Background
Cancer is not only a disease of uncontrolled growth, but in its most aggressive form it is also a disease of uncontrolled cell migration. Ezrin, a member of the ezrin–radixin–moesin (ERM) family of actin cytoskeleton–plasma membrane linker proteins, is involved in multiple aspects of cancer cell migration and its overexpression has been associated with poor prognosis in a variety of solid tumors [
1]. Ezrin plays a critical role in epithelial morphology, adhesion, and migration - all key events that contribute to the invasive phenotype of cancer cells during tumor progression [
2]. It is therefore not surprising that genetic ablation of ezrin or mutation of its key binding sites has been shown to impair metastatic progression in experimental models of a variety of solid cancers [
3].
Lymph nodes (LNs) are the most common sites of solid tumor metastases, and spread of cancer cells to regional LNs is one of the strongest predictors of risk of relapse in BC patients [
4]. In BC, there is extensive evidence for preferential dissemination of cancer cells via the lymphatic rather than blood vascular route [
5]. Furthermore, the addition of regional nodal irradiation to conventional treatment, after mastectomy or breast-conserving surgery in node-positive patients, reduces the rate of locoregional and systemic recurrence [
6,
7]. Despite this clinical importance, and partly due to a lack of appropriate single-cell resolution imaging models of LN metastasis, we have a very limited understanding of the fate of cancer cells within LNs and of the efficacy of therapeutic targeting of prometastatic molecules to prevent further metastatic spread of cancer cells beyond regional LNs. We have previously shown that tumor ezrin levels correlate with lymphovascular invasion in a locally accrued BC cohort and that ezrin acts cooperatively with Src in regulating tumor lymphangiogenesis [
8]. Clinically, ezrin overexpression has also been reported to correlate with the presence of lymph node metastasis in breast cancer [
9], pancreatic cancer [
10], and nonsmall-cell lung cancer [
11]. Together, these findings prompted us to examine whether pharmacological inhibition of ezrin could have therapeutic benefits by suppressing the spread of highly metastatic cancer cells from lymph node micrometastases.
Ezrin’s interactions are dependent on conformational activation of the molecule. In its inactive state, ezrin binding sites are masked by intramolecular interaction between its N-terminal 4.1/ezrin/radixin/moesin (FERM) domain and the C-terminus. Upon phosphorylation of a conserved C-terminal threonine residue (T567) by protein kinase C or Rho kinase and subsequent binding with membrane-associated phosphatidylinositol 4,5-biphosphate (PIP2), the intramolecular masking of its binding sites is removed and interaction with binding partners can take place [
2]. Bulut and colleagues have recently discovered several small molecule inhibitors with high binding affinity to ezrin and their ability to block T567 phosphorylation [
12‐
14]. These authors demonstrated a reduction in the invasive phenotype of cancer cells and inhibition of lung metastases in mice treated with ezrin inhibitors in osteosarcoma experimental models [
12].
In this study, we have shown that elevated tumor ezrin protein expression is associated with increased risk of relapse in node-positive and high-risk node-negative BC patients. We developed a novel qIVM approach using tumor-bearing lymphatic reporter mice to track metastatic cancer cell migration in vivo in real time. We observed a significant reduction in migration and invasion capacity of cancer cells in tumor-draining inguinal lymph node metastases following pharmacological inhibition of ezrin, with a concomitant decrease in metastatic burden in the draining axillary node.
Discussion
In the present study, we have identified ezrin as an independent prognostic marker for relapse in node-positive and high-risk node-negative BC patients. Together, with elevated ezrin expression in LN metastases compared to matched primary tumors, these data support our contention that ezrin overexpression confers a metastatic advantage to BC cells during tumor progression. To study the effect of ezrin inhibition on cancer cells in vivo, we have developed a novel intravital model using a lymphatic reporter mouse to directly track cancer cell migration within lymph node metastatic nodules. A growing body of evidence implicates ezrin as a key promoter of the metastatic process in solid tumors (reviewed in [
3]). Ezrin plays a critical role in maintaining epithelial integrity and in focal adhesion and invadopodia turnover, both key processes in metastatic progression of cancer cells [
2,
20,
24,
25]. Data from our laboratory and other groups have shown that ezrin overexpression in breast cancer cells increases cell scattering and invasion [
26,
27], whereas knockdown or mutational inactivation of ezrin reduces cancer cell migration and diminishes the metastatic potential of cancer cells [
20,
28‐
30]. The data presented here, to our knowledge, are the first to show that pharmacological inhibition of ezrin can effectively impede migratory capacity of cancer cells in vivo and reduce metastatic burden in LNs and lungs in BC models.
