p38ß (MAPK11) mediates gemcitabine-associated radiosensitivity in sarcoma experimental models.

BACKGROUND AND PURPOSE: Gemcitabine is an antitumour agent currently used in the treatment of several types of cancer with known properties as a radiosensitizer. p38MAPK signalling pathway has been shown to be a major determinant in the cellular response to gemcitabine in different experimental models. However, the molecular mechanism implicated in gemcitabine-associated radiosensitivity remains unknown. MATERIALS AND METHODS: The human sarcoma cell lines A673 and HT1080, and a mouse cell line derived from a 3-methylcholanthrene induced sarcoma were used as experimental models. Modulation of p38MAPKs was performed by pharmacological approaches (SB203580) and genetic interference using lentiviral vectors coding for specific shRNAs. Viability was assessed by MTT. Gene expression was evaluated by western blot and RT-qPCR. Induction of apoptosis was monitored by caspase 3/7 activity. Response to ionizing radiation was evaluated by clonogenic assays. RESULTS: Our data demonstrate that chemical inhibition of p38MAPK signalling pathway blocks gemcitabine radiosensitizing potential. Genetic interference of MAPK14 (p38α), the most abundantly expressed and best characterized p38MAPK, despite promoting resistance to gemcitabine, it does not affect its radiosensitizing potential. Interestingly, specific knockdown of MAPK11 (p38ß) induces a total loss of the radiosensitivity associated to gemcitabine, as well as a marked increase in the resistance to the drug. CONCLUSION: The present work identifies p38ß as a major determinant of the radiosensitizing potential of gemcitabine without implication of p38α, suggesting that p38ß status should be analysed in those cases in which gemcitabine is combined with ionizing radiation.

Mutant p53 inhibits miRNA biogenesis by interfering with the microprocessor complex.

Downregulation of microRNAs (miRNAs) is commonly observed in cancers and promotes tumorigenesis suggesting that miRNAs may function as tumor suppressors. However, the mechanism through which miRNAs are regulated in cancer, and the connection between oncogenes and miRNA biogenesis remain poorly understood. The TP53 tumor-suppressor gene is mutated in half of human cancers resulting in an oncogene with gain-of-function activities. Here we demonstrate that mutant p53 (mutp53) oncoproteins modulate the biogenesis of a subset of miRNAs in cancer cells inhibiting their post-transcriptional maturation. Interestingly, among these miRNAs several are also downregulated in human tumors. By confocal, co-immunoprecipitation and RNA-chromatin immunoprecipitation experiments, we show that endogenous mutp53 binds and sequesters RNA helicases p72/82 from the microprocessor complex, interfering with Drosha-pri-miRNAs association. In agreement with this, the overexpression of p72 leads to an increase of mature miRNAs levels. Moreover, functional experiments demonstrate the oncosuppressive role of mutp53-dependent miRNAs (miR-517a, -519a, -218, -105). Our study highlights a previously undescribed mechanism by which mutp53 interferes with Drosha-p72/82 association leading, at least in part, to miRNA deregulation observed in cancer.

Genetic epistasis between killer immunoglobulin-like receptors and human leukocyte antigens in Kawasaki disease susceptibility.

Kawasaki disease (KD) is a pediatric acute multisystemic vasculitis complicated by development of coronary artery lesions. The breakthrough theory on KD etiopathogenesis points to pathogens/environmental factors triggered by northeastern wind coming from China. Natural Killer cells and T lymphocytes express the inhibitory/activating Killer Immunoglobulin-like Receptors (KIR) to elicit an immune response against pathogens by binding to human leukocyte antigens (HLA) class I epitopes. We first report on the role of KIR/HLA genetic epistasis in a sample of 100 Italian KD children. We genotyped KIR, HLA-A, HLA-B and HLA-C polymorphisms, and compared KD data with those from 270 Italian healthy donors. The HLA-A*11 ligand for KIR2DS2/2DS4/3DL2 was a KD susceptibility marker by itself (odds ratio (OR)=3.85, confidence interval (CI)=1.55-9.53, P=0.004). Although no epistasis between HLA-A*11 and KIR2DS2/S4 emerged, HLA-A*11 also engages KIR3DL2, a framework gene encoding for a pathogen sensor of CpG-oligodeoxynucleotides (CpG-ODN), and KD blood mononuclear cells are actually prone to pathogen CpG-ODN activation in the acute phase. Moreover, carriers of KIR2DS2/HLA-C1 and KIR2DL2/HLA-C1 were more frequent among KD, in keeping with data demonstrating the involvement of these HLA/KIR couples in autoimmune endothelial damage. The highest KD risk factor was observed among carriers of KIR2DL2 and two or more HLA ligands (OR=10.24, CI=1.87-56.28; P=0.007).

