Tripartite split-GFP assay to identify selective intracellular nanobody that suppresses GTPase RHOA subfamily downstream signaling.

Strategies based on intracellular expression of artificial binding domains present several advantages over manipulating nucleic acid expression or the use of small molecule inhibitors. Intracellularly-functional nanobodies can be considered as promising macrodrugs to study key signaling pathways by interfering with protein-protein interactions. With the aim of studying the RAS-related small GTPase RHOA family, we previously isolated, from a synthetic phage display library, nanobodies selective towards the GTP-bound conformation of RHOA subfamily proteins that lack selectivity between the highly conserved RHOA-like and RAC subfamilies of GTPases. To identify RHOA/ROCK pathway inhibitory intracellular nanobodies, we implemented a stringent, subtractive phage display selection towards RHOA-GTP followed by a phenotypic screen based on F-actin fiber loss. Intracellular interaction and intracellular selectivity between RHOA and RAC1 proteins was demonstrated by adapting the sensitive intracellular protein-protein interaction reporter based on the tripartite split-GFP method. This strategy led us to identify a functional intracellular nanobody, hereafter named RH28, that does not cross-react with the close RAC subfamily and blocks/disrupts the RHOA/ROCK signaling pathway in several cell lines without further engineering or functionalization. We confirmed these results by showing, using SPR assays, the high specificity of the RH28 nanobody towards the GTP-bound conformation of RHOA subfamily GTPases. In the metastatic melanoma cell line WM266-4, RH28 expression triggered an elongated cellular phenotype associated with a loss of cellular contraction properties, demonstrating the efficient intracellular blocking of RHOA/B/C proteins downstream interactions without the need of manipulating endogenous gene expression. This work paves the way for future therapeutic strategies based on protein-protein interaction disruption with intracellular antibodies.

Uncoupling Splicing From Transcription Using Antisense Oligonucleotides Reveals a Dual Role for I Exon Donor Splice Sites in Antibody Class Switching.

Class switch recombination (CSR) changes antibody isotype by replacing Cµ constant exons with different constant exons located downstream on the immunoglobulin heavy (IgH) locus. During CSR, transcription through specific switch (S) regions and processing of non-coding germline transcripts (GLTs) are essential for the targeting of activation-induced cytidine deaminase (AID). While CSR to IgG1 is abolished in mice lacking an Iγ1 exon donor splice site (dss), many questions remain regarding the importance of I exon dss recognition in CSR. To further clarify the role of I exon dss in CSR, we first evaluated RNA polymerase II (RNA pol II) loading and chromatin accessibility in S regions after activation of mouse B cells lacking Iγ1 dss. We found that deletion of Iγ1 dss markedly reduced RNA pol II pausing and active chromatin marks in the Sγ1 region. We then challenged the post-transcriptional function of I exon dss in CSR by using antisense oligonucleotides (ASOs) masking I exon dss on GLTs. Treatment of stimulated B cells with an ASO targeting Iγ1 dss, in the acceptor Sγ1 region, or Iµ dss, in the donor Sµ region, did not decrease germline transcription but strongly inhibited constitutive splicing and CSR to IgG1. Supporting a global effect on CSR, we also observed that the targeting of Iµ dss reduced CSR to IgG3 and, to a lesser extent, IgG2b isotypes. Altogether, this study reveals that the recognition of I exon dss first supports RNA pol II pausing and the opening of chromatin in targeted S regions and that GLT splicing events using constitutive I exon dss appear mandatory for the later steps of CSR, most likely by guiding AID to S regions.

Transcriptional orchestration of mitochondrial homeostasis in a cellular model of PGC-1-related coactivator-dependent thyroid tumor.

