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Tumors with microsatellite instability (MSI) are caused by a defective DNA mismatch repair system that leads to the accumulation of mutations within microsatellite regions. Indels in microsatellites of coding genes can result in the synthesis of frameshift peptides (FSP). FSPs are tumor-specific neoantigens shared across patients with MSI. In this study, we developed a neoantigen-based vaccine for the treatment of MSI tumors. Genetic sequences from 320 MSI tumor biopsies and matched healthy tissues in The Cancer Genome Atlas database were analyzed to select shared FSPs. Two hundred nine FSPs were selected and cloned into nonhuman Great Ape Adenoviral and Modified Vaccinia Ankara vectors to generate a viral-vectored vaccine, referred to as Nous-209. Sequencing tumor biopsies of 20 independent patients with MSI colorectal cancer revealed that a median number of 31 FSPs out of the 209 encoded by the vaccine was detected both in DNA and mRNA extracted from each tumor biopsy. A relevant number of peptides encoded by the vaccine were predicted to bind patient HLA haplotypes. Vaccine immunogenicity was demonstrated in mice with potent and broad induction of FSP-specific CD8 and CD4 T-cell responses. Moreover, a vaccine-encoded FSP was processed in vitro by human antigen-presenting cells and was subsequently able to activate human CD8 T cells. Nous-209 is an “off-the-shelf” cancer vaccine encoding many neoantigens shared across sporadic and hereditary MSI tumors. These results indicate that Nous-209 can induce the optimal breadth of immune responses that might achieve clinical benefit to treat and prevent MSI tumors.Significance:These findings demonstrate the feasibility of an “off-the-shelf” vaccine for treatment and prevention of tumors harboring frameshift mutations and neoantigenic peptides as a result of microsatellite instability.

Top Nous-209-derived peptides predicted to bind to HLA-A*02:01.

International audience; Safe and effective vaccines against coronavirus disease 2019 (COVID-19) are essential for ending the ongoing pandemic. Although impressive progress has been made with several COVID-19 vaccines already approved, it is clear that those developed so far cannot meet the global vaccine demand alone. We describe a COVID-19 vaccine based on a replication-defective gorilla adenovirus expressing the stabilized prefusion severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein named GRAd-COV2. We assessed the safety and immunogenicity of a single-dose regimen of this vaccine in healthy younger and older adults to select the appropriate dose for each age group. For this purpose, a phase 1, dose-escalation, open-labeled trial was conducted including 90 healthy participants (45 aged 18 to 55 years old and 45 aged 65 to 85 years old) who received a single intramuscular administration of GRAd-COV2 at three escalating doses. Local and systemic adverse reactions were mostly mild or moderate and of short duration, and no serious adverse events were reported. Four weeks after vaccination, seroconversion to spike protein and receptor binding domain was achieved in 43 of 44 young volunteers and in 45 of 45 older participants. Consistently, neutralizing antibodies were detected in 42 of 44 younger-age and 45 of 45 older-age volunteers. In addition, GRAd-COV2 induced a robust and T helper 1 cell (T H 1)-skewed T cell response against the spike protein in 89 of 90 participants from both age groups. Overall, the safety and immunogenicity data from the phase 1 trial support the further development of this vaccine.

Safe and effective vaccines against coronavirus disease 2019 (COVID-19) are urgently needed to control the ongoing pandemic. Although impressive progress has been made with several COVID-19 vaccines already approved, it is clear that those developed so far cannot meet the global vaccine demand. We have developed a COVID-19 vaccine based on a replication-defective gorilla adenovirus expressing the stabilized pre-fusion SARS-CoV-2 Spike protein, named GRAd-COV2. We aimed to assess the safety and immunogenicity of a single-dose regimen of this vaccine in healthy younger and older adults to select the appropriate dose for each age group. To this purpose, a phase 1, dose-escalation, open-label trial was conducted including 90 healthy subjects, (45 aged 18-55 years and 45 aged 65-85 years), who received a single intramuscular administration of GRAd-CoV2 at three escalating doses. Local and systemic adverse reactions were mostly mild or moderate and of short duration, and no serious AE was reported. Four weeks after vaccination, seroconversion to Spike/RBD was achieved in 43/44 young volunteers and in 45/45 older subjects. Consistently, neutralizing antibodies were detected in 42/44 younger age and 45/45 older age volunteers. In addition, GRAd-COV2 induced a robust and Th1-skewed T cell response against the S antigen in 89/90 subjects from both age groups. Overall, the safety and immunogenicity data from the phase 1 trial support further development of this vaccine.One Sentence SummaryGRAd-COV2, a candidate vaccine for COVID-19 based on a novel gorilla adenovirus, is safe and immunogenic in younger and older adults

