The endosialin/CD248/TEM1 receptor is expressed on the cell surface of tumor-associated stroma cells as well as in sarcoma and neuroblastoma cells. This receptor is emerging as an attractive molecule in diagnostics and therapeutics because of its expression across the stroma of many human tumors, the low to absent expression in normal tissues and accessibility from the vascular circulation. In this study, we present evidence of the preclinical efficacy of a novel Antibody-Drug Conjugate (ENDOS/ADC). It consists of a humanized endosialin monoclonal antibody, named hMP-E-8.3, conjugated to a potent duocarmycin derivative. In endosialin expressing cancer cell lines, this ENDOS/ADC showed a powerful, specific and target-dependent killing activity. High expression levels of endosialin in cells correlated with efficient internalization and cytotoxic effects in vitro. Efficacy studies demonstrated that ENDOS/ADC treatment led to a long-lasting tumor growth inhibition of a cell line-based model of human osteosarcoma. Taken together, our results demonstrate that endosialin is an attractive target in sarcoma and suggest that ENDOS/ADC has the potential to be developed into a bio-therapeutic agent for these malignancies.

Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6) is frequently amplified in breast cancer, with an increased incidence in the triple-negative breast cancer (TNBC) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three-dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6-depleted, triple-negative MDA-MB-231 cells rearranged the actin cytoskeleton and restored epidermal growth factor-mediated invasion. In patients with localized, lymph node-negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse-free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6-fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC. (c) 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

P63 is a transcription factor belonging to the family of p53, essential for the development and differentiation of epithelia. In recent years, it has become clear that altered expression of the different isoforms of this gene can play an important role in carcinogenesis. The p63 gene encodes for two main isoforms known as TA and ΔN p63 with different functions. The role of these different isoforms in sustaining tumor progression and metastatic spreading however has not entirely been clarified. Here we show that breast cancer initiating cells express ΔNp63 isoform that supports a more mesenchymal phenotype associated with a higher tumorigenic and metastatic potential. On the contrary, the majority of cells within the tumor appears to express predominantly TAp63 isoform. While ΔNp63 exerts its effects by regulating a PI3K/CD44v6 pathway, TAp63 modulates this pathway in an opposite fashion. As a result, tumorigenicity and invasive capacity of breast cancer cells is a balance of the two isoforms. Finally, we found that tumor microenvironmental cytokines significantly contribute to the establishment of breast cancer cell phenotype by positively regulating ΔNp63 and CD44v6 expression.

Abstract Background: In the human mammary gland p63 isoforms control mammary epithelial cells fate and DNp63, which lacks the amino-terminal trans-activation domain, counterbalances the full-length isoform TAp63 to allow maturation of epithelia and the maintenance of the myoepithelial/basal cells features. Mammary gland tissue's adult stem cells retain self-renewal and multi-lineage differentiation ability and, by acquiring a malignant behavior, are in charge of tumor seeding. A correlation between Cancer Stem Cells (CSCs) in primary lesions and increased metastatic dissemination has been reported, however, still little is known about the unique profile of distant metastasis’ cell origin. Herein, we show for the first time that breast CSCs (BCSCs) expressing DNp63 are capable to generate metastasis in a preclinical model, while TAp63 can be solely tumorigenic and unable to serial xenografting. We anticipate novel farsighted implications for a prognostic role of p63 and for its targeting to prevent tumor growth and metastatic disease. Methods: BCSCs were freshly isolated from the histological uninvolved resection of breast cancer tissues. Obtained cells were plated in ultra-low flask in serum-free media in presence of bFGF and EGF. Cells were treated with 1mmol/l doxorubicin hydrochloride. Synthetic genes encoding DNp63 and TAp63 were inserted into the p-TWEEN EGFP lentiviral expression vector. Stable p63 knockdown was produced by lentiviral transduction of the pLentilox 3.7 vector carrying shp63 and scramble sequences. For the tumorigenic and metastagenic assay BCSCs (3×105) were suspended in matrigel and injected orthotopically or in the sub-renal capsule of NOD/SCID mice, respectively. Tumors were measured with a caliper each week and volume was calculated by the formula: π/6 x larger diameter x (smaller diameter)2. Metastasis formation was followed over time and visualized by in vivo imaging. Summary: Herein, we demonstrate that luminal and basal BCSCs differ in the content of p63 isoforms, DNp63expression was higher in the stem-like compartment of basal BCSCs, compared to TAp63 mainly expressed in bulk primary cells. DNp63 increased the G0-G1 phase in both luminal and basal BCSCs, enhanced doxorubicin resistance and induced a mesenchymal switch. Despite both TAp63 and DNp63 overexpressing BCSCs formed subcutaneous xenografts in NOD/SCID mice, DNp63 cells exclusively retain capabilities of serial xenografting. Thus, suggesting that exogenous expression of TAp63 could promote transition from stem cells to progenitor cells. In a metastatic preclinical model TAp63 overexpression hampered metastatic potential of BCSCs, while DNp63 overexpressing cells gained capabilities to colonize distant organs such as lung and kidney. Conclusions: These findings state DNp63 as a major regulator of stemness and invasiveness in malignant breast tissues and offer new insights on p63 role as a prognostic biomarker and therapeutic target. Citation Format: Alice Turdo, Simone Di Franco, Antonina Benfante, Maria Luisa Colorito, Marco Bonanno, Miriam Gaggianesi, Daniela Barcaroli, Francesco Dieli, Jan Paul Medema, Vincenzo De Laurenzi, Giorgio Stassi, Matilde Todaro. DNp63 governs metastatic outgrowth of breast cancer stem cells. [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 LB-143. doi:10.1158/1538-7445.AM2015-LB-143

