Hsf-1 and POB1 Induce Drug Sensitivity and Apoptosis by Inhibiting Ralbp1

Abstract

Hsf-1 (heat shock factor-1) is a transcription factor that is known to regulate cellular heat shock response through its binding with the multispecific transporter protein, Ralbp1. Results of present studies demonstrate that Hsf-1 causes specific and saturable inhibition of the transport activity of Ralbp1 and that the combination of Hsf-1 and POB1 causes nearly complete inhibition through specific bindings with Ralbp1. Augmentation of cellular levels of Hsf-1 and POB1 caused dramatic apoptosis in non-small cell lung cancer cell line H358 through Ralbp1 inhibition. These findings indicate a novel model for mutual regulation of Hsf-1 and Ralbp1 through Ralbp1-mediated sequestration of Hsf-1 in the cellular cytoskeleton and Hsf-1-mediated inhibition of the transport activity of membranebound Ralbp1.In response to heat stress, human cells respond by activation of Hsf-1 (heat shock factor-1), a transcription factor that binds to NGAAN repeats of the promoter of heat shock genes, augmenting transcription (1-5). Considered the master regulator of the heat shock response (1-3), Hsf-1 binds DNA constitutively, and its binding affinity is based upon its phosphorylation in response to heat shock (1-5). In the unstressed state, Hsf-1 is sequestered in a complex with tubulin, HSP90, and Ralbp1 (6). Stress or constitutively active Ral-GTP binding to Ralbp1 triggers the release of Hsf-1 and its migration to the nucleus, where its transcription factor activity is important for the expression of heat shock proteins (6, 7). Although these studies focused on Ralbp1 present in the cytoplasm bound to the cytoskeleton and nuclear membrane, several previous and subsequent reports have clearly demonstrated the presence of Ralbp1 in nuclear as well as plasma membranes (8 -12). In several recent studies, we have conclusively demonstrated that Ralbp1 is a transmembrane protein with a defined cell surface domain (8 -11) and that it catalyzes in ATP hydrolysis-dependent trans-membrane anti-gradient efflux of toxic xenobiotics as well as endogenous metabolites. The preferred physiological substrates for transport by Ralbp1 are glutathione-electrophile conjugates of electrophilic lipid metabolites that arise from stress or heat shockinduced lipid peroxidation (13). The cell surface domain of Ralbp1 can be targeted by highly specific antibodies that inhibit the transport activity of Ralbp1 and result in dramatic regression of tumor in syngeneic and xenograft models of melanoma, lung cancer, and colon cancer (14, 15). The membrane functionality of Ralbp1 is also evident from its crucial role in endocytosis as a rate-regulatory element (12, 16 -18).An endocytosis-linked protein POB1 that binds Ralbp1 in a similar region as Hsf-1 has been shown to be a specific and saturable inhibitor of the glutathione-electrophile conjugates and doxorubicin (DOX) 2 transport activity of membrane-reconstituted purified Ralbp1 (19). We proposed that just as POB1 could function as an inhibitor of the transport activity of Ralbp1, Hsf-1 could also function as a tr...

REPS2 (RALBP1 associated Eps domain containing 2)

“…Hsf-1 also causes specific and saturable inhibition of the transport activity of RalBP1. The combined augmentation of Hsf-1 and REPS2/POB1 causes nearly complete inhibition of RalBP1 and a dramatic apoptosis in NSLC (non-small cell lung cancer) cell line H358 through Ralbp1 binding (Singhal et al, 2008). The marked apoptotic effect caused by the increase of Hsf-1 and REPS2/POB1 in lung cancer cells, suggests a novel targeted therapy in which liposomally encapsulated Hsf-1 and POB1 could be used clinically as a therapeutic agent.…”

“…Shingal et al show a ternary complex formation between RalBP1, Hsf-1, and REPS2/POB1. RalBP1, Hsf1, HSP90 and tubulin make a complex in cell (Singhal et al, 2008). Hsf-1 and REPS2/POB1 induce drug sensitivity and apoptosis by inhibiting RalBP1.…”

Role of RLIP76 in doxorubicin resistance in lung cancer (Review)

“…1B). These structural features link RLIP76 to a confounding array of physiological functions including clathrin-coated-pit-mediated receptor-ligand endocytosis of epidermal growth factor receptor (EGFR), insulin receptor (IR) and transforming growth factor ß (TGF-ß) as well as mitosis signaling (8,16,(21)(22)(23)(24)(25)(26)(27). In a series of experiments over the last decade, an important role for RLIP76 in malignant cell survival and drug resistance and normal tissue response to oxidative stress has been confirmed.…”

Expression and Clinical Significance of REPS2 in Human Esophageal Squamous Cell Carcinoma

“…Recently, it has been shown that overexpression of REPS2 and its binding with RalBP1 induce apoptosis and loss of REPS2 expression results in dysregulation of growth factor signaling in prostate cancer cells (Oosterhoff et al, 2003;Oosterhoff et al, 2005). Likewise, augmentation of cellular levels of REPS2 and Hsf-1 result in dramatic apoptosis in non-small cell lung cancer cell line H358 through RalBP1 inhibition (Singhal et al, 2008). What is more, the binding of REPS2 to PAG2 inhibits cell migration (Oshiro et al, 2002).…”