Periodic Reporting for period 1 - HspAdhesion (Role of cell membrane associated Hsp70 in cancer cell adhesion and metastasis)
Reporting period: 2015-05-01 to 2017-04-30
High levels of Hsp70-1A have been reported to promote cell motility and the transition of across membranes. These processes are highly relevant for tumor invasion and the development of metastasis. We hypothesized that the presence of mHsp70 indicates alterations in differential adhesion and compared the interaction of mHsp70 positive and mHsp70 negative tumor cells with endothelial cells that line the inner surface of blood and lymphatic vessels. We attached individual tumor cells to a micro-cantilever to bring them into contact with the lining cells and measured the interaction forces during separation (Figure 1B). Our data revealed strong interaction of mHsp70 negative cells with blood vessel lining cells and measurably lower interaction for mHsp70 positive cancer cells. Low interaction with blood vessel represents an advantage for circulating tumor cells and could contribute to the development of distant metastasis.
The mechanism of cancer exclusive anchorage of mHsp70 on the cell surface is currently unknown and discussed controversially. Hsp70-1A lacks conventional cues for translocation from the inner of the cell to the surface. The membrane anchorage may thus follow a new paradigm that could possibly apply to other cancer markers. We believe that the anchoring mechanism involves specific protein-lipid interactions and hold the key for deciphering the role of mHsp70 in cancer. We conducted investigations with the atomic force microscope on model membranes which mimicked the inner and outer leaflet of the cell membrane. We observed clusters of Hsp70-1A in very specific lipid environments that may be found predominantly in certain tumors (Figure 1C). Under normal conditions these lipids are restricted to the inner leaflet of the cell membrane. Once they translate to the surface recognition procedures are triggered that result in removal of the cell by macrophages and production of autoantibodies. Hypoxia in stressed tumor cells, e.g. in response to radiation treatment may lead to the export of these lipids. However, in association with Hsp70-1A they remain undetected by the immune system leading to the survival of cancer cells despite radiation treatment. Our findings conclusively link a possible mechanisms for anchorage of mHsp70 to a potential role in the development of resistances to radiation and chemotherapy.
In WP2 we studied the insertion of mHsp70 into the plasma membrane by AFM and confocal fluorescence microscopy on the single protein level. We prepared model lipid membranes that mimicked the outside and insdide of the cell membrane in normal and cancer cells. Detailed investigations of a previously proposed anchoring mechanism that suggested a rare glycosphingolipid (Gb3) as interaction partner revealed shortcomings of this model. We then tested different lipid environments and conditions to induce insertion of Hsp70-1A into model lipid membranes and explored the influence of cholesterol, which is often enriched in cancer cells. We found that Hsp70-1A did not insert into the outer leaflet of the membrane under normal condition. Instead, we observed a concentration dependent in membranes that contained saturated phosphatidylserine (PS). Cancer cells are often enriched in saturated phospholipids, which protect them from oxidative damage and inhibit uptake of chemotherapeutics. PS is mostly present on the indide of the membrane but radiation treatment and chemotherapy can lead to the export of PS to the surface. Under normal condition the presence of PS on the surface triggers an immune response and the removal of the cell, but association with Hsp70-1A can mask PS. Thus the enrichment of cancer cells with saturated PS in combination with over-exessive production of Hsp70-1A and treatment with therapeutics and/or radiation may potentially result in the exhibition of mHsp70 in certain cancer cells and cause immunity.
The work performed in HspAdhesion a resulted in two book chapters and four journal manuscripts, of which one is already published. The project and its achievements were presented to the scientific community at 3 international conferences and to the general public.
We reproduced different environments and conditions of the cell membrane and directly visualized the insertion of Hsp70-1A on the single protein level. Based on our results we suggest a conclusive scenario that links for the first time the mechanism for transport of Hsp70-1A from the cytosol to the membrane, its anchorage on the surface and potential role in the development of tumour resistance to radiation and chemotherapy.
We applied biophysical methods on the single molecule and single cell level to explore parameters and key questions that have been inaccessible by standard techniques in medical research. The results of this project advance our understanding of the modes of action of Hsp70-1A in tumor progression and demonstrated the complexity of functional consequences of the plasma membrane association of Hsp70-1A in cancer diseases.