have an increased mitochondrial oxidative metabolism compared to stromal cells. Conclusions: PTC has a very active tricarboxylic acid cycle, continuously replenished by a PC-mediated anaplerosis. This is specifically observed in the tumor cells.

Thyroid follicular adenomas and carcinomas: molecular profiling provides evidence for a continuous evolution.

Non-autonomous thyroid nodules are common in the general population with a proportion found to be cancerous. A current challenge in the field is to be able to distinguish benign adenoma (FA) from preoperatively malignant thyroid follicular carcinoma (FTC), which are very similar both histologically and genetically. One controversial issue, which is currently not understood, is whether both tumor types represent different molecular entities or rather a biological continuum. To gain a better insight into FA and FTC tumorigenesis, we defined their molecular profiles by mRNA and miRNA microarray. Expression data were analyzed, validated by qRT-PCR and compared with previously published data sets. The majority of deregulated mRNAs were common between FA and FTC and were downregulated, however FTC showed additional deregulated mRNA. Both types of tumors share deregulated pathways, molecular functions and biological processes. The additional deregulations in FTC include the lipid transport process that may be involved in tumor progression. The strongest candidate genes which may be able to discriminate follicular adenomas and carcinomas, CRABP1, FABP4 and HMGA2, were validated in independent samples by qRT-PCR and immunohistochemistry. However, they were not able to adequately classify FA or FTC, supporting the notion of continuous evolving tumors, whereby FA and FTC appear to show quantitative rather than qualitative changes. Conversely, miRNA expression profiles showed few dysregulations in FTC, and even fewer in FA, suggesting that miRNA play a minor, if any, role in tumor progression.

Distinctive Desmoplastic 3D Morphology Associated With BRAFV600E in Papillary Thyroid Cancers.

Context: Although 60% of papillary thyroid carcinomas are BRAFV600E mutant (PTCV600E), the increased aggressiveness of these cancers is still debated. Objective: For PTCV600E we aimed to further characterize the extent of the stroma and its activation, the three-dimensional (3D) tumor-stroma interface, and the proliferation rates of tumor and stromal fibroblasts. Design: We analyzed exomes, transcriptomes, and images of 364 papillary thyroid carcinoma (PTCs) from The Cancer Genome Atlas (TCGA), including 211 PTCV600E; stained 22 independent PTCs for BRAFV600E and Ki67; sequenced the exomes and stained BRAFV600E in 5 primary tumor blocks and 4 nodal metastases from one patient with PTCV600E; and reconstructed the 3D volumes of one tumor and one metastatic block at histological resolution. Results: In TCGA, BRAFV600E was associated with higher expression of proliferation markers and lower expression of thyroid differentiation markers, independently of tumor purity. Moreover, PTCV600E, in line with their overall lower purity, also had higher expression of fibroblast- and T cell-associated genes and presented more fibrosis. Tumor cells that appeared disconnected on two-dimensional histological slices were revealed to be part of a unique tumor component in the 3D reconstructed microvolumes, and they formed a surprisingly complex connected space, infiltrating a proliferative stroma. Finally, in our PTC set, both stromal fibroblasts and tumor cells presented higher proliferation rates in PTCV600E. Conclusions: Our results support the increased aggressiveness associated with BRAFV600E in PTC and shed light on the important role of the stroma in tumor expansion. The greater and more active fibrotic component predicts better efficiency of combined targeted treatments, as previously proposed for melanomaV600E.

miRNA expression and function in thyroid carcinomas: a comparative and critical analysis and a model for other cancers.

As in many cancer types, miRNA expression profiles and functions have become an important field of research on non-medullary thyroid carcinomas, the most common endocrine cancers. This could lead to the establishment of new diagnostic tests and new cancer therapies. However, different studies showed important variations in their research strategies and results. In addition, the action of miRNAs is poorly considered as a whole because of the use of underlying dogmatic truncated concepts. These lead to discrepancies and limits rarely considered. Recently, this field has been enlarged by new miRNA functional and expression studies. Moreover, studies using next generation sequencing give a new view of general miRNA differential expression profiles of papillary thyroid carcinoma. We analyzed in detail this literature from both physiological and differential expression points of view. Based on explicit examples, we reviewed the progresses but also the discrepancies and limits trying to provide a critical approach of where this literature may lead. We also provide recommendations for future studies. The conclusions of this systematic analysis could be extended to other cancer types.

ApolipoproteinL1 is expressed in papillary thyroid carcinomas.

