ROS-induced near-homozygous genomes in thyroid cancer

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   Figure 1

   Lesser-allele intensity-ratio analysis (LAIR) of XTC.UC1, FTC-236 follicular thyroid cancer cells and FTC-236 subclones shows whole-chromosome missegregations. (A) LAIR analysis of XTC.UC1 and FTC-236. Note that many autosomes (1–4, 6, 8, 10, 11, 13, 15 and 17) showed a homozygous state (AA), highly comparable to clinical FTC-OV samples after endoreduplication of the entire near-haploid genome. (B) Flow cytometric DNA content analysis of, respectively, the oncocytic follicular thyroid cancer cell line XTC.UC1 (left panels) and the follicular thyroid cancer cell line FTC-236 (right panels). Two cycling populations were observed in FTC-236, with a DI of 1.22 and 1.31. (C) Selection of seven FTC-236 subclones after limiting dilution showing subtle intra-subclone chromosomal differences in chromosomes 5, 12, 18 and 20. A reciprocal event was observed for chromosome 18: compare clone A (AB), clones B, E, G, H and I (AAB) and clone C (A). (D) A model is proposed for asymmetric mitotic events in FTC-236, possibly caused by merotelic-attached kinetochores or excessive stability of the kinetochore–microtubule attachment. i, allelic state; ii, copy number; iii, 0 < allelic ratio < 1; (AB) or (AABB), heterozygous; (AAB) or (AAAB), imbalance; (AA) or (AAA), etc., homozygous; ChT, chromotripsis; TRBC, trout red blood cells; DI, DNA index; G1₁, G1 of the major cycling population; G1₂, G1 of the minor cycling population.
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   Figure 2

   A low-energetic phenotype corresponds to follicular thyroid cancer cell lines with an NHG but not with mtDNA mutations. (A) Energy profiles of FTC-133, -236 and -238, XTC.UC1 (all showing an NHG) vs BHP 2–7, SW579 and HeLa after challenging with FCCP. Abscissa: ECAR, ordinate: OCR. Open squares: ECAR/OCR untreated cells, closed squares ECAR/OCR FCCP treated cells. Note that FTC-133 (dark red), -236 (green) and -238 (purple), XTC.UC1 (gray) show a relative quiescent profile (cluster 1, blue-dotted circle), while BHP 2–7 (dark gray/blue), SW579 (orange) and HeLa (light gray/blue) show a strong increase in both glycolysis and oxygen consumption (cluster 2, red-dotted circle). (B) Damaging/disruptive mtDNA mutations in all thyroid cancer cell lines tested except for BHP 2–7. FTC-133, -236 and -238, derived from different metastasis from the same patient, share the same mutation. The known mutations in XTC.UC1 were confirmed (Table 2). Also note that SW579 shows a relative strong energetic response when challenged with FCCP, despite a damaging mtDNA mutation.
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   Figure 3

   ROS is linked to missegregations via CHK2 activation. (A) Examples of spontaneous missegregations in XTC.UC1 showing: a lagging chromosome (upper row), a chromatin bridge (middle row) and an acentric chromosome (lower row). From left to right: kinetochore (red, AF594 fluorescence), α-tubulin (green, AF488 fluorescence), DNA (cyan, Hoechst33342 fluorescence), merged image. All images were captured with a 40× dry objective. An identical digital zoom was applied to all images. (B) XTC.UC1 and FTC-236 show a significant higher number of lagging chromosomes than BHP 2–7 and SW579 (*P = 0.0004) (n = 300). Strong reduction (**P < 0.0001) of the percentage of mainly lagging chromosomes in XTC.UC1, FTC-236 and BHP 2–7 after treatment the cells for 2 h with the ROS scavenger NAC. Black: lagging chromosomes, gray: chromosome bridge, white: acentric chromosomes. (C) Western blot showing auto-phosphorylation of CHK2 in XTC.UC1 cells (in green), α-tubulin internal control (red). CHK2 can be further activated by H₂O₂, while glutathione treatment reduced the amplitude of phosphorylation. An increase in pCHK2 was shown after incubating the cells with antimycin A. (D) XTC.UC1 cells were treated with antimycin A (lower panels) or left untreated (upper panels) and stained for ROS, pCHK2 and DNA. Increased ROS activity (MitoSOX Red, in green) was observed in many metaphase cells (see red arrows), which coincided with an increased pCHK2 fluorescence (in yellow, AF647 fluorescence). DNA was stained with Hoechst33342 (cyan). All images were captured with a 20× dry objective. An identical digital zoom was applied to all images.
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   Figure 4

   Genomic ranking and proposed tumor progression model for oncocytic follicular thyroid cancer. (A) Ranking of oncocytic follicular thyroid tumors based on whole chromosome losses or gains. The DNA index was obtained by multiparameter flow cytometry of cell suspension derived from FFPE tissue punches. Seven samples were bimodal (B) and samples 3, 10, 15 19, 20, 23 and 24 show endoreduplication (genome doubling). (B) Sum of the relative allelic score showing progressive whole-chromosome losses or gains. Note the relative sharp transition from FA-OV to FTC-OV. (C) Newly proposed model for FTC-OV tumor progression based on the activation of CHK2 via direct ATM oxidation by high ROS. This results in increased tension of the kinetochore/α-tubulin network leading to whole-chromosome segregation errors (Bakhoum et al. 2014). The progression from FA-OV seems to be driven by a stepwise loss of whole chromosomes, but with retention of chromosome 7 due to maternal- and paternal-imprinted genes important for survival (Boot et al. 2016). The loss of chromosome 22 is an early event. Blue, heterozygous (AB) or (AABB), score 2.0; light blue, imbalance (AAB), score 3.0; purple, imbalance composed of a mixture of status (AB) and (A), score 1.5; red, homozygous (A) or (AA), etc., score 1.0; B, bimodal DNA histogram (underlined: major population); U, unimodal DNA histogram.

• © 2018 Society for Endocrinology