Chemotherapy-Regulated microRNA-125-HER2 Pathway as a Novel Therapeutic Target for Trastuzumab-Mediated Cellular Cytotoxicity in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) accounts for 15% of all lung cancer cases and is a highly lethal disease. For the last several decades, the standard treatment for SCLC has been deadlocked, and new therapeutic strategies are urgently needed. HER2 is a member of the HER family and has been reported to be overexpressed in 30% of SCLC cases with poor prognosis. However, the clinical relevance of HER2-targeted therapy for SCLC remains unclear. Here, we first identify that cytotoxic drugs induce significant HER2 overexpression through microRNA-125a (miR-125a) and miR-125b downregulation, which in turn act as a novel therapeutic target for trastuzumab-mediated cellular cytotoxicity in SCLC. In this study, we showed that treatment of the HER2-positive SCLC cells, SBC-3 and SBC-5, with cytotoxic drugs induced a significant upregulation of HER2. Cisplatin (CDDP) treatment of SCLC cells resulted in a significant downregulation of miR-125a and miR-125b. We confirmed that miR-125a and miR-125b bound to the 3'-untranslated regions of HER2 mRNA, and that downregulation of miR-125a and miR-125b resulted in upregulation of HER2 in SCLC cells, suggesting a relationship between cytotoxic drug exposure and miR-125/HER2 dysregulation. Furthermore, using a calcein assay, we demonstrated a significantly enhanced cytotoxic effect of CDDP and trastuzumab that was mediated via antibody-dependent cellular cytotoxicity. Finally, we clearly demonstrated the enhanced antitumor effect of these agents in an orthotopic lung cancer model in vivo. Our results offer a novel therapeutic strategy for HER2-positive SCLCs by using trastuzumab combined with cytotoxic drugs.

Generation of a monoclonal antibody against the extracellular domain of IGF-1R.

Type I insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase that is involved in the transformation of cells, cancer proliferation, and metastatic events in various types of cancer. The present study reports on the generation of a mouse monoclonal antibody (MAb) to IGF-1R using the mouse lymph node method. MAb 1B1, which reacts specifically with the extracellular domain of IGF-1R, was obtained using flow cytometry (FCM) screening using MCF-7 cells. Using immunostaining, MAb 1B1 detected IGF-1R mainly on the plasma membrane of MCF-7 cells. MAb 1B1 would be useful in FCM and immunofluorescence assays to detect IGF-1R-expressing cells for basic and clinical research.

KRAS mutation confers resistance to antibody-dependent cellular cytotoxicity of cetuximab against human colorectal cancer cells.

Cetuximab is a chimeric IgG1 monoclonal antibody (mAb) that targets the extracellular domain of epidermal growth factor receptor (EGFR). Oncogenic KRAS mutations in tumors have been shown to be a negative predictor of the response of colorectal cancer (CRC) to cetuximab treatment. Cetuximab exerts its therapeutic effects through several mechanisms including antibody-dependent cellular cytotoxicity (ADCC). However, the influence of KRAS mutations on cetuximab-mediated ADCC is not fully understood. Here, we investigated cetuximab-mediated ADCC in two pairs of isogenic CRC cells with or without a KRAS mutation. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and NK92, a natural killer (NK) cell line that exogenously expresses FcγRIIIa (CD16a), were used as effector cells. In an ADCC assay, perforin-dependent target cell lysis was not affected by the KRAS mutation status. On the other hand, perforin-independent ADCC was observed only in CRC cells with wild-type KRAS, but not in cells with mutant KRAS. Neutralizing experiments revealed that the Fas-Fas ligand (FasL) interaction was responsible for the induction of apoptosis and perforin-independent ADCC. Furthermore, the presence of effector cells clearly enhanced the growth-inhibitory effect of cetuximab only in CRC cells with wild-type KRAS, but not in those with mutant KRAS. These findings suggest that ADCC is an important mode of action of cetuximab and that KRAS mutation impairs the therapeutic effect exerted by cetuximab-mediated ADCC.

Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells to radiation through the abrogation of DNA damage checkpoint.

Poly(ADP-ribose) glycohydrolase (PARG) is a major enzyme that plays a role in the degradation of poly(ADP-ribose) (PAR). PARG deficiency reportedly sensitizes cells to the effects of radiation. In lung cancer, however, it has not been fully elucidated. Here, we investigated whether PARG siRNA contributes to an increased radiosensitivity using 8 lung cancer cell lines. Among them, the silencing of PARG induced a radiosensitizing effect in 5 cell lines. Radiation-induced G2/M arrest was largely suppressed by PARG siRNA in PC-14 and A427 cells, which exhibited significantly enhanced radiosensitivity in response to PARG knockdown. On the other hand, a similar effect was not observed in H520 cells, which did not exhibit a radiosensitizing effect. Consistent with a cell cycle analysis, radiation-induced checkpoint signals were not well activated in the PC-14 and A427 cells when treated with PARG siRNA. These results suggest that the increased sensitivity to radiation induced by PARG knockdown occurs through the abrogation of radiation-induced G2/M arrest and checkpoint activation in lung cancer cells. Our findings indicate that PARG could be a potential target for lung cancer treatments when used in combination with radiotherapy.

Generation of a rat monoclonal antibody specific for glyoxalase I.

Glyoxalase I (GLO1) is a key enzyme that plays a role in the detoxification of methylglyoxal (MG), a toxic cellular metabolite produced during glycolysis. The present study reports on the preparation and properties of a monoclonal antibody (MAb) directed against mouse GLO1. The antibody was produced by hybridization of mouse myeloma cells with lymph node cells from an immunized rat. The MAb 6F10 specifically recognized GLO1, as evidenced by immunoblotting using a variety of extracts from cultured cells. In immunostaining using MAb 6F10, a diffuse cytoplasmic and nuclear staining pattern was observed. The MAb 6F10 promises to be useful in immunoblotting and immunostaining experiments in various cells and tissues to determine the expression levels of GLO1, as well as to further analyze the biological function of this protein.

The formation of argpyrimidine, a methylglyoxal-arginine adduct, in the nucleus of neural cells.

Methylglyoxal (MG) is an endogenous metabolite in glycolysis and forms stable adducts primarily with arginine residues of intracellular proteins. The biological role of this modification in cell function is not known. In the present study, we found that a MG-detoxification enzyme glyoxalase I (GLO1) is mainly expressed in the ventricular zone (VZ) at embryonic day 16 which neural stem and progenitor cells localize. Moreover, immunohistochemical analysis revealed that argpyrimidine, a major MG-arginine adduct, is predominantly produced in cortical plate neurons not VZ during cerebral cortex development and is exclusively located in the nucleus. Immunoblotting experiment showed that the formation of argpyrimidine occurs on some nuclear proteins of cortical neurons. To our knowledge, this is first report of the argpyrimidine formation in the nucleus of neuron. These findings suggest that GLO1, which is dominantly expressed in the embryonic VZ, reduces the intracellular level of MG and suppresses the formation of argpyrimidine in neural stem and progenitor cells. Argpyrimidine may contribute to the neural differentiation and/or the maintenance of the differentiated state via the modification of nuclear proteins.