Michela Lupia

Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer

Summary Our understanding of the molecular determinants of cancer is still inadequate because of cancer heterogeneity. Here, using epithelial ovarian cancer (EOC) as a model system, we analyzed a minute amount of patient-derived epithelial cells from either healthy or cancerous tissues by single-shot mass-spectrometry-based phosphoproteomics. Using a multi-disciplinary approach, we demonstrated that primary cells recapitulate tissue complexity and represent a valuable source of differentially expressed proteins and phosphorylation sites that discriminate cancer from healthy cells. Furthermore, we uncovered kinase signatures associated with EOC. In particular, CDK7 targets were characterized in both EOC primary cells and ovarian cancer cell lines. We showed that CDK7 controls cell proliferation and that pharmacological inhibition of CDK7 selectively represses EOC cell proliferation. Our approach defines the molecular landscape of EOC, paving the way for efficient therapeutic approaches for patients. Finally, we highlight the potential of phosphoproteomics to identify clinically relevant and druggable pathways in cancer.

Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VIshort and myosin VIlong, which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VIlong and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VIshort in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VIshort. Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VIlong) or migratory (myosin VIshort) functional roles.

Ovarian cancer stem cells: still an elusive entity?

The cancer stem cell (CSC) model proposes that tumor development and progression are fueled and sustained by undifferentiated cancer cells, endowed with self-renewal and tumor-initiating capacity. Ovarian carcinoma, based on its biological features and clinical evolution, appears as a prototypical example of CSC-driven disease. Indeed, ovarian cancer stem cells (OCSC) would account not only for the primary tumor growth, the peritoneal spread and the relapse, but also for the development of chemoresistance, thus having profound implication for the treatment of this deadly disease. In the last decade, an increasing body of experimental evidence has supported the existence of OCSC and their pathogenic role in the disease. Nevertheless, the identification of OCSC and the definition of their phenotypical and functional traits have proven quite challenging, mainly because of the heterogeneity of the disease and of the difficulties in establishing reliable biological models. A deeper understanding of OCSC pathobiology will shed light on the mechanisms that underlie the clinical behaviour of OC. In addition, it will favour the design of innovative treatment regimens that, on one hand, would counteract the resistance to conventional chemotherapy, and, on the other, would aim at the eradication of OC through the elimination of its CSC component.

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI‐1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR. Moreover, we show that PAI‐1 counteracts the negative feedback and behaves as a proteolysis‐triggered stabilizer of uPAR‐mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N‐terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process.

CD73 Regulates Stemness and Epithelial-Mesenchymal Transition in Ovarian Cancer-Initiating Cells

Summary Cancer-initiating cells (CICs) have been implicated in tumor development and aggressiveness. In ovarian carcinoma (OC), CICs drive tumor formation, dissemination, and recurrence, as well as drug resistance, thus accounting for the high death-to-incidence ratio of this neoplasm. However, the molecular mechanisms that underlie such a pathogenic role of ovarian CICs (OCICs) remain elusive. Here, we have capitalized on primary cells either from OC or from its tissues of origin to obtain the transcriptomic profile associated with OCICs. Among the genes differentially expressed in OCICs, we focused on CD73, which encodes the membrane-associated 5′-ectonucleotidase. The genetic inactivation of CD73 in OC cells revealed that this molecule is causally involved in sphere formation and tumor initiation, thus emerging as a driver of OCIC function. Furthermore, functional inhibition of CD73 via either a chemical compound or a neutralizing antibody reduced sphere formation and tumorigenesis, highlighting the druggability of CD73 in the context of OCIC-directed therapies. The biological function of CD73 in OCICs required its enzymatic activity and involved adenosine signaling. Mechanistically, CD73 promotes the expression of stemness and epithelial-mesenchymal transition-associated genes, implying a regulation of OCIC function at the transcriptional level. CD73, therefore, is involved in OCIC biology and may represent a therapeutic target for innovative treatments aimed at OC eradication.

Extensive and systematic rewiring of histone post-translational modifications in cancer model systems

Abstract Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations.

VE-Cadherin–Mediated Epigenetic Regulation of Endothelial Gene Expression

Rationale: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. Objective: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. Methods and Results: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and &bgr;-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/&bgr;-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. Conclusions: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.

