In addition to the above mentioned functions, YBX1 also interacts with a diverse range of biologic pathways and molecules, many of which are key factors implicated in cancer pathogenesis. Interactions relevant to a role in cancer are discussed below.
TP53
Tumour protein 53, or TP53, is one of the best described human tumour suppressor genes and is implicated in a wide variety of human cancers. Its known functions include cell cycle regulation, induction of growth arrest or apoptosis and it is induced in response to various cellular stresses. Mutation of this gene at the germ-line or somatic level is known to be a key factor in the pathogenesis of multiple cancer types. YBX1 was shown early on to interact directly with TP53 through three independent domains of YBX1 and also to facilitate binding of TP53 to its consensus DNA sequence (Okamoto et al, 2000). Interestingly however, YBX1 has also been shown to be negative regulator of TP53 and to repress transcription of the TP53 promoter, with YBX1 inhibition resulting in induction of TP53 in a
56 variety of cell lines and resultant apoptosis via a TP53-dependent pathway (Lasham et al, 2003; Shiota et al, 2008a)
Epidermal Growth Factor Receptor (EGFR)
The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein and a member of the receptor tyrosine kinase ErbB family. EGFR (also known as ERbB1 or HER1) is a cell surface receptor and its ligand is epidermal growth factor. Binding of the ligand leads to receptor dimerization, tyrosine autophosphorylation and cellular proliferation. The activated receptor complex activates at least four downstream signalling cascades, including RAS-RAF-MEK-ERK, PI3 kinase-AKT, PLCgamma-PKC, and STATs modules. Other members of this group of receptors include HER2, HER3 and HER4.
YBX1 has been shown to regulate EGFR in several papers. The first reports of the relationship of YBX1 and EGFR were reported by the team led by Sandra Dunn from Vancouver. YBX1 overexpression in HMEC breast cancer cells leads to overexpression and constitutive phosphorylation of EGFR and growth factor independence (Berquin et al, 2005). This work was complemented by a paper the following year showing that overexpression of YBX1 in a breast cancer cell line was associated with overexpression of EGFR and HER-2, while knockdown suppressed expression of these proteins (Wu et al, 2006). This paper also showed that YBX1 mutated at Ser(102) was unable to bind the promoters for EGFR and HER-2. This work outlined the key role of YBX1 and EGFR in driving the growth of breast tumour cells. Work by the same team identified two YBX1 responsive elements in the EGFR promoter at -940 and -968 using ChiP and gel shift assays, and also showed that inhibition of EGFR and YBX1 suppression inhibited anchorage-independent growth of human basal-like breast cancer cells (Stratford et al, 2007). In lung cancer cell lines, YBX1 nuclear overexpression has been shown to be positively correlated with HER-2 expression in non- small cell lung cancer cell lines (Hyogotani et al, 2012; Kashihara et al, 2009). In PC-3 cells, use of an integrin-linked kinase inhibitor was shown to facilitate the dephosphorylation of Akt and inhibit the expression of YBX1 and EGFR, leading to decreased proliferation (Lee et al, 2011b).
Fas (CD95/Apo-1), Collagen alpha 1
Fas is a member of the TNF-receptor superfamily and contains a death domain. It has been shown to play a central role in the physiological regulation of programmed cell death
57 and has been implicated in the causation of various malignancies and immune diseases. YBX1 has been shown to be a transcriptional repressor of Fas, and overexpression of YBX1 in Jurkat cells (immortalized T lymphocytes) is associated with decreased Fas surface staining (Lasham et al, 2000). YBX1 may therefore assist tumour cells in escaping immune- mediated cell death triggers by repressing a key protein in the apoptotic cascade.
Collagen is a key component of the extracellular matrix and presents part of the barrier to cell invasion and migration. Collagen alpha 1 is a key component of bone and cartilage. YBX1 has been reported to transcriptionally repress human collagen alpha 1 expression (Norman et al, 2001). Theoretically this may allow YBX1 overexpressing cells to facilitate the establishment of bone metastases by disrupting the local bone environment and preventing normal bone collagenisation and structural integrity.
1.6.12 Summary and discussion of YBX1 functions and interactions
As can be appreciated from the above literature review, YBX1 has many disparate roles in eukaryotic cells. It has major functions in DNA and RNA chaperoning, including the modulation of mRNA transcription and translation, DNA repair, and resistance to DNA- damaging agents, particularly chemotherapeutics. It has also been found to interact with numerous cellular pathways and control systems, including the cell-cycle control proteins, TP53, EGFR and Fas. It has also been shown in multiple publications that YBX1 overexpression correlates with an adverse prognosis and early recurrence in multiple human cancers, particularly breast, ovarian, brain and colorectal cancers. For breast cancer in particular, the evidence is fairly robust that YBX1 overexpression is a reliable indicator of early relapse and adverse outcomes, even outperforming HER2, which is a standard component of prognostic panels currently. The reasons why YBX1 has not been adopted into routine clinical use for some of these cancer types is not clear. From the perspective of a clinician and surgeon, the candidate would comment that YBX1 appears to possess many favourable features of a prognostic biomarker, including expression in a significant proportion of patients. One can only speculate that it has not been developed into a commercial assay either due to breast surgeons and physicians being unaware of the data, or difficulties experienced with older commercial antibodies in delivering reliable and repeatable staining of YBX1 in tissue samples. Given the above however, our insights into how YBX1 confers an advantage to particular cancers is limited. Aside from inferring effects based on its known biological functions, we have no direct evidence for how it promotes
58 aggression or survival of certain cancers. In prostate cancer for example, as discussed in section 1.6.10, we know from in-vitro and mice studies that YBX1 expression increased with a progression to an androgen-independent cancer phenotype, and that YBX1 levels are higher in some high Gleason grade tumours. However, the evidence for YBX1 having a causative effect in progression to androgen-independent disease was lacking until recently. Important evidence in this regard emerged recently showing that YBX1 regulates AR expression (Shiota et al, 2011a) and that YBX1 overexpressing cells were resistant to castration-induced growth suppression. The same group also showed that YBX1 induction after chemotherapy led to induction of clusterin, which protects cells from chemotherapy and is implicated in treatment resistance (Shiota et al, 2011c). Aside from these two recent papers, evidence is scant on how YBX1 mechanistically confers an advantage to prostate cancers.
Another important problem the candidate was alerted to early on during this thesis was the presence and expression of YBX1 pseudogenes. The issue has confounded design of exclusive primers owing to the high sequence homology of YBX1 genomic DNA and the pseudogene (PG) DNA. It became clear that it would be important to address this problem thoroughly in order to avoid the confounding effects of PG interference in various assays and also to prevent these interfering with identification of new target genes with which YBX1 may interact. This problem is expanded upon in the next section, and current knowledge about pseudogenes and YBX1 pseudogenes is presented.