Apoptosis, a process of programmed cell death, can occur in response to extrinsic or intrinsic signals (see Hart and El-Deiry). Overexpression of Par-4 (prostate apoptosis response-4), a proapoptotic protein with various cytoplasmic and nuclear binding partners and functions, induces apoptosis in cancer cells but not in nonmalignant cells. The apoptotic effects of Par-4 in cancer cells are known to depend on intracellular Par-4. However, when Burikhanov et al. transfected cultured PC-3 (prostate cancer) cells with fluorescently labeled Par-4 [or its fluorescently labeled SAC (selective for apoptosis in cancer cells) domain] and used immunocytochemical analysis to identify activated caspase-3 as a marker of apoptosis, they found that even untransfected cells underwent apoptosis. Conditioned medium derived from transfected cells was found to contain the transfected proteins; moreover, endogenous Par-4 was secreted from various transformed and untransformed mammalian cells (not just prostate cells). Conditioned medium from PC-3 transfectants elicited apoptosis in cancer cells but not in untransformed cells, as did recombinant Par-4 or SAC. Brief treatment with TRAIL (tumor necrosis factor–related apoptosis-inducing ligand, which acts extrinsically to stimulate apoptosis) increased Par-4 secretion without increasing its abundance in cell extracts. TRAIL-mediated Par-4 secretion did not depend on apoptosis (which requires longer treatment); was inhibited by brefeldin A, which blocks the secretory pathway; and was mimicked by agents that, like TRAIL, elicit the endoplasmic reticulum (ER) stress response. TRAIL increased the abundance of the ER stress response protein GRP78 (glucose-regulated protein 78), as did treatment with Par-4 and SAC; furthermore, GRP78 bound Par-4 and SAC in pull-down assays and coimmunoprecipitated with endogenous Par-4. Immunocytochemical analysis revealed that Par-4 colocalized with GRP78 in the ER and that TRAIL stimulated their translocation to the plasma membrane. Neutralizing antibodies directed against the N-terminal region of GRP78 blocked Par-4-, SAC-, and TRAIL-dependent apoptosis; TRAIL-dependent apoptosis was also inhibited by antibodies directed against Par-4. Knockdown of endogenous Par-4 with RNA interference decreased cell-surface localization of GRP78 and inhibited apoptosis in response to treatment with external Par-4 or SAC, as well as their ability to increase GRP78 abundance. Transfection of Par-4-knockdown cells with membrane-directed GRP78 restored the ability of external Par-4 to elicit apoptosis; indeed, its transfection enabled Par-4 to elicit apoptosis in untransformed cells. Thus, Par-4 appears to function extracellularly as well as intracellularly to elicit apoptosis in malignant cells.
R. Burikhanov, Y. Zhao, A. Goswami, S. Qiu, S. R. Schwarze, V. M. Rangnekar, The tumor suppressor Par-4 activates an extrinsic pathway for apoptosis. Cell 138, 377–388 (2009).[PubMed]
L. S. Hart, W. S. El-Deiry, Cell death: A new Par-4 the TRAIL. Cell 138, 220–222 (2009).[PubMed]