Tumors with microsatellite instability (MSI) are caused by a defective DNA mismatch repair system that leads to the accumulation of mutations within microsatellite regions. Indels in microsatellites of coding genes can result in the synthesis of frameshift peptides (FSP). FSPs are tumor-specific neoantigens shared across patients with MSI. In this study, we developed a neoantigen-based vaccine for the treatment of MSI tumors. Genetic sequences from 320 MSI tumor biopsies and matched healthy tissues in The Cancer Genome Atlas database were analyzed to select shared FSPs. Two hundred nine FSPs were selected and cloned into nonhuman Great Ape Adenoviral and Modified Vaccinia Ankara vectors to generate a viral-vectored vaccine, referred to as Nous-209. Sequencing tumor biopsies of 20 independent patients with MSI colorectal cancer revealed that a median number of 31 FSPs out of the 209 encoded by the vaccine was detected both in DNA and mRNA extracted from each tumor biopsy. A relevant number of peptides encoded by the vaccine were predicted to bind patient HLA haplotypes. Vaccine immunogenicity was demonstrated in mice with potent and broad induction of FSP-specific CD8 and CD4 T-cell responses. Moreover, a vaccine-encoded FSP was processed in vitro by human antigen-presenting cells and was subsequently able to activate human CD8 T cells. Nous-209 is an “off-the-shelf” cancer vaccine encoding many neoantigens shared across sporadic and hereditary MSI tumors. These results indicate that Nous-209 can induce the optimal breadth of immune responses that might achieve clinical benefit to treat and prevent MSI tumors. Significance: These findings demonstrate the feasibility of an “off-the-shelf” vaccine for treatment and prevention of tumors harboring frameshift mutations and neoantigenic peptides as a result of microsatellite instability.

Background Over the past decade, newly designed cancer therapies have not significantly improved the survival of patients diagnosed with Malignant Pleural Mesothelioma (MPM). Among a limited number of genes that are frequently mutated in MPM several of them encode proteins that belong to the HIPPO tumor suppressor pathway. Methods The anticancer effects of the top flower standardized extract of Filipendula vulgaris (Dropwort) were characterized in “in vitro” and “in vivo” models of MPM. At the molecular level, two “omic” approaches were used to investigate Dropwort anticancer mechanism of action: a metabolomic profiling and a phosphoarray analysis. Results We found that Dropwort significantly reduced cell proliferation, viability, migration and in vivo tumor growth of MPM cell lines. Notably, Dropwort affected viability of tumor-initiating MPM cells and synergized with Cisplatin and Pemetrexed in vitro. Metabolomic profiling revealed that Dropwort treatment affected both glycolysis/tricarboxylic acid cycle as for the decreased consumption of glucose, pyruvate, succinate and acetate, and the lipid metabolism. We also document that Dropwort exerted its anticancer effects, at least partially, promoting YAP and TAZ protein ubiquitination. Conclusions Our findings reveal that Dropwort is a promising source of natural compound(s) for targeting the HIPPO pathway with chemo-preventive and anticancer implications for MPM management. Electronic supplementary material The online version of this article (10.1186/s13046-019-1352-3) contains supplementary material, which is available to authorized users.

The impact of gender and diet on the proteome of Longissimus dorsi was addressed by 2D-PAGE analysis of male and female pigs, fed with a barley-based control diet and a diet enriched with extruded linseed and plant extracts. No statistically significant difference in protein number between female and male samples was found. Furthermore, PCA excluded gender-dependent protein clusters. For both the control and enriched diet, several spots exhibited at least a 1.5-fold intensity difference, but none showed a statistically relevant variation. Protein profiles PCA for both diets indicated that the first two principal components account up to 47% of total variance, with two diet-dependent separated clusters. Among 176 common spots, 29 exhibited >1.5 fold change, mostly more abundant in the control diet. PMF identified 14 distinct proteins, including myofibrillar proteins, glycolytic enzymes and myoglobin, thus suggesting a diet-dependent meat quality. A statistically significant increase in carbonylated proteins of enriched diet samples was detected using the 2,4-dinitrophenylhydrazine method but not using fluorescein-5-thiosemicarbazide-labeled bands. ROS induction and DNA oxidative damage, detected in a human cell line exposed to digested meat from both diets, further support the notion that the enriched diet does not protect against oxidative stress. SIGNIFICANCE: The comparison of the protein profile of female and male Longissimus dorsi from pigs fed by a control diet and a diet enriched with polyphenols, indicate no gender effect, whereas diet affects the abundance of several proteins, possibly linked to meat quality. Protein carbonylation was statistically higher in meat from the enriched diet, suggesting that polyphenols at the concentration present in the diet did not exert a protective effect against oxidation.

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.

Processed meat, representing about 22% of per capita meat consumption, undergoes technological transformations, such as salting, drying, cooking, or frying. Proteomic methods have been applied to monitor the effects of the processing on meat from cow, yak, pig, lamb, goat, and chicken with the aim of correlating protein pattern with meat quality as well as of identifying adulterations.

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-145's 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.

// Federica Mori 1 , Andrea Sacconi 2 , Valeria Canu 2 , Federica Ganci 2 , Mariangela Novello 3 , Vincenzo Anelli 4 , Renato Covello 5 , Virginia Ferraresi 6 , Paola Muti 7 , Roberto Biagini 8 , Giovanni Blandino 2,7 and Sabrina Strano 1,7 1 Molecular Chemoprevention Unit, Regina Elena National Cancer Institute, Rome, Italy 2 Translational Oncogenomics, Regina Elena National Cancer Institute, Rome, Italy 3 Department of Pathology, Catholic University, Rome, Italy 4 UOC Radiology, Regina Elena National Cancer Institute, Rome, Italy 5 UOC Department of Pathology, Regina Elena National Cancer Institute, Rome, Italy 6 Medical Oncology A, Regina Elena National Cancer Institute, Rome, Italy 7 Department of Oncology, McMaster University, Hamilton, ON, Canada 8 UOC Orthopedic Surgery, Regina Elena National Cancer Institute, Rome, Italy Correspondence: Giovanni Blandino, email: // Keywords : giant cell tumor, osteosarcoma, miRNA profiling, relapse Received : December 11, 2014 Accepted : February 17, 2015 Published : March 12, 2015 Abstract High-grade osteosarcoma (OS) is characterized by low incidence, high aggressiveness and moderate 5-years survival rate after aggressive poly-chemotherapy and surgery. Here we used miRNA profiling as a tool to possibly predict and monitor OS’s development and therapeutic outcome. First, we evaluated the altered expression of selected miRNAs from a case of Giant Cell Tumor (GCT) apparently evolved into an OS. We found that most of modulated miRs were associated with pathways of bone resorption and osteogenesis. miRNA expression also revealed that GCT and OS were distinct tumors. Second, we validated the observed miRNA profile in two independent casuistries of ten GCT (not evolved into malignant tumors) and sixteen OS patients. Interestingly, we found that miR-181c and other three miRNAs identified in the first step of the study were also consistently de-regulated in all OS patients. Ectopic expression of miR-181c reduced cell viability and enhanced chemotherapeutic-induced cell death of U2OS and SAOS2 cells. These findings indicate that: i) miRNAs aberrantly modulated in GCT could be predictive of its development into OS and ii) miRNAs expression could be useful to monitor the OS therapeutic outcome.

Metformin is a commonly prescribed type II diabetes medication that exhibits promising anticancer effects. Recently, these effects were found to be associated, at least in part, with a modulation of microRNA expression. However, the mechanisms by which single modulated microRNAs mediate the anticancer effects of metformin are not entirely clear and knowledge of such a process could be vital to maximize the potential therapeutic benefits of this safe and well-tolerated therapy. Our analysis here revealed that the expression of miR-21-5p was downregulated in multiple breast cancer cell lines treated with pharmacologically relevant doses of metformin. Interestingly, the inhibition of miR-21-5p following metformin treatment was also observed in mouse breast cancer xenografts and in sera from 96 breast cancer patients. This modulation occurred at the levels of both pri-miR-21 and pre-miR-21, suggesting transcriptional modulation. Antagomir-mediated ablation of miR-21-5p phenocopied the effects of metformin on both the clonogenicity and migration of the treated cells, while ectopic expression of miR-21-5p had the opposite effect. Mechanistically, this reduction in miR-21-5p enhanced the expression of critical upstream activators of the AMP-activated protein kinase, calcium-binding protein 39-like and Sestrin-1, leading to AMP-activated protein kinase activation and inhibition of mammalian target of rapamycin signaling. Importantly, these effects of metformin were synergistic with those of everolimus, a clinically relevant mammalian target of rapamycin inhibitor, and were independent of the phosphatase and tensin homolog status. This highlights the potential relevance of metformin in combinatorial settings for the treatment of breast cancer.

Background Thymic epithelial tumors (TET) are the most frequent human primary mediastinal tumors in adults. A deep biological characterization of the processes at the basis of the transformed phenotype could strongly improve our understanding of the morphological and clinical heterogeneity of these diseases. MicroRNAs (miRNAs) are non-coding RNAs involved in post-transcriptional regulation and their altered expression accounts for the pathogenesis of several tumors. Objectives The aim of this study was to identify the miRNAs that are differentially expressed in tumor vs normal thymic tissues or among the different tumor histotypes and that could impact on the biology of TET. Materials and methods microRNAs expression profiling was performed by microarray analysis of formalin-fixed paraffin embedded (FFPE) tissue from 54 thymic tumor samples and 12 normal counterparts, derived from two patient cohorts. Results and conclusion We identified groups of miRNAs differentially expressed between: (i) TET and normal thymic tissues, (ii) thymomas and thymic carcinomas, (iii) histotype groups. Moreover, we identified putative molecular pathways targeted by these differentially expressed miRNAs that could be involved in thymic carcinogenesis and in the maintenance and spreading of this tumor.

