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Study Points to New 'Pioneer Factor' Role in ER+ Breast Cancer Proliferation

By Andrea Anderson

NEW YORK (GenomeWeb News) – By profiling genomic patterns of a DNA-binding protein called PBX1 in breast cancer cells, Dartmouth Medical School researchers have shown that it serves as a "pioneer factor," paving the way for the estrogen receptor-positive breast cancer proliferation in response to the estrogen hormone.

As they reported online last night in PLoS Genetics, the group used a combination of chromatin immunoprecipitation sequencing, or ChIP-Seq, and formaldehyde assisted isolation of regulatory elements-based sequencing, called FAIRE-Seq, to map PBX1 binding across the genome and to determine the effects that this binding has on chromatin structure.

Based on their findings, investigators propose that PBX1 sets up shop at many estrogen receptor-alpha (ER-alpha) binding sites prior to the ER-alpha transcription factors arrival. The presence of PBX1 appears to open chromatin, they found, recruiting ER-alpha to these more open sites in ER-positive breast cancer cells in response to estrogen. Without this so-called "pioneer factor" activity, on the other hand, ER-alpha's ability to turn on certain genes in response to estrogen seems to be altered.

From analyses of available breast cancer transcriptomes, the team also found evidence that higher PBX1 expression in ER-positive breast cancers corresponds to more aggressive tumors and higher metastasis risk.

"We know already that estrogen receptor-positive patients do much better compared to estrogen receptor-negative patients," first author Luca Magnani, a post-doctoral researcher in study leader Mathieu Lupien's Dartmouth lab, told GenomeWeb Daily News. "But when we specifically look at the estrogen receptor-positive group, we see that PBX1 gives you an additional layer of prediction."

Pioneer factors are a recently identified set of transcription factors that recognize certain epigenetic marks, bind to compacted regions of chromatin, and open up these chromatin regions, making the chromatin accessible to other transcription factors that they help recruit, Magnani explained.

"They can bind chromatin even if it is compacted," he said. "They don't have chromatin remodeling abilities, per se, but they can still bind very tightly to this chromatin. Other transcription factors normally only bind regions of chromatin that are wide open."

Only a handful of these pioneer factors have been identified so far, he added, including FoxA1, a pioneer factor that facilitates some ER-alpha interactions in the genome. But because ER-alpha is found at many sites in the genome where FoxA1 is absent, the team decided to screen for other pioneer or transcription factors with possible roles in ER-positive breast cancer — a search that helped them narrow in on PBX1.

From their initial analyses, along with expression database information and published data, researchers found that PBX1 expression and ER-alpha expression were well correlated in breast cancer, hinting that the two factors might work together, Magnani explained.

By curbing PBX1 protein expression in an ER-positive MCF7 breast cancer cell line, on the other hand, researchers prevented proliferation of these cancer cells in response to estrogen, even though the estrogen receptor itself was still expressed.

To learn more about PBX1's apparent role in ER-positive breast cancer response, researchers used ChIP-Seq and FAIRE-Seq to look at PBX1 binding sites and chromatin patterns at these sites, respectively.

The estrogen receptor is only recruited to binding sites in the genome when cells are exposed to the hormone, Magnani said.

But the team's ChIP-Seq and other experiments uncovered PBX1 at the majority of ER-alpha binding sites, including some sites where FoxA1 is also found. PBX1 binding at these sites occurred both before and after cells were exposed to estrogen.

"This supported the idea that PBX1 binds in any case and then following estradiol stimulation the estrogen receptor goes to PBX1-marked chromatin," Magnani explained.

When cells were exposed to estrogen, though, removing PBX1 interfered with estrogen receptor binding.

Consistent with its proposed role as a pioneer factor promoting chromatin openness, the researchers' FAIRE-Seq experiments suggested that breast cancer cells missing PBX1 had much more compact chromatin.

When they brought ChIP-Seq and FAIRE-Seq information together, Magnani noted, researchers saw that PBX1 binding sites in the genome also tend to have a relatively open chromatin structure.

Finally, through a meta-analysis of data from Compendia Bioscience's cancer transcriptome database, Oncomine, the team found clues that PBX1 levels may provide prognostic information for breast cancer cases involving ER-positive tumors, since high PBX1 expression correspond to elevated metastasis risk and to the expression of other genes linked to poor outcomes and aggressive cancers.

"Together, our results reveal that PBX1 is a novel pioneer factor defining aggressive ER-alpha-positive breast tumors, as it guides ER-alpha genomic activity to unique genomic regions promoting a transcriptional program favorable to breast cancer progression," the researchers wrote.

The researchers are currently doing additional PBX1 ChIP-Seq experiments in a "long term estrogen-deprived" breast cancer cell line — estrogen-independent cells that were derived from ER-positive MCF7 cells that were grown in the absence of estrogen over long periods of time.

Such cells serves as a model for resistance to aromatase inhibitor treatments for breast cancer, Magnani explained, since they still express high levels of the estrogen receptor but are able to grow without estrogen. Along with studies of PBX1 patterns in these estrogen-independent cells, the team is testing compounds that may curb PBX1 activity.