Luigi Marchionni

Notch signaling can promote tumorigenesis in the nervous system and plays important roles in stem-like cancer cells. However, little is known about how Notch inhibition might alter tumor metabolism, particularly in lesions arising in the brain. The gamma-secretase inhibitor MRK003 was used to treat glioblastoma neurospheres, and they were subdivided into sensitive and insensitive groups in terms of canonical Notch target response. Global metabolomes were then examined using proton magnetic resonance spectroscopy, and changes in intracellular concentration of various metabolites identified which correlate with Notch inhibition. Reductions in glutamate were verified by oxidation-based colorimetric assays. Interestingly, the alkylating chemotherapeutic agent temozolomide, the mTOR-inhibitor MLN0128, and the WNT inhibitor LGK974 did not reduce glutamate levels, suggesting that changes to this metabolite might reflect specific downstream effects of Notch blockade in gliomas rather than g...

Cellular function and diversity are orchestrated by complex interactions of fundamental biomolecules including DNA, RNA and proteins. Technological advances in genomics, epigenomics, transcriptomics and proteomics have enabled massively parallel and unbiased measurements. Such high-throughput technologies have been extensively used to carry out broad, unbiased studies, particularly in the context of human diseases. Nevertheless, a unified analysis of the genome, epigenome, transcriptome and proteome of a single human cell type to obtain a coherent view of the complex interplay between various biomolecules has not yet been undertaken. Here, we report the first multi-omic analysis of human primary naïve CD4+ T cells isolated from a single individual. Integrating multi-omics datasets allowed us to investigate genome-wide methylation and its effect on mRNA/protein expression patterns, extent of RNA editing under normal physiological conditions and allele specific expression in naïve CD4+ T cells. In addition, we carried out a multi-omic comparative analysis of naïve with primary resting memory CD4+ T cells to identify molecular changes underlying T cell differentiation. This analysis provided mechanistic insights into how several molecules involved in T cell receptor signaling are regulated at the DNA, RNA and protein levels. Phosphoproteomics revealed downstream signaling events that regulate these two cellular states. Availability of multi-omics data from an identical genetic background also allowed us to employ novel proteogenomics approaches to identify individual-specific variants and putative novel protein coding regions in the human genome. We utilized multiple high-throughput technologies to derive a comprehensive profile of two primary human cell types, naïve CD4+ T cells and memory CD4+ T cells, from a single donor. Through vertical as well as horizontal integration of whole genome sequencing, methylation arrays, RNA-Seq, miRNA-Seq, proteomics, and phosphoproteomics, we derived an integrated and comparative map of these two closely related immune cells and identified potential molecular effectors of immune cell differentiation following antigen encounter.

Prostate cancers incite tremendous morbidity upon metastatic growth. We previously identified Asporin (ASPN) as a potential mediator of metastatic progression found within the tumor microenvironment. ASPN contains an aspartic acid (D)-repeat domain and germline polymorphisms in D-repeat-length have been associated with degenerative diseases. Associations of germline ASPN D polymorphisms with risk of prostate cancer progression to metastatic disease have not been assessed. Germline ASPN D-repeat-length was retrospectively analyzed in 1600 men who underwent radical prostatectomy for clinically localized prostate cancer and in 548 non-cancer controls. Multivariable Cox proportional hazards models were used to test the associations of ASPN variations with risk of subsequent oncologic outcomes including metastasis. Orthotopic xenografts were used to establish allele- and stroma-specific roles for ASPN D variants in metastatic prostate cancer. Variation at the ASPN D locus was differentia...

Lungs resected for adenocarcinomas often harbour minute discrete foci of cytologically atypical pneumocyte proliferations designated as atypical adenomatous hyperplasia (AAH). Evidence suggests that AAH represents an initial step in the progression to adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and fully invasive adenocarcinoma. Despite efforts to identify predictive markers of malignant transformation, alterations driving this progression are poorly understood. Here we perform targeted next-generation sequencing on multifocal AAHs and different zones of histologic progression within AISs and MIAs. Multiregion sequencing demonstrated different genetic drivers within the same tumour and reveal that clonal expansion is an early event of tumorigenesis. We find that KRAS, TP53 and EGFR mutations are indicators of malignant transition. Utilizing droplet digital PCR, we find alterations associated with early neoplasms in paired circulating DNA. This study provide...

