Integrin regulation by tissue factor promotes cancer stemness and metastatic dissemination in breast cancer

Tissue factor, initiator of blood clotting, promotes early phases of metastatic dissemination and cancer stemness via the regulation of β-integrins. Inhibition of tissue factor results in mesenchymal-to-epithelial transition and less stemness behavior.
Integrin regulation by tissue factor promotes cancer stemness and metastatic dissemination in breast cancer
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Breast cancer progression is heavily dependent on the tumor micro-environment and data from in vitro and animal experiments in the past 20 years have shown that hemostasis (i.e. the process to stop bleeding from a damaged blood vessel) is one of those mediators. Proteins such as thrombin are known inducers of de novo blood vessel development in certain types of cancer and blood platelets have a clear but distinct effect on cancer metastasis.

Nevertheless, the on-and-off switch of blood clotting, tissue factor, has also been in the center of attention of cancer biologists over the years. (My PhD motto: “Tissue factor is the center of the universe”). Current dogma's dictate that tissue factor fulfills two major roles in cancer progression. First of all, it serves as a signal transduction receptor to promote blood vessel formation in tumors and consequently tumor growth. Second, upon entering the blood vessel, tissue factor activates the blood coagulation and forms a protective shield around the tumor cells and thus promotes survival of metastatic cells. Our previous work has demonstrated how the procoagulant feature of tissue factor was involved in metastasis [1]. However, the signaling axis of TF during the early phases leading to metastasis has not been studied before.

Importantly, our study indicates high tissue factor expression promotes metastasis of breast cancer cells. The clinical relevance of our study is, that we do see more metastasis in women with breast cancer that express high levels of tissue factor in estrogen and progesterone receptor negative tumors, and their survival is reduced (Fig. 1A). To understand if the signaling axis of tissue factor was involved in metastatic dissemination, we decided to conduct in vivo experiments. When the signaling -but not clotting- function of tissue factor was inhibited with a specific tissue factor antibody, we observed less metastasis in our mice studies (Fig. 1B-C). We were also able to demonstrate that this was solely dependent on the signaling axis, and independent of the protective shield around the circulating tumor cell. Searching for the mechanism behind the early phases of metastatic dissemination, we focused on the shift in epithelial-to-mesenchymal transition. Prior to metastasis, tumor cells start losing contact with neighboring cells and adopt a more spindle-like morphology. After extravasation at distant organs, cells undergo the opposite transition. Indeed, we observed less expression of mesenchymal markers in tumors isolated from our mouse experiments once tissue factor signaling was inhibited. Nowadays, it becomes more evident that there is a relationship between mesenchymal-to-epithelial transition and cancer stem cells for successful metastasis. Cancer stem cells are a part of the tumor that are able to self-renew and seed new tumors.

Currently, it is unknown whether after successful metastasis cells become more epithelial and then de-differentiate into cancer stem cells or if cancer stem cells start expressing epithelial-to-mesenchymal associated markers. A collaboration, with the group of Cliona Kirwan shed a first glance for the role of tissue factor on cancer stem cell activity in vitro [2]. Here, tissue factor promotes increased stem cell activity, with increased spheroid forming capacity and expression of a specific breast cancer stem cell marker ALDH1. Coupling this back to clinical data, we stained tumor specimens derived from breast cancer patients, and we did confirm an association between high tissue factor expression and the breast cancer stem cell marker ALDH1. Based on these findings, we wondered if the opposite would be true, i.e. does inhibition of tissue factor lead to diminished cancer stem cell numbers. Thus, we decided to repeat our in vivo experiment, and this time take the tumors out and place them back in cell culture. This led to a remarkable observation in the lab: tissue factor-inhibited tumor cells had lost their spindle-like morphology and gained a cobble-stone-like epithelial shape. This observation was corroborated not only by decreased expression of mesenchymal- and cancer stem cell markers, but also by reduced invasive capacity and spheroid forming efficiency. To rule out any artifacts of the ex vivo model, and to be certain if these effects were caused by inhibition of tissue factor signaling, we repeated these experiments in vitro. Likewise, we were able to reproduce the same results, suggesting that tissue factor signaling is involved in the expression profiles of epithelial-to-mesenchymal transition and cancer stemness.