Critical role of zinc finger protein 521 in the control of growth, clonogenicity and tumorigenic potential of medulloblastoma cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The stem cell-associated transcription co-factor ZNF521 has been implicated in the control of haematopoietic, osteogenic and neural progenitors. Very high expression of this factor is present in cerebellum and particularly in the granule layer of neonatal cerebellum, that contains candidate cells-of-origin of medulloblastoma. Here we have explored the possible involvement of ZNF521 in the development of this tumour. As an experimental system we used the human medulloblastoma cell line, DAOY, and primary cells from medulloblastomas occurring in Ptc1-/+ mice. To investigate the effect of ZNF521 on the growth and tumourigenic potential of these cells, its expression was modulated using lentiviral vectors carrying the ZNF521 cDNA, or containing shRNAs that silence its expression. Enforced overexpression of ZNF521 in DAOY cells, that normally produce relatively low amounts of this protein, was associated with a significant increase in their proliferation rate. This was mirrored by an increase in the ability to grow as spheroids and clonogenicity in single-cell cultures and in semisolid media, and accompanied by an enhanced migratory capacity in wound-healing assays. Finally, ZNF521-expressing DAOY cells demonstrated a greatly enhanced tumourigenic potential in nude mice. All these activities required the presence of an N-terminal domain of ZNF521 that recruits the nucleosome remodeling and histone deacetylase (NuRD) complex. Consistently with the effects of ZNF521 overexpression in DAOY, silencing of Zfp521 in Ptc1-/+ medulloblastoma cells resulted in a drastic decrease in their proliferation and tumourigenic potential, lending further support to the notion that zinc finger protein 521 may contribute to the generation and/or maintenance of the cancer-initiating cell compartment in this cancer. Preliminary experiments detected a selective up-regulation of HES5 mRNA in DAOY overexpressing ZNF521, raising the possibility that some of the effects illustrated here may at least in part be mediated by the co-operation of ZNF521 with the Notch pathway. Citation Format: Raffaella Spina, Gessica Filocamo, Enrico Iaccino, Stefania Scicchitano, Michela Lupia, Emanuela Chiarella, Tiziana Mega, Daniela Pelaggi, Maria Mesuraca, Eli E. Bar, Heather M. Bond, Charles G. Eberhart, Christian Steinkuhler, Gianni Morrone. Critical role of zinc finger protein 521 in the control of growth, clonogenicity and tumorigenic potential of medulloblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5045. doi:10.1158/1538-7445.AM2013-5045 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Platinum sensitivity and DNA repair in a recently established panel of patient-derived ovarian carcinoma xenografts

A xenobank of patient-derived (PDX) ovarian tumor samples has been established consisting of tumors with different sensitivity to cisplatin (DDP), from very responsive to resistant. As the DNA repair pathway is an important driver in tumor response to DDP, we analyzed the mRNA expression of 20 genes involved in the nucleotide excision repair, fanconi anemia, homologous recombination, base excision repair, mismatch repair and translesion repair pathways and the methylation patterns of some of these genes. We also investigated the correlation with the response to platinum-based therapy. The mRNA levels of the selected genes were evaluated by Real Time-PCR (RT-PCR) with ad hoc validated primers and gene promoter methylation by pyrosequencing. All the DNA repair genes were variably expressed in all 42 PDX samples analyzed, with no particular histotype-specific pattern of expression. In high-grade serous/endometrioid PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and POLB. High-grade serous/endometrioid PDXs with TP53 mutations had significantly higher levels of POLQ, FANCD2, RAD51 and POLB than high-grade TP53 wild type PDXs. The mRNA levels of CDK12, PALB2 and XPF inversely associated with the in vivo DDP antitumor activity; higher CDK12 mRNA levels were associated with a higher recurrence rate in ovarian patients with low residual tumor. These data support the important role of CDK12 in the response to a platinum based therapy in ovarian patients.

Abstract A66: The CD73+/CD24- subpopulation of ovarian cancer cells is enriched in cancer stem cells.

Recent data suggest that drug resistance and/or disease recurrence in ovarian cancer (OC) are driven by a subpopulation of cells in human tumors with stem-like characteristics (cancer stem cells, CSCs). CSCs are defined as a small subpopulation of cells within the tumor bulk that possess the capacity, on one hand, to self-renew and, on the other hand, to give rise to all heterogeneous cancer cell lineages that compose the tumor of origin. The CSC hypothesis provides an attractive cellular mechanism to explain the therapeutic refractoriness, dormant behavior, and relapse of OC. Our study aims at assessing ovarian cancer stem cells (OCSC) as causal players in OC etiology and progression and at defining their molecular and functional traits. Specifically, we are pursuing these objectives through the accomplishment of the following milestones: 1) collection of normal and pathological samples; 2) identification of OCSC based on functional properties; 3) comparison of gene expression profiles between cancer stem cells and their normal counterpart; 4) characterization of novel genes/pathways involved in OCSC function (clonogenicity, tumorigenicity, quiescence, chemoresistance, etc.). This workflow has been applied to surgical samples of OC as well as to normal ovarian surface epithelium (OSE) and fallopian tube epithelium (FTE), namely the tissues of origin of OC. OCSC have been derived from primary cultures exploiting their ability to resist anoikis and form monoclonal spheroids when cultured under nonadherent conditions. The transcriptome of OC-derived spheroids was compared with that of their normal counterparts, yielding a set of differentially expressed genes. Among these, for the initial characterization we have selected two cell surface markers CD73, upregulated in spheroids, and CD24, downregulated in spheroids. The CD73+/CD24- subpopulation was enriched in sphere-forming cells and, most importantly, exhibited higher tumorigenic capacity when xenografted in immunocompromised mice. Thus, by utilizing clinically relevant samples we have characterized the transcriptome of OCSC and of their normal counterparts. Furthermore, we found that the CD73+/CD24- phenotype in ovarian cancer is associated to CSC-like traits. On one hand, our approach revealed novel OCSC biomarkers that can be exploited for imaging and purification purposes. On the other hand, we have identified potential therapeutic targets, such as CD73, that might set the stage for innovative treatments aimed at the eradication of OC through the elimination of OCSC. Citation Format: Michela Lupia, Giovanni Bertalot, Pier Paolo Di Fiore, Nicoletta Colombo, Fabrizio Bianchi, Ugo Cavallaro. The CD73+/CD24- subpopulation of ovarian cancer cells is enriched in cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A66.