Genomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. KCNK9 encodes for TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. However, KCNK9 gene amplification accounts for increased expression in <10% of these breast cancers.
Epigenetic adaptations in response to in utero nutritional and environmental factors are hypothesized to play an important role in developmental plasticity and human disease susceptibility 
. Diet-derived methyl donors and co-factors are necessary for the synthesis of S-adenosylmethionine (SAM), the methyl group donor for DNA methylation. Thus, environmental factors that alter early nutrition and/or SAM production can potentially influence adult disease risk by altering CpG methylation at critically important, epigenetically labile regulatory regions 
Genomic imprinting is an inherited form of parent-of-origin dependent epigenetic gene regulation that renders autosomal genes functionally haploid in a species, developmental stage, and tissue dependent manner 
. There is evidence that epigenetic modifications in the genome link environmental exposures to adult disease susceptibility 
, including cancer 
. Moreover, imprinting can be dysregulated not only in somatic cells, but also in germ cells, potentially affecting offspring never subject to the parental exposure 
. Since imprinted genes are frequently clustered and coordinately regulated by differentially methylated regions (DMRs), changes in a single DMR can disrupt the expression of more than one imprinted gene 
. Disease susceptibility due to epigenetic deregulation also has specific windows of vulnerability, including embryogenesis, puberty, pregnancy, and old age 
In a computational model, KCNK9
was predicted to be regulated by imprinting 
and its gene product, TASK3, is of interest for human health studies, as overexpression is strongly tied to cancer. KCNK9
is maternally expressed in the human brain, as well as in the mouse brain 
. The KCNK9
gene encodes for the pH sensitive potassium channel protein, TASK3. TASK3 is present at the plasma membrane and regulates membrane depolarization in response to acidosis via inhibition of the background potassium-current 
. Inactivation of the expressed maternal copy of KCNK9
results in Birk-Barel syndrome 
. Overexpression of TASK3 in cell lines promotes tumor formation and hypoxia-resistance 
. Blocking the TASK3 channel protein, either chemically or by mutation, reduces cell proliferation and increases apoptosis, by unknown mechanisms 
. However, TASK3 has been observed to also be localized in the mitochondria, in addition to the plasma membrane 
, and inhibition of TASK3 function has been shown to lead to mitochondrial dysfunction 
TASK3 is overexpressed in >40% of breast cancers, but genomic amplification of KCNK9
only accounts for TASK overexpression in <10% of breast cancers 
is known to be epigenetically regulated. Consequently, we hypothesized that overexpression of TASK3 protein (in the absence of KCNK9
duplication) could be due to epigenetic dysregulation, specifically the loss of parental silencing methylation or imprinting.
2. Epigenetic Dysregulation of KCNK9 Imprinting
Loss of normal imprinting (1) occurs due to poor prenatal nutrition or exposure to heavy metals (e.g., cadmium, lead, arsenic) and (2) is linked to obesity, autism, and cancer 
. The KCNK9
gene-product TASK3 is a pH-regulated, potassium channel membrane protein that we, and others 
, show regulates mitochondrial membrane potential and apoptosis. This study provides the first demonstration that KCNK9
is imprinted and monoallelically expressed in mammary epithelial cells. It also identifies a DMR that likely regulates imprinting at this locus in human breast tissue. In addition to the DMR identification, we demonstrated, by NOMe-Seq, a region of differential chromatin structure related to the methylation status of the DMR. The relationship between DNA methylation and condensed chromatin structure is consistent with a model in which methylation at the DMR silences gene expression by impacting chromatin accessibility and preventing transcription factor binding. This model also supports an epigenetic mechanism for KCNK9
/TASK3 overexpression in breast cancer.
Hypomethylation of the KCNK9
DMR was observed more frequently in African-American women with TNBC (p
= 0.006) and less frequently in European-American women with TNBC (p
= 0.70). Hypomethylation of the KCNK9
DMR was observed concurrently in TNBC and normal-appearing adjacent breast tissue. Abnormal KCNK9
imprinting was associated with increased mitochondrial membrane potential in live TNBC cells and non-cancerous mammary epithelial cells from high-risk women (p
< 0.001). These results are consistent with a study by Nagy et al. who showed that, in melanoma, TASK3 regulates apoptosis and mitochondrial function 
. The finding that KCNK9
-US1 DMR hypomethylation occurs preferentially in African-Americans suggests that KCNK9
/TASK3 may provide a new target for prevention of TNBC.
While 50% of Ashkenazi European women with TNBC have a germline mutation of BRCA1
, only 20% of African-American women with TNBC have a BRCA1
. This indicates that other mechanism(s) beyond germline mutation of BRCA1
are responsible for the etiology of TNBC in African-American women. African-American women experience disparities in income, access to care, and an unequal burden of environmental exposures 
. Given that imprinting is dysregulated by poor nutrition and environmental toxicants, our findings provide a potential mechanistic link between disparities and TNBC in African-American women who do not have germline BRCA1
mutations. A limitation of this study is that it was conducted in a single institution in a restricted number of women; multi-institutional testing with an expanded test set and validation set is required to validate KCNK9
/TASK3 as a potential risk biomarker.
The hypomethylation at the KCNK9 DMR was observed in both non-cancerous and cancerous breast tissue, but it is rare in the WBC of at-risk individuals. These findings indicate that epigenetic alterations occurring at the KCNK9 locus do not typically form at the time of fertilization and implantation. In contrast, they are consistent with alterations occurring in later epigenetically vulnerable developmental windows. These windows include during tissue differentiation, early childhood, or puberty. Identification of hypomethylation of the KCNK9 DMR in both breasts of at-risk individuals is indicative of a relatively early developmental change with large spatial distribution. Hypomethylation seen in only one breast would indicate epigenetic alteration occurring later in development, in a more specific location or cell type. The epialleles identified here are quantifiable markers for association studies between the environmental factors and the critical exposure timing that contribute to breast cancer risk.
The DMR methylation and NOMe-Seq chromatin data provide intriguing targets for future studies to better understand the origins and progression of TNBC. This will include further investigation of DMR methylation, chromatin structure, transcription factor binding, and gene/protein expression in this aggressive form of breast cancer. As TNBC has rapid-onset, aggressive growth, and resistance to treatment, patient survival could be improved through better classification of risk status, early detection, and better treatment targets. Furthermore, since genomic imprinting is dysregulated by poor nutrition and exposure to environmental toxicants, the results here provide support for the importance of good nutrition and a healthy environment in the prevention of TNBC.
Other recent work into the epigenetic regulation of KCNK9
has identified long distance cis-interactions between the promoter CpG island of KCNK9
, which was found to be hypomethylated, and the PEG13 DMR 
. As mentioned previously, parent-of-origin specific methylation in the KCNK9
CpG island was not detected, so any parental-specific regulation by this interaction would be due solely to the PEG13 DMR. Thus, it will be important to determine how CpG methylation and chromatin structure in the KCNK9
promoter influence long-range interactions with PEG13. Furthermore, it will be of interest to determine whether interactions between the KCNK9
-US1 DMR and the KCNK9
promoter are involved in gene regulation. Such increased understanding of the epigenetic regulation of KCNK9
will be of great value in determining the role that expression of this gene has in the development and progression of cancers and in developing new treatment methods.