Contractile dysfunction and increased deposition of O-GlcNAcylation in cardiac proteins are a hallmark of the diabetic heart. However, whether and how this post-translational alteration contributes to lower cardiac function remains unclear. Using a refined β-elimination/Michael addition with Tandem Mass Tags (TMT) labeling proteomic technique, here we show that CpOGA, a bacterial analogue of O-GlcNAcase (OGA) that cleaves O-GlcNAc in vivo, removes site-specific O-GlcNAcylation from myofilaments, restoring Ca(2+) sensitivity in STZ-diabetic cardiac muscles. We report that in control rat hearts, O-GlcNAc and O-GlcNAc Transferase (OGT) are mainly localized at the Z-line, whereas OGA is at the A-band. Conversely, in diabetic hearts O-GlcNAc levels are increased, and OGT and OGA delocalized. Consistent changes were found in human diabetic hearts. STZ-diabetic hearts display increased physical interactions of OGA with α-actin, tropomyosin, and myosin light chain1, along with reduced OGT a...

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations resulting in constitutive kinase activity are common in acute myeloid leukemia (AML) and carry a poor prognosis. Several agents targeting FLT3 have been developed, but their limited clinical activity suggests that the inhibition of other factors contributing to the malignant phenotype is required. We examined gene expression data sets as well as primary specimens and found that the expression of GLI2, a major effector of the Hedgehog (Hh) signaling pathway, was increased in FLT3-ITD compared to wild-type FLT3 AML. To examine the functional role of the Hh pathway, we studied mice in which Flt3-ITD expression results in an indolent myeloproliferative state and found that constitutive Hh signaling accelerated the development of AML by enhancing signal transducer and activator of transcription 5 (STAT5) signaling and the proliferation of bone marrow myeloid progenitors. Furthermore, combined FLT3 and Hh pathwa...

Radical prostatectomy (RP) is a primary treatment option for men with intermediate- and high-risk prostate cancer. Although many are effectively cured with local therapy alone, these men are by definition at higher risk of adverse pathologic features and clinical disease recurrence. It has been shown that the Decipher test predicts metastatic progression in cohorts that received adjuvant and salvage therapy following RP. To evaluate the Decipher genomic classifier in a natural history cohort of men at risk who received no additional treatment until the time of metastatic progression. Retrospective case-cohort design for 356 men who underwent RP between 1992 and 2010 at intermediate or high risk and received no additional treatment until the time of metastasis. Participants met the following criteria: (1) Cancer of the Prostate Risk Assessment postsurgical (CAPRA-S) score ≥3; (2) pathologic Gleason score ≥7; and (3) post-RP prostate-specific antigen nadir <0.2 ng/ml. The primary endpoint was defined as regional or distant metastases. Time-dependent receiver operating characteristic (ROC) curves, extension of decision curve analysis to survival data, and univariable and multivariable Cox proportional-hazards models were used to measure the discrimination, net benefit, and prognostic potential of genomic and pathologic risk factors. Cumulative incidence curves were constructed using Fine-Gray competing-risks analysis with appropriate weighting of the controls to account for the case-cohort study design. Ninety six patients had unavailable tumor blocks or failed microarray quality control. Decipher scores were then obtained for 260 patients, of whom 99 experienced metastasis. Decipher correlated with increased cumulative incidence of biochemical recurrence, metastasis, and prostate cancer-specific mortality (p<0.01). The cumulative incidence of metastasis was 12% and 47% for patients with low and high Decipher scores, respectively, at 10 yr after RP. Decipher was independently prognostic of metastasis in multivariable analysis (hazard ratio 1.26 per 10% increase;…

