The data presented above indicate that HNSCC and NSCLC undergo activation of candidate proto-oncogenes with associated demethylation in a coordinated fashion in individual tumors. We were able to demonstrate transformation-associated effects of BORIS expressed ectopically in BORIS-negative cell lines as well as growth effects with individual target genes that have been shown to be epigenetically activated and expressed by BORIS. However, this does not rule out the contribution of as yet unidentified genes to BORIS related effects or a cooperative effect between identified target genes. The lack of direct correlation of demethylation of promoter and increased expression in some of these targets () may simply be a reflection of the small cohort used to test this association in HNSCC, but may also be due to alternate mechanisms of transcriptional control of these genes other than promoter methylation status, including other promoters, repressors, or mediators (e.g. BORIS). Cancer testes antigens include four of our genes, MAGE A2
, are part of the melanoma antigen family A (MAGE-A) family of genes initially discovered as targets for immunotherapy due to their near exclusive tumor-specific expression, but the MAGE-A family plays a functional role in cancer development 
. MAGEA2 binds to p53-responsive promoters and leads to assembly of a p53/MAGEA2/HDAC3 protein complex, resulting in transcriptional silencing of genes ordinarily activated by p53 because of histone deacetylation. Additionally, different MAGE-A family members can repress downstream targets of p53, and studies have also linked MAGE-A family overexpression to chemo-resistance
, and MAGE family members have been shown to increase cell growth and inactivate TSG activity 
. Recently, MAGEA has been shown to repress p53-dependent apoptosis, and has been associated with resistance to taxanes and alkylating agents in gastric cancer 
. We found these MAGE family members to show significant expression in a correlated fashion in HNSCC and NSCLC, and to reexpress in cell lines treated with 5-aza-deoxycytidine. In primary tissue some targets expression level was directly correlated to promoter methylation status.
We found that expression of the MAGE-A family and expression of H19
appeared to be significantly related in our primary tumors, supported by data indicating that these targets are controlled by common methylation-specific transcription factors 
forms half of the best-studied example of imprinted-gene regulation, the IGF2/H19
(insulin-like growth factor 2) is expressed uniquely from the parental allele achieved by monoallelic methylation of the imprinting control region (ICR) at 11p15.5 
. Aberrant hypomethylation at this locus is one cause of Silver-Russell syndrome—a disease of asymmetry or hemihypertrophy associated with increased risk of malignancies including craniopharyngioma, testicular seminoma, hepatocellular carcinoma, and Wilms tumor 
. Additionally, several cases of familial Beckwith-Wiedemann syndrome (BWS), with and without Wilms' tumors, have been shown to be caused by microdeletions of the methylation-specific CTCF binding sites in the H19
ICR, a rare familial cancer syndrome linked to epigenetics 
. H19 and the MAGE family members showed significant correlation in this expression and demethylated promoter status in HNSCC and NSCLC.
Other identified proto-oncogenes in this report have been implicated recently in tumorigenesis. TKTL1
protein expression is correlated to worse outcome in patients with invasive colon and urothelial tumors, and investigators hypothesize that enhanced TKTL1
expression in tumors increases oxygen-independent glucose usage 
. In addition, over-expression of TKTL1 has since been validated as a potential biomarker and treatment target in breast cancer 
have not been implicated in carcinogenesis to date. Although we were unable to demonstrate growth promoting effects of C19ORF28
, this does not exclude the possibility that overexpression of this and any of our other targets may contribute to a malignant phenotype in other cell backgrounds, or via other mechanisms—i.e., motility, invasion, angiogenesis, or apoptosis resistance—or that it may cooperate with other identified targets to produce phenotypic effects.
The epigenetic reactivation of TKTL1
, and C19ORF28
, genes located at diverse chromosomal loci, occurs simultaneously in individual primary tumors from multiple tumor types. This concurrent genome-wide, promoter-specific hypomethylation that results in derepression of many potential oncogenes raises the possibility of a demethylator phenotype analogous to the CpG island methylator phenotype (CIMP) initially noted in colon cancer 
. Many proto-oncogenes are members of the cancer testes antigen family which are ordinarily repressed via epigenetic mechanisms during development. An attractive hypothesis is that this phenomenon represents the coordinated, but pathologic reversal of developmental epigenetic regulatory patterns in cancer cells. The validity of our whole-genome integrative approach to screening for epigenetically-activated genes associated with malignancy is, in fact, confirmed by the appearance of H19
and the MAGE-A family members which have been reported to be controlled by epigenetic activation and show silencing in normal cells. Two separate groups among our nine genes showed statistically significant correlations for patterns of expression: 1) MAGEA family members with H19
and 2) TKTL1
, and GRIN
. We were also able to define these groups according to promoter homology, implicating the participation of promoter-specific binding activity in the coordinated expression of each of these groups and suggesting the existence of additional common transcriptional activators that recognize the specific demethylated promoter sequences of these genes. The strict correlation of BORIS expression with aberrant expression of multiple growth-promoting proto-oncogenes in a variety of solid tumors reinforces the postulated role for BORIS as a key participant in aberrant demethylation and transcriptional activation of putative oncogenes. This concept is supported by cell line experiments demonstrating that BORIS expression by itself is sufficient to simultaneously demethylate and activate the transcription of these genes. However, some reports have shown melanoma tissue samples that express MAGE-A1 in the absence of BORIS activation, suggesting that BORIS is not an obligate factor for activation of these genes 
. It is of great interest to define the factors with which BORIS cooperates to induce these epigenetic and expression changes. Recently, a role for BORIS in histone demethylation and chromatin remodeling has been demonstrated 
. Moreover, regardless of mechanism, our data provide strong evidence for consideration of BORIS as a dominant controlling factor for facilitating epigenetic alterations associated with coordinated demethylation and reactivation of target genes that are of high value as potential therapeutic and diagnostic targets for NSCLC, HNSCC, and other tumors.
This simultaneous reactivation of multiple targets provides a significant challenge to the understanding of the collective, and perhaps cooperative, effects of this phenomenon in cell transformation. In particular, single targets may depend on concurrent activation of, and interaction with, other family members for oncogenic effect. Other investigators have found some evidence of coordination of cancer testes antigen family expression and the possibility of direct interactions 
. In addition, we selected only the top 26/106 possible targets identified after integrative analysis in a single solid tumor type for further analysis. We would expect that future studies of the remaining genes, as well as use of normal cell lines and tumors derived from other tissues in an integrative approach, will allow for discovery of additional, novel, epigenetically-controlled genes that may also act collaboratively to induce malignant transformation.
Due to lack of primary tumor data on a larger array platform we also used a nonintegrative approach, which resulted in ultimate validation of 4.3% of the targets (2/46) compared to the integrative results that produced a 27% hit rate (7/26), reflecting a higher ability to validate targets in primary tumor when these data are included in initial discovery strategies. Additional analysis of other targets that are significantly differentially regulated may also yield additional epigenetically derepressed targets. Finally, these data have therapeutic implications for demethylation therapy and targeting of therapy. The active investigation of pharmacologic demethylating agents as therapy for malignancy based on reversal of silencing of tumor suppressor genes may have unintended effects. It is possible that in certain tissues this may result in reactivation of developmentally repressed proto-oncogene targets, with the unintended effect of promoting late, second primary tumors 
. However, modulation of a pathway that involves the coordinated derepression of a series of growth-promoting proto-oncogene candidates and a key transcriptional effector, BORIS, may provide a significant opportunity for directed therapeutic intervention that simultaneously targets multiple oncogenic pathways.