Search

Abstract Background Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35. Within each repeat unit is a gene, DUX4, that can encode a protein containing two homeodomains. A DUX4 transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how. Methods Using exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods. Results Many of the ~17,000 examined genes were differentially expressed (> 2-fold, p < 0.01) in control myoblasts or myotubes vs. non-muscle cells (2185 and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and 797, respectively). Surprisingly, despite the morphologically normal differentiation of FSHD myoblasts to myotubes, most of the disease-related dysregulation was seen as dampening of normal myogenesis-specific expression changes, including in genes for muscle structure, mitochondrial function, stress responses, and signal transduction. Other classes of genes, including those encoding extracellular matrix or pro-inflammatory proteins, were upregulated in FSHD myogenic cells independent of an inverse myogenesis association. Importantly, the disease-linked DUX4 RNA isoform was detected by RT-PCR in FSHD myoblast and myotube preparations only at extremely low levels. Unique insights into myogenesis-specific gene expression were also obtained. For example, all four Argonaute genes involved in RNA-silencing were significantly upregulated during normal (but not FSHD) myogenesis relative to non-muscle cell types. Conclusions DUX4's pathogenic effect in FSHD may occur transiently at or before the stage of myoblast formation to establish a cascade of gene dysregulation. This contrasts with the current emphasis on toxic effects of experimentally upregulated DUX4 expression at the myoblast or myotube stages. Our model could explain why DUX4's inappropriate expression was barely detectable in myoblasts and myotubes but nonetheless linked to FSHD.

Background: Approximately half of cutaneous melanoma tissues harbor BRAFV600E mutations, resulting in a constitutive activation of the mitogen-activated protein kinase (MAPK) pathway. Nuclear-cytoplasmic transport machinery is dysregulated in neoplastic cells and alters the key regulatory proteins that can lead to tumor progression and drug resistance. The significance of nuclear localization of BRAFV600E has not been fully understood. We examined the clinical significance of intracellular localization of BRAFV600E in cutaneous melanoma.Study Design: Immunohistochemical analysis of BRAFV600E was performed on formalin-fixed, paraffin-embedded specimens of cutaneous melanoma (n = 91). Staining intensity was graded in a blinded manner. Correlations to clinical factors were analyzed by Fisher's exact test and 2-tailed t-test. Localization of BRAFV600E was determined in melanoma cells, and we investigated their resistance to BRAFV600E-specific inhibitor according to nuclear localization in both in vitro and in vivo models.Results: We included 91 patients, of whom 32% (29 of 91) had cytoplasmic BRAFV600E. Nuclear BRAFV600E was observed in 30% (27 of 91). Overall, BRAFV600E expression correlated with TNM stage (p = 0.011), mitotic activity (p = 0.010), and ulceration (p = 0.045). Nuclear BRAFV600E expression correlated with overall clinical stage (p < 0.001), tumor size (p < 0.001), regional lymph node (p < 0.017), depth of invasion (p = 0.005), Clark level (p < 0.001), mitotic activity (p < 0.001), ulceration (p < 0.001), and margin status (p = 0.017). On a cellular level, BRAFV600E was identified in the nucleus, and its translocation was serum dependent. Our in vitro and in vivo data revealed sequestration of BRAFV600E in the cytosol-sensitized resistant cells to vemurafenib; nuclear retention of BRAFV600E was associated with aggressiveness and drug resistance.Conclusions: Nuclear localization of BRAFV600E is associated with melanoma aggressiveness. Further multi-institutional studies are warranted to confirm the clinical relevance of nuclear localization of BRAFV600E. [ABSTRACT FROM AUTHOR]

The presence of mutant BRAF V600E correlates with the risk of recurrence in papillary thyroid cancer (PTC) patients. However, not all PTC patients with BRAF V600E are associated with poor prognosis. Thus, understanding the mechanisms by which certain PTC patients with nuclear BRAF V600E become aggressive and develop resistance to a selective BRAF inhibitor, PLX-4032, is urgently needed. The effect of nuclear localization of BRAF V600E using in vitro studies, xenograft mouse-model and human tissues was evaluated. PTC cells harboring a nuclear localization signal (NLS) of BRAF V600E were established and examined in nude mice implanted with TPC1-NLS-BRAF V600E cells followed by PLX-4032 treatment. Immunohistochemical (IHC) analysis was performed on 100 PTC specimens previously confirmed that they have BRAF V600E mutations. Our results demonstrate that 21 of 100 (21%) PTC tissues stained with specific BRAF V600E antibody had nuclear staining with more aggressive features compared to their cytosolic counterparts. In vitro studies show that BRAF V600E is transported between the nucleus and the cytosol through CRM1 and importin (α/β) system. Sequestration of BRAF V600E in the cytosol sensitized resistant cells to PLX-4032, whereas nuclear BRAF V600E was associated with aggressive phenotypes and developed drug resistance. Proteomic analysis revealed Arp2/3 complex members, actin-related protein 2 (ACTR2 aliases ARP2) and actin-related protein 3 (ACTR3 aliases ARP3), as the most enriched nuclear BRAF V600E partners. ACTR3 was highly correlated to lymph node stage and extrathyroidal extension and was validated with different functional assays. Our findings provide new insights into the clinical utility of the nuclear BRAF V600E as a prognostic marker for PTC aggressiveness and determine the efficacy of selective BRAF V600E inhibitor treatment which opens new avenues for future treatment options., Competing Interests: None., (AJCR Copyright © 2022.)

Currently, thyroid cancer is one of the most common endocrine cancer in the United States. A recent involvement of sub-population of stem cells, cancer stem cells, has been proposed in different histological types of thyroid cancer. Because of their ability of self-renewal and differentiation into various specialized cells in the body, these putative cells drive tumor genesis, metastatic activity and are responsible to provide chemo- and radioresistant nature to the cancer cells in the thyroid gland. Our Review was conducted from previously published literature to provide latest apprises to investigate the role of embryonic, somatic and cancer stem cells, and discusses the hypothesis of epithelial-mesenchymal transition. Different methods for their identification and isolation through stemness markers using various in vivo and in vitro methods such as flow cytometry, thyrosphere formation assay, aldehyde dehydrogenase activity and ATP-binding cassette sub-family G member 2 efflux-pump mediated Hoechst 33342 dye exclusion have been discussed. The review also outlines various setbacks that still remain to target these tumor initiating cells. Future perspectives of therapeutic strategies and their potential to treat advanced stages of thyroid cancer are also disclosed in this review.

Background/aim: Scientists have been in quest for the best in vivo model to evaluate chemotherapies for radioiodine-resistant metastatic thyroid carcinomas. Human adipose tissue-derived stromal/stem cells (ASCs) have been found to promote in vitro growth and in vivo tumorigenesis. In the present study, we describe a novel model of metastatic human thyroid carcinoma by combining ASCs with the papillary thyroid cancer, K1 cell line., Materials and Methods: Three groups of severe combined immunodefecient mice were investigated. The first group was injected subcutaneously with K1 cells plus ASCs, the second group with K1 cell only, and the last group with ASCs only. Mean tumor volumes and standard deviations were calculated and compared., Results: Concomitant injection of ASCs with the K1 cell line led to the development of significantly larger tumors compared to the other groups (p<0.05). In addition, the lungs of this group demonstrated gross tumor metastasis and pathological features of high-grade neoplasms., Conclusion: In the present study we describe a novel mouse model using ASCs with the potential to be used for assessment of new treatments for the management of metastatic thyroid carcinomas.