Federica Mori

Preventive Vaccination with Telomerase Controls Tumor Growth in Genetically Engineered and Carcinogen-Induced Mouse Models of Cancer

The telomerase reverse transcriptase, TERT, is an attractive target for human cancer vaccination because its expression is reactivated in a conspicuous fraction of human tumors. Genetic vaccination with murine telomerase (mTERT) could break immune tolerance in different mouse strains and resulted in the induction of both CD4+ and CD8+ telomerase-specific T cells. The mTERT-derived immunodominant epitopes recognized by CD8+ T cells were further defined in these mouse strains and used to track immune responses. Antitumor efficacy of telomerase-based vaccination was investigated in two cancer models closely resembling human diseases: the TRAMP transgenic mice for prostate cancer and a carcinogen-induced model for colon cancer. TERT overexpression in tumor lesions was shown in both models by immunohistochemistry, thus reinforcing the similarity of these tumors to their human counterparts. Repeated immunizations with mTERT-encoding DNA resulted in a significant delay of tumor formation and progression in both the prostate cancer and the colon cancer models. Moreover, evaluation of the intratumoral infiltrate revealed the presence of telomerase-specific T cells in vaccinated mice. The safety of vaccination was confirmed by the absence of histomorphologic changes on postnecropsy analysis of several organs and lack of adverse effects on blood cell counts. These results indicate that TERT vaccination can elicit antigen-specific immunosurveillance and imply this antigen as a potential candidate for preventive cancer vaccines.

Revealing New Mouse Epicardial Cell Markers through Transcriptomics

Background The epicardium has key functions during myocardial development, by contributing to the formation of coronary endothelial and smooth muscle cells, cardiac fibroblasts, and potentially cardiomyocytes. The epicardium plays a morphogenetic role by emitting signals to promote and maintain cardiomyocyte proliferation. In a regenerative context, the adult epicardium might comprise a progenitor cell population that can be induced to contribute to cardiac repair. Although some genes involved in epicardial function have been identified, a detailed molecular profile of epicardial gene expression has not been available. Methodology Using laser capture microscopy, we isolated the epicardial layer from the adult murine heart before or after cardiac infarction in wildtype mice and mice expressing a transgenic IGF-1 propeptide (mIGF-1) that enhances cardiac repair, and analyzed the transcription profile using DNA microarrays. Principal Findings Expression of epithelial genes such as basonuclin, dermokine, and glycoprotein M6A are highly enriched in the epicardial layer, which maintains expression of selected embryonic genes involved in epicardial development in mIGF-1 transgenic hearts. After myocardial infarct, a subset of differentially expressed genes are down-regulated in the epicardium representing an epicardium-specific signature that responds to injury. Conclusion This study presents the description of the murine epicardial transcriptome obtained from snap frozen tissues, providing essential information for further analysis of this important cardiac cell layer.

Epigenetic silencing of miR-145-5p contributes to brain metastasis.

Brain metastasis is a major cause of morbidity and mortality of lung cancer patients. We assessed whether aberrant expression of specific microRNAs could contribute to brain metastasis. Comparison of primary lung tumors and their matched metastatic brain disseminations identified shared patterns of several microRNAs, including common down-regulation of miR-145-5p. Down-regulation was attributed to methylation of miR-145s promoter and affiliated elevation of several protein targets, such as EGFR, OCT-4, MUC-1, c-MYC and, interestingly, tumor protein D52 (TPD52). In line with these observations, restored expression of miR-145-5p and selective depletion of individual targets markedly reduced in vitro and in vivo cancer cell migration. In aggregate, our results attribute to miR-145-5p and its direct targets pivotal roles in malignancy progression and in metastasis.

Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

The EphA2 receptor tyrosine kinase is overexpressed in a variety of human epithelial cancers and is a determinant of malignant cellular behavior in pancreatic adenocarcinoma cells. Moreover, it is expressed in tumor endothelium and its activation promotes angiogenesis. To better clarify the therapeutic potential of monoclonal antibodies (mAbs) directed to the EphA2 receptor, we generated a large number of mAbs by differential screening of phage-Ab libraries by oligonucleotide microarray technology and implemented a strategy for the rapid identification of antibodies with the desired properties. We selected two high-affinity and highly specific EphA2 monoclonal antibodies with different in vitro properties on the human pancreatic tumor cell line MiaPaCa2. One is a potent EphA2-agonistic antibody, IgG25, that promotes receptor endocytosis and subsequent degradation, and the second is a ligand antagonist, IgG28, that blocks the binding to ephrin A1 and is cross-reactive with the mouse EphA2 receptor. We measured the effect of antibody treatment on the growth of MiaPaCa2 cells orthotopically transplanted in nude mice. Both IgG25 and IgG28 had strong antitumor and antimetastatic efficacy. In vivo treatment with IgG25 determined the reduction of the EphA2 protein levels in the tumor and the phosphorylation of FAK on Tyr576 while administration of IgG28 caused a decrease in tumor vascularization as measured by immunohistochemical analysis of CD31 in tumor sections. These data show that in a pancreatic cancer model comparable therapeutic efficacy is obtained either by promoting receptor degradation or by blocking receptor activation.

Multitargeting activity of miR-24 inhibits long-term melatonin anticancer effects

We have previously shown that melatonin exerts tumor suppressor activities by inducing the p38-p53 axis. This occurred within a few hours while no data are available on how melatonin pathway can be sustained on the long term. Here we show that miR-24, which has been demonstrated to target genes involved in the DNA repair process, targets p38, p53, PML and H2AX simultaneously. We show that long-term treatment with melatonin can decrease miR-24 levels post-transcriptionally, which pairs with a long-wave regulation of genes involved in cell proliferation, DNA damage, RNA metabolism and cell shape and transformation. Moreover, we show that melatonin can inhibit cell proliferation and migration, at least in part, by downregulating miR-24. Furthermore, we propose the involvement of hnRNP A1, which is downregulated by melatonin and involved in miRNA processing, in the regulation of miR-24 levels by melatonin. We conclude showing that miR-24 is upregulated in colon, breast and head and neck datasets and its levels negatively correlate with overall survival.

A TLR9 agonist enhances therapeutic effects of telomerase genetic vaccine

The telomerase reverse transcriptase (TERT) is an attractive target for cancer vaccination because its expression is reactivated in most tumors. In this study, we have evaluated the ability of a genetic vaccine targeting murine TERT (mTERT) based on DNA electroporation (DNA-EP) and adenovirus serotype 6 (Ad6) to exert therapeutic effects in combination with a novel TLR9 agonist, referred to as immune modulatory oligonucleotide (IMO), as an adjuvant. IMO was administered to mice at the same time as vaccine. IMO induced dose-dependent cytokine secretion and activation of NK cells. Most importantly, vaccination of mice with IMO in combination with mTERT vaccine conferred therapeutic benefit in tumor bearing animals and this effect was associated with increased NK, DC and T cell tumor infiltration. These data show that appropriate combination of a DNA-EP/Ad6-based cancer vaccine against TERT with IMO induces multiple effects on innate and adaptive immune responses resulting in a significant antitumor efficacy.

A therapeutic cancer vaccine targeting carcinoembryonic antigen in intestinal carcinomas.

A genetic vaccine platform based on DNA electroporation (DNA-EP) and adenovirus (Ad) was used to generate immune response against human carcinoembryonic antigen (CEA) and antitumor effects in murine models with spontaneous tumors arising in an orthotopic location. CEA transgenic (CEA.Tg) mice treated with the carcinogen 1,2-dimethylhydrazine developed CEA-overexpressing tumors that resembled human sporadic colorectal cancer. APC1638N/CEA hybrid mice, generated by crossing mice carrying the adenomatous polyposis coli (Apc1638N) gene mutation with CEA.Tg mice, are representative of human familial polyposis and develop polyps that overexpress the antigen. In both models, the DNA-EP/Ad vaccine succeeded in breaking immune tolerance and achieved significant antitumor effects in therapeutic settings. Our data suggest that genetic vaccines targeting CEA may be feasible strategies against gut tumors that overexpress the antigen. In addition, these models are powerful systems for evaluating antigen-specific tumor immunity and assessing therapeutic vaccine strategies for human colorectal cancer.

