Recent studies identified a poor-prognosis stem/serrated/mesenchymal (SSM) transcriptional subtype of colorectal cancer (CRC). We noted that genes upregulated in this subtype are also prominently expressed by stromal cells, suggesting that SSM transcripts could derive from stromal rather than epithelial cancer cells. To test this hypothesis, we analyzed CRC expression data from patient-derived xenografts, where mouse stroma supports human cancer cells. Species-specific expression analysis showed that the mRNA levels of SSM genes were mostly due to stromal expression. Transcriptional signatures built to specifically report the abundance of cancer-associated fibroblasts (CAFs), leukocytes or endothelial cells all had significantly higher expression in human CRC samples of the SSM subtype. High expression of the CAF signature was associated with poor prognosis in untreated CRC, and joint high expression of the stromal signatures predicted resistance to radiotherapy in rectal cancer. These data show that the distinctive transcriptional and clinical features of the SSM subtype can be ascribed to its particularly abundant stromal component.

Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents an important clinical goal. Erk1,2 signalling is pivotal in the cardiac response to stress, suggesting that its inhibition may be a good strategy to revert heart hypertrophy. In this work, we unveiled the events associated with cardiac hypertrophy by means of a transgenic model expressing activated Met receptor. c-Met proto-oncogene encodes for the tyrosine kinase receptor of Hepatocyte growth factor and is a strong inducer of Ras-Raf-Mek-Erk1,2 pathway. We showed that three weeks after the induction of activated Met, the heart presents a remarkable concentric hypertrophy, with no signs of congestive failure and preserved contractility. Cardiac enlargement is accompanied by upregulation of growth-regulating transcription factors, natriuretic peptides, cytoskeletal proteins, and Extracellular Matrix remodelling factors (Timp1 and Pai1). At a later stage, cardiac hypertrophic remodelling results into heart failure with preserved systolic function. Prevention trial by suppressing activated Met showed that cardiac hypertrophy is reversible, and progression to heart failure is prevented. Notably, treatment with Pimasertib, Mek1 inhibitor, attenuates cardiac hypertrophy and remodelling. Our results suggest that modulation of Erk1.2 signalling may constitute a new therapeutic approach for treating cardiac hypertrophies.

We developed a selectable marker rendering human cells resistant to Diphtheria Toxin (DT). The marker (DT(R)) consists of a primary microRNA sequence engineered to downregulate the ubiquitous DPH2 gene, a key enzyme for the biosynthesis of the DT target diphthamide. DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth. DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers. As mice are insensitive to DT, DT(R)-based selection can be also applied in vivo. Direct injection of a GFP-DT(R) lentiviral vector into human cancer cell-line xenografts and patient-derived tumorgrafts implanted in mice, followed by systemic DT administration, yielded tumors entirely composed of permanently transduced cells and detectable by imaging systems. This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.

We propose a new multi-network-based strategy to integrate different layers of genomic information and use them in a coordinate way to identify driving cancer genes. The multi-networks that we consider combine transcription factor co-targeting, microRNA co-targeting, protein-protein interaction and gene co-expression networks. The rationale behind this choice is that gene co-expression and protein-protein interactions require a tight coregulation of the partners and that such a fine tuned regulation can be obtained only combining both the transcriptional and post-transcriptional layers of regulation. To extract the relevant biological information from the multi-network we studied its partition into communities. To this end we applied a consensus clustering algorithm based on state of art community detection methods. Even if our procedure is valid in principle for any pathology in this work we concentrate on gastric, lung, pancreas and colorectal cancer and identified from the enrichment analysis of the multi-network communities a set of candidate driver cancer genes. Some of them were already known oncogenes while a few are new. The combination of the different layers of information allowed us to extract from the multi-network indications on the regulatory pattern and functional role of both the already known and the new candidate driver genes.

Colorectal cancer (CRC) transcriptional subtypes have been recently identified by gene expression profiling. Here we describe an analytical pipeline, microRNA master regulator analysis (MMRA), developed to search for microRNAs potentially driving CRC subtypes. Starting from a microRNA-mRNA tumour expression data set, MMRA identifies candidate regulator microRNAs by assessing their subtype-specific expression, target enrichment in subtype mRNA signatures and network analysis-based contribution to subtype gene expression. When applied to a CRC data set of 450 samples, assigned to subtypes by 3 different transcriptional classifiers, MMRA identifies 24 candidate microRNAs, in most cases downregulated in the stem/serrated/mesenchymal (SSM) poor prognosis subtype. Functional validation in CRC cell lines confirms downregulation of the SSM subtype by miR-194, miR-200b, miR-203 and miR-429, which share target genes and pathways mediating this effect. These results show that, by combining statistical tests, target prediction and network analysis, MMRA effectively identifies microRNAs functionally associated to cancer subtypes.

