Approximately 30 million years ago, a retrovirus integrated into the germline of a common ancestor of baboons, gorillas, chimpanzees and humans. That endogenous retrovirus, now known as gammaretrovirus human endogenous retrovirus 1 (HERV-1), may provide clues about the aberrant regulation of gene transcription that enables tumor cells to grow and survive.
Understanding the Mechanism Behind Cancer Gene Expression
Scientists have long described the striking differences in gene expression, signaling activity and metabolism between cancer cells and normal cells, but the underlying mechanisms that cause these differences are not fully understood. In a recent Science Advances article, published by Ivancevic et al., researchers from the University of Colorado, Boulder; the University of Colorado Anschutz Medical Campus, and the University of Colorado School of Medicine report their efforts to identify endogenous retrovirus elements that might be part of the answer to the complex question of what biological events are responsible for the changes in gene expression in cancer cells.
The researchers hypothesized that transposable elements (TEs), specifically those associated with endogenous retroviruses could be involved in cancer-specific gene regulation. Endogenous retroviruses (ERVs) are the remnants of ancient retroviral infections that have integrated into the germline of the host.
Identifying Endogenous Retrovirus Elements That Affect Cancer Gene Expression
To identify potential ERV TEs involved in cancer gene transcription, the researchers analyzed epigenetic modifications across 21 cancer types using chromatin accessibility maps generated by the Cancer Genome Atlas Projects. They subtracted activity of regions that are involved in normal, healthy adult gene expression from the cancer atlas information. Based on this study, they found 23 subfamilies of repeat elements that were significantly enriched in at least one cancer type (out of 1315 repeat subfamilies studied). Of those, 19 corresponded to long terminal repeats of primate-specific ERVs. They decided to focus their attention on LTR10 elements because these elements were enriched for several types of epithelial cancers—including colorectal cancer (CRC), stomach cancer, prostate cancer and lung cancer (figure 1.C and S1 of the paper). Furthermore, they found that LTR10 elements were active in CRC tumors from multiple individuals.
Using the HCT116 CRC cell line, the researchers next looked at data sets describing DNA epigenetic modifications. They found that both LTR10A and LTR10F showed markers associated with element activity, while epigenomic analysis from normal tissues showed no markers of enhancer activity, but rather was associated with markers of transposon silencing.
Assessing the Role of LTR10 Elements in Gene Regulation with Dual Luciferase Reporter Assays
Next, Ivancevic and colleagues conducted a series of functional studies to understand the role of LTR10 elements in transcriptional regulation. Because their analyses revealed that the transcription factor, AP1 binds to LTR10 elements and that LTR10 transcriptional activity correlates with the API factor FOSLI (second transcription factor implicated in several cancers), they used reporter assays to test whether LTR10 activity is affected when API/MAPK signaling pathways are disrupted.
The researchers used NanoLuc® Luciferase and the pGL4.50 Luciferase Reporter Vector to study the enhancer activity of LTR10 elements. They transfected HCT116 CRC cells with plasmids containing LTR10 sequences using a Fugene® Transfection Reagent. Luminescence was measured using the Nano-Glo® Dual-Luciferase® Reporter Assay System. Luminescence readings were normalized to co-transfected constitutively active firefly reporters and then data were presented as a fold-change against cells transfected with an empty minimal promoter, pNL3.3.
These cells were treated with either TNFα or cobimetinib (MEK1 inhibitor) to either stimulate or inhibit signaling, respectfully. The assays revealed that LTR10 elements have significant enhancer activity, driving gene expression in the cancer cells. Constructs with shuffled AP1 motifs served as controls and showed reduced activity, highlighting the importance of AP1 motifs in LTR10 enhancer function.
Understanding How LTR10 Elements Regulate Gene Expression with CRISPR Knockouts
Building on the gene expression studies, they then used CRISPR knockout studies, to investigate how LTR10 elements in HCT116 CRC cells regulate gene expression and play a role in cancer progression. Using CRISPR/Cas9 technology, they created targeted knockouts of specific LTR10 elements within HCT116 cells. Changes in gene expression levels of genes near the LTR10 elements helped identify genes that might be regulated by the LTR10 enhancers.
To get more information on potential regulatory targets of LTR10 enhancers, they used RNA sequencing to look for changes in gene expression following LTR10 knockout. These experiments identified genes such as ATG12, XRCCR and VCAN, which are known to be associated with cancer development and progression, as targets of LTR10 regulation.
ATG12 encodes a protein that is required for macroautophagy, mitochondrial homeostasis and apoptosis. Expression of ATG12 is associated with cancer development and drug resistance. To determine if the LTR10.ATG12 enhancer regulated ATG12 gene expression in HCT116 cells, the researchers first asked if silencing the enhancer caused ATG12 protein levels in the cells to decrease, which it did.
They next treated the cells with staurosporine (STS) to trigger mitochondrial apoptosis. Cells where the enhancer was silenced showed reduced caspase-3/7 activity as assessed by the Caspase-Glo® 3/7 Assay. Caspases 3 and 7 are enzymes involved in the initiation and execution of apoptosis.
XRCCR is a DNA repair gene that has been implicated in tumor resistance to chemo- and radiation therapy. VCAN encodes an extracellular matrix protein that has been implicated in tumor metastasis. Looking at the LTR10.XRCC4 enhancer, which regulates expression of both genes, the researchers asked if knockout of the enhancer influences the response of cells to irradiation. Using the CellTiter-Glo® Luminescent Cell Viability Assay, they assessed viability of the knockout versus wildtype cells exposed to radiation and found that the knockout cells had decreased viability compared to wildtype cells.
Summary
These experiments provided compelling evidence that LTR10 elements, through their enhancer activity, significantly influence gene expression and cell behavior in colorectal cancer. This work highlights how transposable elements in the genome that are normally silent can become reactivated resulting in aberrant gene regulation, cancer development and progression, and therapeutic resistance, and identifies a potential new target in cancer therapy research.
Citation
Ivancevic, A. et al. (2024) Endogenous retroviruses mediate transcriptional rewiring in response to oncogenic signaling in colorectal cancer. Science Advances 10(29).
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