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Role of cell membrane associated Hsp70 in cancer cell adhesion and metastasis

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

Hsp70-1A is the major stress-inducible member of the HSP70 chaperone family. The protein is being implicated in cancer diseases with the development of resistances to standard therapies, increased invasiveness and metastasis, and poor prognosis for the patient. In normal cells Hsp70-1A is expressed only in response to external stimuli such as physical exertion and heat to deal with denatured proteins and prevent toxic aggregations in the cell. A majority of human tumors produce Hsp70-1A permanently and in high amounts. In a growing number of these tumors a significant fraction is also found associated with the cell surface (mHsp70). In this project we studied the role of mHsp70 in metastasis and the mechanism of its anchorage to the cell surface on the single cell and single molecule level. We employed a biophysical perspective to a medical problem to advance our understanding of the development of cancer diseases. (Figure 1A)

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.
The scientific objectives were organized in two work packages WP1 and WP2. In WP1 we used atomic force microscopy (AFM) based single-cell force spectroscopy (SCFS) to measure and compare the interaction of cancer cells with high and low amount of mHsp70. We implemented and validated our approach by comparing the adhesion to unspecific substrates of two pairs of breast cancer cells and colon cancer cell lines that varied in their expression of mHsp70. We then explored their interaction with lining cells of the inner wall of blood and lymphatic vessels. The autologous colon cancer cell lines CX+ (mHsp70 positive) and CX- (mHsp70 negative) showed no differences in their adhesion to unspecific substrates, but signifficant differences in their interaction with the lining cells. MHsp70 positive CX+ cells adhere much less to blood vessel lining cells than mHsp70 negative CX- cells. Stronger adhesion indicates a higher tendency to remain at the primary site. Reduced adhesion presents and advantage dfor circulation through the blood stream and could contribute to the development of distant metastasis.

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.
In a first of its kind study we were looking at the interaction of individual cancer cells from the same type of tumour with blood vessel lining cells. We observed 70% less binding when Hsp70-1A was present on the cell surface. This reduced interaction presents a possible advantage for circulating tumour cells to reach distant organs and metastasize.

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.
Figure 1