Surmounting Chemoresistance by Targeting the Y-Box Binding Protein-1

Abstract

The Y-box binding protein 1 (YB-1), characterized by the presence of the cold shock domain, has been reported to induce chemoresistance in cancer therapy. Chemotherapy is one of the main therapeutic strategies in the treatment of cancer, in addition to surgery, radiation therapy, and hormonal therapy. However, chemoresistance remains a key obstacle to successful cancer management. In this review, we will focus on the role of YB-1, an important regulator of gene transcription, in cancer therapy and chemoresistance. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.

Cancer is one of the leading causes of death globally. Management of cancer includes surgical intervention, chemotherapy, radiotherapy, hormonal treatment, and biological therapy. Chemotherapy, which is given systematically to stop cancer growth by either killing the cells or halting the cells from dividing, has been beneficial in treating both early and advanced cancers. However, chemoresistance, defined as the resistance by components of a cancer cell to the action of a specific chemotherapeutic agent (Mosby,2009), is known to be induced by a number of genes and their gene products, such as the Y-box binding protein-1 (YB-1), multidrug-resistance (MDR1), glutathione-S-transferase pi, dihydrofolate reductase, and topoisomerase II (Mousseau et al.,1993; Bargou et al.,1997; Janz et al.,2002; Kuwano et al.,2004). In this review, we will focus on chemoresistance induced by YB-1, a potential biomarker in cancer (Janz et al.,2002; Gessner et al.,2004; Huang et al.,2005). Although the official symbol of the Y-box binding protein is YBX1, for the purpose of this review, we have opted to retain the YB-1 nomenclature, which is commonly used in the scientific literature.

Y-BOX BINDING PROTEIN-1 (YB-1)

YB-1 was first isolated in 1988 as a transcription factor which binds to the Y-box of MHC class II promoters (Didier et al.,1988; Sakura et al.,1988). YB-1 is also known as CCAAT-binding transcription factor I subunit A (CBF-A) and by other names, such as DNA-binding protein B (dpbB), p50 and Enhancer factor I subunit A (EF1A) (Matsumoto and Wolffe,1998; Kohno et al.,2003). YB-1 is a member of the cold-shock containing proteins and RNA binding protein superfamily by virtue of the ∼40% identity to a cold-shock protein from bacteria which interacts with the sequence motif CTGATTGG, also known as the Y-box (Izumi et al.,2001; Kohno et al.,2003). All living organisms except Sacharomyces cerevisiae contain proteins belonging to the Y-box family with YB-1, being the best-characterized member in somatic mammalian cells (Evdokimova et al.,2006).

The YB-1 gene which is situated on chromosome 1 p 34, consists of 8 exons with 19 kb of genomic DNA (Toh et al.,1998). The YB-1 gene has a very long 5′ UTR that is probably involved in the regulation of gene expression (Kohno et al.,2003). The mRNA of YB-1, which is about 1.5 kb long, encodes for a protein of 324 amino acids with a molecular weight of 36 kDa (Didier et al.,1988; Kuwano et al.,2004). The YB-1 protein comprises three domains, namely, the variable amino-terminal domain, a highly conserved cold shock domain and carboxyl-terminal tail domain (Fig. 1A). The amino-terminal domain, which is rich in proline and alanine, is believed to be involved in trans-activation, whereas the carboxyl-terminal is composed of alternating clusters (about 30 amino acids) of basic and acidic amino acids, also known as a B/A repeat that is a unique feature in vertebrates (Kohno et al.,2003). The carboxyl-terminal domain is involved in protein–protein interactions yielding homomultimeric and heteromultimeric complexes (Okamoto et al.,2000; Shnyreva et al.,2000) and can bind specifically to single-stranded DNA and/or RNA in vitro (Wolffe et al.,1992; Wolffe,1994). The carboxyl-terminal domain also has multiple phosphorylation sites involved in the regulation of various protein functions, in addition to other modifications, such as acetylation and ubiquitylation. The cold shock domain consists of a five-stranded β-barrel (Fig. 1B) and binds to RNA via the RNA-binding motifs RNP-1 and RNP-2 in addition to DNA (Kloks et al.,2002).

