Abstract
Ovarian cancers are frequently not diagnosed until advanced stages, resulting in a high case fatality rate. Because of this, more tumor markers, in addition to CA125, for detecting and monitoring ovarian cancer are needed. During a systematic search for potential biomarkers of ovarian cancer, we compared the protein profiles between tumor interstitial fluid and normal interstitial fluid of ovaries, rationalizing that abnormal levels of proteins in tumor interstitial fluid may be detected in peripheral blood and thus serve as easily accessible tumor markers. Here, we show that stress-induced phosphoprotein 1 (STIP1) was secreted by ovarian cancer tissues into the peripheral blood of patients, resulting in a significant increase of serum levels of STIP1 in cancer patients compared with those in age-matched normal controls. Our results further indicated that combined use of CA125 and STIP1 may increase early detection of ovarian cancer. Functionally, recombinant STIP1 significantly induced ERK phosphorylation, promoted DNA synthesis, and increased Ki-67 immunoreactivity in ovarian cancer cells, suggesting that STIP1 in vitro promotes cell proliferation. Colocalization of STIP1 and phospho-ERK in human ovarian cancer tissues also supports an in vivo activation of ERK by STIP1. Further understanding of molecular roles of STIP1 in human ovarian cancer may shed light on its pathophysiology and development of novel therapeutic strategies.
Highlights
Ovarian cancers are frequently not diagnosed until advanced stages, resulting in a high case fatality rate
stress-induced phosphoprotein 1 (STIP1) protein contains nine tetratricopeptide repeat (TPR) motifs, which are clustered into domains each consisting of three TPRs, and a nuclear localization signal from amino acid residues 223 to 238 (Human Protein Reference Database)
ELISAc a Data are presented as means Ϯ S.E. b brain-derived neurotrophic factor (BDNF) results of tumor interstitial fluids (TIFs) and nonmalignant interstitial fluid (NIF) were normalized by the protein concentration of each specimen, thereby presented as pg/mg. c Transferrin results of TIF and NIF were normalized by the protein concentration of each specimen, thereby presented as ng/mg
Summary
Study Design for Analysis of TIF and NIF—Ovarian tumors and normal ovarian tissues were obtained during surgery for suspected ovarian malignancies and for benign gynecological conditions such as adenomyosis or myoma uteri. Immunohistochemistry (IHC)—Using procedures that were reported previously [21], we did IHC studies on cryosections of human ovarian cancer tissues and normal ovaries from which TIF and NIF were derived, respectively. STIP1 Detection in Cell Culture Supernatant—For protein identification in culture supernatant, ovarian cancer cells were cultured overnight in serum-free Opti-MEM (Invitrogen). After centrifugation at 1600 rpm for 10 min to remove cell debris, 45 l of culture supernatant was analyzed by Western blot with a chicken anti-STIP1 polyclonal antibody (1:1000; Abcam) followed with goat antichicken IgY horseradish peroxidase-conjugated antibody (1:3000; Abcam) using the same procedure described above. Following the manufacturer’s protocol, a stable clone of copper-inducible, STIP1-secreting S2 cells was generated by cotransfection of 19 g of pMT/Bip/STIP1 expression vector and 1 g of pCoHygro selecting vector (Invitrogen) by using a calcium phosphate transfection kit (Invitrogen) and selected in culture medium containing 500 g/ml hygromycin (Invitrogen). P values less than 0.05 were considered statistically significant
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