Annalisa Pianta

Nucleophosmin is overexpressed in thyroid tumors

Nucleophosmin (NPM) is a protein that contributes to several cell functions. Depending on the context, it can act as an oncogene or tumor suppressor. No data are available on NPM expression in thyroid cells. In this work, we analyzed both NPM mRNA and protein levels in a series of human thyroid tumor tissues and cell lines. By using immunohistochemistry, NPM overexpression was detected in papillary, follicular, undifferentiated thyroid cancer, and also in follicular benign adenomas, indicating it as an early event during thyroid tumorigenesis. In contrast, various levels of NPM mRNA levels as detected by quantitative RT-PCR were observed in tumor tissues, suggesting a dissociation between protein and transcript expression. The same behavior was observed in the normal thyroid FRTL5 cell lines. In these cells, a positive correlation between NPM protein levels, but not mRNA, and proliferation state was detected. By using thyroid tumor cell lines, we demonstrated that such a post-mRNA regulation may depend on NPM binding to p-Akt, whose levels were found to be increased in the tumor cells, in parallel with reduction of PTEN. In conclusion, our present data demonstrate for the first time that nucleophosmin is overexpressed in thyroid tumors, as an early event of thyroid tumorigenesis. It seems as a result of a dysregulation occurring at protein and not transcriptional level related to an increase of p-Akt levels of transformed thyrocytes.

Fludarabine-based induction therapy does not overcome the negative effect of ABCG2 (BCRP) over-expression in adult acute myeloid leukemia patients

Over-expression of multidrug resistance (MDR) proteins PGP and BCRP has a negative prognostic impact in acute myeloid leukemia (AML) patients. Inclusion of fludarabine in induction chemotherapy increases remission rate in PGP over-expressing cases. We investigated the role of BCRP in 138 adult AML patients receiving induction therapy with fludarabine. None of the MDR-related proteins influenced complete remission attainment. Conversely, high levels of BCRP significantly affected disease-free survival, as higher relapse rates (48.5% vs 28.5%) and earlier relapse occurred in BCRP+ patients. Also overall survival was affected by BCRP positivity, and survival significantly worsened in case of concomitant PGP and BCRP over-expression.

Functional characterization of the common c.-32-13T>G mutation of GAA gene: identification of potential therapeutic agents

Glycogen storage disease type II is a lysosomal storage disorder due to mutations of the GAA gene, which causes lysosomal alpha-glucosidase deficiency. Clinically, glycogen storage disease type II has been classified in infantile and late-onset forms. Most late-onset patients share the leaky splicing mutation c.-32-13T>G. To date, the mechanism by which the c.-32-13T>G mutation affects the GAA mRNA splicing is not fully known. In this study, we demonstrate that the c.-32-13T>G mutation abrogates the binding of the splicing factor U2AF65 to the polypyrimidine tract of exon 2 and that several splicing factors affect exon 2 inclusion, although the only factor capable of acting in the c.-32-13 T>G context is the SR protein family member, SRSF4 (SRp75). Most importantly, a preliminary screening using small molecules described to be able to affect splicing profiles, showed that resveratrol treatment resulted in a significant increase of normal spliced GAA mRNA, GAA protein content and activity in cells transfected with a mutant minigene and in fibroblasts from patients carrying the c-32-13T>G mutation. In conclusion, this work provides an in-depth functional characterization of the c.-32-13T>G mutation and, most importantly, an in vitro proof of principle for the use of small molecules to rescue normal splicing of c.-32-13T>G mutant alleles.

Concomitant ABCG2 overexpression and FLT3‐ITD mutation identify a subset of acute myeloid leukemia patients at high risk of relapse

ABCG2 protein overexpression and FLT3 internal tandem duplication (ITD) correlate with higher relapse rate and shorter disease‐free survival (DFS) in acute myeloid leukemia (AML), but no data are available on the possible effect of concomitant presence of these 2 factors.