Notch signalling is implicated in the pathogenesis of a variety of cancers, but its role in prostate cancer is poorly understood. However, selected Notch pathway members are overrepresented in high-grade prostate cancers. We comprehensively profiled Notch pathway components in prostate cells and found prostate cancer-specific up-regulation of NOTCH3 and HES6. Their expression was particularly high in androgen responsive lines. Up- and down-regulating Notch in these cells modulated expression of canonical Notch targets, HES1 and HEY1, which could also be induced by androgen. Surprisingly, androgen treatment also suppressed Notch receptor expression, suggesting that androgens can activate Notch target genes in a receptor-independent manner. Using a Notch-sensitive Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) reporter assay, we found that basal levels of Notch signalling were significantly lower in prostate cancer cells compared to benign cells. Accordi...

Prostate cancer is one of the most common malignancies and the second leading cause of death from cancer in men. The molecular mechanisms driving prostate carcinogenesis are complex; with several lines of evidence suggesting that the reexpression of conserved developmental programs plays a key role. In this study, we used conditional gene targeting and organ grafting, to describe conserved roles for the transcription factor Sox9 in the initiation of both prostate organogenesis and prostate carcinogenesis in murine models. Abrogation of Sox9 expression prior to the initiation of androgen signaling blocks the initiation of prostate development. Similarly, Sox9 deletion in two genetic models of prostate cancer (TRAMP and Hi-Myc) prevented cancer initiation. Expression profiling of Sox9-null prostate epithelial cells revealed that the role of Sox9 in the initiation of prostate development may relate to its regulation of multiple cytokeratins and cell adherence/ polarity. Due to its esse...

To assess the evidence that three marketed gene expression-based assays improve prognostic accuracy, treatment choice, and health outcomes in women diagnosed with early stage breast cancer. We evaluated the evidence for three gene expression assays on the market; Oncotype DX, MammaPrint and the Breast Cancer Profiling (BCP or H/I ratio) test, and for gene expression signatures underlying the assays. We sought evidence on: analytic performance of tests, clinical validity (i.e., prognostic accuracy and discrimination), clinical utility (i.e., prediction of treatment benefit), harms, impact on clinical decision making and health care costs. Few papers were found on the analytic validity of the Oncotype DX and MammaPrint tests, but these showed reasonable within-laboratory replicability. Pre-analytic issues related to sample storage and preparation may play a larger role than within-laboratory variation. For clinical validity, studies differed according to whether they examined the actu...

Tumor suppressor genes (TSGs) are commonly inactivated by somatic mutation and/or promoter methylation; yet, recent high-throughput genomic studies have not identified key TSGs inactivated by both mechanisms. We pursued an integrated molecular analysis based on methylation binding domain sequencing (MBD-seq), 450K Methylation arrays, whole exome sequencing, and whole genome gene expression arrays in primary head and neck squamous cell carcinoma (HNSCC) tumors and matched uvulopalatopharyngoplasty tissue samples (UPPPs). We uncovered 186 downregulated genes harboring cancer specific promoter methylation including PAX1 and PAX5 and we identified 10 key tumor suppressor genes (GABRB3, HOXC12, PARP15, SLCO4C1, CDKN2A, PAX1, PIK3AP1, HOXC6, PLCB1, and ZIC4) inactivated by both promoter methylation and/or somatic mutation. Among the novel tumor suppressor genes discovered with dual mechanisms of inactivation, we found a high frequency of genomic and epigenomic alterations in the PAX gene family of transcription factors, which selectively impact canonical NOTCH and TP53 pathways to determine cell fate, cell survival, and genome maintenance. Our results highlight the importance of assessing TSGs at the genomic and epigenomic level to identify key pathways in HNSCC, deregulated by simultaneous promoter methylation and somatic mutations.