The PGC-1 (Peroxisome proliferator-activated receptor Gamma Coactivator-1) family of coactivators (PGC-1α, PGC-1ß, and PRC) plays a central role in the transcriptional control of mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) processes. These coactivators integrate mitochondrial energy production into cell metabolism using complementary pathways. The XTC.UC1 cell line is a mitochondria-rich model of thyroid tumors whose biogenesis is almost exclusively dependent on PRC. Here we aim to propose an integrative view of the cellular pathways regulated by PRC through integration of cDNA and miRNA microarray data and chromatin immunoprecipitation results obtained from XTC.UC1 cells invalidated for PRC. This study showes that PRC induces a complex network of cellular functions interacting with at least one to five of the studied transcription factors (Estrogen Related Receptor alpha, ERR1; Nuclear-Respiratory Factors, NRF1 and NRF2; cAMP Response Element Binding, CREB; and Ying Yang, YY1). Our data confirm that ERR1 is a key partner of PRC in the regulation of mitochondrial functions and suggest a potential role of this complex in RNA processing. PRC is also involved in transcriptional regulatory complexes targeting 12 miRNAs, five of which are involved in the control of the OXPHOS process. Our findings demonstrate that the PRC coactivator can act in complex with several transcription factors and regulate miRNA expression to control the fine regulation of main metabolic functions in the cell. Therefore, in PGC-1α/ß-associated pathologies, PRC, as a metabolic sensor, may ensure mitochondrial homeostasis.

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.

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.

Estrogen-related receptor alpha modulates lactate dehydrogenase activity in thyroid tumors.

Metabolic modifications of tumor cells are hallmarks of cancer. They exhibit an altered metabolism that allows them to sustain higher proliferation rates in hostile environment outside the cell. In thyroid tumors, the expression of the estrogen-related receptor α (ERRα), a major factor of metabolic adaptation, is closely related to the oxidative metabolism and the proliferative status of the cells. To elucidate the role played by ERRα in the glycolytic adaptation of tumor cells, we focused on the regulation of lactate dehydrogenases A and B (LDHA, LDHB) and the LDHA/LDHB ratio. Our study included tissue samples from 10 classical and 10 oncocytic variants of follicular thyroid tumors and 10 normal thyroid tissues, as well as samples from three human thyroid tumor cell lines: FTC-133, XTC.UC1 and RO82W-1. We identified multiple cis-acting promoter elements for ERRα, in both the LDHA and LDHB genes. The interaction between ERRα and LDH promoters was confirmed by chromatin immunoprecipitation assays and in vitro analysis for LDHB. Using knock-in and knock-out cellular models, we found an inverse correlation between ERRα expression and LDH activity. This suggests that thyroid tumor cells may reprogram their metabolic pathways through the up-regulation of ERRα by a process distinct from that proposed by the recently revisited Warburg hypothesis.

Nitric oxide and calcium participate in the fine regulation of mitochondrial biogenesis in follicular thyroid carcinoma cells.

Members of the peroxisome proliferator-activated receptor γ coactivator-1 family (i.e. PGC-1α, PGC-1ß, and the PGC-1-related coactivator (PRC)) are key regulators of mitochondrial biogenesis and function. These regulators serve as mediators between environmental or endogenous signals and the transcriptional machinery governing mitochondrial biogenesis. The FTC-133 and RO82 W-1 follicular thyroid carcinoma cell lines, which present significantly different numbers of mitochondria, metabolic mechanisms, and expression levels of PRC and PGC-1α, may employ retrograde signaling in response to respiratory dysfunction. Nitric oxide (NO) and calcium have been hypothesized to participate in this activity. We investigated the effects of the S-nitroso-N-acetyl-DL-penicillamine-NO donor, on the expression of genes involved in mitochondrial biogenesis and cellular metabolic functions in FTC-133 and RO82 W-1 cells by measuring lactate dehydrogenase and cytochrome c oxidase (COX) activities. We studied the action of ionomycin and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM) (i.e. a calcium ionophore and a cytosolic calcium chelator) on whole genome expression and mitochondrial biogenesis in RO82 W-1 cells. COX activity and the dynamics of endoplasmic reticulum and mitochondrial networks were analyzed in regard to calcium-modulating treatments. In the FTC-133 and RO82 W-1 cells, the mitochondrial biogenesis induced by NO was mainly related to PRC expression as a retrograde mitochondrial signaling. Ionomycin diminished COX activity and negatively regulated PRC-mediated mitochondrial biogenesis in RO82 W-1 cells, whereas BAPTA/AM produced the opposite effects with a reorganization of the mitochondrial network. This is the first demonstration that NO and calcium regulate mitochondrial biogenesis through the PRC pathway in thyroid cell lines.