 

Figure 1 Tissue factor signaling is involved in metastasis. A) Kaplan-Meier analysis of metastasis-free survival in breast cancer patients (N=574 breast tumor specimens) with estrogen receptor negative tumors, stratified for high and low tissue factor (TF) expression. B) Breast cancer tumor cells were grafted in the mammary fat pad of mice, in the presence of control (IgG) antibody or tissue factor signaling inhibiting antibody Mab-10H10. Tumor volume was measured until week 7, and tumor weight at day of sacrifice was measured. C) To measure metastasis, the presence of human cells in the lungs of mice were determined by qPCR by measuring the ratio of human GAPDH versus mouse b-Actin mRNA levels.

So now the following question raised: How is tissue factor signaling involved in cancer stemness? Therefore, we focused on integrins which are regulators of cancer stemness. They bind to the extracellular matrix and play a role in migration and apoptosis. Other than tissue factor’s signaling partner PAR2 to promote vessel growth, it can also interact with β1-integrins. Up to now, no clear mechanism of tissue factor/β1-integrin signaling had been elucidated. We postulated that this uncharacterized signaling pathway might affect the epithelial-to-mesenchymal transition, migration and/or cancer stemness. So, when tissue factor signaling was inhibited, we observed an equal overall tissue factor/β1-integrin binding, but a shift of β1-integrin expression from the cholesterol-poor fraction to the cholesterol-rich fraction. Of note, β1-integrin adopted an active conformation in this cholesterol-rich membrane fraction. Investigating the ɑ-subunits of integrin, we found less ɑ2- and ɑ3-integrins in complex with tissue factor, and more ɑ6-integrins in co-complex.

The most outstanding moment of this project, or the “Eureka!” moment, happened in March 2017, when Bierie and colleagues published an article where they present β4-integrin expression as a cancer stem cell surface marker for partially mesenchymal cells[3]. In MDA-MB-231 cells the β4-integrin-positive cells associate with a more epithelial morphology that has less tumorigenic properties. This made us question whether a similar effect was ongoing in our cells when tissue factor signaling was inhibited. And in line with their work, we observed a tremendous increase in β4-integrin expression with a persistent epithelial-like morphology in cell culture. (I am not going to lie, but after triple checking that paper, I did a small happy dance in cell culture after seeing my results).

To conclude, tissue factor promotes metastatic dissemination and cancer stemness via the regulation of β-integrins. When we inhibited tissue factor signaling, the mesenchymal profile and cancer stemness was downregulated, that resulted in less metastatic features. Furthermore, we have shown the underlying mechanism of action, where uncoupling of tissue factor/β1-integrin signaling pathway results in elevated β4-integrin expression, that eventually leads to suppressed metastasis (Fig. 2). As our clinical data shows that tissue factor expression was associated with poor survival in estrogen receptor negative tumors, we believe that disruption of tissue factor signaling in triple-negative breast tumors may help prevent relapse and increase the overall survival of these breast cancer patients.

 

Figure 2 Systematic overview of inhibition of tissue factor signaling in breast cancer. The tissue factor signaling inhibitor, Mab-10H10, disrupts the tissue factor/β1-integrin complex, increases tissue factor/β4-integrin complex formation, and results active conformation of integrins with FAK-activation. This promotes epithelial-like morphology with decreased breast cancer stemness and metastasis to the lungs.

References 

1          Versteeg HH, Schaffner F, Kerver M, Petersen HH, Ahamed J, Felding-Habermann B et al. Inhibition of tissue factor signaling suppresses tumor growth. Blood 2008; 111: 190-199. 

2          Shaker H, Harrison H, Clarke R, Landberg G, Bundred NJ, Versteeg HH et al. Tissue Factor promotes breast cancer stem cell activity in vitro. Oncotarget 2017; 8: 25915-25927.

3          Bierie B, Pierce SE, Kroeger C, Stover DG, Pattabiraman DR, Thiru P et al. Integrin-beta4 identifies cancer stem cell-enriched populations of partially mesenchymal carcinoma cells. Proc Natl Acad Sci U S A 2017; 114: E2337-E2346.

 

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