The apolipoprotein L (apoL) family has not yet been ascribed any definite patho-physiological function although the conserved BH3 protein domain suggests a role in programmed cell death. As repression of the regular apoptotic program is considered a hallmark of tumor progression, we investigated apoL expression in cancer. We show that the levels of one member of the family, apolipoprotein L1 (apoL1) is higher in papillary thyroid carcinoma compared to normal tissue. A combination of qRTPCR, immunohistochemistry and in situ hybridization allowed us to ascribe this increase to endogenous overexpression in carcinoma cells. Whether apoL1 plays an instrumental role in refraining cell death is the subject of ongoing molecular biology experiments.

Gene signature of the post-Chernobyl papillary thyroid cancer.

PURPOSE: Following the nuclear accidents in Chernobyl and later in Fukushima, the nuclear community has been faced with important issues concerning how to search for and diagnose biological consequences of low-dose internal radiation contamination. Although after the Chernobyl accident an increase in childhood papillary thyroid cancer (PTC) was observed, it is still not clear whether the molecular biology of PTCs associated with low-dose radiation exposure differs from that of sporadic PTC. METHODS: We investigated tissue samples from 65 children/young adults with PTC using DNA microarray (Affymetrix, Human Genome U133 2.0 Plus) with the aim of identifying molecular differences between radiation-induced (exposed to Chernobyl radiation, ECR) and sporadic PTC. All participants were resident in the same region so that confounding factors related to genetics or environment were minimized. RESULTS: There were small but significant differences in the gene expression profiles between ECR and non-ECR PTC (global test, p < 0.01), with 300 differently expressed probe sets (p < 0.001) corresponding to 239 genes. Multifactorial analysis of variance showed that besides radiation exposure history, the BRAF mutation exhibited independent effects on the PTC expression profile; the histological subset and patient age at diagnosis had negligible effects. Ten genes (PPME1, HDAC11, SOCS7, CIC, THRA, ERBB2, PPP1R9A, HDGF, RAD51AP1, and CDK1) from the 19 investigated with quantitative RT-PCR were confirmed as being associated with radiation exposure in an independent, validation set of samples. CONCLUSION: Significant, but subtle, differences in gene expression in the post-Chernobyl PTC are associated with previous low-dose radiation exposure.

New global analysis of the microRNA transcriptome of primary tumors and lymph node metastases of papillary thyroid cancer.

BACKGROUND: Papillary Thyroid Cancer (PTC) is the most prevalent type of endocrine cancer. Its incidence has rapidly increased in recent decades but little is known regarding its complete microRNA transcriptome (miRNome). In addition, there is a need for molecular biomarkers allowing improved PTC diagnosis. METHODS: We performed small RNA deep-sequencing of 3 PTC, their matching normal tissues and lymph node metastases (LNM). We designed a new bioinformatics framework to handle each aspect of the miRNome: whole expression profiles, isomiRs distribution, non-templated additions distributions, RNA-editing or mutation. Results were validated experimentally by qRT-PCR on normal samples, tumors and LNM from 14 independent patients and in silico using the dataset from The Cancer Genome Atlas (small RNA deepsequencing of 59 normal samples, 495 PTC, and 8 LNM). RESULTS: We performed small RNA deep-sequencing of 3 PTC, their matching normal tissues and lymph node metastases (LNM). We designed a new bioinformatics framework to handle each aspect of the miRNome: whole expression profiles, isomiRs distribution, non-templated additions distributions, RNA-editing or mutation. Results were validated experimentally by qRT-PCR on normal samples, tumors and LNM from 14 independent patients and in silico using the dataset from The Cancer Genome Atlas (small RNA deep-sequencing of 59 normal samples, 495 PTC, and 8 LNM). We confirmed already described up-regulations of microRNAs in PTC, such as miR-146b-5p or miR-222-3p, but we also identified down-regulated microRNAs, such as miR-7-5p or miR-30c-2-3p. We showed that these down-regulations are linked to the tumorigenesis process of thyrocytes. We selected the 14 most down-regulated microRNAs in PTC and we showed that they are potential biomarkers of PTC samples. Nevertheless, they can distinguish histological classical variants and follicular variants of PTC in the TCGA dataset. In addition, 12 of the 14 down-regulated microRNAs are significantly less expressed in aggressive PTC compared to non-aggressive PTC. We showed that the associated aggressive expression profile is mainly due to the presence of the BRAF V600E mutation. In general, primary tumors and LNM presented similar microRNA expression profiles but specific variations like the down-regulation of miR-7-2-3p and miR-30c-2-3p in LNM were observed. Investigations of the 5p-to-3p arm expression ratios, non-templated additions or isomiRs distributions revealed no major implication in PTC tumorigenesis process or LNM appearance. CONCLUSIONS: Our results showed that down-regulated microRNAs can be used as new potential common biomarkers of PTC and to distinguish main subtypes of PTC. MicroRNA expressions can be linked to the development of LNM of PTC. The bioinformatics framework that we have developed can be used as a starting point for the global analysis of any microRNA deep-sequencing data in an unbiased way.

microRNA expression in autonomous thyroid adenomas: Correlation with mRNA regulation.

