Here we address the mechanism by which highly conserved, non-coding DNA elements within the 8q24 gene desert influence cancer risk. Previously we identified a subregion of 8q24, AcP10, that contains a risk SNP and has enhancer activity in cultured cells (Jia et al., 2009
). In the present study, we show that a 1.5 kb fragment, including a highly conserved DNA sequence in which the SNP is embedded, directs reporter gene expression to a subset of lumenal epithelial cells in the prostate. These include some cells that persist after androgen ablation and express the transcription factor Nkx3.1. Shen and colleagues demonstrated that these cells, designated castration-resistant Nkx3.1-positive (CARN) cells, are both stem cells and potential cells of origin of prostate cancer (Wang et al., 2009
). We show, moreover, that AcP10 is active in cells of prostate tumors developing in mice in which the Pten
gene is inactivated in the prostate. Our results thus provide a plausible mechanism by which the AcP10 enhancer element can influence prostate cancer risk.
Accumulating evidence shows that highly conserved, non-coding elements (CNEs) can have gene regulatory functions (Bulger and Groudine, 2011
). The human genome contains many thousands of such elements, which are likely to have originated as retroposons and in some instances have acquired a function in the host genome, a process known as exaptation (Santangelo et al., 2007
). Some CNEs have been shown to function as tissue-specific enhancers. In most but not all cases they are also defined by their extremely high degree of DNA sequence conservation across vertebrates (Birney et al., 2007
). At least some CNEs are under negative selection, implying that the extremely high degree of sequence conservation is required for function.
The AcP10 element has a central region approximately 450 bp in length that is conserved across terrestrial vertebrates, exhibiting approximately 67% identity to a sequence in lizards. This high degree of conservation suggests a fundamental and slowly evolving function in vertebrates. Such a function might include controlling gene expression in the prostate or in tissues from which the prostate evolved, such as the Mullerian duct (Wake, 1981
We note that the AcP10 element drives expression in epithelial tissues of the breast, colon and skin, but not in epithelia generally. We have not yet identified the specific cell types in these tissues in which the AcP10 transgene is expressed. This is likely to be of interest at least in the colon because AcP10 is associated with risk of colon cancer as well as prostate cancer.
The fact that the high-risk (G) allele of the SNP rs6983267 is the ancestral allele is evident in comparisons across vertebrates showing the G allele in all groups. It is also apparent in the geographic distribution of the two alleles in humans, which shows the high risk G allele in African populations and the low-risk T allele outside Africa, consistent with the T allele emerging as ancestral humans migrated out of Africa. The T allele might have conferred a selective advantage, perhaps reducing the risk of cancer. Grandparent effects (Hawkes, 2010
) on allele frequency – i.e. selection acting at the level of extended family or group – could explain fixation of an allele that does not provide a direct advantage to individuals of reproductive age.
An approach that has been used by several groups to identify stem cells in the mouse prostate is to reduce androgen levels by physical or chemical castration and examine the cells remaining after this treatment (Wang et al., 2009
). When androgens are withdrawn, the prostate involutes to a small size. Resupplying androgens causes the gland to regenerate. All the cells required to regenerate the gland are most likely present in the involuted gland following androgen ablation. Using this approach, Shen and colleagues (Wang et al., 2009
) showed that a subset of lumenal epithelial cells remain in androgen-depleted prostates. These CARN cells are capable of giving rise to an entire prostate, and are also a cell of origin of prostate cancer.
As we noted previously (Jia et al., 2009
), AcP10 has chromatin marks consistent with enhancer activity. Moreover, it can function as an enhancer in cultured cells. Thus, we expected AcP10 to drive reporter gene expression in transgenic mice. We were surprised, however, at the remarkable specificity of transgene expression in a subset of prostate epithelial cells. Marker analysis showed that the AcP10–β-gal-positive cells are lumenal. Positive cells tend to be clustered, implying that such cells are clonally related.
