Identification of a recurrent STRN/ALK fusion in thyroid carcinomas.

Thyroid carcinoma is the most common endocrine malignant tumor and accounts for 1% of all new malignant diseases. Among all types and subtypes of thyroid cancers that have been described so far, papillary thyroid carcinoma is the most frequent. The standard management treatment of these tumors consists of surgery, followed by radioiodine treatment in case of high risk of relapse. The most aggressive forms are commonly treated by chemotherapy, radiotherapy or experimental drug testing. We recently reported the case of a patient presenting an anaplastic thyroid carcinoma with lung metastases. Fluorescence in situ hybridization analysis allowed us to detect a rearrangement of the anaplastic lymphoma kinase (ALK) gene in both tumors. The patient was treated with crizotinib and presented an excellent drug response. We present here the subsequent investigations carried out to further characterize this genetic alteration and to assess the prevalence of ALK rearrangements in thyroid lesions. High resolution array-comparative genomic hybridization data complemented by RT-PCR and sequencing analyses, allowed us to demonstrate the presence of a STRN/ALK fusion. The STRN/ALK transcript consisted of the fusion between exon 3 of STRN and exon 20 of ALK. Subsequent screening of 75 various thyroid tumors by RT-PCR revealed that 2 out of 29 papillary thyroid carcinomas exhibited the same fusion transcript. None was detected in other types of malignant or benign thyroid lesions analyzed. These findings could pave the way for the development of new targeted therapeutic strategies in the treatment of papillary thyroid carcinomas and point to ALK inhibitors as promising agents that merit rapid evaluation

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

<i>ALK</i>, <i>STRN</i> and <i>STRN/ALK</i> fusion status at mRNA and genomic DNA levels in positive PTC.

<p>(A) Expression profiles of <i>ALK</i>, <i>STRN</i> and <i>STRN/ALK</i> fusion transcript obtained by RT-PCR in two PTC samples and in one control sample (C1) are presented. L: molecular weight ladder, bp: base-pair. (B) Chromatogram showing the sequence of <i>STRN/ALK</i> fusion transcript at the breakpoint observed in the two PTC samples. The <i>STRN</i> exon 3 (NM_003162) at the 5′ part of the transcript is fused to the <i>ALK</i> exon 20 (NM_004304) at the 3′ portion. (C) Products obtained after PCR on genomic DNA using first <i>ALK</i> and <i>STRN</i> forward and reverse primers and then the combination of a <i>STRN</i> forward primer with an <i>ALK</i> reverse primer for case 5 and one control sample (C1). (D) Chromatogram showing the sequence of <i>STRN/ALK</i> fusion at the genomic breakpoint observed in the case 5. One nucleotide at the intronic junction (C) is identical in both fused genes and might be contributed by either of them.</p

ALK Interphase fluorescence <i>in situ</i> hybridization and immunohistochemistry.

<p>(A) On the representative picture of the FISH, realized using the LSI ALK Dual Color Break Apart Rearrangement Probe, we could observe <i>ALK</i> probe break-aparts highlighted by arrows in the two positive PTC cases. Magnification: X1000. (B) Pictures of immunohistochemical labeling for ALK in the two PTC samples. Pictures of hematoxylin, eosin and safran (HES) staining of the two samples are also presented. Magnification for HES: X200, Magnification for IHC: X400.</p

Potential ERRα response elements in <i>LDHB</i> and <i>LDHA</i> promoters (A) Potential ERRα binding sites numbered relative to the transcription starting site (TSS) (B) Chromatin ImmunoPrecipitation (ChIP) assay for <i>LDH</i> promoters in XTC.UC1 cells using a polyclonal ERRα antibody.

<p>Chromatin was immunoprecipitated with the indicated antibody and submitted to quantitative PCR. Results are expressed as fold change of enrichment compared to control IgG immunoprecipitated material. ERRα-IP was realized in duplicate and each sample was tested in triplicate for quantitative PCR. TFBS: transcription factor binding site.</p

ERRα modulates expression and activity of LDH. <i>LDHA</i> and <i>LDHB</i> expression levels were measured by quantitative real-time PCR.