The objective of the study was to identify the deregulated miRNA in autonomous adenoma and to correlate the data with mRNA regulation. Seven autonomous adenoma with adjacent healthy thyroid tissues were investigated. Twelve miRNAs were downregulated and one was upregulated in the tumors. Combining bioinformatic mRNA target prediction and microarray data on mRNA regulations allowed to identify mRNA targets of our deregulated miRNAs. A large enrichment in mRNA encoding proteins involved in extracellular matrix organization and different phosphodiesterases were identified among these putative targets. The direct interaction between miR-101-3p and miR-144-3p and PDE4D mRNA was experimentally validated. The global miRNA profiles were not greatly modified, confirming the definition of these tumors as minimal deviation tumors. These results support a role for miRNA in the regulation of extracellular matrix proteins and tissue remodeling occurring during tumor development, and in the important negative feedback of the cAMP pathway, which limits the consequences of its constitutive activation in these tumors.

Intratumor heterogeneity and clonal evolution in an aggressive papillary thyroid cancer and matched metastases.

The contribution of intratumor heterogeneity to thyroid metastatic cancers is still unknown. The clonal relationships between the primary thyroid tumors and lymph nodes (LN) or distant metastases are also poorly understood. The objective of this study was to determine the phylogenetic relationships between matched primary thyroid tumors and metastases. We searched for non-synonymous single-nucleotide variants (nsSNVs), gene fusions, alternative transcripts, and loss of heterozygosity (LOH) by paired-end massively parallel sequencing of cDNA (RNA-Seq) in a patient diagnosed with an aggressive papillary thyroid cancer (PTC). Seven tumor samples from a stage IVc PTC patient were analyzed by RNA-Seq: two areas from the primary tumor, four areas from two LN metastases, and one area from a pleural metastasis (PLM). A large panel of other thyroid tumors was used for Sanger sequencing screening. We identified seven new nsSNVs. Some of these were early events clonally present in both the primary PTC and the three matched metastases. Other nsSNVs were private to the primary tumor, the LN metastases and/or the PLM. Three new gene fusions were identified. A novel cancer-specific KAZN alternative transcript was detected in this aggressive PTC and in dozens of additional thyroid tumors. The PLM harbored an exclusive whole-chromosome 19 LOH. We have presented the first, to our knowledge, deep sequencing study comparing the mutational spectra in a PTC and both LN and distant metastases. This study has yielded novel findings concerning intra-tumor heterogeneity, clonal evolution and metastases dissemination in thyroid cancer.

MiRNA expression may account for chronic but not for acute regulation of mRNA expression in human thyroid tumor models.

BACKGROUND: For thyroid tumorigenesis, two main human in vitro models are available: primary cultures of human thyrocytes treated with TSH or EGF/serum as models for autonomous adenomas (AA) or papillary thyroid carcinomas (PTC) respectively, and human thyroid tumor derived cell lines. Previous works of our group have assessed properties of those models, with a special emphasis on mRNA regulations. It is often assumed that miRNA may be one of the primary events inducing these mRNA regulations. METHODS: The purpose of this study was to investigate the representativity of those models to study microRNA regulations and their relation with mRNA expression. To achieve this aim, the miRNA expressions profiles of primary cultures treated with TSH or EGF/serum and of 6 thyroid cancer cell lines were compared to the expression profiles of 35 tumor tissues obtained by microarrays. RESULTS: Our data on primary cultures have shown that the TSH or EGF/serum treatment did not greatly modify the microRNA expression profiles, which is contrary to what is observed for mRNA expression profiles, although they still evolved differently according to the treatment. The analysis of miRNA and mRNA expressions profiles in the cell lines has shown that they have evolved into a common, dedifferentiated phenotype, closer to ATC than to the tumors they are derived from. CONCLUSIONS: Long-terms TSH or EGF/serum treatments do not mimic AA or PTC respectively in terms of miRNA expression as they do for mRNA, suggesting that the regulations of mRNA expression induced by these physiological agents occur independently of miRNA. The general patterns of miRNA expression in the cell lines suggest that they represent a useful model for undifferentiated thyroid cancer. Mirna probably do not mediate the rapid changes in gene expression in rapid cell biology regulation.

miRNA expression in anaplastic thyroid carcinomas.