Until recently, only basal epithelial cells were thought to contain stem populations and to serve as cells of origin of prostate cancer (Goldstein et al., 2010a
). New findings by the Shen group (Wang et al., 2009
) that the lumenal compartment might also have stem cells prompted us to investigate whether the AcP10–β-gal-positive cells exhibit molecular characteristics of lumenal epithelial stem cells. Shen and colleagues showed that the transcription factor Nkx3.1 is expressed in all or most lumenal epithelial cells in control mice (Wang et al., 2009
). Upon androgen depletion by castration, the prostate is greatly reduced in size, and Nkx3.1 expression is retained in a small subset of lumenal cells. Our results show that about 8.5% of AcP10-positive cells are also positive for Nkx3.1, compared with 3.6% of AcP10-negative cells (). Chi-square analysis suggests that this overlap in expression patterns is significant (P
That AcP10 is not expressed in all Nkx3.1-positive cells suggests that there is not a simple relationship between the enhancer activity of AcP10 and the identity of such cells or the regulation of Nkx3.1. Thus it is not likely that the main function of AcP10 is to regulate the expression of Nkx3.1 in lumenal epithelial cells; nor is it probable that Nkx3.1 is a main determinant of the activity of AcP10 in such cells. Rather, the increased likelihood that AcP10 is expressed in Nkx3.1-positive cells suggests that AcP10 and Nkx3.1 are linked in a regulatory network, although perhaps distantly. Even a distant link, however, could be part of the explanation of how AcP10 increases the risk of prostate cancer. Shen and colleagues (Wang et al., 2009
) showed that Nkx3.1-positive cells are also potential cells of origin of prostate cancer in a Pten
mouse model. Thus, our results connect the AcP10 enhancer to a population of cells that can self renew, produce prostate epithelial cell types after injection into the mouse kidney capsule, and give rise to prostate tumors (Wang et al., 2009
Our overall hypothesis is that AcP10 controls the expression of a gene(s) whose activity is crucial for tumorigenesis. One possible AcP10-regulated gene is Myc
. We showed previously that AcP10 loops to Myc
, and that the risk allele of rs6983267 enhances TCF7L2 binding to the enhancer (Pomerantz et al., 2009a
). This result led us to predict that Myc
expression in prostate tumors would correlate with the SNP genotype. We found that this was not the case, however. There was no eQTL relationship between SNP genotype and Myc
expression in tumors. It remains possible that AcP10 modulates Myc
expression levels transiently in a tumor precursor cell, an idea also suggested by Wasserman and colleagues (Wasserman et al., 2010
). We carried out immunostaining with an anti-Myc antibody and found that Myc protein was expressed widely in the prostate epithelium (data not shown), consistent with the findings of Wasserman and colleagues (Wasserman et al., 2010
). Although there was some overlap with AcP10 expression, the broad distribution of Myc made it impossible to determine whether this overlap occurred by chance. Clearly, because AcP10 is expressed in few Myc-positive cells, it is not likely to be a principal determinant of Myc expression in the prostate. Nevertheless, AcP10 might have a quantitative effect on Myc expression in a subset of cells. Such an effect could be sufficient to predispose such cells to oncogenic transformation and thus explain the elevated risk of prostate cancer associated with the rs6983267 genotype.
Highly conserved, non-coding elements such as AcP10 are thought to be derived from retroposons (Bejerano et al., 2006
). Although the function of such elements is unclear, at least some undergo exaptation, a process by which they are co-opted as participants in a cellular regulatory process that is beneficial to the host organism (Bejerano et al., 2006
). Such a process might include the acquisition of an ability to regulate gene expression in a tissue-specific manner and thus control cellular identity. AcP10 could be such an element, and AcP10-expressing cells could have a distinct identity and set of properties, perhaps related to prostate function or development. A key unanswered question is whether the association of AcP10 with oncogenesis is related to such a function in normal prostate development, or is a consequence of a fortuitous ability of AcP10 to direct gene expression to the prostate lumenal epithelium.