SMPD1 Mutation Update: Database and Comprehensive Analysis of Published and Novel Variants

Niemann–Pick Types A and B (NPA/B) diseases are autosomal recessive lysosomal storage disorders caused by the deficient activity of acid sphingomyelinase (ASM) because of the mutations in the SMPD1 gene. Here, we provide a comprehensive updated review of already reported and newly identified SMPD1 variants. Among them, 185 have been found in NPA/B patients. Disease‐causing variants are equally distributed along the SMPD1 gene; most of them are missense (65.4%) or frameshift (19%) mutations. The most frequently reported mutation worldwide is the p.R610del, clearly associated with an attenuated NP disease type B phenotype. The available information about the impact of 52 SMPD1 variants on ASM mRNA and/or enzymatic activity has been collected and whenever possible, phenotype/genotype correlations were established. In addition, we created a locus‐specific database easily accessible at http://www.inpdr.org/genes that catalogs the 417 SMPD1 variants reported to date and provides data on their in silico predicted effects on ASM protein function or mRNA splicing. The information reviewed in this article, providing new insights into the genotype/phenotype correlation, is extremely valuable to facilitate diagnosis and genetic counseling of families affected by NPA/B.

Nucleophosmin Delocalization in Thyroid Tumour Cells

Nucleophosmin (NPM) is a multifunctional nucleolar protein that, depending on the context, can act as oncogene or tumour suppressor. Mutations of the NPM1 gene induce delocalization of NPM in acute myeloid leukaemia. Differently, in solid tumours, only NPM overexpression, but not delocalization, has been so far reported. Here, NPM localization in thyroid tumours was investigated. By using immunohistochemistry, we show increase of NPM cytoplasmic localization in follicular adenomas and papillary carcinomas compared to normal thyroid tissue (p = 0.0125 and <0.0001, respectively). NPM1 mutations commonly found in human leukaemia are not present in thyroid tumours. Immunofluorescence in cultured cell lines was utilized to discriminate between nucleolar and nuclear localization. We show that in thyroid cancer cell lines NPM localizes both in the nucleolus and in nucleus, while in non-tumorigenic thyroid cell lines localizes only in nucleolus. Either presence of the histone deacetylase inhibitor trichostatin A or absence of thyroid-stimulating hormone induces NPM nuclear localization in non-tumorigenic thyroid cell lines.

Molecular analysis of a human PAX6 homeobox mutant

Pax6 controls eye, pancreas and brain morphogenesis. In humans, heterozygous PAX6 mutations cause aniridia and various other congenital eye abnormalities. Most frequent PAX6 missense mutations are located in the paired domain (PD), while very few missense mutations have been identified in the homeodomain (HD). In the present report, we describe a molecular analysis of the human PAX6 R242T missense mutation, which is located in the second helix of the HD. It was identified in a male child with partial aniridia in the left eye, presenting as a pseudo-coloboma. Gel-retardation assays revealed that the mutant HD binds DNA as well as the wild-type HD. In addition, the mutation does not modify the DNA-binding properties of the PD. Cell transfection assays indicated that the steady-state levels of the full length mutant protein are higher than those of the wild-type one. In cotransfection assays a PAX6 responsive promoter is activated to a higher extent by the mutant protein than by the wild-type protein. In vitro limited proteolysis assays indicated that the presence of the mutation reduces the sensitivity to trypsin digestion. Thus, we suggest that the R242T human phenotype could be due to abnormal increase of PAX6 protein, in keeping with the reported sensitivity of the eye phenotype to increased PAX6 dosage.

