Combined effects of FH (E404D) and ACOX2 (R409H) cause metabolic defects in primary cardiac malignant tumor

Abstract

Primary malignant cardiac tumors (PMCTs) are extremely rare. The apparent immunity of the heart to invasive cancer has attracted considerable interest given the continuously rising incidence of cancer in other organs. This study aims to determine the conditions that could result in cardiac carcinoma and expand our understanding of cardiac tumor occurrence. We report two cases: a male (Patient-1) with primary cardiac malignant fibrous histiocytoma (MFH) and a female (Patient-2) with primary cardiac angiosarcoma. Merged genome-wide analyses of aCGH, Exome sequencing, and RNA-sequencing were performed on Patient-1 using peripheral blood, carcinoma tissue, and samples of adjacent normal tissue. Only whole-transcriptome analysis was carried out on Patient-2, due to insufficient quantities of sample from Patient-2. We identified a novel inherited loss of functional mutation of FH (Glu404Asp), a recurrent somatic hotspot mutation of PIK3CA (His1047Arg) and a somatic duplication in copy number of HIF1A. FH (E404D) severely compromised FH enzyme activity and lead to decreased protein expression in cardiac tumor tissues. We previously reported a functional mutation ACOX2 (R409H), which is potentially associated with decreased β-oxidation of fatty acids in the cardiac tumor tissue. Results of transcriptome analyses on two patients further revealed that the RNA expression of genes in the TCA cycle and beta-oxidation were uniformly downregulated. In this study, combined effects of FH (E404D) and ACOX2 (R409H) on metabolic switch from fatty acids to glucose were remarkably distinct, which might be an essential precondition to trigger the occurrence of PMCTs and mimic the Warburg effect, a hallmark of cancer metabolism.

Fig. 1. A 40-year-old male was diagnosed as having cardiac MFH (Patient-1).

a The pedigree of the Patient-1's family. (1) Unknown reason death; (2) esophageal carcinoma; (3) neuroglioma; (4) acute myeloid leukemia. A sibling (54s) of the patient, who only carries the FH (E404D), once accepted the benign sarcoma resection on the back. In this study, FH (E404D) and ACOX2 (R409H) were paternally and maternally derived, respectively. b Imaging modalities for identification and characterization of a primary cardiac tumor in Patient-1. Transthoracic echocardiogram and chest X-ray revealed increased cardio-thoracic ratio due to a 10 × 5.5 cm² tumor in the right ventricle (RV). c The irregular and heterogenous pieces of sarcoma were resected from RV during the surgery

Fig. 2. Pathological diagnosis of primary cardiac malignant fibrous histiocytoma (MFH).

Light microscopy (hematoxylin–eosin, HE) presenting the morphology of tumor cells. Immunohistochemical staining positive for Vim (vimentin), CK (cytokeratin), and WT-1 (Wilms' tumor gene 1) and negative for MyoD-1 (myogenic differentiation antigen 1), CD31 (platelet endothelial cell adhesion molecule-1, PECAM-1/CD31), Desmin, S100 (calcium-binding protein), SMA (smooth muscle actin), MSA (muscle-specific Actin), EMA (epithelial membrane antigen), Myogenin, CK5/6 (cytokeratin 5/6), CD34 (hematopoietic progenitor cell antigen CD34), HBME-1 (human mesothelial cell), and PGM-1 (phosphoglucomtase-1). Representative images of the antibody staining are displayed. Scale bars, 100 µm

Fig. 3. FH (E404D) was identified in Patient-1 by exome sequencing.

a Sanger sequencing and sequencing alignment of FH (E404D) (NCBI reference sequences are shown on the right). Black arrow indicated that the heterozygous mutation in FH (c.1212G>C, p.Glu404Asp) was further confirmed by Sanger sequencing. C.elegans Caenorhabditis elegans, D.melanogaster Drosophila melanogaster, E.coli Escherichia coli, S.cerevisiae Saccharomyces cerevisiae, S.tuberosum Solanum tuberosum. b FH Glu404Asp significantly compromised enzyme activity by overexpressing recombinant FH-Myc protein in HEK 293T cells in vitro (***P < 0.001). An empty vector served as a control (Ctrl). Relative activity is presented as the mean ± SE of four independent experiments; each sample was assayed in quadruplicate in each experiment. c Expression of FH was downregulated, whereas G6PD (glucose-6-phosphate dehydrogenase) were upregulated in the cardiac tumor tissue when compared with adjacent normal tissue. GAPDH and β-ACTIN served as loading controls. Nt adjacent normal tissue, Tt tumor tissue. d FH E404D significantly increased levels of cellular fumarates in vitro (**P < 0.01). HEK 293T cells were transfected and an empty vector served as a control. e FH (E40ED) affected normal tetramer formation. HEK 293T cells were transfected with Myc-tagged wild type or (E40D) FH for recombinant overexpression. A 1:100 dilution of a protein crosslinker (PC) was added to the RIPA buffer before cell lysis. Subsequently, anti-Myc immunoprecipitation and anti-FH immunoblotting were performed following the standard procedures. A 50-KDa band was indicative of FH-Myc recombinant protein. After PC treatment, a 220-KDa band (black arrow) was generated, corresponding to an FH tetramer. This band was significantly decreased in cells transfected with mutant FH. f Native gels assay was performed to test the polymer formation of FH. After anti-Myc immunoprecipitation, two different bands were generated. The higher band, corresponding to polymer, was decreased in the cells transfected with mutant FH

Fig. 4. Somatic HIF1A duplication was identified in cardiac tumors of Patient-1 by aCGH

a Seven somatic CNVs including 42 genes were identified by aCGH (14q23.1–23.2,17p13.2, 5q35.2, 9p21.3, 14q24.2, 14p12, 16p12.2, and 16p13.3). b A larger fragment of somatic duplication including HIF1A on locus 14q23.1-23.2 was identified in both Chip-2 and Chip-3. N adjacent normal tissue, C control for reference labeling, T tumor tissue. c HIF1A expression in cardiac tumor tissues was markedly upregulated. GAPDH served as a loading control. Representative image from three independent experiments was displayed

Fig. 5. Recurrent hotspot PI3KCA (H1047) was first identified in cardiac tumor tissue.

a Sanger sequencing of PIK3CA (His1047Arg) by using blood and tumor tissue samples of Patient-1. b Phosphorylation of Akt Thr 308 site as well as GSK3-beta at Ser9 site was markedly enhanced in cardiac tumor tissues

Fig. 6. Proposed pathogenic model to explain the metabolic defects in cardiac tumor tissues of Patient-1.

Under normal condition, cardiac cells prefer to utilize fatty acids as primary fuels (Left). FH (E404D) and ACOX2 (R409H) could independently take effect in oxidation of fatty acids and TCA cycle as metabolic enzymes, respectively (Middle). These two mutations probably have combined effects on metabolic switch from fatty acids to glycolysis. Green arrows indicate downregulated metabolic processes according to RNA-seq data from Patient-1