MicroRNAs: short non-coding players in cancer chemoresistance

Chemoresistance is one of the main problems in the therapy of cancer. There are a number of different molecular mechanisms through which a cancer cell acquires resistance to a specific treatment, such as alterations in drug uptake, drug metabolism and drug targets. There are several lines of evidence showing that miRNAs are involved in drug sensitivity of cancer cells in different tumor types and by different treatments. In this review, we provide an overview of the more recent and significant findings on the role of miRNAs in cancer cell drug resistance. In particular, we focus on specific miRNA mechanisms of action that in various steps lead from drug cell sensitivity to drug cell resistance. We also provide evidence on how miRNA profiling may unveil relevant predictive biomarkers for therapy outcomes.

Carnitines slow down tumor development of colon cancer in the DMH-chemical carcinogenesis mouse model.

Dietary agents are receiving much attention for the chemoprevention of cancer. While curcumin is known to influence several pathways and affect tumor growth in vivo, carnitin and its congeners play a variety of important metabolic functions: are involved in the oxydation of long‐chain fatty acids, regulate acyl‐CoA levels and influence protein activity and stability by modifying the extent of protein acetylation. In this study we evaluated the efficacy of carnitines in the prevention of cancer development using the 1,2,‐dimethylhydrazine (DMH)‐induced colon carcinogenesis model. We also assessed whether their combination was able to give rise to increased protection from cancer development. Mice treated with DMH were dosed orally with curcumin and/or carnitine and acylcarnitines for 20 weeks. At the end of the treatment colon samples were collected, and scored for multiple ACF and adenomas. We observed that carnitine and acyl‐carnitines had same, if not higher, efficacy than curcumin alone in inhibiting the formation of neoplastic lesions induced by DMH treatment. Interestingly, the combination of curcumin and acetyl‐L‐carnitine was able to fully inhibit the development of advanced adenoma lesions. Our data unveil the antitumor effects of carnitines and warrant additional studies to further support the adoption of carnitines as cancer chemopreventative agents. J. Cell. Biochem. 114: 1665–1673, 2013.

Metallothionein colon crypt immuno-positivity as a rapid in vivo essay for drug efficacy studies