<p>The ratio of LDH activity to CS activity was determined under various conditions. Measurements were made 48 h after transfection or 10 days of treatment with XCT790 and results are presented relative to the control which was assigned a unit value. <i>LDHA</i> and <i>LDHB</i> expression levels, the mean <i>LDHA/LDHB</i> ratio (A) and the relative LDH activity (B) for RO82W-1 cells transfected with 50 ng ERRα or 50 ng ERRα and 50 ng PRC or empty vectors (Control). <i>LDHA, LDHB</i> expression levels and mean of the <i>LDHA/LDHB</i> ratio (C) and relative LDH activity (D) for FTC-133 cells treated with XCT790 or vehicle (Control). <i>LDHA, LDHB</i> expression levels and mean of the <i>LDHA/LDHB</i> ratio (E) and relative LDH activity (F) for FTC-133 cells transfected with control or ERRα siRNA. The results are the mean values±SD of three experiments performed in duplicate relative to controls. *: p≤0.05.</p

ERRα and PRC coregulate the cell cycle and metabolism in thyroid cells.

<p>RO82W-1 cells transfected with ERRα and PRC or empty vector (Control) and analysed 72 h after transfection. Pangenomic microarray results for metabolism genes. Gene-expression levels are grouped by class and ordered according to their mean log level of expression (A) or color-coded in the matrix from green (underexpression) to red (overexpression) (B). Ten main gene ontologies (C). Cell proliferation and lactate levels in media (D).</p

ERRα inhibits <i>LDHB</i> promoter activity.

<p>(A) Different construction of the human LDHB promoter reporter plasmid. (B) RO82W-1 cells were transfected with the indicated promoter constructs together with the expression plasmid of ERRα and/or PRC. Luciferase activity was determined 48 h after transfection and normalized against renilla luciferase activity. Results, presented in Relative Light Units (RLU), are the mean values±SD of three experiments performed in duplicate. <b>*</b>: p≤0.05 versus cells transfected with plasmids controls and no ERRα or PRC.</p

<i>ALK</i>, <i>STRN</i> and <i>STRN/ALK</i> fusion transcript RNA expression and sequencing.

<p>(A) Expression of <i>ALK</i>, <i>STRN</i> and <i>STRN/ALK</i> fusion transcript obtained by RT-PCR in the pulmonary tumor, the thyroid tumor and non tumoral tissue of the initial sample, and in one control sample (C1) are presented. L: molecular weight ladder, bp: base-pair. (B) Expression of <i>STRN/ALK</i> fusion transcript obtained by an <i>ALK</i> specific RT using <i>ALK</i>ex20R1 primer followed by PCR in the same samples. (C) Chromatogram showing the sequence of <i>STRN/ALK</i> fusion transcript at the breakpoint observed in the pulmonary and thyroid carcinomas. The <i>STRN</i> exon 3 (NM_003162) at the 5′ part of the transcript is fused to the <i>ALK</i> exon 20 (NM_004304) at the 3′ portion. (D) Schematic representation of the structure of the fusion transcript, of ALK and STRN proteins and of the predicted fusion protein. For the fusion transcript, initial position of the nucleotides at the fusion are indicated (position on <i>STRN</i> and <i>ALK</i> mRNA). Nu: nucleotide, aa: amino acid, cc: coiled-coil domain. Ensembl genome browser ID: <i>ALK</i> (gene: ENSG00000171094, mRNA: ENST00000389048, prot: ENSP00000373700) <i>STRN</i> (gene: ENSG00000115808, mRNA: ENST00000263918, prot: ENSP00000263918).</p

Genomic profile.

<p>(A) CGH profile obtained from a pulmonary metastasis of the initial case. Genomic alterations are presented and organized on the X axis from chromosome 1 to 22 and X, Y. Log2 ratio values are reported on the Y axis. Significant gains or losses are indicated by blue lines and blue areas above or below each profile, respectively. Chromosome 2 is highlighted with a black box. (B) Enlargements of chromosome 2 and <i>ALK</i> chromosomal region. The chromosome 2 region containing <i>ALK</i> locus is highlighted with a black box. The log2 ratio values of probes covering the proximal regions of <i>ALK</i> and the gene itself are presented. The arrow indicates the breakage region. (C) Enlargements of chromosome 2 and <i>STRN</i> chromosomal region. The chromosome 2 region containing <i>STRN</i> locus is highlighted with a black box. The log2 ratio values of probes covering the proximal regions of <i>STRN</i> and the gene itself are presented. The arrow indicates the breakage region. (D) Schematic representation of potential breakpoints into the two genes at the genomic level (gDNA) according to the CGH data. Agilent CGH probes surrounding the different breakpoints are indicated.</p