October is Breast Cancer Awareness Month in the United States. Breast cancer is the second most common cancer in women, with about 1 in 8 women in the US developing breast cancer at some point in their lives. Biophysical Journal author Niels De Jonge, INM - Leibniz Institute for New Materials, Saarland University, shares information about his research on HER2-overexpressing breast cancer.
What is the connection between your research and breast cancer?
My research program includes an externally-funded project on HER2-overexpressing breast cancer. Two postdocs and a PhD student work on this project, and I also have extensive collaborations with three external groups on this topic. Results have been published in three papers, Sci. Adv. 1:e1500165, 2015; Mol. Biol. Cell 28, 3193-3202, 2017; Biophys. J. 115, 503-513, 2018. See the website: http://www.dejonge.physik.uni-saarland.de
Why is your research important to those concerned about breast cancer?
We have developed a new analytical method with unique capabilities to study membrane protein function and drug response at the single molecule and single cell level, combined with the capability to study hundreds of cells and to detect rare cells “hiding” within the bulk cancer cell population. I expect that breast cancer research will benefit from the new viewing angle provided by our new method, which is explained as follows: The current generation of antibody-based drugs against HER2-overexpressed breast cancer is very sophisticated but nevertheless ineffective within a year due to drug resistance development. Standard analytical methods used in biomedical research are based on population averages and so drug effectiveness is mainly addressing the bulk cancer cell population. But individual, rare cancer cells may not respond as desired to the drug, as is known in literature, and was quantified using our new method (Mol. Biol. Cell 28, 3193-3202, 2017). While they are insignificant early on, those cells may grow into larger populations and use different signaling pathways to trigger their growth, on which the drug has no influence. By addressing not only the bulk cancer cell population but also accounting for cancer cell heterogeneity to find the “exceptions to the rule” using our microscopy method, it may become possible to test new drug combinations on such rare cells, with the aim of providing prolonged drug effectiveness and thus extending the lives of patients.
How did you get into this area of research?
In 2005, I set out to develop a novel electron microscopy method for imaging whole cells in liquid such to be able to study protein function in cells in a state as close as possible to the native state. This research started at Oak Ridge National Laboratory, USA, and later in a joint position with Vanderbilt University School of Medicine, USA. A milestone was the imaging of labeled epidermal growth factor proteins in intact COS7 cells in liquid with 4 nm resolution, published in Proc. Natl. Acad. Sci. 106, 2159-2164, 2009. We also studied live yeast cells (Biophys. J. 100, 2522-2529, 2011), and several other topics. The new field of science opened up by liquid-phase electron microscopy was reviewed in Nature Nanotechnology 6, 695-704, 2011. At that time, I did not want to continue with pure technology development, but aimed instead to apply the technique to challenging topics in medicine. My thought was that a novel analytical method would likely lead to new biomedical insights. I was inspired to enter the field of cancer research when I gave a lecture at a workshop on transient molecular complexes organized by the National Cancer Institute in San Francisco, 2009. I searched for about 4 years to find the right topic that would potentially benefit from my analytical approach. The idea to study breast cancer followed shortly after I obtained a permanent position as senior group leader with excellent base funding :) at the INM-Leibniz Institute for New Materials in Saarbrücken Germany in 2012. Research on the HER2 receptor in breast cancer was established in a collaboration with the German Cancer Research Center in Heidelberg (DKFZ). Accomplishing a breakthrough required combining expertise from several disciplines. The pioneering work was possible because I joined my expertise in physics, biophysics, and electron microscopy with the biomedical expertise of my long-term team member Dr. Diana B. Peckys, who has a biomedical background.
How long have you been working on it?
My first study in cancer involved lung cancer cells, establishing the method to study membrane proteins in cancer (Scientific Reports 3, 2626-1-6, 2013). Together with scientists from the DKFZ, we started to work on HER2 shortly afterwards and published our first exciting biological result in Sci. Adv. 1:e1500165, 2015. But as stated above, the research on liquid-phase electron microscopy already started in 2005. So, it took about 10 years to develop new technology and to obtain scientific results of biomedical relevance.
Do you receive public funding for this work? If so, from what agency?
