Novel HRPT2/CDC73 Gene Mutations and Loss of Expression of Parafibromin in Chinese Patients with Clinically Sporadic Parathyroid Carcinomas

Objective

It is widely recognized that the diagnosis of parathyroid carcinoma (PC) is often difficult because of the overlap of characteristics between malignant and benign parathyroid tumors, especially at an early stage. Based on the identification of tumor suppressor gene HRPT2/CDC73 and its association with hereditary and sporadic PC, screening of gene mutations and detection of parafibromin immunoreactivity have been suggested as diagnostic instruments of PC in Whites. There is little information about HRPT2/CDC73 mutations and its corresponding protein expression in patients with sporadic PC in Chinese population, and the long-term follow-up data is scarce.

Methods

Paraffin-embedded tissues were obtained from 13 patients with PC, 13 patients with parathyroid adenoma (PA) and 7 patients with parathyroid hyperplasia(PH), and 6 normal parathyroid (NP) tissues as controls. Peripheral blood from 11 patients with PC was collected. PCR products using Genomic DNA extracted from tumor tissues or blood as template was sequenced for HRPT2/CDC73 gene. Expression of parafibromin in tumor tissues was evaluated by immunohistochemical analysis.

Results

Six mutations in 6 of 13 patients with PC were identified, with three being novel. Four of them were germ-line mutations. Patients with mutations were susceptible to recurrence of the PC. Complete (8/13, 61.5%) or partial (5/13, 38.5%) loss of parafibromin expression was observed in PC tissues. All of tissue samples from normal parathyroid or benign parathyroid tumors displayed positive immunostaining of parafibromin except one adenoma.

Conclusions

The present study supplies information on the mutations and protein expression of HRPT2/CDC73 gene and phenotypes of parathyroid carcinoma in Chinese population. And the expanded mutation database of this gene may benefit patients in the diagnosis and treatment of this disease.     

Recurrent Hyperparathyroidism and a Novel Nonsense Mutation in a Patient with Hyperparathyriodism-Jaw Tumor Syndrome

ObjectiveTo present the case of a hyperparathyroidism-jaw tumor (HPT-JT) patient with a novel nonsense mutation of the CDC73 gene.MethodsWe present the case of a patient with a history of three prior maxillectomies and two prior parathyroidectomies who presented with recurrent primary hyperparathyroidism (PHPT). We also briefly review the literature pertaining to HPT-JT.ResultsGenetic analysis revealed a novel nonsense mutation (c.85G>T; pGlu29) in exon 1 of CDC73. The patient’s son underwent genetic testing for a CDC73 mutation and was found to be negative.ConclusionHPT-JT is a rare condition characterized by PHPT and benign tumors of the mandible and maxilla. Up to 15% of HPT-JT patients with PHPT have parathyroid carcinoma. HPT-JT is associated with an inactivating mutation of CDC73, a gene that codes for the tumor suppressor protein parafibromin. This report expands our understanding of the genetics underlying this rare disorder and emphasizes the importance of early detection in order to prevent hypercalcemic complications such as parathyroid carcinoma. (Endocr. Pract. 2013;19:e134-e137)     

Retraction Note: Neuropathological and neuroprotective features of vitamin B12 on the dorsal spinal ganglion of rats after the experimental crush of sciatic nerve: an experimental study

The tumor suppressor gene CDC73 was found to be associated with hyperparathyroidism-jaw tumor syndrome (HPT-JT), which is characterized by parathyroid adenoma or carcinoma, ossifying fibroma (OF) of the jaws, and renal and uterine lesions. Mutations in CDC73 have also been frequently detected in sporadic parathyroid carcinomas and renal tumors. However, the prevalence and range of CDC73 mutations in sporadic OFs have not been established. We directly sequenced coding and flanking splice junctional regions of CDC73 in 40 cases of sporadic OF of the jaws. We also used immunohistochemistry to detect parafibromin, the protein product of CDC73, in those cases. Two novel CDC73 mutations were identified in 2 of the 40 cases (5 %). Both were somatic mutations located in exon 1 of the coding region. Strong parafibromin expression was detected in all 40 cases, irrespective of the presence of CDC73 mutations. Mutations inCDC73 were rare in sporadic OF of the jaws, but may affect the pathogenesis of a small subset of tumors of this type.     