Figure 1. Structure and functional domains of human YB-1. A: Amino-terminal domain, cold shock fomain and carboxyl-terminal domain. B: Schematic diagram of structure of YB-1 with a five-stranded antiparallel β-barrel in the cold shock domain (generated using RasMol).

YB-1 has diverse functions, such as gene regulation, DNA replication and repair, drug resistance, cellular response to environmental stimuli, cell proliferation and apoptosis (Okamoto et al.,2000; Izumi et al.,2001; Kohno et al.,2003). Some of the molecular interactions of YB-1 with a wide range of proteins include transcription factors (Chernukhin et al.,2000; Okamoto et al.,2000; Mertens et al.,2002), proteins involved in repair, RNA-binding proteins (Shnyreva et al.,2000; Ashizuka et al.,2002), viral proteins and actin filaments (Matsumoto and Bay,2005). YB-1 has been found to have an affinity for reactive oxygen species (ROS) damaged RNA containing 8-oxoguanine and has the ability to differentiate damaged RNA from normal RNA (Hayakawa et al.,2002), in addition to preferential binding to cisplatin modified and depurinated DNA (Ise et al.,1999). YB-1 is also known to mediate redox dependent activation of gene transcription (Duh et al.,1995). Some of the genes which are susceptible to regulation of gene expression by YB-1 include p21^Cip1, HIV-1, JCV early and late genes, matrix metalloproteinase 2, myosin light chain 2v, cyclins A and B1, and Fas (CD95/Apo-1) genes (Zou and Chien,1995; Mertens et al.,1997; Sawaya et al.,1998; Lasham et al.,2000; Jurchott et al.,2003; Kuwano et al.,2003). YB-1, a general repressor of translation (Nekrasov et al.,2003), has been associated with several translationally inactive mRNAs in mammalian cells and the translational regulation roles of YB-1 appear to have been conserved throughout the process of evolution (Evdokimova et al.,2006).

YB-1 EXPRESSION IN CANCER

Increased YB-1 expression has been observed in cancer cells including primary breast cancer (Bargou et al.,1997). Expression of YB-1 mRNA can be observed in various breast cancer cells in vitro (Fig. 2). The majority of YB-1 proteins (about 90%) are situated in the cytoplasm (Fig. 3), bound to an anchoring protein via a binding site located on the carboxyl-terminal domain even though YB-1 is heavily involved in the regulation of gene expression (Koike et al.,1997). The YB-1 protein has been reported to translocate to the nucleus from the cytoplasm once cells are in contact with heat (Stein et al.,2001), anticancer drugs, and ultraviolet (UV) irradiation (Bargou et al.,1997; Koike et al.,1997; Fujita et al.,2005). Nuclear trafficking of YB-1 has been linked with 20S proteosome-mediated cleavage of the C-terminal of the protein which contains the cytoplasmic retention signal (Sorokin et al.,2005) although a recent study by Braithwaite and coworkers showed that nuclear translocation can occur in the absence of proteolytic processing (Cohen et al.,2010). Nuclear expression of YB-1 has been reported to be a negative prognostic marker which correlated with disease progression in lung cancer (Shibahara et al.,2001; Gessner et al.,2004). Overexpresson of YB-1 was also observed in primary melanoma (Schittek et al.,2007), prostate cancer (Gimenez-Bonafe et al.,2004), glioblastomas (Faury et al.,2007) and neuroblastoma (Wachowiak et al.,2010). A transgenic mouse model has shown that overexpression of YB-1 in mammary gland may result in the formation of breast cancer (Bergmann et al.,2005).4

Figure 2. Relative expression of YB-1 in breast cancer cell lines by quantitative real-time PCR using GAPDH for normalization. MCF 7 breast cancer cell line was used as calibrator. Values are mean ± SEM of three independent experiments. Normal MCF-12A breast epithelial cell line has a marginally lower YB-1 expression than MCF7 breast cancer cell line but significantly lower than the T47D breast cancer cell line.

