TCTN2: a novel tumor marker with oncogenic properties.

Tectonic family member 2 (TCTN2) encodes a transmembrane protein that belongs to the tectonic family, which is involved in ciliary functions. Previous studies have demonstrated the role of tectonics in regulating a variety of signaling pathways at the transition zone of cilia. However, the role of tectonics in cancer is still unclear. Here we identify that TCTN2 is overexpressed in colorectal, lung and ovary cancers. We show that different cancer cell lines express the protein that localizes at the plasma membrane, facing the intracellular milieu. TCTN2 over-expression in cancer cells resulted in an increased ability to form colonies in an anchorage independent way. On the other hand, downregulation of TCTN2 using targeted epigenetic editing in cancer cells significantly reduced colony formation, cell invasiveness, increased apoptosis and impaired assembly of primary cilia. Taken together, our results indicate that TCTN2 acts as an oncogene, making it an interesting cancer-associated protein and a potential candidate for therapeutic applications.

ERMP1, a novel potential oncogene involved in UPR and oxidative stress defense, is highly expressed in human cancer.

Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) are highly activated in cancer and involved in tumorigenesis and resistance to anti-cancer therapy. UPR is becoming a promising target of anti-cancer therapies. Thus, the identification of UPR components that are highly expressed in cancer could offer new therapeutic opportunity.In this study, we demonstrate that Endoplasmic Reticulum Metallo Protease 1 (ERMP1) is broadly expressed in a high percentage of breast, colo-rectal, lung, and ovary cancers, regardless of their stage and grade. Moreover, we show that loss of ERMP1 expression significantly hampers proliferation, migration and invasiveness of cancer cells. Furthermore, we show that this protein is an important player in the UPR and defense against oxidative stress. ERMP1 expression is strongly affected by reticular stress induced by thapsigargin and other oxidative stresses. ERMP1 silencing during reticular stress impairs the activation of PERK, a key sensor of the UPR activation. Loss of ERMP1 also prevents the expression of GRP78/BiP, a UPR stress marker involved in the activation of the survival pathway. Finally, ERMP1 silencing in cells exposed to hypoxia leads to inhibition of the Nrf2-mediated anti-oxidant response and to reduction of accumulation of HIF-1, the master transcription factor instructing cells to respond to hypoxic stress. Our results suggest that ERMP1 could act as a molecular starter to the survival response induced by extracellular stresses. Moreover, they provide the rationale for the design of ERMP1-targeting drugs that could act by inhibiting the UPR initial adaptive response of cancer cells and impair cell survival.

FAT1: a potential target for monoclonal antibody therapy in colon cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the major causes of cancer-associated mortality worldwide. The currently approved therapeutic agents have limited efficacy. METHODS: The atypical cadherin FAT1 was discovered as a novel CRC-associated protein by using a monoclonal antibody (mAb198.3). FAT1 expression was assessed in CRC cells by immunohistochemistry (IHC), immunoblots, flow cytometry and confocal microscopy. In addition, in vitro and in vivo tumour models were done to assess FAT1 potential value for therapeutic applications. RESULTS: The study shows that FAT1 is broadly expressed in primary and metastatic CRC stages and detected by mAb198.3, regardless of KRAS and BRAF mutations. FAT1 mainly accumulates at the plasma membrane of cancer cells, whereas it is only marginally detected in normal human samples. Moreover, the study shows that FAT1 has an important role in cell invasiveness while it does not significantly influence apoptosis. mAb198.3 specifically recognises FAT1 on the surface of colon cancer cells and is efficiently internalised. Furthermore, it reduces cancer growth in a colon cancer xenograft model. CONCLUSIONS: This study provides evidence that FAT1 and mAb198.3 may offer new therapeutic opportunities for CRC including the tumours resistant to current EGFR-targeted therapies.

Angiopoietin-like 7, a novel pro-angiogenetic factor over-expressed in cancer.

Angiopoietin-like (ANGPTL) proteins are secreted proteins showing structural similarity to members of the angiopoietin family. Some ANGPTL proteins possess pleiotropic activities, being involved in cancer lipid, glucose energy metabolisms, and angiogenesis. ANGPTL7 is the less characterized member of the family whose functional role is only marginally known. In this study, we provide experimental evidences that ANGPTL7 is over-expressed in different human cancers. To understand the role played by ANGPTL7 in tumor biology, we asked whether ANGPTL7 is endogenously expressed by malignant cells or in response to environmental stimuli. We found that ANGPTL7 is marginally expressed under standard growth condition while it is specifically up-regulated by hypoxia. Interestingly, the protein is secreted and partially associated with the exosomal fraction, suggesting that it could be found in the systemic circulation of oncologic patients and act in an endocrine way. Moreover, we found that ANGPTL7 exerts a pro-angiogenetic effect on human differentiated endothelial cells by stimulating their proliferation, motility, invasiveness, and capability to form capillary-like networks while it does not stimulate progenitor endothelial cells. Finally, we showed that ANGPTL7 promotes vascularization in vivo in the mouse Matrigel sponge assay, thereby accrediting this molecule as a pro-angiogenic factor.

Identification of new hematopoietic cell subsets with a polyclonal antibody library specific for neglected proteins.

The identification of new markers, the expression of which defines new phenotipically and functionally distinct cell subsets, is a main objective in cell biology. We have addressed the issue of identifying new cell specific markers with a reverse proteomic approach whereby approximately 1700 human open reading frames encoding proteins predicted to be transmembrane or secreted have been selected in silico for being poorly known, cloned and expressed in bacteria. These proteins have been purified and used to immunize mice with the aim of obtaining polyclonal antisera mostly specific for linear epitopes. Such a library, made of about 1600 different polyclonal antisera, has been obtained and screened by flow cytometry on cord blood derived CD34+CD45dim cells and on peripheral blood derived mature lymphocytes (PBLs). We identified three new proteins expressed by fractions of CD34+CD45dim cells and eight new proteins expressed by fractions of PBLs. Remarkably, we identified proteins the presence of which had not been demonstrated previously by transcriptomic analysis. From the functional point of view, looking at new proteins expressed on CD34+CD45dim cells, we identified one cell surface protein (MOSC-1) the expression of which on a minority of CD34+ progenitors marks those CD34+CD45dim cells that will go toward monocyte/granulocyte differentiation. In conclusion, we show a new way of looking at the membranome by assessing expression of generally neglected proteins with a library of polyclonal antisera, and in so doing we have identified new potential subsets of hematopoietic progenitors and of mature PBLs.

BB14, a Nerve Growth Factor (NGF)-like peptide shown to be effective in reducing reactive astrogliosis and restoring synaptic homeostasis in a rat model of peripheral nerve injury.

Peptidomimetics hold a great promise as therapeutic agents for neurodegenerative disorders. We previously described a Nerve Growth Factor (NGF)-like peptide, now named BB14, which was found to act as a strong TrkA agonist and to be effective in the sciatic nerve injury model of neuropathic pain. In this report we present the effects of BB14 in reducing reactive astrocytosis and reverting neuroplastic changes of the glutamate/GABAergic circuitry in the lumbar spinal cord following spared nerve injury (SNI) of the sciatic nerve. Immunohistochemical analysis of spinal cord sections revealed that SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) responses, indicative of reactive gliosis. These changes were paralleled by (i) decreased glial aminoacid transporters (GLT1 and GlyT1) and increased levels of (ii) neuronal glutamate transporter EAAC1, (iii) neuronal vesicular GABA transporter (vGAT) and (iv) the GABAergic neuron marker GAD65/67. A remarkable increase of the Glutamate/GABA ratio and the reduction of glutathione (GSH) levels were also indicative of modifications of glial function in neuroprotection. All these molecular changes were found to be linked to an alteration of endogenous NGF metabolism, as demonstrated by decreased levels of mature NGF, increase of proNGF and increased activity of NGF-degrading methallo-proteinases (MMPs). Biochemical alterations and SNI-related neuropathic behavior, characterized by allodynia and hyperalgesia, were reversed by 7-days i.t. administration of the NGF-like peptide BB14, as well as by increasing endogenous NGF levels by i.t. infusion of GM6001, a MMPs inhibitor. All together, while confirming the correlation between reactive astrogliosis and perturbation of synaptic circuitry in the SNI model of peripheral nerve injury, these data strongly support the beneficial effect of BB14 in reducing reactive astrogliosis and restoring synaptic homeostasis under pathological conditions linked to alteration of NGF availability and signaling, thereby suggesting a potential role of BB14 as a therapeutic agent.

A novel polyclonal antibody library for expression profiling of poorly characterized, membrane and secreted human proteins.

The YOMICS™ antibody library (http://www.yomics.com/) presented in this article is a new collection of 1559 murine polyclonal antibodies specific for 1287 distinct human proteins. This antibody library is designed to target marginally characterized membrane-associated and secreted proteins. It was generated against human proteins annotated as transmembrane or secreted in GenBank, EnsEMBL, Vega and Uniprot databases, described in no or very few dedicated PubMed-linked publications. The selected proteins/protein regions were expressed in E. coli, purified and used to raise antibodies in the mouse. The capability of YOMICS™ antibodies to specifically recognize their target proteins either as recombinant form or as expressed in cells and tissues was confirmed through several experimental approaches, including Western blot, confocal microscopy and immunohistochemistry (IHC). Moreover, to show the applicability of the library for biomarker investigation by IHC, five antibodies against proteins either known to be expressed in some cancers or homologous to tumor-associated proteins were tested on tissue microarrays carrying tumor and normal tissues from breast, colon, lung, ovary and prostate. A consistent differential expression in cancer was observed. Our results indicate that the YOMICS™ antibody library is a tool for systematic protein expression profile analysis that nicely complements the already available commercial antibody collections.

Mutant prourokinase with adjunctive C1-inhibitor is an effective and safer alternative to tPA in rat stroke.

A single-site mutant (M5) of native urokinase plasminogen activator (prouPA) induces effective thrombolysis in dogs with venous or arterial thrombosis with a reduction in bleeding complications compared to tPA. This effect, related to inhibition of two-chain M5 (tcM5) by plasma C1-inhibitor (C1I), thereby preventing non-specific plasmin generation, was augmented by the addition of exogenous C1I to plasma in vitro. In the present study, tPA, M5 or placebo +/- C1I were administered in two rat stroke models. In Part-I, permanent MCA occlusion was used to evaluate intracranial hemorrhage (ICH) by the thrombolytic regimens. In Part II, thromboembolic occlusion was used with thrombolysis administered 2 h later. Infarct and edema volumes, and ICH were determined at 24 h, and neuroscore pre (2 h) and post (24 h) treatment. In Part I, fatal ICH occurred in 57% of tPA and 75% of M5 rats. Adjunctive C1I reduced this to 25% and 17% respectively. Similarly, semiquantitation of ICH by neuropathological examination showed significantly less ICH in rats given adjunctive C1I compared with tPA or M5 alone. In Part-II, tPA, M5, and M5+C1I induced comparable ischemic volume reductions (>55%) compared with the saline or C1I controls, indicating the three treatments had a similar fibrinolytic effect. ICH was seen in 40% of tPA and 50% of M5 rats, with 1 death in the latter. Only 17% of the M5+C1I rats showed ICH, and the bleeding score in this group was significantly less than that in either the tPA or M5 group. The M5+C1I group had the best Benefit Index, calculated by dividing percent brain salvaged by the ICH visual score in each group. In conclusion, adjunctive C1I inhibited bleeding by M5, induced significant neuroscore improvement and had the best Benefit Index. The C1I did not compromise fibrinolysis by M5 in contrast with tPA, consistent with previous in vitro findings.

A new nerve growth factor-mimetic peptide active on neuropathic pain in rats.

Analysis of the structure of nerve growth factor (NGF)-tyrosine kinase receptor A (TrkA) complex, site-directed mutagenesis studies and results from chemical modification of amino acid residues have identified loop 1, loop 4, and the N-terminal region of the NGF molecule as the most relevant for its biological activity. We synthesized several peptides mimicking the two loops (1 and 4) linked together with an appropriate spacer, with or without the N-terminal region. Two peptides named NL1L4 and L1L4 demonstrated good NGF agonist activity at a concentration as low as 3 mum. They induced differentiation of chick dorsal root ganglia and stimulated tyrosine phosphorylation of TrkA, but not TrkB, receptor. In addition L1L4 was able to induce differentiation of PC12 cells. More interestingly, the peptide with the highest "in vitro" activity (L1L4) was shown to reduce neuropathic behavior and restore neuronal function in a rat model of peripheral neuropathic pain, thereby suggesting a potential therapeutic role for this NGF-mimetic peptide.

Preclinical studies on immunogenicity of the HIV-1 p17-based synthetic peptide AT20-KLH.

Several studies have suggested that HIV-1 p17 matrix protein may play an important role in AIDS pathogenesis, since anti-p17 antibodies represent a serological marker of disease progression during HIV-1 infection both in adults and children. Moreover, it has been recently reported that the viral protein is capable of significantly increasing the proliferation of preactivated T lymphocytes and the release of proinflammatory cytokines. Recombinant HIV-1 p17 also has induced an increased rate of HIV-1 replication in vitro. All p17 biological activities are exerted after its binding to a specific cellular receptor expressed on activated T lymphocytes. The functional p17 epitope involved in receptor binding was found to be located at the NH(2)-terminal region of the viral protein. Immunization of C57BL/6 mice with a 20 amino acid synthetic peptide representative of the HIV-1 p17 functional region (AT20) coupled to the carrier protein keyhole limpet hemocyanin (KLH) and given in Freund's incomplete adjuvant, resulted in the development of p17-neutralizing antibodies capable of blocking p17/p17 receptor interaction, and consequently, all biological activities of the viral protein. Moreover, it was possible to skew the humoral response induced by priming mice with AT20-KLH toward cell-mediated immune responses, boosting animals with p17. Our findings may provide a new strategy to develop a synthetic AIDS vaccine based on a potentially effective and safe subunit vaccine against the HIV-1 cytokine-like matrix protein p17. Preclinical immunogenicity data for AT20-KLH provide the basis for evaluation of the peptide-based vaccine, alone and in combination with p17 or p17 DNA vaccines, in Phase I clinical trials.