Many microarray experiments search for genes with differential expression between a common "reference" group and multiple "test" groups. In such cases currently employed statistical approaches based on t-tests or close derivatives have limited efficacy, mainly because estimation of the standard error is done on only two groups at a time. Alternative approaches based on ANOVA correctly capture within-group variance from all the groups, but then do not confront single test groups with the reference. Ideally, a t-test better suited for this type of data would compare each test group with the reference, but use within-group variance calculated from all the groups.

Overexpression of miR-100 in stem cells derived from basal-like breast cancers causes loss of stemness, induction of luminal breast cancer markers and response to endocrine therapy. We, therefore, explored miR-100 as a novel biomarker in patients with luminal breast cancer.

The MET oncogene encodes a tyrosine kinase receptor involved in the control of a complex network of biological responses that include protection from apoptosis and stimulation of cell growth during embryogenesis, tissue regeneration, and cancer progression. We previously developed an antagonist antibody (DN30) inducing the physical removal of the receptor from the cell surface and resulting in suppression of the biological responses to MET. In its bivalent form, the antibody displayed a residual agonist activity, due to dimerization of the lingering receptors, and partial activation of the downstream signaling cascade. The balance between the two opposing activities is variable in different biological systems and is hardly predictable. In this study, we generated and characterized two single-chain antibody fragments derived from DN30, sharing the same variable regions but including linkers different in length and composition. The two engineered molecules bind MET with high affinity but induce different biological responses. One behaves as a MET-antagonist, promoting programmed cell death in MET "addicted" cancer cells. The other acts as a hepatocyte growth factor (HGF)-mimetic, protecting normal cells from doxorubicin-induced apoptosis. Thus, by engineering the same receptor antibody, it is possible to generate molecules enhancing or inhibiting apoptosis either to kill cancer cells or to protect healthy tissues from the injuries of chemotherapy.

Cutaneous skeletal hypophosphatemia syndrome (CSHS) is caused by somatic mosaic NRAS variants and characterized by melanocytic/sebaceous naevi, eye, and brain malformations, and FGF23-mediated hypophosphatemic rickets. The MEK inhibitor Trametinib, acting on the RAS/MAPK pathway, is a candidate for CSHS therapy. A 4-year-old boy with seborrheic nevus, eye choristoma, multiple hamartomas, brain malformation, pleural lymphangioma and chylothorax developed severe hypophosphatemic rickets unresponsive to phosphate supplementation. The c.182A > G;p.(Gln61Arg) somatic NRAS variant found in DNA from nevus biopsy allowed diagnosing CSHS. We administered Trametinib for 15 months investigating the transcriptional effects at different time points by whole blood RNA-seq. Treatment resulted in prompt normalization of phosphatemia and phosphaturia, catch-up growth, chylothorax regression, improvement of bone mineral density, reduction of epidermal nevus and hamartomas. Global RNA sequencing on peripheral blood mononucleate cells showed transcriptional changes under MEK inhibition consisting in a strong sustained downregulation of signatures related to RAS/MAPK, PI3 kinase, WNT and YAP/TAZ pathways, reverting previously defined transcriptomic signatures. CSHS was effectively treated with a MEK inhibitor with almost complete recovery of rickets and partial regression of the phenotype. We identified "core" genes modulated by MEK inhibition potentially serving as surrogate markers of Trametinib action.

Transcriptional classification has been used to stratify colorectal cancer (CRC) into molecular subtypes with distinct biological and clinical features. However, it is not clear whether such subtypes represent discrete, mutually exclusive entities or molecular/phenotypic states with potential overlap. Therefore, we focused on the CRC Intrinsic Subtype (CRIS) classifier and evaluated whether assigning multiple CRIS subtypes to the same sample provides additional clinically and biologically relevant information.

Blood vessel formation depends on the highly coordinated actions of a variety of angiogenic regulators. Vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1) are both potent and essential proangiogenic factors with complementary roles in vascular development and function. Whereas VEGF is required for the formation of the initial vascular plexus, Ang-1 contributes to the stabilization and maturation of growing blood vessels. Here, we provide evidence of a novel microRNA (miRNA)-dependent molecular mechanism of Ang-1 signalling modulation aimed at stabilizing adult vasculature. MiRNAs are short non-coding RNA molecules that post-trascriptionally regulate gene expression by translational suppression or in some instances by cleavage of the respective mRNA target. Our data indicate that endothelial cells of mature vessels express high levels of miR-126, which primarily targets phosphoinositide-3-kinase regulatory subunit 2 (p85β). Down-regulation of miR-126 and over-expression of p85β in endothelial cells inhibit the biological functions of Ang-1. Additionally, knockdown of miR-126 in zebrafish resulted in vascular remodelling and maturation defects, reminiscent of the Ang-1 loss-of-function phenotype. Our findings suggest that miR-126-mediated phosphoinositide-3-kinase regulation, not only fine-tunes VEGF-signaling, but it strongly enhances the activities of Ang-1 on vessel stabilization and maturation.

Constitutively active receptor tyrosine kinases (RTKs) are known oncogenic drivers and provide valuable therapeutic targets in many cancer types. However, clinical efficacy of RTK inhibitors is limited by intrinsic and acquired resistance. To identify genes conferring resistance to inhibition of the MET RTK, we conducted a forward genetics screen in the GTL-16 gastric cancer cell line, carrying MET amplification and exquisitely sensitive to MET inhibition. Cells were transduced with three different retroviral cDNA expression libraries and selected for growth in the presence of the MET inhibitor PHA-665752. Selected cells displayed robust and reproducible enrichment of library-derived cDNAs encoding truncated forms of RAF1 and BRAF proteins, whose silencing reversed the resistant phenotype. Transduction of naïve GTL-16 cells with truncated, but not full length, RAF1 and BRAF conferred in vitro and in vivo resistance to MET inhibitors, which could be reversed by MEK inhibition. Induction of resistance by truncated RAFs was confirmed in other MET-addicted cell lines, and further extended to EGFR-addicted cells. These data show that truncated RAF1 and BRAF proteins, recently described as products of genomic rearrangements in gastric cancer and other malignancies, have the ability to render neoplastic cells resistant to RTK-targeted therapy.

Patient-Derived Xenografts (PDXs), entailing implantation of cancer specimens in immunocompromised mice, are emerging as a valuable translational model that could help validate biologically relevant targets and assist the clinical development of novel therapeutic strategies for gastric cancer. More than 30% of PDXs generated from gastric carcinoma samples developed human B-cell lymphomas instead of gastric cancer. These lymphomas were monoclonal, Epstein Barr Virus (EBV) positive, originated tumorigenic cell cultures and displayed a mutational burden and an expression profile distinct from gastric adenocarcinomas. The ability of grafted samples to develop lymphomas did not correlate with patient outcome, nor with the histotype, the lymphocyte infiltration level, or the EBV status of the original gastric tumor, impeding from foreseeing lymphoma onset. Interestingly, lymphoma development was significantly more frequent when primary rather than metastatic samples were grafted. Notably, the development of such lympho-proliferative disease could be prevented by a short rituximab treatment upon mice implant, without negatively affecting gastric carcinoma engraftment. Due to the high frequency of human lymphoma onset, our data show that a careful histologic analysis is mandatory when generating gastric cancer PDXs. Such care would avoid misleading results that could occur if testing of putative gastric cancer therapies is performed in lymphoma PDXs. We propose rituximab treatment of mice to prevent lymphoma development in PDX models, averting the loss of human-derived samples.

The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2013 guidelines for HER2 assessment have increased the number of HER2 equivocal breast carcinomas following in situ hybridization reflex testing, that is, HER2 "double equivocal" (equivocal protein expression and equivocal gene copy number). Forty-five double-equivocal carcinomas were subjected to Prosigna analysis. Twenty-seven cases were investigated for the expression of genes found to be differentially expressed between estrogen receptor (ER)-positive/HER2-positive (N=22) and ER-positive/HER2-negative (N=22) control cases. Twenty-nine of the 45 cases were also analyzed by targeted sequencing using a panel of 14 genes. We then explored the pathologic complete response rates in an independent series of double-equivocal carcinoma patients treated with trastuzumab-containing chemotherapy. All cases were ER-positive, with a mean Ki67 of 28%. Double-equivocal carcinomas were predominantly luminal B (76%); 9 cases (20%) were luminal A, and 2 cases (4%) HER2-enriched. The majority (73%) showed a high risk of recurrence by Prosigna, even when the carcinomas were small (<2 cm), node-negative/micrometastatic, and/or grade 2. Double-equivocal carcinomas showed TP53 (6/29, 20%), PIK3CA (3/29, 10%), HER2 (1/29, 3%), and MAP2K4 (1/29, 3%) mutations. Compared with grade-matched ER-positive/HER2-negative breast carcinomas from METABRIC, double-equivocal carcinomas harbored more frequently TP53 mutations and less frequently PIK3CA mutations (P<0.05). No significant differences were observed with grade-matched ER-positive/HER2-positive carcinomas. Lower pathologic complete response rates were observed in double-equivocal compared with HER2-positive patients (10% vs. 60%, P=0.009). Double-equivocal carcinomas are preferentially luminal B and show a high risk of recurrence. A subset of these tumors can be labeled as HER2-enriched by transcriptomic analysis. HER2 mutations can be identified in HER2 double-equivocal cases.

