Sara Volorio

Alterations of the Notch pathway in lung cancer

Notch signaling regulates cell specification and homeostasis of stem cell compartments, and it is counteracted by the cell fate determinant Numb. Both Numb and Notch have been implicated in human tumors. Here, we show that Notch signaling is altered in approximately one third of non–small-cell lung carcinomas (NSCLCs), which are the leading cause of cancer-related deaths: in ≈30% of NSCLCs, loss of Numb expression leads to increased Notch activity, while in a smaller fraction of cases (around 10%), gain-of-function mutations of the NOTCH-1 gene are present. Activation of Notch correlates with poor clinical outcomes in NSCLC patients without TP53 mutations. Finally, primary epithelial cell cultures, derived from NSCLC harboring constitutive activation of the Notch pathway, are selectively killed by inhibitors of Notch (γ-secretase inhibitors), showing that the proliferative advantage of these tumors is dependent upon Notch signaling. Our results show that the deregulation of the Notch pathway is a relatively frequent event in NSCLCs and suggest that it might represent a possible target for molecular therapies in these tumors.

Delocalization and destabilization of the Arf tumor suppressor by the leukemia-associated NPM mutant.

One third of acute myeloid leukemias (AMLs) are characterized by the aberrant cytoplasmic localization of nucleophosmin (NPM) due to mutations within its putative nucleolar localization signal. NPM mutations are mutually exclusive with major AML-associated chromosome rearrangements and are frequently associated with a normal karyotype, suggesting that they are critical during leukemogenesis. The underlying molecular mechanisms are, however, unknown. NPM is a nucleocytoplasmic shuttling protein that has been implicated in several cellular processes, including ribosome biogenesis, centrosome duplication, cell cycle progression, and stress response. It has been recently shown that NPM is required for the stabilization and proper nucleolar localization of the tumor suppressor p19(Arf). We report here that the AML-associated NPM mutant localizes mainly in the cytoplasm due to an alteration of its nucleus-cytoplasmic shuttling equilibrium, forms a direct complex with p19(Arf), but is unable to protect it from degradation. Consequently, cells or leukemic blasts expressing the NPM mutant have low levels of cytoplasmic Arf. Furthermore, we show that expression of the NPM mutant reduces the ability of Arf to initiate a p53 response and to induce cell cycle arrest. Inactivation of p19(Arf), a key regulator of the p53-dependent cellular response to oncogene expression, might therefore contribute to leukemogenesis in AMLs with mutated NPM.

DEK Expression is Controlled by E2F and Deregulated in Diverse Tumor Types

Deregulation of the retinoblastoma (pRB) tumor suppressor pathway associated withaberrant activity of E2F transcription factors is frequently observed in human cancer.Microarray based analyses have revealed a large number of potential downstreammediators of the tumor suppressing activity of pRB, including DEK, a fusion partner ofCAN found in a subset of acute myeloid leukaemia (AML) patients carrying a (6; 9)translocation. Here we report that the expression of DEK is under direct control of E2F transcriptionfactors. Chromatin immunoprecipitation assays show that the DEK promoter is bound byendogenous E2F in vivo. The DEK promoter is transactivated by E2F and mutation ofE2F binding sites eliminates this effect. Expression levels of DEK in human tumors havebeen investigated by tissue micro array analysis. We find that DEK is overexpressed inmany solid tumors such as colon cancer, larynx cancer, bladder cancer, and melanoma.

Cytoplasmic localization of NPM in myeloid leukemias is dictated by gain-of-function mutations that create a functional nuclear export signal

Nucleophosmin (NPM) is a nucleus–cytoplasmic shuttling protein that is implicated in centrosome duplication, cell cycle progression and stress response. At the steady state, NPM localizes mainly in the nucleolus, where it forms a complex with different cellular proteins. One-third of acute myeloid leukemias (AML) are characterized by aberrant cytoplasmic localization of NPM, due to mutations within its last coding exon (exon 12) that cause a frameshift and the formation of novel C-termini. We report here our investigations on the molecular basis for the aberrant localization of mutated NPM. Alignment of the C-terminus of the various NPM mutants revealed the obligatory presence of four amino-acid residues that match a CRM1-dependent nuclear export signal (NES). Single alanine-substitutions at these sites provoked nuclear re-localization, while fusion of the mutated C-terminus to a heterologous nuclear protein induced CRM1-dependent cytoplasmic localization. Molecular characterization of one exceptional AML carrying cytoplasmic NPM and germ line exon 12 revealed a somatic mutation in the splicing donor site of exon 9 that caused the formation of a functional NES. It appears, therefore, that AMLs are frequently characterized by gain-of-function mutations of NPM that create functional NES, suggesting that alterations of nuclear export might represent a general mechanism of leukemogenesis and a novel target for therapeutic intervention.

In vitro and in vivo hematopoietic potential of human stem cells residing in muscle tissue.