Abstract Interactions between cancer cells and stromal cells are critically involved in tumor formation, progression and development of metastasis. Here we report for the first time that human pancreatic adenocarcinoma (PDAC) cells secrete BAG3 that binds and activates macrophages, inducing their activation and the secretion of pancreatic cancer- supporting factors. We also identify IFITM-2 as a BAG3 receptor, by its co-precipitation with BAG3 from macrophage cell membrane and by impairment of BAG3- induced activation in IFITM-2- silenced macrophages. Finally we show the importance of this pathway in vivo since in nu/nu mice carrying orthotopic xenografts of human PDAC (MIA PaCa-2) cells, BAG3 sequestration by an anti-BAG3 antibody results in reduced tumor growth and prevents metastastasis formation. In conclusion we have identified a novel paracrine loop involved in PDAC growth and metastatic spreading and shown that its pharmacological blockage with an anti-BAG3 antibody has therapeutic potential. Citation Format: Alessandra Rosati, Anna Basile, Raffaella D'Auria, Morena d'Avenia, Margot De Marco, Antonia Falco, Michelina Festa, Luana Guerriero, Vittoria Iorio, Maria Pascale, Renato Franco, Claudio Arra, Antonio Barbieri, Domenica Rea, Giulio Menichini, Michael Hahne, Marteen Bijlsma, Daniela Barcaroli, David A. Tuveson, Jelena Todoric, Michael Karin, Vincenzo De Laurenzi, Maria Caterina Turco. PDAC cells release BAG3 that activates a paracrine loop with stromal macrophages. [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 2393. doi:10.1158/1538-7445.AM2015-2393

The incidence and death rate of pancreatic ductal adenocarcinoma (PDAC) have increased in recent years, therefore the identification of novel targets for treatment is extremely important. Interactions between cancer and stromal cells are critically involved in tumour formation and development of metastasis. Here we report that PDAC cells secrete BAG3, which binds and activates macrophages, inducing their activation and the secretion of PDAC supporting factors. We also identify IFITM-2 as a BAG3 receptor and show that it signals through PI3K and the p38 MAPK pathways. Finally, we show that the use of an anti-BAG3 antibody results in reduced tumour growth and prevents metastasis formation in three different mouse models. In conclusion, we identify a paracrine loop involved in PDAC growth and metastatic spreading, and show that an anti-BAG3 antibody has therapeutic potential.

