GRP78/Dna K Is a Target for Nexavar/Stivarga/Votrient in the Treatment of Human Malignancies, Viral Infections and Bacterial Diseases

Sorafenib and regorafenib are multi-kinase inhibitors approved for the treatment of liver and kidney, and colon cancers, respectively (Carr et al., 2013). Sorafenib was originally developed as an inhibitor of RAF-1 in the ERK1/2 pathway. The steady state (7 day) Cmax for sorafenib is ~21 μM in plasma, with ~99% of the drug protein bound based on in vitro human serum binding assays; though it is known that the drug is also rapidly taken up into tissues (with an approximate threefold greater level of drug in tissues), and in addition patient data from clinical trials would argue that a significant amount of the drug has to be bioavailable, at least in the low micro-molar range, in a tumor based on its single agent effects by decreasing both ERK1/2 phosphorylation and reducing MCL-1 protein expression in tumor cells that are not specifically oncogene addicted (Hotte and Hirte, 2002; Elser et al., 2007). Indeed, it has been shown that some sorafenib metabolites such as M2, M4, and M5 can have up to 10-fold greater activity than the parent drug (Li et al., 2010; Pratz et al., 2010; Inaba et al., 2011). Furthermore, in a very recent manuscript we noted that tumor cells grown in 100% human serum were killed by doses of sorafenib and regorafenib in combination with sildenafil (Viagra) at 25% of their respective C max values (Tavallai et al., 2015). Of note, for the studies in the present manuscript, the amount of bioavailable sorafenib in protein rich solid agar for bacterial growth or protein rich liquid bacterial nutrient broth is unknown. Our prior in vitro and in vivo data in tumor cells have tended to argue using several sorafenib + “drug” combinations that PDGFRβ is a major target of sorafenib for its interactions with other agents e.g., with histone deacetylase inhibitors (Park et al., 2010a,b). A major biological effect of sorafenib is the induction of an endoplasmic stress (ER)/unfolded protein response (UPR), with reduced expression of proteins that have short half-lives such as MCL-1 and BCL-XL (e.g., Rahmani et al., 2007; Martin et al., 2009). Reduced MCL-1 levels due to sorafenib exposure have been linked in many tumor types to increased levels of apoptosis. Studies by our group have also linked high dose single agent sorafenib exposure to an increase in the levels of autophagic markers including increased numbers of LC3-GFP vesicles and elevated expression of Beclin1 and ATG5; however, lower sorafenib concentrations only caused a modest transient alteration in autophagy flux (Park et al., 2010a,b). Other studies from our groups have shown that based on the sorafenib dose the induction of ER stress may be a “protective” or a “toxic” event in the cellular response to the drug (e.g., Rahmani et al., 2005). It is very probable that the sorafenib-induced increase in PERK phosphorylation is due to our observation that sorafenib and regorafenib reduce expression of the chaperone GRP78/BiP/HSPA5 (Park et al., 2008; Booth et al., 2012a). ER stress signaling is mediated by three proximal sensors, PERK, the IRE1 (inositol-requiring protein 1α)/XBP1 (X-box binding protein 1) system and ATF6 (activating transcription factor 6) (Pavitt and Ron, 2012; Sano and Reed, 2013). GRP78 plays a key role in regulating the ER stress response; under resting conditions the majority of GRP78 is associated with PERK and IRE1 and keeps these proteins in an inactive state (Gorbatyuk and Gorbatyuk, 2013; Rao et al., 2012; Roller and Maddalo, 2013; Ni and Lee, 2007; Quinones et al, 2008). GRP78, as a chaperone, also plays an important role in the protein folding processes that occur in the ER including cancer, liver disease, and virus replication. The prokaryotic homologue of GRP78, Dna K, also plays an essential role in bacterial cell biology where it chaperones proteins such as Rec A which is essential for bacterial DNA replication and resistance where engulfed to the respiratory burst of macrophages (Noguchi et al, 2014; Roux, 1990; Earl et al., 1991; Anderson et al., 1992; Hogue and Nayak, 1992; Xu et al., 1997; Carleton and Brown, 1997; Xu et al., 1998; Bolt, 2001; Bredeche et al., 2001; Shen et al., 2002; Dimcheff et al., 2004; He, 2006; Spurgers et al., 2010; Moreno and Tiffany-Castiglioni, 2014; Reid et al., 2014). When high levels of unfolded protein are present in the ER, in a tumor cell; in a virally infected cell; or in a rapidly dividing bacterial cell, GRP78 disassociates from PERK and IRE1 resulting in their activation, and GRP78 binds to the unfolded proteins in the ER as a chaperone (Lee, 2007; Luo and Lee, 2013; Chen et al., 2014, and references therein). Activation of PERK-eIF2α signaling acts to prevent the majority of cellular proteins from being synthesized and IRE1 signaling enhances the expression of additional GRP78 protein. Virus infection can cause a profound ER stress response which could by cyto-toxic or prevent virus protein synthesis. Hence, some viruses make targeting proteins, similar to mammalian GADD34 and Nck1, that relocate protein phosphatase 1 with eIF2α thereby preventing eIF2α phosphorylation and high levels of toxic ER stress signaling (Rathore et al., 2013; Yu et al, 2011; Zhang et al, 2014). As GRP78 chaperones the unfolded protein(s), free GRP78 eventually becomes available to re-associate with PERK and IRE1 thereby shutting off the signaling system (Lee, 2007; Luo and Lee, 2013; Chen et al., 2014. Of note, however, is that prolonged ER stress signaling downstream of PERK and IRE1 can facilitate transformed cell killing, though primary cell killing was not observed in our laboratory (see above), arguing that a prolonged reduction of GRP78 in transformed cells reduces cell viability (Booth et al., 2014a,b). GRP78/BiP/HSPA5 and evolutionary conserved homologues of GRP78 play essential roles in the biology and life cycles of viruses, bacteria, protosoal, and yeast cells as well as higher eukaryotic cells, in particular tumor cells that express high levels of many “activated” oncogenic signaling proteins (Roux, 1990; Earl et al., 1991; Anderson et al., 1992; Hogue and Nayak, 1992; Xu et al., 1997; Carleton and Brown, 1997; Xu et al., 1998; Bolt, 2001; Bredeche et al., 2001; Shen et al., 2002; Dimcheff et al., 2004; He, 2006; Spurgers et al., 2010; Moreno and Tiffany-Castiglioni, 2014; Reid et al., 2014). And, the bacterial GRP78/BiP/HSPA5 homologue, called Dna K, is essential for bacterial growth and for bacterial mRNA/protein stability. As sorafenib/regorafenib interact with PDE5 inhibitors to reduce GRP78 expression, the present studies determined whether these established cancer chemotherapy drugs could also alter/prevent viral reproduction and bacterial cell growth.