OBJECTIVE We studied the in vitro and in vivo hematopoietic potential of human stem cells residing in muscle tissue collected from adults with head and neck cancer. MATERIALS AND METHODS Adherent muscle cells were cultured in F12 medium with 10% fetal bovine serum and transplanted into immunodeficient mice. RESULTS On day 12 we obtained a median of 500,000 adherent cells per gram muscle sample. Thy-1, endoglin, HER2/neu, and P1H12 were expressed in the majority of cells. CD34, VEGFR2, c-kit, VCAM-1, and CXCR4 were expressed in 0.5-1.5%, 1-5%, 1-15%, 9-15%, and 30% of cells, respectively. Immunodeficient mice transplanted with fresh muscle cells or less than 500,000 cultured cells showed little or no human engraftment. In mice transplanted with more than 500,000 cultured cells, up to 14% human CD45(+) hematopoietic cells (including myeloid and lymphoid subsets) were detected by flow cytometry. Engraftment was confirmed by polymerase chain reaction, Southern blotting, and DNA sequencing. Liver, muscle, and spleen evaluated for human DNA were positive in the majority of mice showing hematopoietic engraftment in the bone marrow. In vivo hematopoietic engraftment potential was maintained in cultured CD45(-) muscle cells transduced with the green fluorescence protein gene. CONCLUSIONS Human stem cells residing in muscle tissue can generate multilineage hematopoiesis in immunodeficient mice. Surprisingly, this hematopoietic potential increased in cultured versus fresh cells from muscle tissue.

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

Identification of fifteen novel germline variants in the BRCA1 3′UTR reveals a variant in a breast cancer case that introduces a functional miR-103 target site†

Mutations in the BRCA1 gene confer a substantial increase in breast cancer risk, yet routine clinical genetic screening is limited to the coding regions and intron–exon boundaries, precluding the identification of mutations in noncoding and untranslated regions (UTR). As 3′UTR mutations can influence cancer susceptibility by altering protein and microRNA (miRNA) binding regions, we screened the BRCA1 3′UTR for mutations in a large series of BRCA‐mutation negative, population and clinic‐based breast cancer cases, and controls. Fifteen novel BRCA1 3′UTR variants were identified, the majority of which were unique to either cases or controls. Using luciferase reporter assays, three variants found in cases, c.*528G>C, c.*718A>G, and c.*1271T>C and four found in controls, c.*309T>C, c.*379G>A, c.*823C>T, and c.*264C>T, reduced 3′UTR activity (P < 0.02), whereas two variants found in cases, c.*291C>T and c.*1139G>T, increased 3′UTR activity (P < 0.01). Three case variants, c.*718A>G, c.*800T>C, and c.*1340_1342delTGT, were predicted to create new miRNA binding sites and c.*1340_1342delTGT caused a reduction (25%, P = 0.0007) in 3′UTR reporter activity when coexpressed with the predicted targeting miRNA, miR‐103. This is the most comprehensive identification and analysis of BRCA1 3′UTR variants published to date. Hum Mutat 33:1665–1675, 2012.

Ultradeep Sequencing of a Human Ultraconserved Region Reveals Somatic and Constitutional Genomic Instability

Ultradeep sequencing of genomes permits the detection of very low-level genomic instability in non-neoplastic tissues of patients with the most common form of inherited colorectal cancer.

The lymphoma-associated NPM-ALK oncogene elicits a p16INK4a/pRb-dependent tumor-suppressive pathway

Oncogene-induced senescence (OIS) is a barrier for tumor development. Oncogene-dependent DNA damage and activation of the ARF/p53 pathway play a central role in OIS and, accordingly, ARF and p53 are frequently mutated in human cancer. A number of leukemia/lymphoma-initiating oncogenes, however, inhibit ARF/p53 and only infrequently select for ARF or p53 mutations, suggesting the involvement of other tumor-suppressive pathways. We report that NPM-ALK, the initiating oncogene of anaplastic large cell lymphomas (ALCLs), induces DNA damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is caused by activation of the p16INK4a/pRb tumor-suppressive pathway. Analysis of NPM-ALK lymphomagenesis in transgenic mice showed p16INK4a-dependent accumulation of senescent cells in premalignant lesions and decreased tumor latency in the absence of p16INK4a. Accordingly, human ALCLs showed no expression of either p16INK4a or pRb. Up-regulation of the histone-demethylase Jmjd3 and de-methylation at the p16INK4a promoter contributed to the effect of NPM-ALK on p16INK4a, which was transcriptionally regulated. These data demonstrate that p16INK4a/pRb may function as an alternative pathway of oncogene-induced senescence, and suggest that the reactivation of p16INK4a expression might be a novel strategy to restore the senescence program in some tumors.

The p53 Arg72Pro and Ins16bp polymorphisms and their haplotypes are not associated with breast cancer risk in BRCA-mutation negative familial cases

BACKGROUND Germline disease-causing mutations in BRCA1 and BRCA2 genes confer high risk of breast and ovarian cancer, but account approximately for only 15% of familial cases. Theoretical models and experimental observations have indicated that the remaining familial aggregations would be explained by low-penetrance alleles. Moreover, alleles acting as genetic modifiers would modulate the breast cancer risk in carriers of BRCA mutations. The Ins16bp and Arg72Pro polymorphisms of p53 were implicated in breast cancer and recently it has been shown that these polymorphisms could have an effect when combined as specific haplotypes. Here, we investigated the possible role of the Ins16bp and Arg72Pro polymorphisms and their haplotypes as low-penetrance alleles in familial breast cancer. METHODS The Ins16bp and Arg72Pro polymorphisms were genotyped in a total of 350 familial index cases affected with breast cancer and negative for mutations in BRCA genes, and 352 controls. The Ins16bp and Arg72Pro polymorphisms were studied separately, and as haplotypes and haplotypes combinations. RESULTS None of the performed analyses resulted statistically significant. CONCLUSIONS These observations suggested that neither the Ins16bp or Arg72Pro polymorphisms considered separately, nor any related haplotype, were associated with breast cancer risk in BRCA-mutation negative familial cases.