Approximately 5-10% of metastatic colorectal cancers harbor a BRAF-V600E mutation, which is correlated with resistance to EGFR-targeted therapies and worse clinical outcome. Vice versa, targeted inhibition of BRAF-V600E with the selective inhibitor PLX 4032 (Vemurafenib) is severely limited due to feedback re-activation of EGFR in these tumors. Mounting evidence indicates that upregulation of the ErbB-3 signaling axis may occur in response to several targeted therapeutics, including Vemurafenib, and NRG-1β-dependent re-activation of the PI3K/AKT survival pathway has been associated with therapy resistance. Here we show that colon CSCs express, next to EGFR and ErbB-2, also significant amounts of ErbB-3 on their membrane. This expression is functional as NRG-1β strongly induces AKT/PKB and ERK phosphorylation, cell proliferation, clonogenic growth and promotes resistance to Vemurafenib in BRAF-V600E mutant colon CSCs. This resistance was completely dependent on ErbB-3 expression, as evidenced by knockdown of ErbB-3. More importantly, resistance could be alleviated with therapeutic antibody blocking ErbB-3 activation, which impaired NRG-1β-driven AKT/PKB and ERK activation, clonogenic growth in vitro and tumor growth in xenograft models. In conclusion, our findings suggest that targeting ErbB-3 receptors could represent an effective therapeutic approach in BRAF-V600E mutant colon cancer.

BAG3, member the HSP70 co-chaperones family, has been shown to play a relevant role in the survival, growth and invasiveness of different tumor types. In this study, we investigate the expression of BAG3 in 66 specimens from different lung tumors and the role of this protein in small cell lung cancer (SCLC) tumor growth. Normal lung tissue did not express BAG3 while we detected the expression of BAG3 by immunohistochemistry in all the 13 squamous cell carcinomas, 13 adenocarcinomas and 4 large cell carcinomas. Furthermore, we detected BAG3 expression in 22 of the 36 SCLCs analyzed. The role on SCLC cell survival was determined by down-regulating BAG3 levels in two human SCLC cell lines, i.e. H69 and H446, in vitro and measuring cisplatin induced apoptosis. Indeed down-regulation of BAG3 determines increased cell death and sensitizes cells to cisplatin treatment. The effect of BAG3 down-regulation on tumor growth was also investigated in an in vivo xenograft model by treating mice with an adenovirus expressing a specific bag3 siRNA. Treatment with bag3 siRNA-Ad significantly reduced tumor growth and improved animal survival. In conclusion we show that a subset of SCLCs over express BAG3 that exerts an anti-apoptotic effect resulting in resistance to chemotherapy.

p63 is an important regulator of epithelial development expressed in different variants containing (TA) or lacking (ΔN) the N-terminal transactivation domain. The different isoforms regulate stem-cell renewal and differentiation as well as cell senescence. Several studies indicate that p63 isoforms also play a role in cancer development; however, very little is known about the role played by p63 in regulating the cancer stem phenotype. Here we investigate the cellular signals regulated by TAp63 and ΔNp63 in a model of epithelial cancer stem cells. To this end, we used colon cancer stem cells, overexpressing either TAp63 or ΔNp63 isoforms, to carry out a proteomic study by chemical-labeling approach coupled to network analysis. Our results indicate that p63 is implicated in a wide range of biological processes, including metabolism. This was further investigated by a targeted strategy at both protein and metabolite levels. The overall data show that TAp63 overexpressing cells are more glycolytic-active than ΔNp63 cells, indicating that the two isoforms may regulate the key steps of glycolysis in an opposite manner. The mass-spectrometry proteomics data of the study have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with data set identifiers PXD000769 and PXD000768.

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex-binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex-binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.

Endocannabinoid signaling modulates a variety of neuroinflammatory and neurodegenerative diseases, mainly through the activation of type-1 and type-2 (CB(1)R and CB(2)R) cannabinoid receptors. CB(1)R is negatively regulated by membrane cholesterol, while CB(2)R is unaffected. Here, we identified in the transmembrane helix 7 of human CBRs a consensus sequence already known in other proteins as cholesterol recognition/interaction amino acid sequence and consensus pattern. As this motif is different in the two CBR subtypes, we mutated lysine 402 of CB(1)R into glycine, to obtain a cholesterol recognition/interaction amino acid sequence and consensus similar to that of CB(2)R. Both mutated and wild-type receptors were transiently expressed in human neuronal SH-SY5Y cells, and their localization and functioning were investigated using biochemical assays and immunofluorescence labelling. We found a reduced propensity of the mutant CB(1)R to reside in cholesterol-rich microdomains and, by means of fluorescence recovery after photobleaching analysis, we documented its loss of sensitivity to increased membrane cholesterol content. These results seem to uncover the existence of a new structural determinant in cannabinoid receptors, that is likely implicated in directing their interaction with cholesterol-rich microdomains of cell membranes.