Next-generation sequencing (NGS) studies in cancer are limited by the amount, quality and purity of tissue samples. In this situation, primary xenografts have proven useful preclinical models. However, the presence of mouse-derived stromal cells represents a technical challenge to their use in NGS studies. We examined this problem in an established primary xenograft model of small cell lung cancer (SCLC), a malignancy often diagnosed from small biopsy or needle aspirate samples. Using an in silico strategy that assign reads according to species-of-origin, we prospectively compared NGS data from primary xenograft models with matched cell lines and with published datasets. We show here that low-coverage whole-genome analysis demonstrated remarkable concordance between published genome data and internal controls, despite the presence of mouse genomic DNA. Exome capture sequencing revealed that this enrichment procedure was highly species-specific, with less than 4% of reads aligning to the mouse genome. Human-specific expression profiling with RNA-Seq replicated array-based gene expression experiments, whereas mouse-specific transcript profiles correlated with published datasets from human cancer stroma. We conclude that primary xenografts represent a useful platform for complex NGS analysis in cancer research for tumours with limited sample resources, or those with prominent stromal cell populations.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and protein synthesis. To characterize functions of miRNAs and to assess their potential applications, we carried out an integrated multi-omics analysis to study miR-145, a miRNA that has been shown to suppress tumor growth. We employed gene expression profiling, miRNA profiling and quantitative proteomic analysis of a pancreatic cancer cell line. In our transcriptomic analysis, overexpression of miR-145 was found to suppress the expression of genes that are implicated in development of cancer such as ITGA11 and MAGEA4 in addition to previously described targets such as FSCN1, YES1 and PODXL. Based on miRNA profiling, overexpression of miR-145 also upregulated other miRNAs including miR-124, miR-133b and miR-125a-3p, all of which are implicated in suppression of tumors and are generally co-regulated with miR-145 in other cancers. Using the SILAC system, we identified miR-145-induced downregulation of several ...

Prostate cancer is the second leading cause of cancer death among United States men. However, disease aggressiveness is varied, with low-grade disease often being indolent and high-grade cancer accounting for the greatest density of deaths. Outcomes are also disparate among men with high-grade prostate cancer, with upwards of 65% having disease recurrence even after primary treatment. Identification of men at risk for recurrence and elucidation of the molecular processes that drive their disease is paramount, as these men are the most likely to benefit from multimodal therapy. We previously showed that androgen-induced expression profiles in prostate development are reactivated in aggressive prostate cancers. Herein, we report the down-regulation of one such gene, Sparcl1, a secreted protein, acidic and rich in cysteine (SPARC) family matricellular protein, during invasive phases of prostate development and regeneration. We further demonstrate a parallel process in prostate cancer, with decreased expression of SPARCL1 in high-grade/metastatic prostate cancer. Mechanistically, we demonstrate that SPARCL1 loss increases the migratory and invasive properties of prostate cancer cells through Ras homolog gene family, member C (RHOC), a known mediator of metastatic progression. By using models incorporating clinicopathologic parameters to predict prostate cancer recurrence after treatment, we show that SPARCL1 loss is a significant, independent prognostic marker of disease progression. Thus, SPARCL1 is a potent regulator of cell migration/invasion and its loss is independently associated with prostate cancer recurrence.

To elucidate the role of biological and clinical impact of aberrant promoter hypermethylation (PH) in ovarian cancer (OC). PH of PGP9.5, HIC1, AIM1, APC, PAK3, MGMT, KIF1A, CCNA1, ESR1, SSBP2, GSTP1, FKBP4 and VGF were assessed by quantitative methylation specific PCR (QMSP) in a training set. We selected two genes (VGF and PGP9.5) for further QMSP analysis in a larger independent validation (IV) set with available clinical data. Biologic relevance of VGF gene was also evaluated. PH frequency for PGP9.5 and VGF were 85% (316/372) and 43% (158/366) respectively in the IV set of samples while no PH was observed in controls. In 372 OC cases with available follow up, PGP9.5 and VGF PH were correlated with better patient survival [Hazard Ratios (HR) for overall survival (OS) were 0.59 (95% Confidence Intervals (CI)  = 0.42-0.84, p = 0.004), and 0.73 (95%CI = 0.55-0.97, p = 0.028) respectively, and for disease specific survival (DSS) were 0.57 (95%CI 0.39-0.82, p = 0.003) and 0.72 (95%CI 0.54-0.96, p = 0.027). In multivariate analysis, VGF PH remained an independent prognostic factor for OS (HR 0.61, 95%CI 0.43-0.86, p<0.005) and DSS (HR 0.58, 95%CI 0.41-0.83, p<0.003). Furthermore, PGP9.5 PH was significantly correlated with lower grade, early stage tumors, and with absence of residual disease. Forced expression of VGF in OC cell lines inhibited cell growth. Our results indicate that VGF and PGP9.5 PH are potential biomarkers for ovarian carcinoma. Confirmatory cohorts with longitudinal follow-up are required in future studies to define the clinical impact of VGF and PGP9.5 PH before clinical application.

