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  • br Materials and methods br Results

    2019-08-05


    Materials and methods
    Results
    Discussion This study is the first to our knowledge that describes upstream cis-elements and transcription factors that are important for Crm1 expression and that drive its high expression in cancer and transformed cells. Using deletion and mutation analysis we identified the −175 to +99 region of the Crm1 promoter as responsible for the differential expression of Crm1 in normal and cancer cells, and identified within this region two CCAAT boxes and a GC box as important regulatory elements. Moreover, we identified binding of NFY/CBP and Sp1 transcription factors to these respective sites in transformed and cancer cells. We propose that the elevated levels of NFY and Sp1 proteins in transformed and cancer cells may be in part responsible for overexpression of Crm1 in these cells. Furthermore, our study is a first to describe a role for p53 in repressing the Crm1 promoter in response to DNA damage; a finding which could also contribute to the high levels of Crm1 in cancer cells, since most cancer cells either contain low levels of p53 protein (through enhanced p53 degradation) or harbour p53 mutations [28]. There is increasing evidence implicating NFY as a key player in cancer development. NFY binds the CCAAT box which is present in about 30% of eukaryotic promoters [23]. It is reported that promoters that lack a TATA box rely greatly on the CCAAT box for transcriptional activation [18], and we show that the Crm1 promoter lacks a TATA box but contains two functional CCAAT boxes. The presence of more than one CCAAT box in the promoter of a gene is not uncommon, especially in the promoters of G2/M-associated genes; genes that are reported to rely greatly on NFY for activation [25], [29], [30]. Interestingly, Crm1 mRNA is reported to reach a peak at G2/M [26] and Crm1 is known to play a key role in the control of mitosis [31], likely contributing to its reliance on NFY for transcriptional activation. Literature reports that NFY plays an important role in proliferation [21] and the Ciprofibrate [32], and its own activity is regulated in a cell cycle-dependent manner [33], [34]. It is believed that NFY is a major contributor to the high expression of many cell cycle-regulated genes in cancer cells [35], [36] and Niida et al. found that the NFY motif is significantly enriched in genes overexpressed in breast cancer, with it being one of the principal regulatory motifs driving breast cancer malignant progression [37]. Furthermore, Pang et al. found that NFY binding to the thymidine kinase promoter was at levels 5- and 15-fold greater in SV-40 virus-transformed human cells and HeLa cells, compared to normal cells, respectively, and that the binding activity of NFY changes from a cell cycle-dependent one in normal cells to a constitutive one in transformed cells and tumour cells [38]. They suggest that this is due to a loss of post-translational control during/after cell transformation, as the half-life of NFY in transformed cells was five-fold higher than in normal cells [38]. In this study we have shown that NFY expression is elevated in cervical cancer and transformed human fibroblast cells, resulting in increased binding of NFY to the Crm1 promoter, and thus NFY is very likely a key contributor to Crm1 overexpression in cancer. As well as demonstrating a role for NFY, we identified Sp1 as an important regulator of Crm1 expression in cancer. Interestingly, in an in silico study, Quan et al. predicted that the Sp1 binding site is one of the most common transcription factor binding sites in the promoters of karyopherin β family genes, of which Crm1 is a member [39]. Sp1 regulates a variety of biological functions that are critical to tumour development and progression [19], [24], with its transcriptional activity modulated in a cell cycle-dependent manner [40], and its target genes including many regulators of the cell cycle [41], [42], [43], [44]. It plays an important role in growth regulation, as introduction of a dominant negative Sp1 into HeLa cervical cancer cells significantly inhibited cell proliferation [45]. We identified increased Sp1 protein expression in transformed fibroblasts, SVWI38, and cervical cancer cells, CaSki, compared to normal fibroblasts, WI38. Sp1 expression has previously been shown to be elevated in transformed cells [46], where its increased expression was found to result in increased activation of its target genes [46]. Moreover, elevated Sp1 expression has been associated with several cancers, including gastric cancer [47], pancreatic cancer [48], and skin cancer [49]. We show that inhibition of Sp1 expression in cancer cells leads to diminished Crm1 promoter activity, suggesting that it is, in part, the high expression of Sp1 in cancer cells that maintains high Crm1 expression.