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SciLifeLab Team IDs RARA as Possible Protein Biomarker for Tamoxifen Resistance in Breast Cancer

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A team led by researchers at the Science for Life Laboratory Stockholm has linked the nuclear receptor protein retinoic acid receptor alpha to tamoxifen resistance in breast cancer.

According to Janne Lehtiö, platform manager for mass spectrometry at SciLifeLab and leader of the effort, the findings suggest that RARA could serve as a biomarker for predicting patient response to tamoxifen treatment as well as a therapeutic target.

Detailed in a paper published last week in Nature Communications, the study characterized populations of the breast cancer cell line MCF7 and the tamoxifen-resistant breast cancer cell line MCF7/LCC2 both treated and untreated with the tamoxifen metabolite 4-OHT.

Using a workflow consisting of upfront isoelectric focusing followed by nanoLC and mass spec analysis on an AB Sciex 4800 MALDI TOF/TOF and an Agilent 6530 QTOF, the researchers quantified 830 proteins via iTRAQ isobaric labeling. Upon performing a network analysis of proteins exhibiting altered expression across three different comparisons -- basal level changes between MCF7 and LCC2, and response of MCF7 and LCC2 to 4-OHT – they identified RARA as a central network connector in two of these three comparisons.

This, Lehtiö told ProteoMonitor, led the researchers to focus on RARA as a potential agent involved in tamoxifen resistance. The protein had previously been studied as a biomarker for purposes like breast cancer prediction, he noted, but, there hadn't yet been work linking it specifically to tamoxifen resistance.

Tamoxifen is commonly used to prevent recurrence in estrogen receptor-positive breast cancer patients, but around one-third of patients treated with adjuvant tamoxifen ultimately relapse. Researchers have looked into a variety of mechanisms linked to the development of resistance, such as MAP kinase activation and certain forms of HER2 activation, but, Lehtiö said, he was surprised upon looking into the question at the relatively small amount of proteomics work that had tackled it.

In some ways, he said, tamoxifen resistance provided an excellent model for him and his colleagues to test the ability of their proteomic workflows to identify predictive disease markers.

"There is a long patient follow up and a lot of patient material available on [breast cancer] therapy by tamoxifen," he said. "So that was the rationale" for focusing on this question.

Among existing proteomic research into tamoxifen and breast cancer is work done by Duke University researcher Victoria Seewaldt in collaboration with Theranostics Health using that company's reverse phase protein array technology (PM 11/12/2010).

Lehtiö called the RPPA work a "very interesting way to look at this [question], as well," noting that while the approach doesn't offer the breadth of mass spectrometry, it doesn't suffer from the throughput challenges that still limit mass spec analysis.

"It is still quite difficult to run large series of clinical samples on a mass spectrometer," Lehtiö said. "You want to have comprehensive, in-depth analysis, but that is time consuming and quite expensive. So usually the tradeoff is to either run many samples with quite shallow proteomics or in-depth proteomics on mass spectrometry with only a few samples."

Citing developments like increased isobaric tag multiplexing and combined isotopic-isobaric labeling techniques like the NeuCode method published this year by University of Wisconsin-Madison research Josh Coon (PM 3/1/2013), Lehtiö suggested that "higher throughput clinical proteomics will be in reach within a few years." However, to date, his group has done in-depth mass spec characterization of "at most 24 clinical samples in a single study," he said.

In the Nature Communications study, Lehtiö and his colleagues followed up their mass spec profiling with antibody-based work to look for a link between RARA expression in breast cancer samples and tamoxifen resistance, using Western blotting, ELISA, and immunohistochemistry to measure RARA levels in 8, 72, and 45 clinical samples, respectively.

These measurements found that higher RARA levels were correlated with shorter recurrence-free survival in ER-positive breast cancer patients receiving adjuvant tamoxifen, with patients with high RARA levels showing a 4.1-fold higher rate of relapse. RARA levels were not predictive, however, within the first four years of follow-up – the drug treatment and drug overhang period.

Moving forward, the researchers plan to look at RARA levels and tamoxifen resistance in larger clinical cohorts, Lehtiö said, noting that they have two such studies ongoing.

If those studies return a positive result, they will then try to put together a clinical trial using RARA as a predictive marker to select patients for treatment, he added. Given the follow-up required for such a trial, however, this would be a long-term research goal, he said. "In breast cancer these events are quite slow, so you have to have at least five years to have enough clinical events to conclude anything."

Lehtiö added that the researchers might also expand the scope of the study to investigate whether RARA is predictive of other estrogen-blocking therapies like aromatase inhibitors.

He suggested they could also look further into a finding from the study that while the resistant LCC2 cells were not sensitive to tamoxifen, their proteomes did change in response to treatment with the breast cancer drug fulvestrant – marketed as Faslodex by AstraZeneca – in a way that suggested that agent might prove an effective therapy in such patients.

Citing the considerable amount of previously published work on RARA in breast cancer, Lehtiö said he and his co-authors had no plans to patent any of the paper's findings.

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