Differential loss of heterozygosity in familial, sporadic, and uremic hyperparathyroidism

Various genetic loci harboring oncogenes, tumor suppressor genes, and genes for calcium receptors have been implicated in the development of parathyroid tumors. We have carried out loss of heterozygosity (LOH) studies in chromosomes 1p, 1q, 3q, 6q, 11q, 13q, 15q, and X in a total of 89 benign parathyroid tumors. Of these, 28 were sporadic parathyroid adenomas from patients with no family history of the disease, 41 were secondary parathyroid tumors, 5 were from patients with a history of previous irradiation to the neck, 12 were from patients with a family history of hyperparathyroidism, and 3 were parathyroid tumors related to multiple endocrine neoplasia type 1 (MEN1). In addition, we determined the chromosomal localization of a second putative calcium-sensing receptor, CaS, for inclusion in the LOH studies. Based on analysis of somatic cell hybrids and fluorescent in situ hybridization to metaphase chromsomes, the gene for CaS was mapped to chromosomal region 2q21-q22. The following results were obtained from the LOH studies: (1) out of the 24 tumors that showed LOH, only 4 had more than one chromosomal region involved, (2) in the tumours from uremic patients, LOH of chromosome 3q was detected in a subset of the tumors, (3) LOH of the MEN1 region at 11q13 was the most common abnormality found in both MEN1-related and sporadic parathyroid tumours but was not a feature of the other forms of parathyroid tumors, (4) LOH in 1p and 6q was not as frequent as previously reported, and (5) tumor suppressor genes in 1q and X might have played a role, particularly on the X chromosome, in the case of familial parathyroid adenomas. We therefore conclude that the tumorigenesis of familial, sporadic, and uremic hyperparathyroidism involves different genetic triggers in a non-progressive pattern. Received: 28 October 1996 / Revised: 16 November 1996     

Identification and Functional Characterization of Three NoLS (Nucleolar Localisation Signals) Mutations of the CDC73 Gene

Hyperparathyroidism Jaw-Tumour Syndrome (HPT-JT) is characterized by primary hyperparathyroidism (PHPT), maxillary/mandible ossifying fibromas and by parathyroid carcinoma in 15% of cases. Inactivating mutations of the tumour suppressor CDC73/HRPT2 gene have been found in HPT-JT patients and also as genetic determinants of sporadic parathyroid carcinoma/atypical adenomas and, rarely, typical adenomas, in familial PHPT. Here we report the genetic and molecular analysis of the CDC73/HRPT2 gene in three patients affected by PHPT due to atypical and typical parathyroid adenomas, in one case belonging to familial PHPT. Flag-tagged WT and mutant CDC73/HRPT2 proteins were transiently transfected in HEK293 cells and functional assays were performed in order to investigate the effect of the variants on the whole protein expression, nuclear localization and cell overgrowth induction. We identified four CDC73/HRPT2 gene mutations, three germline (c.679_680delAG, p.Val85_Val86del and p.Glu81_Pro84del), one somatic (p.Arg77Pro). In three cases the mutation was located within the Nucleolar Localisation Signals (NoLS). The three NoLS variants led to instability either of the corresponding mutated protein or mRNA or both. When transfected in HEK293 cells, NoLS mutated proteins mislocalized with a predeliction for cytoplasmic or nucleo-cytoplasmic localization and, finally, they resulted in overgrowth, consistent with a dominant negative interfering effect in the presence of the endogenous protein.     

Dissection of the Candida albicans Cdc4 protein reveals the involvement of domains in morphogenesis and cell flocculation

Background

CDC4, which encodes an F-box protein that is a member of the Skp1-Cdc53/Cul1-F-box (SCF) ubiquitin E3 ligase, was initially identified in the budding yeast Saccharomyces cerevisiae as an essential gene for progression through G1-S transition of the cell cycle. Although Candida albicans CDC4 (CaCDC4) can release the mitotic defect caused by the loss of CDC4 in S. cerevisiae, CaCDC4 is nonessential and suppresses filamentation.