Targeting MKK3 as a novel anticancer strategy: molecular mechanisms and therapeutical implications.

Mitogen-activated protein kinase kinase 3 (MAP2K3, MKK3) is a member of the dual specificity protein kinase group that belongs to the MAP kinase kinase family. This kinase is activated by mitogenic or stress-inducing stimuli and participates in the MAP kinase-mediated signaling cascade, leading to cell proliferation and survival. Several studies highlighted a critical role for MKK3 in tumor progression and invasion, and we previously identified MKK3 as transcriptional target of mutant (mut) p53 to sustain cell proliferation and survival, thus rendering MKK3 a promising target for anticancer therapies. Here, we found that targeting MKK3 with RNA interference, in both wild-type (wt) and mutp53-carrying cells, induced endoplasmic reticulum stress and autophagy that, respectively, contributed to stabilize wtp53 and degrade mutp53. MKK3 depletion reduced cancer cell proliferation and viability, whereas no significant effects were observed in normal cellular context. Noteworthy, MKK3 depletion in combination with chemotherapeutic agents increased tumor cell response to the drugs, in both wtp53 and mutp53 cancer cells, as demonstrated by enhanced poly (ADP-ribose) polymerase cleavage and reduced clonogenic ability in vitro. In addition, MKK3 depletion reduced tumor growth and improved biological response to chemotherapeutic in vivo. The overall results indicate MKK3 as a novel promising molecular target for the development of more efficient anticancer treatments in both wtp53- and mutp53-carrying tumors.

Mutant p53 gains new function in promoting inflammatory signals by repression of the secreted interleukin-1 receptor antagonist.

The TP53 tumor-suppressor gene is frequently mutated in human cancer. Missense mutations can add novel functions (gain-of-function, GOF) that promote tumor malignancy. Here we report that mutant (mut) p53 promotes tumor malignancy by suppressing the expression of a natural occurring anti-inflammatory cytokine, the secreted interleukin-1 receptor antagonist (sIL-1Ra, IL1RN). We show that mutp53 but not wild-type (wt) p53 suppresses the sIL-1Ra production in conditioned media of cancer cells. Moreover, mutp53, but not wtp53, binds physically the sIL-1Ra promoter and the protein-protein interaction with the transcriptional co-repressor MAFF (v-MAF musculoaponeurotic fibrosarcoma oncogene family, protein F) is required for mutp53-induced sIL-1Ra suppression. Remarkably, when exposed to IL-1 beta (IL-1ß) inflammatory stimuli, mutp53 sustains a ready-to-be-activated in vitro and in vivo cancer cells' response through the sIL-1Ra repression. Taken together, these results identify sIL-1Ra as a novel mutp53 target gene, whose suppression might be required to generate a chronic pro-inflammatory tumor microenvironment through which mutp53 promotes tumor malignancy.

Degradation of mutant p53H175 protein by Zn(II) through autophagy.

