Aberrant Calreticulin Expression Is Involved in the Dedifferentiation of Dedifferentiated Liposarcoma Masanori Hisaoka, Atsuji Matsuyama, Mitsuhiro Nakamoto

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Aberrant Calreticulin Expression Is Involved in the Dedifferentiation of Dedifferentiated Liposarcoma Masanori Hisaoka, Atsuji Matsuyama, Mitsuhiro Nakamoto The American Journal of Pathology Volume 180, Issue 5, Pages 2076-2083 (May 2012) DOI: 10.1016/j.ajpath.2012.01.042 Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 1 Immunohistochemical analysis for calreticulin expression in liposarcomas, lipoma, normal fat, and nonlipogenic sarcomas. Unequivocal expression of calreticulin could be seen in the tumor cells lacking an adipocytic phenotype in the dedifferentiated area of dedifferentiated liposarcoma (A) and well-differentiated sclerosing (B) or lipoma-like (C) liposarcoma. Some lipoblasts in well-differentiated liposarcoma and primitive small round or short spindle cells in myxoid liposarcoma (D) were also positively stained. However, the lipoma (E) and normal fat (F) were negative for calreticulin expression. Calreticulin was expressed frequently in undifferentiated high-grade pleomorphic sarcoma (G), angiosarcoma (H), and alveolar rhabdomyosarcoma (I). Scale bars = 50 μm. The American Journal of Pathology 2012 180, 2076-2083DOI: (10.1016/j.ajpath.2012.01.042) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 2 The expression of calreticulin and variable differentiation markers for adipogenesis (C/EBPα, PPARγ, aP2, adipsin, LPL) (A) in liposarcomas and lipomas. A: Calreticulin and adipogenic transcription factors (PPARγ, C/EBPα) were expressed in dedifferentiated liposarcoma, whereas late or terminal adipogenic markers (aP2, adipsin, LPL) were not expressed, suggesting a differentiation arrest. β-Actin served as a loading control. B: Calreticulin was expressed in the dedifferentiated and well-differentiated liposarcomas significantly more than in comparison with lipoma and normal fat, as measured by quantitative RT-PCR (mean ± SD of three examples). *P < 0.01. The American Journal of Pathology 2012 180, 2076-2083DOI: (10.1016/j.ajpath.2012.01.042) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 3 A calreticulin knock-down experiment in FU-DDLS-1 cells. Calreticulin expression was reduced 72 hours after the transfection of siRNA at both the mRNA (mean ± SD of duplicates) (A) and protein (B) levels. In addition to mildly enhanced expression of PPARγ (B), the expression of aP2 and LPL was detectable at 48 and 72 hours. C: Accumulated intracytoplasmic lipid droplets were shown by oil red O staining in the cells 72 hours after the gene silencing, suggesting that adipocyte differentiation was rescued in dedifferentiated liposarcoma cells by the down-regulation of calreticulin. Scale bars = 20 μm. The American Journal of Pathology 2012 180, 2076-2083DOI: (10.1016/j.ajpath.2012.01.042) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 4 The effect of calreticulin knockdown by siRNA on the proliferation of dedifferentiated liposarcoma cells (LPS) and synovial sarcoma cells (SS). The down-regulation of calreticulin repressed the proliferation of LPS but did not affect that of SS (mean ± SD of triplicates). The American Journal of Pathology 2012 180, 2076-2083DOI: (10.1016/j.ajpath.2012.01.042) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 5 Interphase FISH using probes for 19p13.1–13.2 (red) and the centromere of chromosome 19 (green) in dedifferentiated liposarcoma and lipoma cases. Multiple red and green signals, suggesting polysomy of chromosome 19, were detected in the liposarcoma (A) but not the lipoma (B). Scale bars = 5 μm. The American Journal of Pathology 2012 180, 2076-2083DOI: (10.1016/j.ajpath.2012.01.042) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 6 The relative expression levels of candidate miRNAs targeting CALR in dedifferentiated liposarcoma (A) and that of miR-1275 in variable lipogenic tumors and normal fat (B) as determined by quantitative RT-PCR (mean ± SD of triplicates). The repression of miR-1275 expression was identified in the dedifferentiated liposarcoma. The American Journal of Pathology 2012 180, 2076-2083DOI: (10.1016/j.ajpath.2012.01.042) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions