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.
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.
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.
Tissue microarray (TMA) methodology allows the concomitant analysis of hundreds of tissue specimens arrayed in the same manner on a recipient block. Subsequently, all samples can be processed under identical conditions, such as antigen retrieval procedure, reagent concentrations, incubation times with antibodies/probes, and escaping the inter-assays variability. Therefore, the use of TMA has revolutionized histopathology translational research projects and has become a tool very often used for putative biomarker investigations. TMAs are particularly relevant for large scale analysis of a defined disease entity. In the course of these exploratory studies, rare subpopulations can be discovered or identified. This can refer to subsets of patients with more particular phenotypic or genotypic disease with low incidence or to patients receiving a particular treatment. Such rare cohorts should be collected for more specific investigations at a later time, when, possibly, more samples of a rare identity will be available as well as more knowledge derived from concomitant, e.g., genetic, investigations will have been acquired. In this article we analyze for the first time the limits and opportunities to construct new TMA blocks using tissues from older available arrays and supplementary donor blocks. In summary, we describe the reasons and technical details for the construction of rare disease entities arrays.
The identification of markers targetable by specific mAbs represents a high medical need in cancer therapy. Our objective is to discover novel tumor-associated proteins showing promise as targets for monoclonal antibody (mAb) therapy, and to generate and validate highly specific mAbs for therapeutic applications. The approach we used to discover novel tumor markers is based on a high through-put immune-histochemical (IHC) screening of tumor and normal tissues using collections of murine polyclonal and mAbs raised against recombinant human proteins. In the course of such analysis, we discovered and validated different surface exposed proteins over-expressed in one or more cancers. Here, we report a surface exposed protein mainly over-expressed in ovary and breast cancers. Interestingly, the protein is highly expressed in high grade breast cancers. Approximately 40% of cancers in which the protein is over-expressed belong to the triple negative subtype. In line with IHC data, an expression profile analysis in different cells lines showed that this protein is expressed in ovarian and breast cancer cell lines. In breast cell lines, high expression was found in triple negative cells positive to the androgen receptor. Gene silencing experiments combined to phenotypic analysis, showed that loss of protein expression significantly reduces cell proliferation and invasiveness. Several mAbs able to recognize the target protein on the surface of breast and ovary cancer cell lines have been selected and validated in a number of immunoassays. The specificity of the mAb binding was confirmed by gene silencing and competition assays with peptides encompassing the mAb epitopes. Specific mAbs able to detect the protein in cancer cells in IHC are under validation. These antibodies show limited reactivity on normal human tissues and are negative on PBMC from normal donors. Moreover, these mAbs are efficiently internalized by cancer cells, suggesting that they are amenable to the development of Antibody-Drug-Conjugate. Finally, they efficiently recognize the macaca protein ortholog, thus facilitating future safety studies in non-human primates. Overall, results so far accumulated highlight the potential of this novel tumor-associated protein and available mAbs for the development of targeted therapy against ovarian cancer and triple negative breast cancer. Other promising targets and related monoclonal antibodies will be described during the meeting. Citation Format: Matteo Parri, Susanna Campagnoli, Alberto Grandi, Elisa De Camilli, Aurelien Lacombe, Boquan Jin, Serenella Eppenberger-Castori, Giuseppe Viale, Luigi Terracciano, Piero Pileri, Renata Maria Grifantini. Novel targets and monoclonal antibodies for cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3800.
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