TP53, one of the most important oncosuppressors, is frequently mutated in cancer. Several p53 mutant proteins escape proteolytic degradation and are highly expressed in an aberrant conformation often acquiring pro-oncogenic activities that promote tumor progression and resistance to therapy. Therefore, it has been vastly proposed that reactivation of wild-type (wt) function(s) from mutant p53 (mutp53) may have therapeutic significance. We have previously reported that Zn(II) restores a folded conformation from mutp53 misfolding, rescuing wild-type (wt) p53/DNA-binding and transcription activities. However, whether Zn(II) affects mutp53 stability has never been investigated. Here we show that a novel Zn(II) compound induced mutp53 (R175H) protein degradation through autophagy, the proteolytic machinery specifically devoted to clearing misfolded proteins. Accordingly, pharmacological or genetic inhibition of autophagy prevented Zn(II)-mediated mutp53H175 degradation as well as the ability of the Zn(II) compound to restore wtp53 DNA-binding and transcription activity from this mutant. By contrast, inhibition of the proteasome failed to do so, suggesting that autophagy is the main route for p53H175 degradation. Mechanistically, Zn(II) restored the wtp53 ability to induce the expression of the p53 target gene DRAM (damage-regulated autophagy modulator), a key regulator of autophagy, leading to autophagic induction. Accordingly, inhibition of wtp53 transactivation by pifithrin-α (PFT-α) impaired both autophagy and mutp53H175 degradation induced by curcumin-based zinc compound (Zn(II)-curc). Viewed together, our results uncover a novel mechanism employed by Zn(II)-curc to reactivate mutp53H175, which involves, at least in part, induction of mutp53 degradation via wtp53-mediated autophagy.

Inhibition of p85, the non-catalytic subunit of phosphatidylinositol 3-kinase, exerts potent antitumor activity in human breast cancer cells.

The phosphoinositide 3-kinases (PI3Ks) are heterodimers consisting of the catalytic subunit p110 and the regulatory subunit p85. The PI3K/Akt pathway is strongly deregulated in breast cancer (BC) representing one of the mechanisms of resistance to therapies. Therefore, the identification of inhibitors of PI3K components represents one of the main goals to produce therapeutic agents. Here, we evaluated the efficacy of a phosphopeptide 1257 (P-1257) that targeting p85 strongly inhibits PI3K activity. We tested the effects of P-1257 administration in vitro and in vivo using BC cells expressing different levels of ErbB-2 and resistant or responsive to Trastuzumab. We demonstrated that inhibition of p85 activity by P-1257 induces cell death and sensitizes JIMT-1 and KPL-4 ErbB-2-overexpressing BC cells to Trastuzumab treatment. It is noteworthy that P-1257 delivery in vivo by electroporation or liposomes significantly inhibits the proliferation of tumor cells engrafted at subcutaneous and visceral sites. Overall, our data indicate that the p85 subunit is a valid target for therapeutic approaches and suggest that the structure of the peptide used in our study could be utilized for the development of novel drugs to apply in combination with therapies that fail to cure BCs with high PI3K activity.

HER3 targeting of adenovirus by fiber modification increases infection of breast cancer cells in vitro, but not following intratumoral injection in mice.

Despite the tremendous potential of adenovirus (Ad) as a delivery vector for cancer gene therapy, its use in clinical settings has been limited, mainly as a result of the limited infectivity in many tumors and the wide tissue tropism associated with Ad. To modify the tropism of the virus, we have inserted the epidermal growth factor-like domain of the human heregulin-α (HRG) into the HI loop of Ad5 fiber. This insertion had no adverse effect on fiber trimerization nor did it affect incorporation of the modified fiber into infectious viral particles. Virions bearing modified fiber displayed growth characteristics and viral yields indistinguishable from those of wild-type (wt) virus. Most importantly, HRG-tagged virions showed enhanced infection of cells expressing the cognate receptors HER3/ErbB3 and HER4/ErbB4. This was significantly reduced in the presence of soluble HRG. Furthermore, HER3-expressing Chinese hamster ovary (CHO) cells were transduced by the HRG-modified virus, but not by wt virus. In contrast, CHO cells expressing the coxsackie-Ad receptor were transduced with both viruses. However, infection of an in vivo breast cancer xenograft model after intratumoral injection was similar with both viruses, suggesting that the tumor microenvironment and/or the route of delivery have important roles in infection of target cells with fiber-modified Ads.

Long-term clinical profile of children with the low-penetrance R92Q mutation of the TNFRSF1A gene.