Figure 3. Expression of YB-1 protein in MCF7 breast cancer cells. A: Western blot analysis using β-actin for normalization. B: Immunocytochemical staining of YB-1 with DAB as the chromogenic substrate. Counterstain with haematoxylin. C: YB-1 immunofluorescence staining. Micrographs taken at 40× magnification.

Figure 4. Translocation of YB-1 from cytoplasm to nucleus in MDA-MB-231 breast cancer cells in response to treatment of doxorubicin. A: Immunofluorescence staining of cytoplasmic YB-1 in MDA-MB-231 cells before doxorubicin treatment. B: Nuclear staining of translocated YB-1 in response to doxorubicin treatment. Micrographs taken at 40× magnification.

Sequence analysis of the gene promoter of the YB-1 gene has shown the presence of several GC-boxes and E-boxes (Makino et al.,1996). Binding of c-Myc (a proto-oncogene product) to the E-boxes transactivates the YB-1 gene promoter and increases expression of the YB-1 protein which is known to regulate cell proliferation (Jurchott et al.,2003). In this regard, p73 has also been observed to interact with c-Myc in stimulating YB-1 gene transcription (Uramoto et al.,2002). Targeted disruption of an allele of YB-1 (Chk-YB-1) in DT40 cells has been shown to induce slower cellular growth (Swamynathan et al.,2002). Phosphorylation of the YB-1 protein at Ser102 by Akt promotes nuclear translocation of YB-1 and affects growth of breast cancer cells (Sutherland et al.,2005). The inhibition of YB-1 has been shown to deregulate the cell cycle by upregulating RAD9A and CDKN3 genes leading to G1-arrest and downregulating proliferation genes, such as CDC6, CDC20, CKS2, and CCNC (Basaki et al.,2010; Yu et al.,2010). Recently, Jurchott et al. (2010) observed that proliferation in colorectal cancer is associated with YB-1 mediated RAS/MEK/ERK-signaling. However, Evdokimova et al. (2009a) argued that the hyperproliferation of cancer cells may actually be detrimental to the survival of the tumor cells during certain stages of cancer like the dissemination from primary tumors and the establishment of metastatic outgrowth. They observed an increased level of expression of the YB-1 protein associated with decreased proliferation rates in metastatic breast cancer cells exhibiting epithelial-mesenchymal transition (Evdokimova et al.,2009b).

Other functions of YB-1 include acting as a transcription inducer of EGFR (Stratford et al.,2007) and stimulating Twist-promoted cancer cell growth (Shiota et al.,2008). YB-1 has been reported to be a tumor-associated antigen, which can elicit a T cell immune response in neuroblastoma in vivo (Zheng et al.,2009). In a recent study, YB-1 has also been found to be expressed in the angiogenic endothelial cells of several types of cancer cells such as gastric cancer, lung cancer, esophageal cancer, colon cancer, and glioblastoma, playing a crucial role in the growth of the endothelial cells in the region of the cancer cells (Takahashi et al.,2010).