New therapies are critically needed to improve the outcome for patients with small cell lung cancer (SCLC). Insulin-like growth factor 1 receptor (IGF-1R) inhibition is a potential treatment strategy for SCLC: the IGF-1R pathway is commonly upregulated in SCLC and has been associated with inhibition of apoptosis and stimulation of proliferation through downstream signaling pathways, including phosphatidylinositol-3-kinase-Akt and mitogen-activated protein kinase. To evaluate potential determinants of response to IGF-1R inhibition, we assessed the relative sensitivity of 19 SCLC cell lines to OSI-906, a small molecule inhibitor of IGF-1R, and the closely related insulin receptor. Approximately one third of these cell lines were sensitive to OSI-906, with an IC50 < 1 μmol/L. Cell line expression of IGF-1R, IR, IGF-1, IGF-2, IGFBP3, and IGFBP6 did not correlate with sensitivity to OSI-906. Interestingly, OSI-906 sensitive lines expressed significantly lower levels of baseline phospho-ERK relative to resistant lines (P = 0.006). OSI-906 treatment resulted in dose-dependent inhibition of phospho-IGF-1R and phospho-Akt in both sensitive and resistant cell lines, but induced apoptosis and cell-cycle arrest only in sensitive lines. We tested the in vivo efficacy of OSI-906 using an NCI-H187 xenograft model and two SCLC patient xenografts in mice. OSI-906 treatment resulted in 50% tumor growth inhibition in NCI-H187 and 30% inhibition in the primary patient xenograft models compared with mock-treated animals. Taken together our data support IGF-1R inhibition as a viable treatment strategy for a defined subset of SCLC and suggest that low pretreatment levels of phospho-ERK may be indicative of sensitivity to this therapeutic approach.

Resting memory CD4(+) T cells are the largest reservoir of persistent infection in HIV-1-positive subjects. They harbor dormant, stably integrated virus despite suppressive antiretroviral therapy, posing an obstacle to a cure. Surface markers that identify latently infected cells remain unknown. Microarray analyses comparing resting latently infected and uninfected CD4(+) T cells generated in vitro showed profound differences in the expression of gene programs related to transcriptional and posttranscriptional regulation, cell proliferation, survival, cycle progression, and basic metabolism, suggesting that multiple biochemical and metabolic blocks contribute to preventing viral production in latently infected cells. We identified 33 transcripts encoding cell surface markers that are differentially expressed between latently infected and uninfected cells. Quantitative reverse transcriptase PCR (RT-QPCR) and flow cytometry analyses confirmed that the surface marker CD2 was expressed at higher levels on latently infected cells. To validate this result in vivo, we sorted resting memory CD4(+) T cells expressing high and low surface levels of CD2 from six HIV-1-infected subjects successfully treated with antiretroviral drugs for at least 3 years. Resting memory CD4(+) CD2(high) T cells from all subjects harbored higher HIV-1 DNA copy numbers than all other CD4(+) T cell subsets. Moreover, after ex vivo viral reactivation, robust viral RNA production was detected only from resting memory CD4(+) CD2(high) T cells but not from other cell subsets. Altogether, these results show that a high CD2 expression level is a hallmark of latently infected resting memory CD4(+) T cells in vivo.