[MiRNAs in follicular thyroid tumors]. / Micro-ARNs dans les tumeurs folliculaires de la thyroïde.

MiRNAs are small noncoding RNA ensuring post-transcriptional regulation of gene expression. Their expression is tissue-specific and some miRNAs have diagnostic and/or prognostic interest for tumor classes. MiRNAs are involved in tumoral process in quantitative (amplification, deletion of chromosomal regions) or qualitative terms (mutation in the miRNA or in the corresponding site of interaction in the mRNA). Overexpression of three miRNAs (miR-146b, miR-221 and miR-222) correlates with the development of papillary thyroid tumors. Polymorphisms in the c-kit gene or in that encoding miR-146a are susceptibility factors for the development of papillary thyroid tumors.

Estrogen-related receptor alpha and PGC-1-related coactivator constitute a novel complex mediating the biogenesis of functional mitochondria.

Mitochondrial biogenesis, which depends on nuclear as well as mitochondrial genes, occurs in response to increased cellular ATP demand. The nuclear transcriptional factors, estrogen-related receptor alpha (ERRalpha) and nuclear respiratory factors 1 and 2, are associated with the coordination of the transcriptional machinery governing mitochondrial biogenesis, whereas coactivators of the peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family serve as mediators between the environment and this machinery. In the context of proliferating cells, PGC-1-related coactivator (PRC) is a member of the PGC-1 family, which is known to act in partnership with nuclear respiratory factors, but no functional interference between PRC and ERRalpha has been described so far. We explored three thyroid cell lines, FTC-133, XTC.UC1 and RO 82 W-1, each characterized by a different mitochondrial content, and studied their behavior towards PRC and ERRalpha in terms of respiratory efficiency. Overexpression of PRC and ERRalpha led to increased respiratory chain capacity and mitochondrial mass. The inhibition of ERRalpha decreased cell growth and respiratory chain capacity in all three cell lines. However, the inhibition of PRC and ERRalpha produced a greater effect in the oxidative cell model, decreasing the mitochondrial mass and the phosphorylating respiration, whereas the nonphosphorylating respiration remained unchanged. We therefore hypothesize that the ERRalpha-PRC complex plays a role in arresting the cell cycle through the regulation of oxidative phosphorylation in oxidative cells, and through some other pathway in glycolytic cells.

PGC-1-related coactivator modulates mitochondrial-nuclear crosstalk through endogenous nitric oxide in a cellular model of oncocytic thyroid tumours.

BACKGROUND: The PGC-1 related coactivator (PRC), which shares structural and functional features with PGC-1alpha, is believed to regulate several metabolic pathways as well as mitochondrial biogenesis. Its involvement in the early programming of cell proliferation suggests the existence of finely regulated crosstalk between mitochondrial functions and the cell cycle status. METHODOLOGY/PRINCIPAL FINDINGS: PRC-regulated pathways were explored in a cell-line model derived from mitochondrial-rich tumours with an essentially oxidative metabolism and specifically high PRC expression. The functional status of mitochondria was compared to the results of microarray analysis under conditions of temporal PRC inhibition. To specify the fine PRC regulation, the expression levels of the genes and proteins involved in the oxidative phosphorylation process were studied by real time quantitative PCR and western blotting. As in earlier studies on PGC-1alpha, we investigated the role of nitric oxide in PRC-regulated mitochondrial biogenesis and determined its action in the control of the phosphorylation status of the mitogen-activated protein kinase pathway. CONCLUSION/SIGNIFICANCE: We found that nitric oxide rapidly influences PRC expression at the transcriptional level. Focusing on mitochondrial energetic metabolism, we observed that PRC differentially controls respiratory chain complexes and coupling efficiency in a time-dependent manner to maintain mitochondrial homeostasis. Our results highlight the key role of PRC in the rapid modulation of metabolic functions in response to the status of the cell cycle.