Doxorubicin anti-cancer therapy is associated with cardiotoxicity, resulting from DNA damage response (DDR). Hepatocyte growth factor (HGF) protects cardiomyocytes from injury, but its effective use is compromised by low biodistribution. In this study, we have investigated whether the activation of the HGF receptor-encoded by the Met gene-by an agonist monoclonal antibody (mAb) could protect against doxorubicin-induced cardiotoxicity.

In colorectal cancer (CRC), WNT pathway activation by genetic rearrangements of RSPO3 is emerging as a promising target. However, its low prevalence severely limits availability of preclinical models for in-depth characterization. Using a pipeline designed to suppress stroma-derived signal, we find that RSPO3 "outlier" expression in CRC samples highlights translocation and fusion transcript expression. Outlier search in 151 CRC cell lines identified VACO6 and SNU1411 cells as carriers of, respectively, a canonical PTPRK(e1)-RSPO3(e2) fusion and a novel PTPRK(e13)-RSPO3(e2) fusion. Both lines displayed marked in vitro and in vivo sensitivity to WNT blockade by the porcupine inhibitor LGK974, associated with transcriptional and morphological evidence of WNT pathway suppression. Long-term treatment of VACO6 cells with LGK974 led to the emergence of a resistant population carrying two frameshift deletions of the WNT pathway inhibitor AXIN1, with consequent protein loss. Suppression of AXIN1 in parental VACO6 cells by RNA interference conferred marked resistance to LGK974. These results provide the first mechanism of secondary resistance to WNT pathway inhibition.

Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.

Aberrant activation of the MET receptor in cancer is sustained by genetic alterations or, more frequently, by transcriptional upregulations. A fraction of MET-amplified or mutated tumors are sensible to MET targeting agents, but their responsiveness is typically short-lasting, as secondary resistance eventually occurs. Since in the absence of genetic alterations MET is usually not a tumor driver, MET overexpressing tumors are not/poorly responsive to MET targeted therapies. Consequently, the vast majority of tumors exhibiting MET activation still represent an unmet medical need.

The mitotic spindle assembly checkpoint (SAC) is an essential control system of the cell cycle that contributes to mantain the genomic stability of eukaryotic cells. SAC genes expression is often deregulated in cancer cells, leading to checkpoint impairment and chromosome instability. The mechanisms responsible for the transcriptional regulation and deregulation of these genes are still largely unknown. Herein we identify the nonhistone architectural nuclear proteins High Mobility Group A1 (HMGA1), whose overexpression is a feature of several human malignancies and has a key role in cancer progression, as transcriptional regulators of SAC genes expression. In particular, we show that HMGA1 proteins are able to increase the expression of the SAC genes Ttk, Mad2l1, Bub1 and Bub1b, binding to their promoter regions. Consistently, HMGA1-depletion induces SAC genes downregulation associated to several mitotic defects. In particular, we observed a high number of unaligned chromosomes in metaphase, a reduction of prometaphase time, a delay of anaphase, a higher cytokinesis time and a higher percentage of cytokinesis failure by using live-cell microscopy. Finally, a significant direct correlation between HMGA1 and SAC genes expression was detected in human colon carcinomas indicating a novel mechanism by which HMGA1 contributes to cancer progression.

In this study we investigated the contribution of gender to global gene expression in peripheral blood mononuclear cells from multiple sclerosis (MS) patients and healthy controls. We observed that, in contrast to the conventional approach, gender-based case-control comparisons resulted in genelists with significantly reduced heterogeneity in human populations. In addition, MS was characterized by significant changes both in the quantity and in the quality of the sex-specific genes. Application of stringent statistics defined gender-based signatures which classified a second independent MS population with high precision. The global unsupervised cluster analyses for 60 subjects showed that 29/31 female and 27/29 male samples were properly identified. Notably, MS was associated in women and in men with distinct gene signatures which however shared several molecular functions, biological processes and interactors. Issues regarding epigenetic control of gene expression appeared as the main common theme for disease, with a central role for the functional modules related to histone deacetylase, NF-kappaB and androgen receptor signaling. Moreover, in silico analyses predicted that the differential expression in MS women and men were depending on the transcription factor SP1. Specific targeting of this pathway by the bis-anthracycline WP631 impaired T cell responses in vitro and in vivo, and reduced the incidence and the severity of experimental autoimmune encephalomyelitis, indicating that SP1 dependent gene transcription sustains neuroinflammation. Thus, the gender-based approach with its reduced heterogeneity and the systems biology tools with the identification of the molecular and functional networks successfully uncovered the differences but also the commonalities associated to multiple sclerosis in women and men. In conclusion, we propose gender-based systems biology as a novel tool to gain fundamental information on disease-associated functional processes.