Unexpected phenotype of a typical NPM1 mutant

NPM1 mutations are the most frequently known gene alterations in cytogenetically normal acute myeloid leukaemia patients (CN-AML), and are considered as positive prognostic factors (Mrozek et al, 2007). All of the NPM1 mutations identified so far in CN-AML patients generate a Nuclear Export Signal (NES) and cause the loss of one or two tryptophan residues. This feature is believed to be necessary for the mutated npm protein to play a role in leukaemia (Falini et al, 2006a; Liso et al, 2008). NPM1 mutations were investigated in 103 AML patients referred to the Haematology Unit of Udine University Hospital. RNA extraction from bone marrow and peripheral blood mononucleated cells was performed by RNABlood Mini Kit (Qiagen, Hilden, Germany); cDNA was generated by standard techniques and amplified to obtain NPM1 polymerase chain reaction (PCR) products, as previously described (Noguera et al, 2002; Pitiot et al, 2007). Exons 11 and 12 of NPM1 were amplified together using the following primers: 5¢-6FAM-GGTGGTTCTCTTCCCAAAGT-3¢ and 5¢-AACATT TATCAAACACGGTA-3¢; the amplified products were analysed by DNA sequencing, using standard procedures. Two novel NPM1 mutations were identified in two patients; one was in exon 11 and the other was in exon 12. Patient 1 was diagnosed with AML in June 2007. Bone marrow aspirate was diagnostic for AML, French-AmericanBritish classification subtype M2. Cytogenetic and molecular analysis showed a normal karyotype, internal tandem duplication (ITD) mutation of FLT3 and overexpression of BAALC. This patient was heterozygous for a double mutation in NPM1, namely an insertion (976_985insGGAATCTCCG) and a deletion (976_977delTC) in exon 11, that caused a frameshift, leading to the creation of a truncated protein (274 aa instead of 294 aa). However, as shown in Fig 1, the mutant allele may be due to a double insertion (i.e. 976_979insGGAA and 982_985insTCCG). The sizing of PCR fragments showed that the two mutations were in cis because of the presence of only two alleles, wild-type (243 bp) and mutant (251 bp) (data not shown). The mutant protein was characterized by a putative NES motif with the VxxxFxxLxI sequence at its C-terminal (Albiero et al, 2007), according to the nesbase version 1.1 programme (http://www.cbs.dtu.dk prediction server). This mutation differed from the other two that have been identified so far in exon 11 (Albiero et al, 2007; Pitiot et al, 2007), but produced a protein with an amino acid sequence identical to the one that has already been described (Albiero et al, 2007), with the exception of two amino acids (Fig 1). Patient 2 was also diagnosed with AML in June 2007. Cytogenetic analysis displayed a normal chromosomal asset. The patient was positive for the FLT3 ITD mutation and overexpression of BAALC. This patient was heterozygous for a novel insertion (1023_1026insAGGC) in exon 12, which determined the loss of W290 (but retention of W288) and created a putative NES motif with the LxxxLxxVxL sequence, which was different to that observed in Patient 1 (Fig 1). This type of NES motif has already been described and, as in our case, has been found in association with W288 (Bolli et al, 2007). Both patients are currently in complete remission with no NPM1 mutation (data not shown). In order to detect the effects of mutations on the npm phenotype, npm localization in mutant blasts was analysed by immunofluorescence. As expected, a sample from an AML patient without the npm mutation was characterized by complete npm nucleolar localization, while blasts from an AML patient carrying the npm type ‘A’ mutation (the most frequent NPM1 mutation in AML-CN patients) showed the typical npm cytoplasmic localization. While the blasts of Patient 2 displayed a nuclear/nucleolar localization, in addition to cytoplasmic npm, the sample from Patient 1 showed a complete nucleolar localization of npm (Fig 2A). The latter phenotype was unexpected on the basis of current models of npm localization (Mrozek et al, 2007). To better investigate the results obtained from the patients’ blasts, we used a transfection approach. Mutant constructs were generated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) and the plasmid template that was adopted for this purpose was pcDNAHANPM, containing full length NPM1 cDNA, with the HA flag at the N-terminal. HeLa cells were transfected with wild-type and mutant constructs using the calcium-phosphate method. Localization of npm proteins produced by transfected constructs was performed by immunofluorescence using an anti-HA flag antibody. Different types of mutants were generated: (i) Mutant 1 from Patient 1’s mutation; (ii) Mutant 2 from Patient 2’s mutation; (iii) Mutant 3 consisting of mutant 1 reverted to an already described NPM1 mutant (Albiero et al, 2007); and (iv) Mutant A, containing the well-known NPM1 type ‘A’ mutation, used as positive control for npm delocalization (Mrozek et al, 2007). All these mutant constructs were transfected in HeLa cells and npm localization was detected by immunofluorescence (Fig 2B, C). As expected, the wild-type protein had correspondence