Humans only need one active X chromosome, which means that people with two must shut one down to avoid doubling up on its protein products. The gene tasked with inactivating one X, called XIST, codes for a long noncoding RNA that coats and epigenetically silences the extra X chromosome early in the development of people with two X chromosomes. XIST has previously been linked to cancer in females, as well as to testicular cancer. Now, in a study published November 9 in Cell Systems, researchers have found XIST activity in cancers in various somatic tissues of males, too.
Irregular XIST activity has been linked to testicular cancers, which arise from male germ cells. But in the new study, the researchers found high levels of XIST RNA in male somatic cells sampled from cancerous tissues. While the study doesn’t show a causal link been XIST expression and male cancers, it finds that XIST shuts down X chromosome gene expression in adult male somatic cells, which are thought to need the proteins encoded on the X chromosome. This silencing likely has deleterious effects, the researchers say.
“We’ve known about the testicular cancers having XIST, and we’ve probably suspected that there’s XIST in some other [cancers], but to actually do such a detailed study across all types of cancers—that’s really fascinating,” says Carolyn Brown, a geneticist at the University of British Columbia who was not involved in the study. “It shows that we miss things.”
Study coauthor Srinivas Viswanathan, a medical oncologist at Dana Farber Cancer Institute in Boston, explains that the discovery occurred “a bit incidentally.”
He and his team had been analyzing transcriptome information for cells in The Cancer Genome Atlas Program (TCGA) database for the presence of XIST RNA. They expected to see XIST in tumors, but only in female cells and testicular cancers. The researchers “happened to notice that the samples that were annotated as male expressed [XIST] as well,” Viswanathan says. Looking further, they found that 4 percent of all cancers in males expressed XIST, and of those cancers, 74 percent were testicular. But the other 26 percent were from other tissues, including lung, kidney, heart, and liver. “The conventional thought is that XIST is a female-exclusive transcript,” says Viswanathan. “[Males] aren’t supposed to have X inactivation. [That was] quite surprising.”
To make sure that XIST expression wasn’t coming from healthy cells, the team used RNAseq data to measure the amount of XIST RNA normally present in the healthy tissues of people of both sexes. When they compared the aggregate data, they saw that on average cancer cells expressed higher levels of XIST than healthy male tissues—an amount that was comparable to that of female tissue. Cells were labeled as XIST+ if they expressed XIST at a similar level as female tissue. In a few cases, the researchers had samples from both healthy and cancerous tissue from the same individual. In these cases, they only found XIST expression in cancerous cells.
Using the TCGA’s genomic sequencing data, the researchers found that XIST expression was associated in many samples with X chromosome aneuploidy, a phenomenon where cells contain multiple copies of a chromosome. DNA in cancer cells is notoriously unstable, and these cells have high rates of aneuploidy from incomplete cell division. Only roughly half of the XIST+ cancers had multiple X chromosomes, however. The researchers are still searching for a mechanism to explain XIST’s connection to cancer.
XIST RNA also carried out its usual role of silencing X chromosome gene expression when it appeared in cancer cells, according to analyses of RNAseq and ATACseq (Assay for Transposase-Accessible Chromatin sequencing, a technique to assess how accessible DNA is to transcriptional proteins) data. The researchers found fewer genes from the X chromosome expressed in XIST-containing cancer cells, indicating that XIST was likely silencing X-linked genes. In another experiment, they used a cancer cell line known to express XIST and knocked out the transcript, finding that they could reverse the silencing effect and restore gene expression. This indicated that XIST is indeed responsible for the silencing effect.
Brown says that the study “provides [evidence] for a very interesting concept: that XIST, acquired somatically, can still be perhaps functioning to silence a chromosome.” She says that this raises the question, “Which genes are sensitive in the somatic cells to the presence of XIST?”
Study coauthor and Harvard Medical School computational biologist Cheng-Zhong Zhang says that while the findings don’t have any direct clinical impact, the study highlights the need for researchers to take sex into account when searching for new therapeutic targets. He adds that researchers often ignore sex when they analyze the gene expression of cancer cells, which is likely why researchers missed the XIST connection previously.
“Sex itself is a biomarker. I think that’s been underappreciated,” says Viswanathan. “This is one example of a biological baseline difference between males and females leading to cancer-specific differences."