Results

To further elucidate the function of CaCDC4, a C. albicans strain, with one CaCDC4 allele deleted and the other under the repressible C. albicans MET3 promoter (CaMET3p) control, was made before introducing cassettes capable of doxycycline (Dox)-induced expression of various C. albicans Cdc4 (CaCdc4) domains. Cells from each strain could express a specific CaCdc4 domain under Dox-induced, but CaMET3-CaCDC4 repressed conditions. Cells expressing domains without either the F-box or WD40-repeat exhibited filamentation and flocculation similarly to those lacking CaCDC4 expression, indicating the functional essentiality of the F-box and WD40-repeat. Notably, cells expressing the N-terminal 85-amino acid truncated CaCdc4 partially reverse the filament-to-yeast and weaken the ability to flocculate compared to those expressing the full-length CaCdc4, suggesting that N-terminal 85-amino acid of CaCdc4 regulates both morphogenesis and flocculation.

Conclusions

The F-box and the WD40-repeat of CaCdc4 are essential in inhibiting yeast-to-filament transition and flocculation. The N-terminal region (1–85) of CaCdc4 also has a positive role for its function, lost of which impairs both the ability to flocculate and to reverse filamentous growth in C. albicans.     

MIR34A regulates autophagy and apoptosis by targeting HMGB1 in the retinoblastoma cell

MIR34A (microRNA 34a) is a tumor suppressor gene, but how it regulates chemotherapy response and resistance is not completely understood. Here, we show that the microRNA MIR34A-dependent high mobility group box 1 (HMGB1) downregulation inhibits autophagy and enhances chemotherapy-induced apoptosis in the retinoblastoma cell. HMGB1 is a multifaceted protein with a key role in autophagy, a self-degradative, homeostatic process with a context-specific role in cancer. MIR34A inhibits HMGB1 expression through a direct MIR34A-binding site within the HMGB1 3′ untranslated region. MIR34A inhibition of HMGB1 leads to a decrease in autophagy under starvation conditions or chemotherapy treatment. Inhibition of autophagy promotes oxidative injury and DNA damage and increases subsequent CASP3 activity, CASP3 cleavage, and PARP1 [poly (ADP-ribose) polymerase 1] cleavage, which are important to the apoptotic process. Finally, upregulation of MIR34A, knockdown of HMGB1, or inhibition of autophagy (e.g., knockdown of ATG5 and BECN1) restores chemosensitivity and enhances tumor cell death in the retinoblastoma cell. These data provide new insights into the mechanisms governing the regulation of HMGB1 expression by microRNA and their possible contribution to autophagy and drug resistance.     

Targeting of EIF4EBP1 by miR-99a-3p affects the functions of B lymphocytes via autophagy and aggravates SLE disease progression

Excessive activation of immune cells plays a key role in the pathogenesis of systemic lupus erythematosus (SLE). The regulation of immune cells by miRNAs is a research hotspot. In this study, second-generation high-throughput sequencing revealed a reduction in miR-99a-3p expression in patients with SLE; however, the specific mechanism underlying this phenomenon remains unclear. After transfection with an miR-99a-3p agomir, the proliferation of Ball-1 cells decreased and the levels of their apoptosis increased. The opposite effects were observed in cells transfected with the miR-99a-3p antagomir. Luciferase reporter assay indicated that miR-99a-3p directly targeted EIF4EBP1. Rescue experiments confirmed the proposed interaction between miR-99a-3p and EIF4EBP1. In vitro, in vivo and clinical investigations further confirmed that the miR-99a-3p agomir reduced the expression of EIF4EBP1, LC3B and LAMP-2A. In the in vivo experiments, serum levels of anti-nuclear antibodies, double-stranded DNA, IgE, IgM, IL-6, IL-10 and B lymphocyte stimulator were higher in mice from the antagomir group than those in mice from the MRL/lpr group. Furthermore, the protein and mRNA levels of EIF4EBP1, LC3B and LAMP-2A, the intensity of immunohistochemical staining of EIF4EBP1, LC3B and LAMP-2A, the urinary protein levels, and the C3 immunofluorescence deposition increased in mice from the antagomir group. The upregulation of miR-99a-3p expression protected B cells from EIF4EBP1-mediated autophagy, whilst the downregulation of miR-99a-3p expression induced autophagy via the EIF4EBP1-mediated regulation of the autophagy signalling pathway in B cells isolated from individuals with SLE. Based on these results, miR-99a-3p and EIF4EBP1 may be considered potential targets for SLE treatment.     

