NEW YORK (GenomeWeb) – Harvard University researchers have completed a proteomic analysis of circulating breast tumor cells that suggests new approaches to treating certain forms of the disease.
In a study published this week in Nature, the researchers analyzed hormone receptor-positive, HER2-positive cell populations that developed from HR-positive, HER2-negative primary tumors. They found that although these cells were not susceptible to HER2-targeted therapies, particular combination therapies were effective.
The work builds on an "underlying observation, that has been made by other groups, as well, that if you have hormone receptor-positive breast cancer patients who don't have HER2 expression, after several cycles of treatment, if you look at their CTCs, some will have acquired HER2 expression," Shyamala Maheswaran, an associate professor of surgery at Harvard Medical School and author on the study, told GenomeWeb.
This finding, she noted, has generated excitement among clinicians who hoped it might mean these patients were now sensitive to HER2-targeted treatments like Genentech's Herceptin (trastuzumab) or GlaxoSmithKline's Tykerb (lapatinib). The Harvard researchers' analysis indicates, though, that while these cells do start expressing HER2, this expression remains lower than in other HER2-positive cells, and that these cells are not, in fact, susceptible to HER2-targeted therapies.
The researchers' mass spec analysis did, however, identify differences between the HER2-positive and HER2-negative populations that could be used to inform treatment of these tumors, Maheswaran noted.
"The HER2-positive cells were basically not addicted to HER2, which is why they don't respond to HER2 therapy," she said. "However, they are driven by multiple signaling pathways that allow them to proliferate relatively quickly compared to the HER2-negative cells, and as a result, they are quite sensitive to chemotherapeutic drugs."
The HER2-negative cells, meanwhile, showed activation of Notch and DNA damage signaling pathways, and while they were resistant to chemotherapy, they were sensitive to Notch inhibition.
To get at the differences between these two breast cancer cell subpopulations, Maheswaran and her colleagues used isobaric tandem mass tag (TMT) reagents to quantify their proteomes, measuring the levels of more than 6,300 proteins. For this, they used the MultiNotch MS3 multiplexing method developed by Harvard researchers Steven Gygi and Wilhelm Haas (a co-author on this week's Nature paper), which resolves precursor interference issues associated with isobaric tagging without significant losses in sensitivity.
Isobaric labeling uses stable isotope tags attached to peptides of interest to enable relative or absolute quantitation of proteins via tandem mass spectrometry. Digested peptides are labeled with tags that fragment during the second stage of fragmentation, MS2, to produce signals corresponding to the amount of peptide present in a sample.
Because quantitation is done at the MS2 level, precursor interference can be a problem. With isobaric tagging, researchers isolate a precursor peptide and fragment it with MS2, causing the tag's reporter to fall off, with the proportion of these reporter signals correlating to the proportions of a given peptide in the tagged sample.
However, other precursors can fall into the fragmentation window for a given peptide, and the reporters released by these additional precursors can interfere with the reporters released by the target peptide, making quantitation unreliable in some cases.
One approach researchers including Gygi and Haas have taken to dealing with this problem is to do quantification at the MS3 level, which adds another level of ion isolation and fragmentation, reducing the precursor interference issue. Proteome Sciences, which manufactures the TMT reagents, has developed three-stage TMTs for use in such workflows. The MultiNotch method used in the new Nature paper improves upon conventional MS3 TMT workflows by increasing the amount of reporter ions present in the MS3 spectrum, which improves the accuracy and sensitivity of quantitation.
Using the MultiNotch MS3 method on a Thermo Fisher Scientific Orbitrap Fusion instrument to perform 10-plex TMT assays, the researchers identified the aforementioned protein differences between the HER2-negative and HER2-positive cell populations. They then followed up on these findings by screening these cells against a panel of 55 drugs to see which might be able to target the pathways identified as differentially regulated by their mass spec analysis.
From this they found, as Maheswaran noted, that the HER2-positive cells were not susceptible to HER2-targeted treatment but did respond to chemotherapy, and that the HER2-negative cells responded to Notch inhibitors but not to chemotherapy.
The researchers also observed that CTC populations not only shifted from HER2-negative to HER2-positive in response to treatment, but also from HER2-positive to HER2-negative.
"The concept is that here is a kind of mechanism where the cells are able to spontaneously oscillate between these two states, and thereby not only can they evade therapy, but have slightly different tumorigenic properties," Maheswaran said. As the authors noted, this ability of breast cancer cell subpopulations to switch between HER2 statuses could explain the limited success of clinical trials using inhibitors pathways like Notch signaling to prevent cancer growth after chemotherapy.
Instead, Maheswaran suggested, simultaneous treatment with chemotherapy and, for instance, a Notch inhibitor could be necessary. To test this notion, she and her colleagues established mouse models with tumors containing both HER2-negative and acquired HER2-positive cells and treated them with this sort of combination therapy.
"When we treated these mice with either the Notch inhibitors or the chemotherapeutic drugs [separately], the tumors were [still] partially active," she said. "But when we treated them with the combination, it eliminated the entire tumor and the recurrence occurred very, very slowly."
She added that while the Nature study focused on HER2-based heterogeneity, it is likely that there are other forms of heterogeneity that could be relevant to treatment. "My feeling is that we have just uncovered the tip of the iceberg," she said.
Maheswaran said she and her colleagues are now trying to determine what mechanisms trigger the switch from HER2-negative to HER2-positive status.
"We are interested in looking at the differences in the epigenetic landscape of these two populations, hoping that that might give us an idea of what the difference is due to," she said. "We are also planning to look at the different secretions from the different populations to see if there is something that might be going between these two cells to get them into the different molecular states."
"We know that if we subject the cells to stress they do switch, but we don't know exactly what this spontaneous switch is due to," she said. "So that is the next level of investigation."