// Mario Cioce 1* , MariaCristina Valerio 2* , Luca Casadei 2 , Claudio Pulito 3 , Andrea Sacconi 4 , Federica Mori 3 , Francesca Biagioni 4 , Cesare Manetti 2 , Paola Muti 5 , Sabrina Strano 3 , and Giovanni Blandino 4 . 1 Department of Cardiothoracic Surgery, NYU Langone Medical Center, New York, NY USA 2 Department of Chemistry, University of Rome ‘La Sapienza’, 00185 Rome, Italy 3 Molecular Chemoprevention Group, Italian National Cancer Institute “Regina Elena”, Rome, Italy 4 Translational Oncogenomic Unit, Italian National Cancer Institute “Regina Elena”, Rome, Italy 5 Department of Oncology, McMaster University, Hamilton, Ontario, L8V 5C2, Canada * These two authors contributed equally Correspondence: Giovanni Blandino , email: // Keywords : Metformin, metabolism, chemoresistance, ALDH, metabolic reprogramming, cancer Received : January 22, 2014 Accepted : March 24, 2014 Published : March 26, 2014 Abstract Metabolic remodeling is a hallmark of cancer progression and may affect tumor chemoresistance. Here we investigated by 1H-NMR/PCA analysis the metabolic profile of chemoresistant breast cancer cell subpopulations (ALDH bright cells) and their response to metformin, a promising anticancer metabolic modulator. The purified ALDH bright cells exhibited a different metabolic profile as compared to their chemosensitive ALDH low counterparts. Metformin treatment strongly affected the metabolism of the ALDHbright cells thereby affecting, among the others, the glutathione metabolism, whose upregulation is a feature of progenitor-like, chemoresistant cell subpopulations. Globally, metformin treatment reduced the differences between ALDH bright and ALDH low cells, making the former more similar to the latter. Metformin broadly modulated microRNAs in the ALDH bright cells, with a large fraction of them predicted to target the same metabolic pathways experimentally identified by 1H-NMR. Additionally, metformin modulated the levels of c-MYC and IRS-2, and this correlated with changes of the microRNA-33a levels. In summary, we observed, both by 1H-NMR and microRNA expression studies, that metformin treatment reduced the differences between the chemoresistant ALDH bright cells and the chemosensitive ALDH low cells. This works adds on the potential therapeutic relevance of metformin and shows the potential for metabolic reprogramming to modulate cancer chemoresistance.

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.

Melatonin has been known to be a chemopreventive agent since its levels inversely correlate with the risk of developing cancer. We have recently shown that melatonin induces p38-dependent phosphorylation of both p53 and histone H2AX. This is associated with a p53-mediated increase in repair of both endogenous and chemotherapy-induced DNA damage. In addition, the inhibition of p38 activities impairs melatonin's capability to induce a p53-dependent DNA damage response and thus its ability to maintain genome integrity. Since melatonin-induced p53 phosphorylation requires an intact p38 phosphorylation cascade and p38 can be activated by G proteins, we supposed that melatonin's activities could be mediated by its G-protein-coupled membrane receptors, MT1 and MT2. Here, we show that the activation of the p53-dependent DNA damage response by melatonin is indeed mediated by MT1 and MT2. As a result, the absence of either receptor impairs melatonin's ability to reduce both cell proliferation and clonogenic potential of cancer cells. In addition, this causes an impairment of the p53-dependent DNA damage response. By providing molecular insight, our findings might have translational impact, suggesting the involvement of melatonin receptors in tumorigenesis.

Gastric cancer is an important healthcare problem and represents the second leading cause of death for malignant disease worldwide. In the Western world, the diagnosis is done at late stage when treatments can be only palliative. Searches for new therapeutic regimens as well as for new biomarkers are in progress. To reduce cancer mortality is crucial the prevention of the lesion at earlier stages. Therefore, new bullets to prevention are needed. Nowadays, studies relating to different kinds of tumor are unanimous in considering cancer stem cells (CSCs) as "the core" of the tumor and the responsible of tumor chemoresistance and relapse. This chapter aims to provide the instructions to (1) isolate, (2) grow, and (3) validate, both in vivo and in vitro, the gastric CSC subpopulation.

IMPORTANCE: Colorectal carcinomas in patients with Lynch syndrome (LS) arise in a background of mismatch repair (MMR) deficiency, display a unique immune profile with upregulation of immune checkpoints, and response to immunotherapy. However, there is still a gap in understanding the pathogenesis of MMR-deficient colorectal premalignant lesions, which is essential for the development of novel preventive strategies for LS. OBJECTIVE: To characterize the immune profile of premalignant lesions from a cohort of patients with LS. DESIGN, SETTING, AND PARTICIPANTS: Whole-genome transcriptomic analysis using next-generation sequencing was performed in colorectal polyps and carcinomas of patients with LS. As comparator and model of MMR-proficient colorectal carcinogenesis, we used samples from patients with familial adenomatous polyposis (FAP). In addition, a total of 47 colorectal carcinomas (6 hypermutants and 41 nonhypermutants) were obtained from The Cancer Genome Atlas (TCGA) for comparisons. Samples were obtained from the University of Texas MD Anderson Cancer Center and "Regina Elena" National Cancer Institute, Rome, Italy. All diagnoses were confirmed by genetic testing. Polyps were collected at the time of endoscopic surveillance and tumors were collected at the time of surgical resection. The data were analyzed from October 2016 to November 2017. MAIN OUTCOMES AND MEASURES: Assessment of the immune profile, mutational signature, mutational and neoantigen rate, and pathway enrichment analysis of neoantigens in LS premalignant lesions and their comparison with FAP premalignant lesions, LS carcinoma, and sporadic colorectal cancers from TCGA. RESULTS: The analysis was performed in a total of 28 polyps (26 tubular adenomas and 2 hyperplastic polyps) and 3 early-stage LS colorectal tumors from 24 patients (15 [62%] female; mean [SD] age, 48.12 [15.38] years) diagnosed with FAP (n = 10) and LS (n = 14). Overall, LS polyps presented with low mutational and neoantigen rates but displayed a striking immune activation profile characterized by CD4 T cells, proinflammatory (tumor necrosis factor, interleukin 12) and checkpoint molecules (LAG3 [lymphocyte activation gene 3] and PD-L1 [programmed cell death 1 ligand 1]). This immune profile was independent of mutational rate, neoantigen formation, and MMR status. In addition, we identified a small subset of LS polyps with high mutational and neoantigen rates that were comparable to hypermutant tumors and displayed additional checkpoint (CTLA4 [cytotoxic T-lymphocyte-associated protein 4]) and neoantigens involved in DNA damage response (ATM and BRCA1 signaling). CONCLUSIONS AND RELEVANCE: These findings challenge the canonical model, based on the observations made in carcinomas, that emphasizes a dependency of immune activation on the acquisition of high levels of mutations and neoantigens, thus opening the door to the implementation of immune checkpoint inhibitors and vaccines for cancer prevention in LS.