YB-1 MEDIATES CHEMORESISTANCE

The mechanisms leading to chemoresistance usually involve the active efflux of the drugs out of the cell to avoid the toxic effects of the anti-cancer agents to the cell, inactivation of the drugs via conjugation as well as increased activity of DNA repair (Rabik and Dolan,2007; Muñoz-Gámez et al.,2011). The P-glycoprotein, a member of the ATP-binding cassette (ABC) transporter superfamily, is known to mediate the efflux of xenobiotics in the cell (Litman et al.,2001; Kuwano et al.,2003). Upregulation of transcription of the Multidrug Resistance 1 (MDR1) gene located on chromosome 7q21.1 by YB-1, in the presence of genotoxic agents such as anti-cancer drugs and ultraviolet irradiation, results in the increased expression of P-glycoprotein (Uchiumi et al.,1993; Ohga et al.,1998). For instance, when MDA-MB-231 breast cancer cells were treated with doxorubicin, YB-1 was observed to be translocated from the cytoplasm to the nucleus of breast cancers (Fig.4). Nuclear YB-1 could influence the transcription of chemoresistance genes decreasing chemosensitivity to a wide range of anticancer drugs, such as cisplatin (Ohga et al.,1996), doxorubicin (Bargou et al.,1997; Kuwano et al.,2003), paclitaxel (Fujita et al.,2005) and mitomycin C (Shibahara et al.,2004). Cisplatin-resistant human head and neck cancer KB epidermoid cancer cells exhibited higher nuclear expression of YB-1 than the drug-sensitive wild type cancer cells (Ohga et al.,1996). Enhanced nuclear YB-1 expression was also found in cisplatin-resistant ovarian cancer cells when compared with cisplatin-sensitive wild type ovarian cancer cells (Yahata et al.,2002). To et al. (2010) have also recently shown that YB-1 is able to increase the expression of CD44 and CD49f genes (associated with cancer stem cells) which are involved in chemoresistance in MDA-MB-231 breast cancer cells (Fillmore and Kuperwasser,2008; Godar et al.,2008; Li et al.,2008). CD44 interacts with hyaluronan and activates drug-resistant genes such as MDR1 (Bourguignon et al.,2008).

Another protein that is up-regulated in the induction of chemoresistance is the human major vault protein (MVP), a lung resistance-related protein (Scheffer et al.,1995). MVP is the main component of vaults (ribonucleoprotein particles) which could possibly be involved in the cellular defence to xenobiotics (Mossink et al.,2002). Stein et al. (2005) observed that the MVP gene was transcriptionally activated after treatment with doxorubicin, 5-fluorouracil (5-FU), vincristine, and cisplatin in human HCT116 colon cancer cells. The same authors showed that YB-1 was able to enhance the basal and 5-fluorouracil (5-FU)-inducible expression of the MVP gene by controlling the promoter activity of the gene.

YB-1 also binds to DNA repair enzymes, such as human endonuclease III (hNTH1) in vitro, stimulating the activity of hNTH1 which is involved in base excision repair, thereby decreasing the sensitivity of MCF7 breast cancer cells to cisplatin (Guay et al.,2008). The acetylated form of AP-endonuclease (APE1/Ref-1), an enzyme that participates in the repair of oxidative stress induced DNA damage, is known to interact with YB-1 resulting in the activation of the MDR1 gene (Chattopadhyay et al.,2008). YB-1 has also been reported to bind to Proliferating Cell Nuclear Antigen so as to aid in nucleotide excision repair (Ise et al.,1999; Izumi et al.,2001).

In a recent study, Yang et al. (2010) has demonstrated the presence of multinuclear cells in originating from cell fusion in doxorubicin-sensitive MCF 7 cells which was accompanied by regulation of YB-1 and ABCB5 P-glycoprotein, which is known to be fusogenic (Frank et al.,2003). They suggest that reduced accumulation of doxorubicin induced by YB-1 and ABCB5 P-glycoprotein is not the only mechanism for chemoresistance and may involve cell fusion and clonal selection in the acquisition of drug resistance in MCF-7 cells.

A schematic summary of some of the hypothetical pathways linked with YB-1 chemoresistance is shown in Fig. 5.

Figure 5. A schematic summary of the hypothetical pathways involved in YB-1 mediated chemoresistance to therapeutic drugs.

STRATEGIES FOR REVERSING YB-1 ASSOCIATED CHEMORESISTANCE

Downregulating the YB-1 gene or YB-1 protein expression could possibly potentiate chemosensitivity to anticancer drugs. It has been reported by Ohga et al. (1996) that the level of sensitivity to cisplatin could be increased by the addition of antisense YB-1. In a similar vein, Guay et al. (2008) has demonstrated that shRNA-mediated silencing of the YB-1 gene enhanced the sensitivity of cells to cisplatin (a platinum-based drug), ultraviolet light and the cytotoxic alkaloid, camptothecin.