Metallothionein immune positivity indices are considered as representative of crypt stem cell mutations. The frequency and size of MT- immunopositive foci, as well as the total number of MT-immunopositive crypts were assessed here in a short term in vivo assay. Drug efficacy was tested on early mutated crypts in colon of Balb-c mice 30 days after induction with a single dose of the mutagen dimethylhydrazine. The different drugs used (MS 275, vioxx, 5-fluorouracil, aminophylline, 5-azadeoxycytine) affected the metallothionein - immunopositive crypt frequency according to their predicted efficacy on this specific model of mouse colon carcinogenesis. This preliminary validation study of metallothionein - immunopositive crypt frequency strengthens the evidence that metallothionein immunopositivity indices could be used as short-term markers to assess the capability of different pro-drugs to counteract crypt invasion and clonal expansion of mutated stem cell progeny. This rapid in vivo test (30 days) based on metallothionein immunopositivity indices can be assayed in paraffin-fixed tissue sections and has been validated against the Glucose 6 phosphate Dehydrogenase assay. To quantify metallothionein immunopositivity indices, we devised a novel fast analysis protocol based on the Zeiss Axiovision software for image processing.H and development of human haematopoietic stem cells (HSC) are the key role to improve efficient expansion for the transplantations. Successful immune reconstitution with HSC is highly dependent on the number of CD34+ cells transplanted/kg body weight. However, BM and especially single UCB extraction provide insufficient numbers of CD34+ cells for adult transplantation, often requiring the pooling of multiple cord samples to obtain the optimal cell dose for full reconstitution. Complications related to the use of pooled samples include the possibility of graft-versus-graft or multiple graft-versus-host reactions and unpredictable reconstitution of each sample. We report here that activation of human GPIlinked glycoprotein ACA at the surface of human peripheral blood (PB) progenitor cells via PLCγ /IPI3K/Akt promotes selfrenewal and generation of primitive HSCs. Pre-clinical studies reveal, that starting with as little as 40 ml PB, averagely 2-6 x106 progenitor / HSCs (CD34+) cells could be generated, resulting in 63,6 -192 fold increase of these cells compared with the number of these cells that circulate in PB. Evidently, a larger volume of PB, as used in a clinical routine, would give a far greater yield of HSCs. Using xeno-transplantation assay in immunocompromised (NSG) mice, ACA-generated self-renewing cells show long-term multi-lineage repopulation activity in primary and secondary recipients, including T-and natural killer cells. Unmanipulated, steady-state peripheral blood as the strictly autologous source for generation and expansion of early primitive HSCs with long-term functional capacity is an easily accessible and practically infinite source of cells which could circumvent the most confiding limitations of HSCs transplantation, insufficient number of CD34 (CB) or availability of appropriate donor (BM). Accessibility of PB as well as generation and amplification of CD34+ cells by ACA method warrants the major problem in transplantation medicine to be solved.A proper screening for colorectal cancer (CRC) can reduce morbidity and mortality by this disease. However, the current screening modalities are inadequate due to high cost and poor compliance leading to a low participation rate. Therefore, it would be urgently necessary to develop an alternative screening tool such as biomarker-based blood test.We have attempted identify DNA methylation sites which can be utilized as a biomarker for the non-invasive diagnostics of CRC patient at earliest stage.For this, we utilized methylation microarray analysis coupled with enriched methylated DNA bymethylated DNA isolation assay (MeDIA) and discovered differentially methylated genes between tumor and non-tumor tissues.Through step-wise filteringprocedures we then selected an aberrant SDC2 methylation as a potential biomarker for discriminating CRC patients from healthy individuals. When we analyzed methylation status of SDC2 in tumor and non-tumor samples by pyro-sequencing we found that abnormal SDC2 methylation is somatically acquired in the majority of patients with CRC (98%, n=133).We then revealed that the quantitative SDC2 methylation (qMSP) test by real-time PCR in serum DNA can successfully detect of various stage of CRC patients (n=131)with overall high sensitivity (87%, n=131)and specificity (95% n=125). Notably,the sensitivity was 92% for stage I.Currently we extended this to investigate whether aberrant presence of SDC2 methylation in blood can also distinguish patients with adenoma from normal subjects. For quantitative SDC2 methylation assay in heterogeneous and minute amount of serum DNA from precancerous patientswe have optimized detection method of qMSP (meSDC2 IP-qMSP) by degenerated inosine-primer design. The limit of detection for methylated SDC2 test was 100 pg/mL of serum corresponding to ~30 genome copies.A meSDC2 IP-qMSP in serum DNA from independent group of CRC patients(n=146) with various stages (I-IV), 100 adenoma patients and 51 colonoscopy-verified normal subjects showed the overall sensitivities of 91.1% (95% CI, 85.3% to 95.2%) and 71.0% (95% CI, 61.1% to 79.6%) for CRC and adenomatous polyps, respectively with a specificity of 86.3% (95% CI, 73.7% to 94.3%). The sensitivity was 93.5% (29/31) for stage I of CRC.SDC2 methylation level in serum from preoperative patients dramatically disappeared or dropped to lower after curative surgery (P=0.002) indicating this phenomenon is a CRC-specific. We demonstrated that a simple blood DNA test that measures an abnormal presence of SDC2 methylation in serum DNA can successfully distinguish not only CRC patients but also those with adenoma from healthy subjects.P A2 (PLA2) is a universal toxi enzyme present in all the snake venoms causing painful inflammation. PLA 2 forms 60% of the proteins of the venom, hence, preventing PLA2 action is of great importance, first breakthrough came from that, Turmerin: A water soluble antioxidant protein from Turmeric (Curcuma longa Linn) prevents its inflammatory action. Snake venom also induces DNA damage and the damage as assessed by us was much more than be accounted for venom alone. So, we looked for a factor that, may be acting in tandem with the venom in causing apoptosis, such a factor was secreted at 15 minutes after by human lymphocyte treatment with venom PLA2 which can came 44% DNA damage by itself. This factor was purified by several steps was found to be of 6 kDa peptide and its profile was established by using several exocytosis inhibitors. We surmised that, this death peptide signal was of membrane origin. The sequence and homology showed that, it has 40 amino acids MSILPCKNVS IWVIKDTAAS DKEVVLGSDR AIKFLYLATG showing that, it is an apoptosis inducing factor.