Anandamide (AEA) is an endogenous agonist of type 1 cannabinoid receptors (CB1R) that, along with metabolic enzymes of AEA and congeners, compose the “endocannabinoid system.” Here we report the biochemical, morphological, and functional characterization of the endocannabinoid system in human neuroblastoma SH-SY5Y cells that are an experimental model for neuronal cell damage and death, as well as for major human neurodegenerative disorders. We also show that AEA dose-dependently induced apoptosis of SH-SY5Y cells. Through proteomic analysis, we further demonstrate that AEA-induced apoptosis was paralleled by an ∼3 to ∼5-fold up-regulation or down-regulation of five genes; IgG heavy chain-binding protein, stress-induced phosphoprotein-1, and triose-phosphate isomerase-1, which were up-regulated, are known to act as anti-apoptotic agents; actin-related protein 2/3 complex subunit 5 and peptidylprolyl isomerase-like protein 3 isoform PPIL3b were down-regulated, and the first is required for actin network formation whereas the second is still function-orphan. Interestingly, only the effect of AEA on BiP was reversed by the CB1R antagonist SR141716, in SH-SY5Y cells as well as in human neuroblastoma LAN-5 cells (that express a functional CB1R) but not in SK-NBE cells (which do not express CB1R). Silencing or overexpression of BiP increased or reduced, respectively, AEA-induced apoptosis of SH-SY5Y cells. In addition, the expression of BiP and of the BiP-related apoptotic markers p53 and PUMA was increased by AEA through a CB1R-dependent pathway that engages p38 and p42/44 mitogen-activated protein kinases. Consistently, this effect of AEA was minimized by SR141716. In conclusion, we identified BiP as a key protein in neuronal apoptosis induced by AEA.

Aneuploidy is a characteristic of most solid tumors, often associated with negative prognosis. It can arise from two principal mechanisms: from a tetraploid intermediate state, or directly from errors at cell division. The control of cell division, crucial to maintain genomic stability, is still poorly understood in its relationship to aneuploidy. Here we show that the TAp73 alpha isoform induces polyploidy when overexpressed. This is possibly due to the interaction of TAp73 alpha with kinetochore-related proteins leading to the alteration of mitotic checkpoint abilities. TAp73 alpha but not p53 or any of the other p73 isoforms binds Bub1 and Bub3. Since TAp73 alpha is frequently overexpressed in cancer, this interaction may contribute to the aneuploidy observed in cancer progression. Our results suggest a novel molecular mechanism leading to aneuploidy involving interference of TAp73 alpha with Bub1 and Bub3 resulting in an altered mitotic checkpoint.

Cajal Bodies are one of many specialised organelles contained in the eukaryotic cell nucleus, and are involved in a number of functions, including regulation of replication-dependent histone gene transcription. In normal diploid cells their number varies between 0 and 4 depending on the cell cycle phase, although in cancer cell lines their number is extremely variable and it has been suggested that it correlates with cell ploidy.Here we show that in mammalian cells, as in Drosophila, two distinct though functionally related bodies exist: a histone gene locus body and a Cajal Body. The first one can be detected using FLASH or NPAT as markers while the second is labelled using antibodies against Coilin.Only the number of FLASH/NPAT histone gene locus bodies correlates with ploidy and only these organelles appear to be regulated during the cell cycle. Finally, we show that the two organelles completely co-localize during the S phase of the cell cycle.

Anandamide ( arachidonoylethanolamide, AEA) belongs to an important class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids, collectively termed "endocannabinoids." Recently we have shown that AEA inhibits differentiation of human keratinocytes, by binding to type-1 cannabinoid receptors (CB1R). To further characterize the molecular mechanisms responsible for this effect, we investigated the expression of epidermal differentiation-related genes after AEA treatment. We observed that keratin 1 and 10, transglutaminase 5 and involucrin are transcriptionally down-regulated by AEA. Most importantly, we found that AEA is able to decrease differentiating gene expression by increasing DNA methylation in human keratinocytes, through a p38, and to a lesser extent p42/44, mitogen-activated protein kinase-dependent pathway triggered by CB1R. An effect of AEA on DNA methylation because of CB1R-mediated increase of methyltransferase activity is described here for the first time, and we believe that the importance of this effect clearly extends beyond the regulation of skin differentiation. In fact, the modulation of DNA methylation by endocannabinoids may affect the expression of a number of genes that regulate many cell functions in response to these substances.