Modulating genes upstream of YB-1, such as Twist 1 seems to abrogate chemoresistance. Recent research has shown that YB-1 is modulated by p300/CBP-associated factor (PCAF), in a Twist1-dependent way (Shiota et al.,2010). Knockdown of PCAF, a histone acetyltransferase, inhibited YB-1 expression and increased chemosensitivity to cisplatin and doxorubicin in urothelial cancer cells. In another recent study, programmed cell death protein 4 (PCD4) has been found to interact directly with the DNA binding domain of the transcription factor Twist1 to suppress the growth of cancer cells via the downregulation of YB-1, a Twist1 target gene (Shiota et al.,2009). Evidence of the role of PDCD4 in cancer cell proliferation and chemoresistance was exhibited by the sensitivity of cells expressing PDCD4 to cisplatin and paclitaxel.

Chemoresistance induced by YB-1 has also been shown to be reversed by the translocation of YB-1 to the nucleus by an adenovirus known as Xvir03, which expresses the viral proteins E1B55k and E4orf6 during viral replication in PC3 and DU 145 prostate cancer cells and in prostate cancer xenograft mouse models (Mantwill et al.,2006). The authors suggest that YB-1 is recruited for the purpose of viral replication and hence, there is concomitant down-regulation of the MDR1 gene.

It may also be possible to design novel anticancer drugs through the study of specific YB-1 complexes which are formed in situations of cellular stress. The gene promoter of YB-1 has been found to be upregulated by the tumor suppressor gene product, p73 (Uramoto et al.,2002). Because environmental stress, especially in the case of anti-cancer drugs, is able to induce tumor suppressor gene products and c-Myc, YB-1 may be upregulated by these transcription factors to result in drug resistance in cancer cells (Wahl and Carr,2001). More recently, a study has shown that the binding of the YB-1 protein at the last 62 residues to the retinoblastoma binding protein 6 (RBBP6) via the RING finger domain of RBBP6 was able to ubiquitinate YB-1, leading to proteosomal degradation (Chibi et al.,2008). This indicates that RBBP6 may be a potential target for the development of anti-cancer drugs.

CONCLUSIONS

On the whole, it is still early days in the investigation of the role of YB-1 in the induction of chemoresistance in cancer cells although progress has been made in elucidating the physiological functions and pathological significance of YB-1. There remains a host of factors that would have to be considered before we can comprehend the impact of YB-1 on cancer therapy. Moreover, since YB-1 is a protein with pleiotropic functions, it would be a challenging task to devise compounds which are able to specifically inhibit the chemoresistance activity of YB-1. However, YB-1 is a nondruggable molecular target. Nonetheless, therapeutic experimentation with antisense RNA drugs and xenografts are justified to validate YB-1 as a gene target in cancer therapeutics. siRNAs are emerging as a new class of biotherapeutics where the efficacy would be highly dependent on the design of the siRNA itself and the vehicle for delivery of the siRNA (Walton et al.,2010). siRNAs can be conjugated with steroids and lipids (Lorenz et al.,2004) or packaged with nanoparticles (Kam et al.,2005) to enhance cellular uptake and gene knockdown. As a cancer therapeutic, there are still several hurdles to overcome for siRNA related approaches, such as problems associated with efficient delivery and off-target effects. Yet, YB-1 expression in cancer could potentially be used to predict the resistance of tumors to different chemotherapy regimes, and to function as a marker to determine a suitable adjuvant chemotherapy regime for cancer, as exemplified by Tay et al. (2009) and Gluz et al. (2009). Nevertheless, further clinical studies will be required to determine the predictive utility of YB-1 as a marker of chemoresistance.