Gianluca Sala

The Role of Phosphoinositide 3-Kinase C2α in Insulin Signaling

The members of the class II phosphoinositide 3-kinase (PI3K) family can be activated by several stimuli, indicating that these enzymes can regulate many intracellular processes. Nevertheless, to date, there has been no definitive identification of their in vivo product, their mechanism(s) of activation, or their precise intracellular roles. By metabolic labeling, we here identify phosphatidylinositol 3-phosphate as the sole in vivo product of the insulin-dependent activation of PI3K-C2α, confirming the emerging role of such a phosphoinositide in signaling. We demonstrate that activation of PI3K-C2α involves its recruitment to the plasma membrane and that activation is mediated by the GTPase TC10. This is the first report showing a membrane targeting-mediated mechanism of activation for PI3K-C2α and that a small GTP-binding protein can activate a class II PI3K isoform. We also demonstrate that PI3K-C2α contributes to maximal insulin-induced translocation of the glucose transporter GLUT4 to the plasma membrane and subsequent glucose uptake, definitely assessing the role of this enzyme in insulin signaling.

The Role of Phosphoinositide 3-Kinase C2 in Insulin Signaling *□

The members of the class II phosphoinositide 3-kinase (PI3K) family can be activated by several stimuli, indicating that these enzymes can regulate many intracellular processes. Nevertheless, to date, there has been no definitive identification of their in vivo product, their mechanism(s) of activation, or their precise intracellular roles. By metabolic labeling, we here identify phosphatidylinositol 3-phosphate as the sole in vivo product of the insulin-dependent activation of PI3K-C2α, confirming the emerging role of such a phosphoinositide in signaling. We demonstrate that activation of PI3K-C2α involves its recruitment to the plasma membrane and that activation is mediated by the GTPase TC10. This is the first report showing a membrane targeting-mediated mechanism of activation for PI3K-C2α and that a small GTP-binding protein can activate a class II PI3K isoform. We also demonstrate that PI3K-C2α contributes to maximal insulin-induced translocation of the glucose transporter GLUT4 to the plasma membrane and subsequent glucose uptake, definitely assessing the role of this enzyme in insulin signaling.

A Phosphoinositide 3-Kinase/Phospholipase Cgamma1 Pathway Regulates Fibroblast Growth Factor-Induced Capillary Tube Formation

Background The fibroblast growth factors (FGFs) are key regulators of embryonic development, tissue homeostasis and tumour angiogenesis. Binding of FGFs to their receptor(s) results in activation of several intracellular signalling cascades including phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)γ1. Here we investigated the basic FGF (FGF-2)-mediated activation of these enzymes in human umbilical vein endothelial cells (HUVECs) and defined their role in FGF-2-dependent cellular functions. Methodology/Principal Findings We show that FGF-2 activates PLCγ1 in HUVECs measured by analysis of total inositol phosphates production upon metabolic labelling of cells and intracellular calcium increase. We further demonstrate that FGF-2 activates PI3K, assessed by analysing accumulation of its lipid product phosphatidylinositol-3,4,5-P3 using TLC and confocal microscopy analysis. PI3K activity is required for FGF-2-induced PLCγ1 activation and the PI3K/PLCγ1 pathway is involved in FGF-2-dependent cell migration, determined using Transwell assay, and in FGF-2-induced capillary tube formation (tubulogenesis assays in vitro). Finally we show that PI3K-dependent PLCγ1 activation regulates FGF-2-mediated phosphorylation of Akt at its residue Ser473, determined by Western blotting analysis. This occurs through protein kinase C (PKC)α activation since dowregulation of PKCα expression using specific siRNA or blockade of its activity using chemical inhibition affects the FGF-2-dependent Ser473 Akt phosphorylation. Furthermore inhibition of PKCα blocks FGF-2-dependent cell migration. Conclusion/Significance These data elucidate the role of PLCγ1 in FGF-2 signalling in HUVECs demonstrating its key role in FGF-2-dependent tubulogenesis. Furthermore these data unveil a novel role for PLCγ1 as a mediator of PI3K-dependent Akt activation and as a novel key regulator of different Akt-dependent processes.

EV20-Sap, a novel anti-HER-3 antibody-drug conjugate, displays promising antitumor activity in melanoma

Melanoma is the most biologically aggressive skin cancer of well established constitutive and induced resistance to pharmacological treatment. Despite the recent progresses in immunotherapies, many advanced metastatic melanoma patients still face a significant mortality risk. The aggressive nature of this disease sustains an urgent need for more successful, effective drugs. HER-3 - one of the four member of the tyrosin kinase epidermal growth factor receptors (EGFRs) family- is frequently overexpressed in solid tumors, including melanoma. Moreover, up-regulation of HER-3 and its ligand NRGβ-1 are associated with poor prognosis, thus suggesting this receptor as a suitable target for cancer therapy. Several monoclonal antibodies targeting HER-3 are currently available, but preliminary results from clinical testing of these agents reveal a modest efficacy. Thus, a substantial improvement over this immunotherapeutic approach could be offered by an anti-HER-3 based Antibody-Drug Conjugate (ADC). In the present paper, we describe the generation of an ADC obtained by coupling the HER-3 targeting antibody EV20 linked to the plant toxin Saporin (Sap). In vitro, this ADC displays a powerful, specific and target-dependent cytotoxic activity which correlates with the degree of expression and internalization of HER-3 on tumor cells. Furthermore, in a murine melanoma model, EV20-Sap treatment leads to a significant reduction of the number of pulmonary metastasis.

