FXYD3 was originally identified as one of the gene products (mRNAs) highly expressed in murine mammary tumors induced by the transgenic neu or ras but not induced by myc or int-2 oncogenes.1) In human, FXYD3 mRNA was overexpressed in breast cancer cell lines and primary breast cancers, 2) and also in prostate and pancreatic cancers. [3][4][5] Suppression of FXYD3 expression by the small interfering RNA (siRNA) or antisense transfection caused a significant decrease in cellular proliferation of prostate and pancreatic cancer cell lines, respectively. 4,5) Therefore, FXYD3 is involved in proliferation, and is expected to be a tumor marker.FXYD3 is a member of the "FXYD" family proteins, which consist of seven members of small proteins that have a single transmembrane segment, and share a signature sequence of four amino acids "FXYD" (Phe-Xaa-Tyr-Asp) located in the ectodomain close to the transmembrane segment.6) Among the seven members of FXYD proteins, only FXYD2 (Na ϩ ,K ϩ -ATPase g-subunit) and FXYD3 have two isoforms which encode two proteins of different amino acid sequences, respectively. The FXYD3 short-form (FXYD3a) mRNA has an in-frame deletion of 78 nucleotides in the coding sequence compared to the FXYD3 long-form (FXYD3b) mRNA. As a result, the FXYD3b protein has 26 more amino acids in the cytoplasmic domain compared to the FXYD3a protein ( Fig. 1). FXYD family proteins perform fine tuning of ion transport by associating with and modulating the activity of Na ϩ ,K ϩ -ATPase molecule and modifying the activity of ion channels.2,7) FXYD3a slightly decreased the apparent affinity both for intracellular Na ϩ (up to 40%) and extracellular K ϩ (15 to 40%) of Na ϩ ,K ϩ -ATPase whereas FXYD3b slightly increased the apparent affinity for intracellular Na ϩ (about 15%) and decreased the apparent affinity for extracellular K ϩ (up to 50%). 8,9) Both FXYD3 isoforms induced a hyperpolarization-activated chloride current in Xenopus oocytes.2,9) However, physiological roles of FXYD3 isoforms have not been fully understood yet. The expression pattern of FXYD3a and FXYD3b mRNAs has not been cleared. Moreover, it remains to be clear whether the FXYD3 proteins are overexpressed in breast cancers or not. Here, we quantified FXYD3a and FXYD3b mRNAs in human normal tissues and many human cancer cell lines by quantitative reverse transcription polymerase chain reaction (RT-PCR). We prepared two kinds of antibodies against human FXYD3 protein (one antibody against both FXYD3a and 3b; the other against FXYD3b) and examined the expression pattern of FXYD3 proteins in human cancer cell lines and human breast tissues. We also suppressed the expression of FXYD3 proteins by the treatment of siRNA to study the role of FXYD3 on cellular proliferation of a breast cancer cell line, MCF-7. FXYD3, also known as Mat-8 (Mammary tumor 8 kDa), is one of mRNAs highly expressed in mouse and human breast cancers. Here, we newly found that FXYD3 protein was also overexpressed in human breast cancer specimens; invasive ductal carcinomas and intr...

Although embryonal proteins have been used as tumor marker, most are not useful for detection of early malignancy. In the present study, we developed mouse monoclonal antibodies against fetal lung of miniature swine, and screened them to find an embryonal protein that is produced at the early stage of malignancy, focusing on lung adenocarcinoma. We found an antibody clone that specifically stained stroma of lung adenocarcinoma. LC-MS/MS identified the protein recognized by this clone as dimethylarginine dimethylaminohydrolase 2 (DDAH2), an enzyme known for antiatherosclerotic activity. DDAH2 was found to be expressed in fibroblasts of stroma of malignancies, with higher expression in minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma than in adenocarcinoma in situ (AIS). Moreover, tumors with high stromal expression of DDAH2 had a poorer prognosis than those without. In vitro analysis showed that DDAH2 increases expression of endothelial nitric oxide synthase (eNOS), inducing proliferation and capillary-like tube formation of vascular endothelial cells. In resected human tissues, eNOS also showed higher expression in invasive adenocarcinoma than in AIS and normal lung, similarly to DDAH2. Our data indicate that expression of DDAH2 is associated with invasiveness of lung adenocarcinoma via tumor angiogenesis. DDAH2 expression might be a prognostic factor in lung adenocarcinoma.Electronic supplementary materialThe online version of this article (doi:10.1007/s00428-015-1863-z) contains supplementary material, which is available to authorized users.