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.

Gastric cancer is an important healthcare problem and represents the second leading cause of death for malignant disease worldwide. In the Western world, the diagnosis is done at late stage when treatments can be only palliative. Searches for new therapeutic regimens as well as for new biomarkers are in progress. To reduce cancer mortality is crucial the prevention of the lesion at earlier stages. Therefore, new bullets to prevention are needed. Nowadays, studies relating to different kinds of tumor are unanimous in considering cancer stem cells (CSCs) as "the core" of the tumor and the responsible of tumor chemoresistance and relapse. This chapter aims to provide the instructions to (1) isolate, (2) grow, and (3) validate, both in vivo and in vitro, the gastric CSC subpopulation.

Malignant pleural mesothelioma is a poorly treated neoplasia arising from the pleural mesothelial lining. Here we document that the leaf extract of Cynara scolymus exerts broad antitumoral effects both in vitro and in vivo on mesothelioma cell lines. We found that Cynara scolymus treatment affects strongly cell growth, migration and tumor engraftment of mesothelioma cell lines. Strikingly, dietary feeding with Cynara scolymus leaf extract reduces the growth of mesothelioma xenografted tumors similarly to pemetrexed, a commonly employed drug in the treatment of mesothelioma. In aggregate our findings suggest that leaf extract of Cynara scolymus holds therapeutic potential for the treatment of mesothelioma.

We identified a discrete number of microRNAs differentially expressed in benign or malignant mesothelial tissues. We focused on mir-145 whose levels were significantly downregulated in malignant mesothelial tissues and malignant pleural mesothelioma (MPM) cell lines as compared to benign tissues (pleura, peritoneum or cysts). We show that promoter hyper-methylation caused very low levels in MPM cell lines and specimens. Treatment of MPM cell lines with mir-145 agonists negatively modulated some protumorigenic properties of MPM cells, such as clonogenicity, cell migration and resistance to pemetrexed treatment. The main effector mechanism of the clonogenic death induced by mir-145 was that of accelerated senescence. We found that mir-145 targeted OCT4 via specific binding to its 3'-UTR. Increased intracellular levels of mir-145 decreased the levels of OCT4 and its target gene ZEB1, thereby counteracting the increase of OCT4 induced by pemetrexed treatment which is known to favor the development of chemoresistant cells. In line with this, reintroduction of OCT4 into mimic-145 treated cells counteracted the effects on clonogenicity and replicative senescence. This further supports the relevance of the mir-145-OCT4 interaction for the survival of MPM cells. The potential use of mir-145 expression levels to classify benign vs malignant mesothelial tissues and the differences between pemetrexed-induced senescence and that induced by the re-expression of mir-145 are discussed.

Deregulated proliferation is a hallmark of cancer cells. Here, we show that microRNA-10b* is a master regulator of breast cancer cell proliferation and is downregulated in tumoural samples versus matched peritumoural counterparts. Two canonical CpG islands (5 kb) upstream from the precursor sequence are hypermethylated in the analysed breast cancer tissues. Ectopic delivery of synthetic microRNA-10b* in breast cancer cell lines or into xenograft mouse breast tumours inhibits cell proliferation and impairs tumour growth in vivo, respectively. We identified and validated in vitro and in vivo three novel target mRNAs of miR-10b* (BUB1, PLK1 and CCNA2), which play a remarkable role in cell cycle regulation and whose high expression in breast cancer patients is associated with reduced disease-free survival, relapse-free survival and metastasis-free survival when compared to patients with low expression. This also suggests that restoration of microRNA-10b* expression might have therapeutic promise.

