case_images: PMC10883765_01
case_images: Biopsy and autopsy samples of human NUT carcinoma in the retroperitoneum. A: CT-guided biopsy sample of the tumor showed dense proliferation of small atypical cells with a high nucleocytoplasmic ratio. Bx, biopsy. B: A macroscopic view of the autopsied sample demonstrated a very large tumor (24 x 15 x 10 cm) in the retroperitoneum which involved the left kidney and extended to the rectovesical pouch. C: The tumor from the autopsy sample showed a dense proliferation of monomorphic, round tumor cells with a high nucleocytoplasmic ratio. Inset, magnified view of tumor nuclei with fine chromatin and prominent nucleoli. D, E: Tumor cells are immunopositive for cytokeratin (clone CAM5.2) (D) and p40 (E), indicating its histology as poorly differentiated squamous cell carcinoma. F: Tumor cells demonstrated diffuse nuclear reactivity for NUT protein. Inset, magnified view of NUT positivity with a speckled pattern.
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case_images: PMC10883765_01_cureus-0016-00000052814-i01.jpg
case_images: FIG1
case_images: A CT-guided-guided was performed, and a tiny fragment of tumor tissue displayed dense proliferation of small atypical cells with a high nucleocytoplasmic ratio, giving the findings of a so-called small blue round cell tumor (Figure 1A). A very large tumor lesion (24 x 15 x 10 cm) was found in the retroperitoneum, which involved the left kidney and extended to the rectovesical pouch (Figure 1B). Histologically, the tumor showed a sheet-like proliferation of monomorphic, round-to-polygonal tumor cells with a high nucleocytoplasmic ratio, providing an impression of undifferentiated malignant tumors (Figure 1C). Immunostained tumor cells expressed cytokeratin (clones AE1/AE3, CAM5.2) and a squamous cell marker, p40, but were negative for spalt like transcription factor 4, desmin, Wilms tumor 1, melan A, CD3, or CD20, suggesting its histology as poorly differentiated carcinoma with squamous differentiation (Figure 1D-1E). Technology, Danvers, MA) with a pathognomonic, speckled pattern (Figure 1F). Thus, the final pathology diagnosis was a NUT carcinoma, with a presumed primary site of origin as the retroperitoneum.
case_images: Metabolic and epigenetic reprogramming in human NUT carcinoma case. A: Section shows both components of NUT carcinoma tissue and surrounding stromal tissue. B, C: NUT carcinoma cells are strongly immunopositive for glycolytic markers of HK2 (B) and LDHA (C). D-F: In accordance with metabolic shift in human NUT carcinoma tissue, oncogenic histone acetylation marks of H3K9ac (D) and H3K27ac (E) as well as histone methylation marks of H3K27me3 (F) were upregulated in NUT carcinoma. H3K9ac was most prominently expressed in the tumor tissue (D).
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case_images: PMC10883765_01_cureus-0016-00000052814-i02.jpg
case_images: FIG2
case_images: Of note, glycolytic enzymes such as hexokinase 2 (HK2) and lactate dehydrogenase (LDHA) were overexpressed in NUT carcinoma tissue compared with surrounding stroma, suggesting that glycolytic metabolism was activated in NUT carcinoma (Figure 2A-2C). We found that both histone markers of tumor-promoting acetylation and methylation were upregulated in NUT carcinoma tissue in comparison with those in the surrounding stroma, indicating a tumor-specific epigenetic shift in NUT carcinoma (Figure 2D-2F). Of note, H3K9 acetylation (H3K9ac) was most prominently expressed in the tumor tissue (Figure 2D).
case_images: Metabolic and epigenetic reprogramming in NUT-overexpressing cell culture model. A: 293T cells overexpressed with BRD4-NUT plasmid. Successful overexpression of BRD4-NUT was confirmed by staining NUT with immunocytochemistry (red, NUT; blue, 4',6-diamidino-2-phenylindole (DAPI)) and immunoblotting. GFP, GFP-overexpressing control 293T cells; BRD4-NUT, BRD4-NUT-overexpressing 293T cells. B: Immunoblot detection of glycolytic enzymes (HK2) in BRD4-NUT overexpressing cells versus control cells with GFP plasmid. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), loading control. C: Immunoblot detection of c-Myc (MYC) in BRD4-NUT overexpressing cells versus control cells with GFP plasmid. GAPDH, loading control. D: Immunoblot detection of histone H3 acetylation (H3K9ac) in BRD4-NUT overexpressing cells versus control cells with GFP plasmid. Total H3, loading control. E: Cells were seeded at the concentration of 4.0 x 105 cells/well, and cell number was calculated 24 hours after transfection between BRD4-NUT overexpressing cells vs. control cells with GFP plasmid in glucose-containing media (n=6 (six separate transfection experiments on the same day by two people)). ***p<0.001.
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case_images: PMC10883765_01_cureus-0016-00000052814-i03.jpg
case_images: FIG3
case_images: Watertown, MA) in HEK293T embryonic kidney cells (ATCC, Manassas, VA) (Figure 3A) and found that glycolytic enzymes were upregulated in BRD4-NUT overexpressing cells in contrast to control cells with green fluorescent protein (GFP) plasmid, suggesting that BRD4-NUT fusion protein could activate glycolytic metabolism (Figure 3B). We previously demonstrated that c-Myc (MYC) is a master regulator of glycolytic metabolism in malignant tumors, but the overexpression of BRD4-NUT did not have an impact on the induction of MYC in our model, at least at the protein level (Figure 3C). Similar to human NUT carcinoma tissue, the overexpression of BRD4-NUT significantly upregulated histone methylation and acetylation, especially H3K9ac, indicating that a tumor-specific epigenetic shift could be induced by the BRD4-NUT fusion protein (Figure 3D). More importantly, overexpression of NUT could significantly drive cell proliferation in glucose-containing media (Figure 3E).
case_images: BRD4-NUT drives metabolic and epigenetic reprogramming in NUT carcinoma. BRD4-NUT protein, produced by BRD4-NUT fusion genes, could upregulate metabolic genes such as HK2 and LDHA and reprogram glycolytic metabolism, which subsequently shifts tumor epigenome represented by an increase of H3K9ac. Metabolic and epigenetic reprogramming contributes to the aggressive biology of NUT carcinoma but could be exploitable as a novel therapeutic target for the rare and aggressive types of cancer.
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case_images: FIG4