Anaplastic thyroid carcinoma (ATC) is the most lethal form of thyroid neoplasia and represents an end stage of thyroid tumor progression. No effective treatment exists so far. In this study, we analyzed the miRNA expression profiles of 11 ATC by microarrays and their relationship with the mRNA expression profiles of the same 11 ATC samples. ATC show distinct miRNA expression profiles compared to other less aggressive thyroid tumor types. ATC show 18 commonly deregulated miRNA compared to normal thyroid tissue (17 downregulated and 1 upregulated miRNA). First, the analysis of a combined approach of the mRNA gene expression and of the bioinformatically predicted mRNA targets of the deregulated miRNA suggested a role for these regulations in the epithelial to mesenchymal transition (EMT) process in ATC. Second, the direct interaction between one of the upregulated mRNA target, the LOX gene which is an EMT key player, and a downregulated miRNA, the miR-29a, was experimentally validated by a luciferase assay in HEK cell. Third, we confirmed that the ATC tissue is composed of about 50% of tumor associated macrophages (TAM) and suggested, by taking into account our data and published data, their most likely direct or paracrine intercommunication between them and the thyroid tumor cells, amplifying the tumor aggressiveness. Finally, we demonstrated by in situ hybridization a specific thyrocyte localization of 3 of the deregulated miRNA: let-7g, miR-29a and miR-30e and we pointed out the importance of identifying the cell type localization before drawing any conclusion on the physiopathological role of a given gene.

Cancer stem cells, a fuzzy evolving concept: a cell population or a cell property?

The cancer stem cells (CSC) hypothesis represents a pathological extrapolation of the physiological concept of embryonic and somatic stem cells. In its initial definition, it encompassed the hypothesis of a qualitatively distinct population of immortal cancer cells originating from somatic stem cells, which generate in xenotransplants by a deterministic irreversible process, the hierarchy of more differentiated finite lifespan derived cells, which constitute, themselves, the bulk of the cancer. These CSC would express specific biomarkers and gene expressions related to chemo- and radioresistance, stemness, epithelial-mesenchymal transition, etc. No convincing congruence of several of these properties in one cell population has been demonstrated. The concept has greatly evolved with time and with different authors ("the plasticity of cancer stem cells"), leading to a minimal definition of cells generating a hierarchy of derived cells. In this article these concepts are analyzed. It is proposed that stemness is a property, more or less reversible, a hallmark of some cells at some time in a cancer cell population, as immortality, dormancy, chemo- or radioresistance, epithelial-mesenchymal transition etc. These phenotypic properties represent the result of independent, linked, or more or less congruent, genetic, epigenetic, or signaling programs.

5-aza-2'-deoxycytidine has minor effects on differentiation in human thyroid cancer cell lines, but modulates genes that are involved in adaptation in vitro.

BACKGROUND: In thyroid cancer, the lack of response to specific treatment, for example, radioactive iodine, can be caused by a loss of differentiation characteristics of tumor cells. It is hypothesized that this loss is due to epigenetic modifications. Therefore, drugs releasing epigenetic repression have been proposed to reverse this silencing. METHODS: We investigated which genes were reinduced in dedifferentiated human thyroid cancer cell lines when treated with the demethylating agent 5-aza-2'-deoxycytidine (5-AzadC) and the histone deacetylase inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid, by using reverse transcriptase-polymerase chain reaction and microarrays. These results were compared to the expression patterns in in vitro human differentiated thyrocytes and in in vivo dedifferentiated thyroid cancers. In addition, the effects of 5-AzadC on DNA quantities and cell viability were investigated. RESULTS: Among the canonical thyroid differentiation markers, most were not, or only to a minor extent, re-expressed by 5-AzadC, whether or not combined with TSA or forskolin, an inducer of differentiation in normal thyrocytes. Furthermore, 5-AzadC-modulated overall mRNA expression profiles showed only few commonly regulated genes compared to differentiated cultured primary thyrocytes. In addition, most of the commonly strongly 5-AzadC-induced genes in cell lines were either not regulated or upregulated in anaplastic thyroid carcinomas. Further analysis of which genes were induced by 5-AzadC showed that they were involved in pathways such as apoptosis, antigen presentation, defense response, and cell migration. A number of these genes had similar expression responses in 5-AzadC-treated nonthyroid cell lines. CONCLUSIONS: Our results suggest that 5-AzadC is not a strong inducer of differentiation in thyroid cancer cell lines. Under the studied conditions and with the model used, 5-AzadC treatment does not appear to be a potential redifferentiation treatment for dedifferentiated thyroid cancer. However, this may reflect primarily the inadequacy of the model rather than that of the treatment. Moreover, the observation that 5-AzadC negatively affected cell viability in cell lines could still suggest a therapeutic opportunity. Some of the genes that were modulated by 5-AzadC were also induced in nonthyroid cancer cell lines, which might be explained by an epigenetic modification resulting in the adaptation of the cell lines to their culture conditions.