The experiments presented here demonstrate that expression of the L1 retrotransposon protein, ORF1p, can be detected in a variety of cancers and that its nuclear localization correlates with poor outcomes in patients with breast cancers. Understanding whether nuclear localization of L1-ORF1p is causative or coincidental will require additional study. It is clear, however, that L1s display many properties that can alter the evolution of a cancer and affect its outcome.29
For instance, L1s can insert into genes and inactivate them,30
they can increase the transcription rate of neighboring genes,31
and they can promote genomic rearrangements.32,33
Previously, L1s have been linked to cancer through studies on the hypomethylation of L1 promoters in a number of tumors including breast,34
chronic lymphocytic leukemia,38
and chronic myeloid leukemia.40
In some of these studies, a clinical consequence of L1 hypomethylation has been noted, including a link to genomic instability.37,40
Certainly hypomethylation of L1 promoters, which has been shown to correlate with L1-ORF1p expression in cell lines,22
would be consistent with the increases in L1-ORF1p expression that we see in a variety of tumors (). But hypomethylation of L1s could affect the genome in ways other than increasing transcription of the retrotransposon. For example, as highly repetitive elements, L1s could serve as substrates for ectopic recombination events between heterologous portions of the human genome, which would lead to genetic events seen in many cancers including translocations and chromosome loss. Ectopic recombination is presumably inhibited by DNA methylation, which condenses DNA. But it remains unclear whether the extent of L1 hypomethylation observed in tumors is sufficient to enable ectopic recombination. Hypomethylation of L1 elements may also simply be a marker for hypomethylation of other genes, such as oncogenes.
These data presented here, in which breast cancer outcomes are linked to the nuclear localization of L1-ORF1p (), suggest that the L1 retrotransposition reaction itself, rather than global changes in DNA methylation, may play a role in the progression of certain breast tumors. Nuclear localization of L1-ORF1p would presumably bring the L1 mRNA into position for its reverse transcription by L1-ORF2p, as required for L1 retrotransposition. There have been other studies that have suggested that the L1 retrotransposition reaction can affect tumor formation. For instance, it has been reported that tumorigenesis of cell lines in mouse xenograft models can be attenuated by reducing L1-ORF2p activity.41
In one breast tumor in particular, an L1 insertion was discovered that may have upregulated the c-myc oncogene.42
However, it should also be pointed out that, despite intensive sequencing efforts aimed at finding mutations within oncogenes and tumor suppressor genes in many different tumors, only two L1 insertions have ever been found, with the other being an L1 that inactivated the critical tumor suppressor, APC, in a patient with colon cancer.43
This failure to detect a higher rate of L1 retrotransposition in tumors may be due to the fact that sequencing efforts traditionally focus on exons, whereas L1 insertions may be capable of exerting effects if inserted within introns30
by creating new promoters31
altering the rate of transcription or bringing in new polyadenylation sites.44
In addition, it has become clear that not all tumors express L1-ORF1p, and only a subclass of these express the protein in the nucleus. Thus, we have begun efforts to look for L1 insertions within breast tumors that express nuclear L1-ORF1p.
If nuclear localization of L1-ORF1p affects patient outcome through catalysis of L1 retrotransposition, then patients expressing nuclear L1-ORF1p might benefit from treatments that decrease the rate of the retrotransposition reaction. Nevirapine is a small molecule that has been used to treat other diseases and that has been shown to inhibit the L1 reverse transcriptase in whole cell assays.41
Perhaps nevirapine or other small molecules would prove beneficial to patients with nuclear L1-ORF1p. It is also notable that radiation has been shown to accelerate L1 retrotransposition45
and that all of the patients in the breast tumor study were treated with radiation following breast surgery. Within the set of patients expressing nuclear L1-ORF1p, less aggressive radiation therapies, or alternative postoperative treatments, might be considered.
Although nuclear L1-ORF1p appears to have a more severe clinical impact than the cytoplasmic protein in breast cancers, it is not clear that cytoplasmic L1-ORF1p is truly benign. We could not test the clinical effects of cytoplasmic L1-ORF1p in breast tumors, where it is ubiquitous, and we saw no effects of cytoplasmic protein on pancreatic neuroendocrine tumors, but that particular data set may be too recent to yield meaningful information. For this reason, the clinical importance of the expression of this protein in tumors such as bladder or colorectal tumors remains of interest. We also do not know if nucleolar L1-ORF1p, which appears in many cell lines, has any clinical importance. Because of technical limitations, it has not been possible to demonstrate that nucleolar localization of the L1-ORF1p protein occurs in tumors.
Changes in cellular localization of certain proteins, as we show for L1-ORF1p (), are a common theme in tumorigenesis. One well-studied example is β-catenin, which, like L1-ORF1p, is associated with tumor progression upon nuclear localization.46,47
In the case of β-catenin, a variety of mutations and/or extracellular signals can result in nuclear localization.48,49
At this time, we do not know the mechanism by which L1-ORF1p becomes nuclear. Because it does not have a nuclear localization sequence of its own, we assume that it interacts with another protein to enter the nucleus, and expression of this protein may vary among tumors. The L1-ORF2p protein would seem to be the best candidate, and it would be interesting to determine whether the breast tumors that demonstrate nuclear L1-ORF1p also express higher levels of L1-ORF2p. The identification of cell line models in which L1-ORF1p is nuclear (), as well as other cell lines in which it is cytoplasmic (), will help us to address the mechanism by which nuclear localization of the protein occurs and to better understand the consequences of that event.