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Team IDs Population of HER2-Postive Stem Cells in HER2-Negative Breast Cancer Patients

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A team led by researchers at the University of California, Davis, and Lawrence Livermore National Laboratory has identified a population of HER2-overexpressing breast cancer stem cells that they said could be responsible for radioresistance in HER2-negative breast cancer.

In addition, the team performed proteomic analysis of these cells, comparing them to HER2-negative BCSCs to identify proteins potentially involved in the radioresistant phenotype.

The researchers now hope to apply similar proteomic profiling to BCSCs taken from patients at several time points throughout their treatment with the aim of better understanding the mechanisms of resistance, Jian Jian Li, director of translational research in the UC Davis Department of Radiation Oncology and leader of the effort, told ProteoMonitor.

Published in the current edition of Clinical Cancer Research, the study built on previous findings that overexpression of HER2 in HER2-negative MCF7 cells enhances their radioresistance, and that an NFkB-binding site in the HER2 promoter region is linked to HER2 transactivation in radiation-treated HER2-negative breast cancer cells.

"The mechanisms involved in HER2-mediated repopulation of BCSCs under radiation treatment … remain to be elucidated," however, the authors noted.

In the study, Li and colleagues identified HER2-expressing BCSCs in groups of both in vivo and in vitro radiation-treated HER2-negative breast cancer cells and in tumors taken from breast cancer patients. These HER2-positive BCSCs were found in 57.1 percent of primary tumors and 86.4 percent of recurrent tumors.

Their findings, the authors wrote, suggest that HER2 expression in the primary tumors could be induced by treatment, leading to elevated HER2 levels in the recurrent tumors and an increase in their aggressiveness. Alternately, these BCSCs could be lurking pretreatment as a small population of HER2-positive cells that then increase as a proportion of the total tumor cells as the HER2-negative cells are killed during treatment.

Either scenario, Li suggested, could lead to breast cancers that were initially diagnosed as HER2-negative expressing increased levels of HER2, making them potential candidates for treatment with anti-HER2 drugs like trastuzumab – marketed by Roche as Herceptin.

In the Clinical Cancer Research study, the researchers used LC-MS/MS on a Thermo Scientific LTQ Orbitrap XL instrument to compare the proteomes of HER2-positive and HER2-negative BCSCs, looking for factors responsible for the former populations' radioresistant phenotype. They found 499 and 182 proteins upregulated, respectively, in the HER2-positive and HER2-negative cells.

Of particular interest, Li said, was the finding that the proteins STAT3 and Src were expressed in the HER2-positive but not the HER2-negative cells. HER2 activates STAT3 via both JAK2 and Src pathways, leading to increased tumor aggressiveness. He added that his team was now investigating whether STAT3 and HER2 co-expression might prove a useful clinical marker.

The proteomic analysis also identified upregulation in the HER2-positive BCSCs of a number of prosurvival proteins, including those involved in DNA repair, angiogenesis, cell adhesion and proliferation, metastasis, mTOR signaling, mitochondrial ATP generation, and redox balancing.

The study, Li said, suggests that HER2 expression in breast cancer "is a moving target" that can change over the course of a patient's treatment. Given that, Li said he now hopes to expand proteomic study of these BCSCs to track changes in their protein profiles throughout the treatment process.

"I would be very interested to follow this in the same patient before, during, and after treatment," he said. "It would allow dynamic mapping of the signature of the [treatment]-resistant genes."

"Perhaps proteomic profiling can show that in a specific part of the [tumor cell] population, some part of the [protein] profile is changing," Li said. "For example, at three months [into treatment], all the HER2-negative patients may have a particular pattern. Then HER2 is induced, proteins downstream are induced, and maybe during that time, resistance develops in that tumor."

He noted that his lab has been working for the last two years to set up such a project, but has run into significant difficulties, particularly with regard to lining up the necessary patient samples.

The researchers are aiming to get patient samples for proteomic analysis at three time points – before treatment, during treatment, and then several years after treatment once the cancer has recurred. However, Li said, thus far they have managed to obtain only two suitable samples.

The group's findings fit with previous observations that some populations of HER2-negative breast cancer patients receive benefit from anti-HER2 treatment.

In another recent paper in Clinical Cancer Research – this one published in October – scientists from George Mason University also identified a subset of HER2-negative patients that exhibited activation of HER2 pathways (PM 10/12/2012).

Unlike the samples examined by Li and his colleagues, these HER2-negative patients didn't show treatment-induced HER2 expression, but, rather, exhibited phosphorylated HER2 and activation of HER2 signaling pathways despite testing negative for HER2 expression via conventional immunohistochemistry and fluorescence in situ hybridization tests.

"There is data [from past studies] indicating that there are certain patients that are not HER2 positive that are responding to anti-HER2 therapy," Julia Wulfkuhle, a GMU researcher and first author on the paper, told ProteoMonitor at the time. "There have been observations from several trials… that [such] a population of patients might exist."

The GMU team is now collaborating with Soonmyung Paik, director of the Pathology Division at the National Surgical Adjuvant Breast and Bowel Project, to measure HER2 phosphorylation and signaling in sample sets like those from the project's B-31 clinical trial that found evidence of trastuzumab response in HER2-negative patients.

GMU researcher Emanuel Petricoin, one of Wulfkuhle's co-authors on the HER2 phosphorylation study, told ProteoMonitor this week that while Li's study highlighted a different phenomenon than the GMU team's HER2 phosphorylation findings, it potentially fit with observations the group had made during the Biomarkers Consortium's I-SPY breast cancer study that increasing HER2 expression post-treatment in HER2-negative patients was a negative prognostic.

"That increase indicated basically a lack of response," Petricoin said. "That fits nicely with what Li described in that perhaps [via treatment] we are selecting out this [HER2-positive] stem cell population."

He added that he and his colleagues have been in touch with Li about possibly looking into this question together.

"We'd like to go back and see if those cells [with increased HER2 expression] had increased [expression of the stem cell marker] CD44, because that could suggest that this is the stem cell population that Li describes is expanding," he said. "And that could potentially be a really interesting [negative] prognostic marker."

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