Enhancer of zeste homolog 2 activates wnt signaling through downregulating CXXC finger protein 4

Through silencing tumor suppressor genes, epigenetic changes can activate signaling pathways important to cancer development. In this report, we found an epigenetic contribution to the aberrant activation of wnt signaling in human gastric cancer. CXXC4 (CXXC finger protein 4) was identified as a novel target of EZH2 (enhancer of zeste homolog 2), and EZH2 promotes the activation of wnt singaling by downregulating CXXC4 expression. CXXC4 inhibits the growth of gastric cancer cells both in vitro and in vivo through inactivating wnt signaling. In contrast, depletion of CXXC4 activates wnt signaling and promotes the anchorage-independent growth of nontumor gastric epithelial cells. CXXC4 is downregulated in gastric carcinoma tissues and its downregulation is associated with poor outcome of gastric cancer patients (hazard ratio: 5.053, P<0.05). Through its binding to dishevelled (Dvl), CXXC4 stabilizes the destruction complex of β-catenin to inhibit wnt signaling. Two critical amino acid residues in CXXC4, K161 and T162 were found to be important to its binding to Dvl and the growth inhibitory effect of CXXC4. In summary, EZH2 promotes the activation of wnt signaling in gastric carcinogenesis through the downregulation of CXXC4 expression. CXXC4 is a novel potential tumor suppressor directly regulated by EZH2, and its expression is a significant prognosis factor for patients with early stages of gastric cancer.     

Characterization of a New CDC73 Missense Mutation that Impairs Parafibromin Expression and Nucleolar Localization

Mutations of the Cell Division Cycle 73 (CDC73) tumor suppressor gene (previously known as HRPT2), encoding for parafibromin, are associated with the Hyperparathyroidism-Jaw Tumor (HPT-JT) syndrome, an autosomal dominant disease whose clinical manifestations are mainly parathyroid tumors and, less frequently, ossifying fibromas of the jaws, uterine and renal tumors. Most mutations of CDC73 are nonsense or frameshift, while missense mutations are rare and generally affect the N-terminal domain of parafibromin, a region that is still poorly characterized. The aim of this study was to characterize a novel somatic CDC73 missense mutation (Ile60Asn) identified in the mandibular tumor of a HPT-JT patient carrying a germline CDC73 inactivating mutation. Immunostaining of the tumor showed reduced nuclear parafibromin immunoreactivity. Western blotting and confocal microscopy of transfected cells demonstrated that the Ile60Asn mutant parafibromin was less expressed than the wild-type protein and exhibited impaired nucleolar localization. Treatment of transfected cells with translation and proteasome inhibitors demonstrated a decreased stability of the Ile60An mutant, partially due to an increase in proteasomal degradation. Overexpression of the Ile60Asn mutant led to increased cell proliferation and to accumulation in the G2/M phase of cell cycle. Moreover, mutant parafibromin lost the ability to down-regulate c-myc expression. In conclusion, our study shows that a missense mutation in the N-terminus of parafibromin, identified in an ossifying fibroma from a HPT-JT patient, stimulated cell proliferation and impaired parafibromin expression and nucleolar localization, suggesting a relevant role of the N-terminal domain for parafibromin function.     