Diabetic patients treated with metformin have a reduced incidence of cancer and cancer-related mortality. Here we show that metformin affects engraftment and growth of breast cancer tumours in mice. This correlates with the induction of metabolic changes compatible with clear anticancer effects. We demonstrate that microRNA modulation underlies the anticancer metabolic actions of metformin. In fact, metformin induces DICER expression and its effects are severely impaired in DICER knocked down cells. Conversely, ectopic expression of DICER recapitulates the effects of metformin in vivo and in vitro. The microRNAs upregulated by metformin belong mainly to energy metabolism pathways. Among the messenger RNAs downregulated by metformin, we found c-MYC, IRS-2 and HIF1alpha. Downregulation of c-MYC requires AMP-activated protein kinase-signalling and mir33a upregulation by metformin. Ectopic expression of c-MYC attenuates the anticancer metabolic effects of metformin. We suggest that DICER modulation, mir33a upregulation and c-MYC targeting have an important role in the anticancer metabolic effects of metformin.

Here we show that pemetrexed-treated mesothelioma cells undergo accelerated senescence. This is characterized by the secretion of proinflammatory and mitogenic cytokines, reminiscent of an SASP (senescence-associated secretory phenotype). Conditioned media from senescent MPM (malignant pleural mesothelioma) cells trigger the emergence of EMT (epithelial-to-mesenchymal)-like, clonogenic and chemoresistant cell subpopulations, expressing high levels of ALDH (aldehyde dehydrogenase) activity (ALDH(bright) cells). We show by fluorescence-activated cell sorting of purified ALDH(bright) and ALDH(low) cells, that both cell-autonomous and cell-non-autonomous mechanisms converge to maintain the SASP-induced, EMT-like cell subpopulations. Chemoresistant ALDH(bright) cells exist within primary MPM specimens and enrichment for ALDH(bright) cells correlates with an earlier tumor onset into NOD/SCID mice. We show that RAS(v12) expression induces SASP-like changes in untransformed human mesothelial cells, and that p53 ablation increases the effect of RAS(v12) expression. We identify STAT3 activation as a crucial event downstream to SASP signaling. In fact, small hairpin RNA-mediated ablation of STAT3 deeply attenuates the induction of EMT genes and the increase of ALDH(bright) cells induced by SASP-cytokines. This strongly affects the chemoresistance of MPM cells in vitro and leads to anticancer effects in vivo.

Purpose: Matrix metalloproteinases (MMP) are zinc-dependent endopeptidases that mediate numerous physiologic and pathologic processes, including matrix degradation, tissue remodeling, inflammation, and tumor metastasis. To develop a vaccine targeting stromal antigens expressed by cancer-associated fibroblasts, we focused on MMP11 (or stromelysin 3). MMP11 expression correlates with aggressive profile and invasiveness of different types of carcinoma. Experimental Design: To show the efficacy of a vaccine targeting MMP11, we constructed a series of plasmid DNA vectors expressing murine MMP11. Mice were vaccinated by i.m. injection followed by in vivo DNA electroporation. A chemically induced, MMP11-overexpressing colon cancer model was established and characterized. Antibody and T-cell responses were determined, and immunoreactive epitopes were characterized. To analyze the possible use of MMP11 as tumor-associated antigen in cancer patients, HLA-A2.1 transgenic mice (HHD) were used to identify reactive epitopes as tools to assess immunogenicity in humans. Results: Using microarray, we confirmed the overexpression of MMP11 mRNA in a large panel of human tumor samples. MMP11 vaccine induced cell mediated and antibody immune response and exerted significant antitumoral protection in mice with colon cancer in prophylactic and therapeutic settings. HHD transgenic mice were vaccinated with a plasmid encoding human MMP11, and a HLA-A2.1–restricted epitope (hMMP237) was identified. hMMP237 was shown to be immunogenic in human peripheral blood mononuclear cells (PBMC) by in vitro priming. Conclusion: Our study describes the identification of MMP11 as a novel broadly expressed tumor associated antigen as target candidate for cancer immunotherapy.

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.

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. [Cancer Res 2008;68(23):9865–74]

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that mediate a number of physiological and pathological processes, such as matrix degradation, tissue remodeling, inflammation and tumor metastasis. The objective of this study was to develop and characterize a vaccine targeting stromal antigens expressed by cancer associated fibroblasts (CAFs). We focused on MMP11 (or stromelysin 3, ST3), that has been detected primarily in CAFs and its expression correlates with aggressive clinical behavior and invasiveness of different types of carcinoma. We demonstrated that intramuscular injection of genetic vectors such as Adenovirus (Ad) and plasmid DNA encoding MMP11 engineered variants followed by in vivo electroporation (DNA-EP) resulted in breakage of immune tolerance and induction of both cell mediated and humoral immune response. Importantly, MMP11 vaccine was able to confer significant antitumoral protection in a chemically induced, MMP11 overexpressing colon cancer model both in prophylactic and therapeutic settings. To determine the mechanism of action of MMP11 vaccine, we have utilized IFNgamma- and μmicroMT-knock out mice, devoid of T- and B-cell immune response, respectively. Our results show that both arms of the immune response are important to confer the therapeutic effect. Moreover, MMP11 stromal vaccine showed synergic effects when combined with genetic vaccines targeting classic tumor associated antigens, such as telomerase reverse transcriptase (TERT) and carcinoembryonic antigen (CEA). Finally, to assess the immunogenicity and the safety of MMP11 vaccine in a large animal model, nonhuman primates have been vaccinated with Ad and DNA-EP. A strong immune response was measured with no detectable side effects. In addition, MMP11 detection and spontaneous immune responses in the blood of breast and prostate cancer patients further corroborate MMP11 as a valid target for immune intervention. Taken together, these data support the use of MMP11 as a potential candidate for cancer immunotherapy in human clinical trials. Citation Format: Laura Luberto, Rita Mancini, Arianna Di Napoli, Daniela Peruzzi, Federica Mori, Giuseppe Roscilli, Emanuele Marra, Manuela Cappelletti, Gennaro Ciliberto, Luigi Aurisicchio. Targeting the stroma to hit the tumor: MMP11 as a novel target for cancer immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 291. doi:10.1158/1538-7445.AM2015-291