I received a grant for the Else Kröner-Fresenius-Stiftung in Germany for the project entitled: “Investigation of the Influence of Breast Cancer Drugs on HER2 Dimerization at the Molecular Level in Individual Cells Aiming to Find Clues for Causes of Drug Resistance: HERe”. The project aims to gain better understanding of the role of cancer heterogeneity in mechanism of the drug trastuzumab in cell lines and in biopsy samples. I hope to discover a molecular signature in cancer cells from patients, serving as predictor for drug resistance. Cooperation partners include the German Cancer Research Center, Roche Diagnostics GmbH, and the University Hospital of Saarland. I also received various other grants not listed here for completed projects.
Have you had any surprise findings thus far?
Most striking to me was to discover that despite the large body of literature on HER2, still key open questions exist about the functioning of this receptor and about the mechanisms of the HER2-targeting drug trastuzumab. My explanation is that most research is based on indirect analytical methods in which the membrane proteins are extracted from cells and material is pooled from many cells, for example in biochemical methods, in X-ray crystallography, and in cryo-electron microscopy. One then loses the cellular context and also any information about diversity between membrane areas in individual cells, and between cells. Even the question of whether HER2 forms signaling active homodimers (pairs) was not conclusively answered in literature because the HER2 protein is not stable when extracted from the plasma membrane. With our method we were able to directly look at the spatial organization of the protein-subunits at a single cell level, while at the same time being able to study hundreds of cells. We not only confirmed the presence of HER2 homodimers but also found an interesting phenomenon that these homodimers are only present in highly dynamic, ruffled membrane regions and that resting cells do not appear to have the receptor in the signaling active homodimeric state (Sci. Adv. 1:e1500165, 2015). In our next study, we found that the drug trastuzumab induced HER2 down-regulation by uptake into the cell, which would explain that reduced cell growth signaling is responsible for drug action. Most importantly, we also discovered a small subpopulation of cells in which HER2 was only present as signaling inactive monomer and those were not up-taken upon drug incubation (Mol. Biol. Cell 28, 3193-3202, 2017). We now speculate that we are onto something important that could possible provide an important piece of the puzzle in understanding drug resistance development. Most recently, we found HER2 to be oriented in linear chains and this was a big surprise to us as that would possibly indicate the presence of larger oligomeric structures connected to the cytoskeleton (Biophys. J. 115, 503-513, 2018).
What is interesting about the work from the perspective of the other researchers?
Liquid-phase electron microscopy presents a paradigm change in studies of protein function and drug response in cells. Notwithstanding a huge body of literature using a wide variety of methods, the capability to probe the functional state of an endogenous protein via its subunit configuration in intact cells and with physiologically relevant protein densities is extremely difficult if not impossible with other methods discussed elsewhere (Sci. Adv. 1:e1500165, 2015). The fact that open questions and even contradiction exist about HER2 and the corresponding prescription drug emphasizes the limitations of the current analytical methods, and the need for new approaches. As we have already demonstrated, it will help cancer research to obtain single molecule and single cell data and at the same time collecting data from hundreds of cells thus addressing cancer cell heterogeneity, as is provided by our new method. Also, beyond cancer, our approach provides unique information about the functioning of membrane proteins in cells for a wide variety of proteins and cells.
What is particularly interesting about the work from the perspective of the public?
Novel insights into to functioning of HER2 and other proteins, and drug responses in cancer cells will likely lead to new ideas for the development of new drugs against cancer. HER proteins are major targets for antibody-based drugs, the market of which is predicted to reach sales of $120 billion by 2021. Worldwide, an estimated 500,000 women died from breast cancer in 2011, many due to HER2-overexpressing cancer. But despite widescale efforts in research and the development of cancer medications, the problem of drug resistance in tumors is of unchanged urgency, leading to severe suffering of patients and causing tremendous costs associated with dysfunctional therapy. The annual costs for a trastzumab therapy are currently ~50,000 € per patient (in Germany). Our research will provide novel insights into the molecular functioning of the anti-HER drug trastuzumab, and possibly demonstrate that HER2 cancer drug therapy should be supplemented to work effectively not only for the average cell but also for low abundance cells with different drug response to avoid the development of drug resistance.