In this study, we provide insight into the mechanisms controlling expression of the CT-RCC HERV-E in kidney cancer. In our prior analysis, we found this HERV-E was expressed in the majority of RCC cell lines and fresh tumors and was highly immunogenic (Takahashi et al., 2008
). The CT-RCC-1 peptide derived form this provirus was found to encode an antigen that stimulates cytotoxic T-cells that kill RCC cells in vitro
and in vivo
in a patient with metastatic kidney cancer who had durable tumor regression following an allogeneic hematopoietic stem cell transplant. Remarkably, this provirus was found to have expression restricted to kidney cancer tumors, with no detectable expression in normal tissues or non kidney cancer tumors. Renal neoplasms are divided into distinct histological subtypes and include clear cell carcinoma, papillary RCC, chromophobe tumors, collecting duct tumors and medullary RCC (Cohen and McGovern, 2005
). Here we show expression of this HERV-E is restricted to clear cell carcinoma, which, interestingly, appears to be the subtype of kidney cancer that is immuno-responsive. Although expression levels varied among tumors, a majority of ccRCCs (80%) were found to express CT-RCC transcripts. Furthermore, proviral expression occurred at the earliest stages of carcinogenesis, with CT-RCC transcripts being detected in small (<3cm) primary tumors with no correlation between tumor lesion size and level of expression.
Clear cell carcinoma is the most common subtype of RCC, accounting for approximately 70–80% of all RCC tumors. In most clear cell cancers, the tumor suppressor gene VHL
is inactivated from either gene mutation or promoter hypermethylation. In accordance, we found VHL
was deficient in all the ccRCC cell lines used in this analysis. Sequencing analysis showed 12/14 ccRCC lines contained VHL
mutations or promoter methylation. The remaining two ccRCC lines did not contain any genetic or epigenetic alterations in the VHL
gene. Nevertheless, both had strongly reduced levels of VHL
, which may have occurred as a consequence of other non-genetic alterations which lead to loss of VHL
expression (i.e. micro-RNA reducing VHL
expression in CLL B cells; Ghosh et al., 2009
). Among tumors where VHL
was found to be mutated, we found that the majority (31/37; 84%) expressed the CT-RCC HERV-E including 11/14 (79%) ccRCC cell lines and 20/23 (87%) primary tumors obtained from patients with VHL
disease. Importantly, transfection of wt VHL
deficient ccRCC cell lines dramatically reduced the proviral expression. Taken together, these data suggest inactivation of VHL
is an essential event required for the CT-RCC HERV-E expression in RCC.
Genetic mutations that disrupt the function of VHL protein in clear cell tumors induce the activation of HIF-mediated hypoxia-induced gene pathways during normoxia (reviewed by Kaelin, 2009
). HIF is a basic heterodimeric transcription factor consisting of a constitutively expressed beta subunit and one of three oxygen sensitive alpha subunits (HIF-1α, HIF-2α, or HIF-3α). The HIF-1α and HIF-2α are known to be widely expressed in different types of cancer and play a role in angiogenesis and tumor apoptosis resistance. HIF-2α was reported to be expressed in ccRCC, but its expression was variable in non-clear cell RCC tumors and absent or present at very low levels in the normal kidney cortex (Turner et al., 2002
; Kim et al., 2006
; Sandlund et al., 2009
). Furthermore, VHL
-defective RCC cells seem to show a bias toward HIF-2α rather than HIF-1α expression (Krieg et al., 2000
; Turner et al., 2002
; Kim et al., 2006
; Sandlund et al., 2009
). In accordance with these findings, all the ccRCC lines studied in this work demonstrated elevated expression of HIF-2α subunits, either alone or in combination with HIF-1α. Because VHL
inactivation was found to be associated with HERV-E expression, we evaluated whether HIF promotes proviral expression in ccRCC. HIF-1α was found to be absent in some ccRCC cell lines that had high levels of the HERV-E expression. In contrast, HIF-2α subunits were detected in all HERV-E expressing ccRCC tumors, with HIF-2α mRNA levels linearly correlating with the levels of proviral transcripts. The observation that siRNA silencing of HIF-2α dramatically decreased proviral expression firmly establishes that HIF-2α plays a role in promoting expression of CT-RCC HERV-E.
HERV LTRs are known to function as promoters, with some being enriched with binding sites for transcription factors such as p53, CTCF, Pou5F1-Sox2, and ESR1 (Cohen et al., 2009
). We identified core HRE sequences in the 5′LTR of the CT-RCC HERV-E, leading us to hypothesize that HIF-2α binds directly to the 5′LTR promoting proviral expression. Indeed, chromatin immunoprecipitation using HIF-2α antibodies demonstrated an interaction between HIF-2α and the proviral LTR in HERV-E expressing ccRCC cell lines. We found the HRE motif as well as other surrounding CpGs in the HERV-E 5′LTR were hypomethylated in all HERV-E expressing ccRCCs. In contrast, this genomic region was highly methylated in normal tissues, non-kidney tumors, non clear cell RCC cell lines, and the small subset of ccRCC cell lines that were found to be HERV-E negative. Using ChIP analysis we observed substantially lower levels of HIF-2α/5′LTR interaction in HERV-E-negative ccRCC cell lines that possessed hypermethylated HERV-E LTRs. These data are consistent with prior data showing that methylation of a cytosine nucleotide in the core HRE blocks binding of HIF alpha subunits inhibiting transcription (Wenger et al., 1998
). Taken together, these data suggest methylation of CpGs in the HERV-E 5′LTR silences proviral expression, even when the transcriptional factor HIF-2α is present, potentially explaining why a minority of ccRCC tumors had no CT-RCC HERV-E expression. Consistent with this observation, we found treatment of ccRCC tumors with the demethylating agent DAC or the histone deacetylase inhibitor depsipeptide, given alone or together, increased proviral expression in ccRCC cells that possessed hypermethylated HERV-E 5′LTRs. Remarkably, the upregulation in proviral expression by combined DAC/depsipeptide treatment decreased substantially when wt VHL
was transfected into the 786-0 ccRCC line, providing further evidence that functional pVHL suppresses activation of CT-RCC HERV-E by destabilization of the transcriptional factor HIF-2α.
Although the mechanisms involved are not totally understood, deregulation of DNA methylation is thought to contribute to genomic instability in tumors. Global hypomethylation of CpG dinucleotides is characteristic of many cancers and there is evidence for reactivation of a number of different types of HERVs in a variety of cancer histologies due to the liberation of their LTRs from epigenetic constraints (Florl et al., 1999
; Menendez et al., 2004
; Lavie et al., 2005
; Szpakowski et al., 2009
; Gimenez et al., 2010
). However, limited data exists on the factors controlling locus-specific transcription of HERVs that are selectively expressed in specific histological subtypes of malignancies. Hsiao et al.
recently characterized the mechanism through which HERV-K18 transactivation occurs in EBV-infected B lymphocytes (Hsiao et al., 2006
). Our study provides the first insights into the mechanisms accounting for the selective expression of a HERV in a solid tumor, demonstrating that inactivation of a tumor suppressor gene can result in aberrant expression of a highly immunogenic provirus in ccRCC. The transcriptional up-regulation of HERV-E appears to be related to three critical events: (i) VHL
inactivation, (ii) HIF-2α overexpression, and (iii) hypomethylation of the HERV-E 5′LTR. This work provides insights needed for research investigating a potential oncogenic role of CT-RCC HERV-E in kidney cancer.