Background and Aims Gastric cancer is the fourth most common malignancy and the second leading cause of cancer death in both sexes worldwide. Despite a global decrease in incidence, gastric cancer remains a disease of prominent morbidity and mortality, especially in Eastern Asia. The clinical outcome of gastric cancer has gradually improved, but the survival rate of patients with advanced gastric cancer is still disappointing. Achieving a better understanding of molecular aberrations associated with gastric carcinogenesis might identify new diagnostic and therapeutic strategies for this disease.MicroRNAs (miRNAs) recently emerged as prominent regulators of cancer processes. MicroRNAs are small, 18- to 24-nucleotide noncoding RNAs, with profound impact on many processes that are frequently disrupted during malignant transformation, including cell proliferation, apoptosis, stress responses, maintenance of stem cell potency and metabolism. Here we aim to identify miRs whose deregulated expression leads to the activation of oncogenic pathways in human gastric cancers. Methods 39 out of 123 tumoral and matched peritumoral gastric specimens from three independent Europian subsets of patients were analyzed for the expression of 851 human miRs using Agilent Platform. The remaining 84 samples were used to validate miRs differentially expressed between tumoral and matched peritumoral specimens by qPCR. In vitro and in vivo experimental assays were employed to test the tumor suppressor activities of miR-204 and to functionally validate its mRNAs targets. Results miR-204 falls into a cluster of 8 miRs differentially expressed between tumoral and peritumoral samples in all three analyzed subsets. Its downregulation has prognostic value and correlates with T status. Ectopic expression of miR-204 inhibited colony forming ability, migration and tumor engraftment of gastric cancer cells. Conclusions microRNAs are differentially expressed in gastric tumors and underline specific genetic alterations. MiR 204 is significantly down-regulated in gastric tumor specimens compared to matched peritumoral tissues. These findings strongly indicate that down-regulation of miR-204 in gastric tumors might coincide with loss of tumor suppressor activity and consequently favor gastric transformation.

Deregulation of the DNA damage response (DDR) is a main feature of cancer progression and a common finding in breast cancer. While a clear correlation between mutations of BRCA1/2 and ATM in cancer development has been found in hereditary breast cancers, the molecular basis of DDR deregulation of sporadic breast cancers is still unclear. Triple-negative (TNBC) and basal-like (BLBC) breast cancers are very aggressive sporadic tumors that relapse very frequently. Only 2% of sporadic tumors bear detectable BRCA1/2 and ATM mutations. Interestingly, TNBCs and BLBCs present a histoclinical phenotype comparable to that observed in patients with hereditary germline BRCA1/2 mutations (“BRCAness” phenotype). However, while BRCA1 mutated breast cancers are sensitive to PARP inhibitors, both TNBCs and BLBCs are poorly responsive to anticancer treatments. Recent evidence has shown that the altered expression of microRNA genes can be associated with diverse types of cancers. microRNAs are small non-coding RNA molecules that negatively regulate gene expression at the post-transcriptional level. They bind through partial sequence homology to the 3′-untranslated region of target mRNAs and cause translational inhibition and/or mRNA degradation. In the June 1, 2013 issue of Cell Cycle, Del Sal’s group surveyed public breast cancer data sets for miR-181a/b expression.1 They found that increased expression of miR-181a/b correlated with shorter disease-free survival and early onset of metastatic disease. The expression of miR-181a/b was also assessed on more than 100 primary breast cancer samples and was found upregulated in tumor specimens when compared with normal tissues. This also correlated with tumor aggressiveness. miR-181a/b have been shown to promote migration and invasion of breast cancer cells as well as the expansion of breast cancer stem-like cells.2 miR181a/b expression is regulated by transforming growth factor-b,3-5 whose pathway is aberrantly activated in breast cancer transformation.6,7 At the molecular level, Del Sal group showed that miR-181a/b targeted ATM mRNA, thereby impinging severely on DDR response of breast cancer cells. BRAC1 phosphorylation was reduced in miR181a/b-overexpressing cells, suggesting that miR-181a/b increased expression may dampen DDR and DNA double-strand breaks repair, leading to the accumulation of unfixed DNA lesions in breast cancer cells. This might unveil the oncogenic value of miR-181a/b overexpression and lead to the development of potential therapeutic strategies aimed at overcoming its pro-tumorigenic effects in breast cancers. It is becoming increasingly clear that a detailed molecular cancer portrait of a single patient is required to design the most effective cancer treatment. This is ineludible, as the development of resistance to target therapy for diverse types of tumors, including breast cancer, significantly increases. Indeed, chemoresistance remains a major clinical obstacle to successful treatment of breast cancer and a source of poor prognosis. The power of cancer patient response prediction to given treatments needs to be strongly improved. It is reasonable to think that the more molecular variables we can include in the prediction model the better prediction we may expect. Notably, in this issue of Cell Cycle, Bisso and colleagues reported that miR-181a/b overexpressing cells are sensitive to pharmacological inhibition of PARP. miR-181a/b overexpression sensitized breast cancer cells to PARP inhibition, as for Olaparib (AstraZeneca). This raises the intriguing possibility that overexpression of miR-181a/b could be regarded as a potential biomarker that allows the identification of a subgroup of TNBC and BLBC patients who may benefit from the treatment with PARPi. The miR-181a/b overexpression Del Sal’s group observed in diverse tumors types could also have major implications for other types of human cancers. In light of these findings, additional work attempting the functional validation of novel miR-181a/b mRNA targets might provide novel insight into the molecular characterization of aggressive cancer subtypes and hold important therapeutic potential. (Fig 1) Figure 1. Hereditary breast cancers and sporadic breast cancers presenting BRCA1 mutations account for the 12% of the total breast tumors. Basal-like and triple-negative breast cancers among the sporadic breast tumors (88%) that present “BRCAness” ...