OBJECTIVE: To analyze the long-term impact of the R92Q mutation of TNFRSF1A in children with periodic fever, in comparison with children with tumor necrosis factor receptor-associated periodic syndrome (TRAPS) with TNFRSF1A structural mutations and children with periodic fever of unknown origin fulfilling the criteria for periodic fever, aphthosis, pharyngitis, and adenitis syndrome (PFAPA). METHODS: The extracellular region of TNFRSF1A was analyzed in 720 consecutive children with periodic fever, using denaturing high-performance liquid chromatography and DNA sequencing. Followup data on 11 pediatric patients with TNFRSF1A structural mutations (cysteine or T50M), 23 pediatric patients with an R92Q substitution, and 64 pediatric patients with PFAPA were collected during routine clinic visits. The 50-item Child Health Questionnaire was used to assess health-related quality of life (HRQOL). RESULTS: The frequency of typical TRAPS-related clinical manifestations was significantly lower and the impact of the disease on HRQOL was significantly reduced in patients with the R92Q mutation compared with TRAPS patients carrying structural mutations of TNFRSF1A. Followup data on 11 TRAPS patients with TNFRSF1A structural mutations (mean followup 7.9 years), 16 patients with theR92Q substitution (mean followup 7.3 years), and 64 patients with PFAPA (mean followup 5.2 years) were available. Patients with R92Q mutations and patients with PFAPA displayed a higher rate of self-resolution or amelioration of the fever episodes than did TRAPS patients with structural mutations. CONCLUSION: Although some cases may progress to a more chronic disease course, the majority of children with an R92Q mutation of the TNFRSFA1 gene show a milder disease course than that in children with TNFRSFA1 structural mutations and have a high rate of spontaneous resolution and amelioration of the recurrent fever episodes.

A prospective study on children with initial diagnosis of transient hypogammaglobulinemia of infancy: results from the Italian Primary Immunodeficiency Network.

Transient hypogammaglobulinemia of infancy (THI) is a heterogeneous disorder characterized by reduced serum IgG levels in early infancy. A putative diagnosis is initially made after exclusion of other causes of hypogammaglobulinemia while a definitive diagnosis of THI can only be made a posteriori in patients with normalization of IgG levels. The aim of this study is to characterize clinical and immunological features of children with an initial diagnosis of THI in correlation to natural outcome, and to assess predictive laboratory parameters of clinical evolution for this disorder. We prospectively analysed clinical and immunological characteristics of 77 THI children at initial diagnosis and of 57 patients at follow-up. Memory B cell subsets and in vitro immunoglobulin production were evaluated. Seventy patients (91 percent) showed clinical symptoms. Patients suffered from infections (91 percent), allergies (47 percent) and autoimmune disease (4 percent). During follow-up 41/57 children (72 percent) normalized IgG values, mostly within 24 months of age (p less than 0.001), allowing the diagnosis of THI. The 16 children who did not normalize their IgG levels showed a higher frequency of severe infections and autoimmune disease (p less than 0.01). Moreover, they expressed a reduced frequency of IgM and switched memory B cells (p less than 0.01) and an inability to produce IgG in vitro (p less than 0.02). We conclude that most patients with an initial diagnosis of THI spontaneously recover within 24 months of age and have a benign clinical course, while a subgroup of children with undefined hypogammaglobulinemia share a clinical and immunological profile with other primary immunodeficiencies. Early recognition of children with hypogammaglobulinemia during infancy who are likely to suffer from permanent immunodeficiencies later in life would allow prompt and appropriate laboratory and clinical interventions.

A diagnostic score for molecular analysis of hereditary autoinflammatory syndromes with periodic fever in children.

