Circulating serpin tumor markers SCCA1 and SCCA2 are not actively secretedbut reside in the cytosol of squamous carcinoma cells

An elevation in the circulating level of the squamous-cell carcinoma antige n (SCCA) can be a poor prognostic: indicator in certain types of squamous-c ell cancers. Total SCCA in the circulation comprises 2 nearly identical, si milar to 45 kDa proteins, SCCA1 and SCCA2. Both proteins are members of the high-molecular weight serine proteinase inhibitor (serpin) family with SCC A1 paradoxically inhibiting lysosomal cysteine proteinases and SCCA2 inhibi ting chymotrypsin-like serine proteinases. Although SCCA1 and SCCA2 are det ected in the cytoplasm of normal squamous epithelial cells, neither serpin is detected normally in the serum. Thus, their presence in the circulation at relatively high concentrations suggests that malignant epithelial cells are re-directing serpin activity to the fluid phase via an active secretory process. Because serpins typically inhibit their targets by binding at 1:1 stoichiometry, a change in the distribution pattern of SCCA1 and SCCA2 (i. e., intracellular to extracellular) could indicate the need of tumor cells to neutralize harmful extracellular proteinases. The purpose of our study w as to determine experimentally the fate of SCCA1 and SCCA2 in squamous carc inoma cells. Using subcellular fractionation, SCCA-green fluorescent fusion protein expression and confocal microscopy, SCCA1 and SCCA2 were found exc lusively in the cytosol and were not associated with nuclei, mitochondria, lysosomes, microtubules, actin or the Golgi. In contrast to previous report s, metabolic labeling and pulse-chase experiments showed that neither non-s timulated nor TNF alpha/PMA-stimulated squamous carcinoma cells appreciably secreted these ov-serpins into the medium. Collectively, these data sugges t that the major site of SCCA1 and SCCA2 inhibitory activity remains within the cytosol and that their presence in the sera of patients with advanced squamous-cell carcinomas may be due to their passive release into the circu lation. (C) 2000 Wiley-Liss, Inc.