Micro RNAs (miRs) are small non-coding RNAs aberrantly expressed in human tumors. Here, we aim to identify miRs whose deregulated expression leads to the activation of oncogenic pathways in human gastric cancers (GCs). Thirty nine out of 123 tumoral and matched uninvolved peritumoral gastric specimens from three independent European subsets of patients were analyzed for the expression of 851 human miRs using Agilent Platform. The remaining 84 samples were used to validate miRs differentially expressed between tumoral and matched peritumoral specimens by qPCR. miR-204 falls into a group of eight miRs differentially expressed between tumoral and peritumoral samples. Downregulation of miR-204 has prognostic value and correlates with increased staining of Bcl-2 protein in tumoral specimens. Ectopic expression of miR-204 inhibited colony forming ability, migration and tumor engraftment of GC cells. miR-204 targeted Bcl-2 messenger RNA and increased responsiveness of GC cells to 5-fluorouracil and oxaliplatin treatment. Ectopic expression of Bcl-2 protein counteracted miR-204 pro-apoptotic activity in response to 5-fluorouracil. Altogether, these findings suggest that modulation of aberrant expression of miR-204, which in turn releases oncogenic Bcl-2 protein activity might hold promise for preventive and therapeutic strategies of GC.

Giant cell tumor of bone (GCT) is a relatively uncommon tumor that, although generally benign, may rarely become malignant. We report a case of osteosarcoma arising 5 months after surgery for GCT.On September 2011, a 22-year-old girl presented with a left hip pain.Radiology revealed a proximal left femur osteolytic lesion highly suggestive for GCT as confirmed by biopsy. She underwent curettage and bone grafting. 4 months after surgery, the pain came back and the X-rays demonstrated a local relapse. A first biopsy confirmed the GCT diagnosis and the immunohistochemistry revealed a high level of bHCG positive cells (patient not pregnant). As imaging and clinical evolution suggested a malignant diagnosis, several biopsies were performed till the high grade osteosarcoma diagnosis. The patient was treated with neoadiuvant chemotherapy (MTX/DDP/ADN/ x2) and on September 2011 a hip resection (extra-articular) was performed. On February 2012 the patient died due to lung metastases.To investigate the correlation between the GCT and the subsequent osteosarcoma we compared the microRNA profiles from FFPE histological samples ( Table 1 ).We added two other independent patients, corresponding to a primary GCT that did not develop in osteosarcoma (B) and to a primary osteosarcoma, not derived from GCT (C).PCA analysis of the microRNA signal intensity distribution revealed that osteosarcoma and GCT are independent lesions ( Figure 1 ). Moreover, a stepwise change in microRNA expression was observed in the lesions which developed into osteosarcoma. We focused on microRNAs differentially down-regulated in osteosarcoma as compared to GCT. Interestingly, we found that most of them were endowed with tumor suppressor activity. This finding is relevant in order to identify i) microRNAs whose expression in GCT could be predictive of its development into osteosarcoma and ii) microRNAs whose expression, if restored, may revert or arrest osteosarcoma development. Sources of funding: New Idea Award; AIRC-ROC Download : Download full-size image Fig. 1 . PCA analysis. Table 1 . Histological samples Derivation Reference Histology Origin Surgery GCT Giant cell tumor Patient A (case report patient) Biopsy GCT beta Giant cell tumor with bHCG positive cells Biopsy OST beta Osteosarcoma with bHCG positive cells Surgery OST Osteosarcoma Surgery CONTR gct Giant cell tumor Patient B Surgery CONTR ost Osteosarcoma Patient C