OBJECTIVE: To identify a set of clinical parameters that can predict the probability of carrying mutations in one of the genes associated with hereditary autoinflammatory syndromes. METHODS: A total of 228 consecutive patients with a clinical history of periodic fever were screened for mutations in the MVK, TNFRSF1A, and MEFV genes, and detailed clinical information was collected. A diagnostic score was formulated based on univariate and multivariate analyses in genetically positive and negative patients (training set). The diagnostic score was validated in an independent set of 77 patients (validation set). RESULTS: Young age at onset (odds ratio [OR] 0.94, P = 0.003), positive family history of periodic fever (OR 4.1, P = 0.039), thoracic pain (OR 4.6, P = 0.05), abdominal pain (OR 33.1, P < 0.001), diarrhea (OR 3.3, P = 0.028), and oral aphthosis (OR 0.2, P = 0.007) were found to be independently correlated with a positive genetic test result. These variables were combined in a linear score whose ability to predict a positive result on genetic testing was validated in an independent data set. In this latter set, the diagnostic score revealed high sensitivity (82%) and specificity (72%) for discriminating patients who were genetically positive from those who were negative. In patients with a high probability of having a positive result on genetic testing, a regression tree analysis provided the most reasonable order in which the genes should be screened. CONCLUSION: The proposed approach in patients with periodic fever will increase the probability of obtaining positive results on genetic testing, with good specificity and sensitivity. Our results further help to optimize the molecular analysis by suggesting the order in which the genes should be screened.

Novel Munc13-4 mutations in children and young adult patients with haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disorder characterised by constitutive defects in cellular cytotoxicity resulting in fever, hepatosplenomegaly and cytopenia, and the outcome is fatal unless treated by chemoimmunotherapy followed by haematopoietic stem-cell transplantation. Since 1999, mutations in the perforin gene giving rise to this disease have been identified; however, these account only for 40% of cases. Lack of a genetic marker hampers the diagnosis, suitability for transplantation, selection of familial donors, identification of carriers, genetic counselling and prenatal diagnosis. Mutations in the Munc13-4 gene have recently been described in patients with FHL. We sequenced the Munc13-4 gene in all patients with haemophagocytic lymphohistiocytosis not due to PRF1 mutations. In 15 of the 30 families studied, 12 novel and 4 known Munc13-4 mutations were found, spread throughout the gene. Among novel mutations, 2650C-->T introduced a stop codon; 441del A, 532del C, 3082del C and 3226ins G caused a frameshift, and seven were mis sense mutations. Median age of diagnosis was 4 months, but six patients developed the disease after 5 years of age and one as a young adult of 18 years. Involvement of central nervous system was present in 9 of 15 patients, activity of natural killer cells was markedly reduced or absent in 13 of 13 tested patients. Chemo-immunotherapy was effective in all patients. Munc13-4 mutations were found in 15 of 30 patients with FHL without PRF1 mutations. Because these patients may develop the disease during adolescence or even later, haematologists should include FHL2 and FHL3 in the differential diagnosis of young adults with fever, cytopenia, splenomegaly and hypercytokinaemia.

Mutant p53 gain of function: reduction of tumor malignancy of human cancer cell lines through abrogation of mutant p53 expression.

Mutations in the TP53 tumor suppressor gene are the most frequent genetic alteration in human cancers. These alterations are mostly missense point mutations that cluster in the DNA binding domain. There is growing evidence that many of these mutations generate mutant p53 proteins that have acquired new biochemical and biological properties. Through this gain of function activity, mutant p53 is believed to contribute to tumor malignancy. The purpose of our study was to explore mutant p53 as a target for novel anticancer treatments. To this aim, we inhibited mutant p53 expression by RNA interference in three different cancer cell lines endogenously expressing mutant p53 proteins, and evaluated the effects on the biological activities through which mutant p53 exerts gain of function. We found that depletion of mutant p53 reduces cell proliferation, in vitro and in vivo tumorigenicity, and resistance to anticancer drugs. Our results demonstrate that mutant p53 knocking down weakens the aggressiveness of human cancer cells, and provides further insight into the comprehension of mutant p53 gain of function activity in human tumor.

The immunological synapse of CTL contains a secretory domain and membrane bridges.

Cytotoxic T lymphocytes (CTL) rapidly destroy their targets. Here we show that although target cell death occurs within 5 min of CTL-target cell contact, an immunological synapse similar to that seen in CD4 cells rapidly forms in CTL, with a ring of adhesion proteins surrounding an inner signaling molecule domain. Lytic granule secretion occurs in a separate domain within the adhesion ring, maintaining signaling protein organization during exocytosis. Live and fixed cell studies show target cell plasma membrane markers are transferred to the CTL as the cells separate. Electron microscopy reveals continuities forming membrane bridges between the CTL and target cell membranes, suggesting a possible mechanism for this transfer.