The role of phospholipase Cγ1 in breast cancer and its clinical significance

Breast cancer, the most common malignancy among women, is usually detected at an early stage and has a low risk of relapse. Nevertheless, a significant number of patients cannot be cured solely by local treatment. Distinguishing between patients who are of low risk of relapse from those who are of high risk may have important implications to improve treatment outcomes. The PLC-γ1 signaling pathway promotes many physiological processes, including cell migration and invasion. Increasing evidence shows aberrant PLC-γ1 signaling implication in carcinogenesis including breast cancer. In this review, the role of PLC-γ1 in breast cancer and its clinical implications will be discussed, as well as its potential as a prognostic factor and a therapeutic target.

Abstract 40: Development of a novel antibody-drug conjugate targeting endosialin/TEM-1: potent antitumor activity in sarcoma

The TEM-1/Endosialin/CD248 receptor is expressed in 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 (ADC). It consists of a humanized TEM-1 monoclonal antibody (E.8-3) conjugated to a highly potent payload (TEM-1-ADC). In TEM-1 expressing cancer cell lines, this TEM-1-ADC demonstrated a powerful, specific and target-dependent killing activity. High expression levels of TEM-1 in cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro. Efficacy studies demonstrated that TEM-1-ADC treatment leads to a long lasting tumor growth inhibition of cell line-based models of human sarcoma. Taken together, our results demonstrated that TEM-1 is an attractive target in sarcoma and suggest that TEM-1-ADC has the potential to be developed into a biotherapeutic agent in these malignancies. Citation Format: Gianluca Sala, Stefano Iacobelli, Emily Capone, Enza PIccolo, Jean-Fred Sauniere, Vanessa Vannucci Douet. Development of a novel antibody-drug conjugate targeting endosialin/TEM-1: potent antitumor activity in sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 40. doi:10.1158/1538-7445.AM2017-40

Abstract 41: An Antibody Drug Conjugate targeting HER-3 demonstrates promising antitumor efficacy in a wide range of human cancer

The HER-3 receptor is emerging as an attractive molecule in therapeutics because of its overexpression across many human cancers and because of its role in in several compensatory processes that underlay emergence of resistance to certain cancer drugs. In this study, we present evidence of the preclinical efficacy of a novel Antibody-Drug Conjugate (ADC) targeting HER-3. It consists of a humanized HER-3 monoclonal antibody (mAb EV20), which recognizes the HER-3 extracellular domain, conjugated to different payloads (HER-3-ADC)s. In HER-3 expressing cancer cell lines, these HER-3-ADCs demonstrated a powerful, specific and target-dependent killing activity. High expression levels of HER-3 in tumor cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro. Efficacy studies demonstrated that HER-3-ADCs treatment leads to a long lasting tumor growth inhibition of cell line-based models of human head and neck, breast, pancreatic, prostatic, lung, stomach cancers and melanoma. Overall, these findings validate HER-3 as an attractive therapeutic target in multiple solid tumors and support further clinical development and application of HER-3 targeting ADCs. Citation Format: Gianluca Sala, Manuela Iezzi, Alessia Lamolinara, Emily Capone, Stefano Iacobelli, Jean-Fred Sauniere, Sara Ponziani, Francesco Giansanti, Rodolfo Ippoliti. An Antibody Drug Conjugate targeting HER-3 demonstrates promising antitumor efficacy in a wide range of human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 41. doi:10.1158/1538-7445.AM2017-41

Abstract LB-A14: Targeting BAG3-dependent paracrine loop reduces growth and metastatic spreading of Pancreatic adenocarcinoma

Incidence and death rate of Pancreatic Ductal Adenocarcinoma (PDAC) has 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 tumor formation and development of metastasis. We have recently shown (manuscript in press) that PDAC cells secrete BAG3 that binds and activates macrophages trough a specific receptor (IFITM2), inducing their activation and the secretion of PDAC supporting factors. Treatment with a murine monoclonal antibody (AC-2) targeting BAG3 reduced tumor growth and impaired metastasis formation in mouse models. Here we report further characterization of the in vivo activity of the antibody. Moreover, we have now successfully generated humanized variants of the AC-2 antibody and report the in vitro and in vivo activity of the humanized antibodies. Both appear to have the ability to inhibit macrophage activation in vitro and reduce tumor growth in PDAC models to an extent comparable to the murine parental antibody (AC-2). 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 for PDAC treatment. Citation Format: Vincenzo De Laurenzi, Alessandra Rosati, Gianluca Sala, Maria Caterina Turco. Targeting BAG3-dependent paracrine loop reduces growth and metastatic spreading of Pancreatic adenocarcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A14.