Genome-Wide and Locus Specific Alterations in CDC73/HRPT2-Mutated Parathyroid Tumors

Mutations in the hyperparathyroidism type 2 (HRPT2/CDC73) gene and alterations in the parafibromin protein have been established in the majority of parathyroid carcinomas and in subsets of parathyroid adenomas. While it is known that CDC73-mutated parathyroid tumors display specific gene expression changes compared to CDC73 wild-type cases, the molecular cytogenetic profile in CDC73-mutated cases compared to unselected adenomas (with an expected very low frequency of CDC73 mutations) remains unknown. For this purpose, nine parathyroid tumors with established CDC73 gene inactivating mutations (three carcinomas, one atypical adenoma and five adenomas) were analyzed for copy number alterations and loss of heterozygosity using array-comparative genomic hybridization (a-CGH) and single nucleotide polymorphism (SNP) microarrays, respectively. Furthermore, CDC73 gene promoter methylation levels were assessed using bisulfite Pyrosequencing. The panel included seven tumors with single mutation and three with double mutations of the CDC73 gene. The carcinomas displayed copy number alterations in agreement with previous studies, whereas the CDC73-mutated adenomas did not display the same pattern of alterations at loci frequently deleted in unselected parathyroid tumors. Furthermore, gross losses of chromosomal material at 1p and 13 were significantly (p = 0.012) associated with parathyroid carcinomas as opposed to adenomas. Quantitative PCR-based copy number loss regarding CDC73 was observed in three adenomas, while all the carcinomas were diploid or showed copy number gain for CDC73 gene. Hypermethylation of the CDC73 gene promoter was not observed. Our data could suggest that CDC73-mutated parathyroid adenomas exhibit a partly unique cytogenetic profile in addition to that of carcinomas and unselected adenomas. Furthermore, CDC73-mutated carcinomas displayed losses at 1p and 13 which are not seen in CDC73-mutated adenomas, making these regions of interest for further studies regarding malignant properties in tumors from CDC73-mutated cases. However, due to the small sample size, validation of the results in a larger cohort is warranted.     

EIF4EBP1 Overexpression Is Associated with Poor Survival and Disease Progression in Patients with Hepatocellular Carcinoma

ObjectiveEIF4EBP1 acts as a crucial effector in mTOR signaling pathway. Studies have suggested that EIF4EBP1 plays a critical role in carcinogenesis. However, the clinical significance and biological role of EIF4EBP1 in hepatocellular carcinoma (HCC) have not been elucidated. Therefore, we aimed to investigate the clinical significance of EIF4EBP1 in HCC.MethodsTotal 128 cases of HCCs were included in this study. EIF4EBP1 expression in HCC tissues was detected by qRT-PCR, Western blot and immunohistochemistry, respectively. Then the relationships between EIF4EBP1 expression and clinical features as well as survival were analyzed.ResultsThe expression level of EIF4EBP1 mRNA is significantly higher in 60% (24/40) of fresh HCC tissues than that in the matched adjacent nontumor liver (NCL) tissues (P = 0.044). Similarly, EIF4EBP1 protein is notably upregulated in 8 HCC tissues (randomly selected from the 40 HCCs) measured by Western blot and is significantly increased in another 88 paraffin-embedded HCCs (53%, 47/88) by immunohistochemistry compared with the matched NCLs (P < 0.001). EIF4EBP1 protein expression in HCC tissues is significantly correlated with serum AFP (P = 0.003) and marginally significantly associated with pathological grade (P = 0.085), tumor number (P = 0.084), tumor embolus (P = 0.084) and capsulation (P = 0.073). Patients with higher EIF4EBP1 protein expression have a much worse 5-year overall survival (40.3% vs 73.6%) and 5-year disease-free survival (33.0% vs 49.0%) than those with low expression. Furthermore, Cox regression analysis shows that EIF4EBP1 protein is an independent prognostic factor for overall survival (HR, 2.285; 95% CI, 1.154–4.527; P = 0.018) and disease-free survival (HR, 1.901; 95% CI, 1.067–3.386; P = 0.029) in HCC patients.ConclusionsOur results demonstrate for the first time that EIF4EBP1 mRNA and protein are markedly up-regulated in HCC tissues, and the protein overexpression is significantly associated with poor survival and progression, which provide a potential new prognostic marker and therapeutic target for HCC patients.     

Morphogenesis beyond Cytokinetic Arrest in Saccharomyces cerevisiae

The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37°C, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother–daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.