Fas ligand is targeted to secretory lysosomes via a proline-rich domain in its cytoplasmic tail.

Fas ligand (FasL) induces apoptosis through its cell surface receptor Fas. T lymphocytes and natural killer cells sort newly synthesised FasL to secretory lysosomes but, in cell types with conventional lysosomes, FasL appears directly on the plasma membrane. Here, we define a proline-rich domain (PRD) in the cytoplasmic tail of FasL that is responsible for sorting FasL to secretory lysosomes. Deletion of this PRD results in cell surface expression of FasL in cells with secretory lysosomes. Positively charged residues flanking the PRD are crucial to the sorting motif and changing the charge of these residues causes mis-sorting to the plasma membrane. In cells with conventional lysosomes, this motif is not recognised and FasL is expressed at the plasma membrane. The FasL PRD is not required for endocytosis in any cell type, as deletion mutants lacking this motif are endocytosed efficiently to the lysosomal compartment. Endogenous FasL cannot internalise extracellular antibody, demonstrating that FasL does not transit the plasma membrane en route to the secretory lysosomes. We propose that an interaction of the PRD of FasL with an SH3-domain-containing protein, enables direct sorting of FasL from the Golgi to secretory lysosomes.

Echocardiographic evaluation of patients cured of childhood cancer: a single center study of 117 subjects who received anthracyclines.

BACKGROUND: The risk of cardiomyopathy following exposure to anthracycline in asymptomatic long-term survivors of childhood cancer is still hard to predict and precisely quantify. To identify the impact of different cumulative doses, even within a non-high dose range, and the echocardiographic parameters suitable for evaluating cardiac function, we studied diastolic and systolic echocardiographic parameters in a cohort of patients followed in a single center. PROCEDURE: A total of 117 subjects were studied at a median time of 7 years after treatment completion. A complete M-mode, two-dimensional and Doppler echocardiographic study was obtained at rest in all patients according to the standard recommendations of the American Society of Echocardiography. RESULTS: Ninety-nine patients (85%) had completely normal cardiac function, while 18 had abnormal echocardiographic findings: 12 had one abnormal value, 5 had two, and 1 had three abnormal values. All the changes were in left ventricular dimensions, wall thickness or indices of systolic function; no alterations in left ventricular diastolic function parameters were found. None of the echocardiographic parameters correlated significantly with the cumulative dose of anthracyclines administered either at univariate analysis or after adjusting for sex, body surface area or considered risk factors. CONCLUSIONS: Subjects exposed to a median cumulative dose of 214 mg/m(2) had no echographic abnormalities a median of 7 years later. We did not find any correlation between cumulative anthracycline dose and the echocardiographic parameters tested. We now offer echocardiographic follow-up to patients with mildly reduced fractional shortening and/or ejection fraction to rule out late onset dysfunction.

Neonatal screening program for congenital adrenal hyperplasia: adjustments to the recall protocol.

OBJECTIVE: To evaluate the influence of gestational age (GA) and birth weight (BW) on 17 alpha-OH-progesterone (17-OHP) levels with respect to their impact on the recall rate of neonatal screening programs for congenital adrenal hyperplasia (CAH). PATIENTS AND METHODS: In June 1997 we began a pilot screening program for CAH measuring 17-OHP using a fluoroimmunoassay method (DELFIA) on dried blood spots. Until September 1999, 24,153 babies were screened. Among them, we analyzed the levels of 17-OHP in 1,313 samples from healthy preterm babies (23-36 weeks) and 1,500 term babies (>37 weeks), grouped according to GA and BW. All preterm babies underwent another sampling in their 2nd week of life. RESULTS: 5 CAHs were detected. The 30-nmol/l cutoff limit for 17-OHP in blood corresponded to the calculated 99th percentile in term newborns, while in preterm babies higher levels were found. GA and BW correlated inversely with 17-OHP levels. CONCLUSION: GA and BW were useful tools to adjust cutoff levels, obtaining a significant reduction in follow-up testing and psychological stress for families. The high false-positive recall rate in preterm babies can be substantially lowered with adjusted GA and/or BW criteria.