Abstract 5437: Dual targeting of ErbB-2 and ErbB-3: A new potential strategy for the treatment of pancreatic cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Pancreatic cancer (PC) is one of the most lethal cancers and there is an urgent need to find out new therapeutic approaches. Deregulated signaling through ErbB-1 and ErbB-2, members of the epidermal growth factor receptor family, frequently occurs in PC. However, attempts aiming at inhibiting ErbB-1 and ErbB-2 have revealed only a modest impact on disease outcome. A growing body of evidence suggests that ErbB-3 and its ligand NRG-1β are key players in driving oncogenic signaling and resistance to targeted therapy in PC. We have recently developed a novel humanized ErbB-3 antibody, named EV20, which displayed a potent antitumor activity by promoting receptor downregulation in vitro and in vivo. Here we hypothesized that targeting of ErbB-3 with EV20 would enhance the therapeutic effect of ErbB-2 inhibitors in PC. Materials and Methods: Three different PC cell lines (BxPC-3, HPAF II and SW1990) were chosen based on their metastatic origin, genetic background (KRAS status) and responsiveness to erlotinib. The expression of ErbB-1, ErbB-2 and ErbB-3 was analyzed by FACS and WB. The effects of trastuzumab, cetuximab, EV20 and their combinations on NRG-1β-induced activation of the ErbB3/PI3K/Akt axis, cell proliferation, and in vivo tumor growth were evaluated by standard procedures. Results: ErbB-1 was overexpressed by all the cell lines, whereas the expression levels of ErbB-2 and ErbB-3 were moderate in HPAFII cells and low in BxPC-3 and SW1990 cells. NRG-1β was more effective than EGF (an ErbB-1 ligand) in activating ErbB-3 downstream signaling, which was not inhibited by cetuximab, despite a high ErbB-1 expression in the cells. Conversely, inhibition of ErbB-2 and/or ErbB-3 resulted in a marked suppression of NRG-1β-driven Akt activation. Finally, EV20 in combination with trastuzumab exerted a superior antitumor activity compared to single agent alone in terms of cell proliferation and growth of PC xenografts in nude mice. Conclusions: Dual targeting of ErbB-2 and ErbB-3 could represent a newer therapeutic approach in PC. Note: This abstract was not presented at the meeting. Citation Format: Gianluca Sala, Reza Ghasemi, Emily Capone, Ilario Giovanni Rapposelli, Sara Traini, Cosmo Rossi, Pier Giorgio Natali, Stefano Iacobelli. Dual targeting of ErbB-2 and ErbB-3: A new potential strategy for the treatment of pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5437. doi:10.1158/1538-7445.AM2014-5437

Abstract C58: Humanization and characterization of an anti-ErbB-3 murine monoclonal antibody.

Background: The signaling network downstream of the human epidermal growth factor receptors ErbB-1 and ErbB-2 have been extensively targeted by cancer therapeutics. Although initially efficacious in a subset of patients, drugs targeting these receptors led invariably to resistance which is often associated with reactivation of the ErbB-3/PI3K-Akt axis. This may be overcome by ErbB-3 ligands that abrogates receptor-mediated signaling. Methods: the murine MP-RM-1 monoclonal antibody that specifically binds ErbB-3 ectodomain was chimerized by replacing the murine constant regions with the human constant regions and humanized by gratfing Complementarity Determining Regions from the murine into a human antibody framework using a computer assisted molecular modelling. Recombinant genes were placed into the pCDNA3.1 expression vector and transfected into Chinese Hamster Ovary-S cells. Antibodies were screened on the basis of their ability to inhibit Neuregulin-1 (NRG-1)-induced activation of the ErbB-3/PI3K-Akt axis in IR-8 human melanoma cells (Table 1). Four antibody variants, i.e., cMP-RM-1 #1, hMP-RM-1 #6, hMP-RM-1 #10, and hMP-RM-1 #20, were selected and further tested for their ability to promote ErbB-3 downregulation and to inhibit in vitro and in vivo growth of tumor cells. Results: Four chimeric (c) and sixteen humanized (h) antibody variants were obtained. All variants showed to possess antigen binding affinity comparable to that of their parental antibody. Initial screening identified one chimeric (cMP-RM-1 #1) and three humanized (hMP-RM-1 #6, hMP-RM-1 #10, hMP-RM-1 #20) antibodies as the most effective in inhibiting ErbB-3/Akt phosphorylation and promotion of ErbB-3 downregulation. Further testing revealed the antibodies potently inhibited colony formation ability of tumor cells and halted growth of melanoma and ovarian cancer xenografts in nude mice. Treatment with the antibodies was associated with a decreased ErbB-3 and Akt phosphorylation and ErbB-3 expression in the excised tumor tissue. Conclusions: This successful production of chimeric/humanized antibodies possessing antitumor activity in vitro and in vivo may provide novel candidates for ErbB-3-targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C58.