Metastasis is a complex process involving tumor cell motility, intravasation, circulation in the blood or lymph system, extravasation, and growth in distant sites [
31]. The increase in metastatic cancer cell motility and invasiveness is a prerequisite for cancer progression and metastasis. Therefore, one can predict that a decrease in cancer cell motility in ezrin inhibitor-treated mice would contribute to the reductions in LN and lung metastatic burden observed in this study. However, it is important to acknowledge the existence of non-ezrin-dependent mechanisms of cell migration that may play an important role in metastatic progression. The elongated mode of cell motility (or mesenchymal-like motility) has been shown to be associated with Rac-dependent F-actin-rich protrusions independent of ezrin function [
32]. We cannot exclude the contribution of alternate modes of cancer cell motility in our model. In fact, we do not observe complete inhibition of cell migration in vitro or full block of distant metastases in vivo following ezrin knockdown or inhibition, suggesting that non-ezrin-dependent pathways also play a role in our model. Furthermore, clinical evidence suggests that increased lymphatic vascular density in primary tumors is associated with increased lymphatic metastasis and poor outcome in BC [
5]. We have previously shown that ezrin knockdown in breast cancer cells leads to reduced lymphangiogenic activity in a Matrigel plug xenograft model in mice [
8]. However, in the present study we did not observe a reduction in peritumoral vascular density in mice treated with ezrin inhibitor. It is plausible that ezrin knockdown targets functions that are independent of the p-T567 (pTERM) activation site (Fig.
2b) effected by ezrin inhibitor. Recently, Celik et al. have described a novel role for ezrin in the regulation of transcription factor DDX3 that is independent of ezrin’s activation and membrane-localized open conformation initiated by phosphorylation of T567 site. This could suggest that decreasing migration capacity of cancer cells in the presence of ezrin inhibitor could play a more prominent role in reducing distant metastasis than contribution from alterations in peritumoral vascular density in our model. Further studies are required to examine the exact mechanism of action for NSC668394 ezrin inhibitor in metastatic cascade.
The relevance of LN metastasis in the progression of metastatic disease has been a subject of considerable debate, partly due to limited models to study the dynamics of cancer cell invasion and response to anticancer therapy within metastatic sites [
33,
34]. The intravital model presented in this study provides direct evidence that a subset of metastatic cells maintain their invasive capacity within LN metastases. Our observations are further supported by two recent studies on the fate of metastatic cells within LNs. Pereira et al. traced the fate of cancer cells expressing a photoconvertible protein and reported that a fraction of metastatic cells were able to invade the LN blood vessels, enter the blood circulation, and colonize the lungs in tumor-bearing mice [
35]. Brown et al. also demonstrated that cancer cells microinfused into mouse afferent lymphatic vessels were able to disseminate via LN blood vessels and metastasize to the lungs without involvement of the thoracic duct [
36]. Together, these observations in mouse models provide evidence that LN metastases can be a source of cancer cells for distant metastases and should therefore be part of the treatment protocol to prevent cancer progression and eliminate all disease from the patient. In fact, findings from a number of clinical trials support this theory by showing reduced rates of locoregional and systemic recurrence following addition of regional node radiation therapy to the standard of care in node-positive BC patients [
6,
7].
Despite the emergence of new prognostic biomarkers and genomic profiling, the nodal status remains a key factor in BC prognosis and has critical therapeutic implications. Moreover, the incidence of lymph node-negative invasive BC has been on the rise due to advances in early detection technologies [
37]. This fact, combined with less aggressive surgical biopsy and treatment of localized disease [
38] plus the recent evidence that systemic spread is an early event in BC [
39], highlights a need for novel prognostic factors for relapse and improved therapeutics to prevent spread of invasive cancer cells and occult micrometastases [
40] in high-risk node-negative patients. The clinical evidence in our study points to ezrin as a potential independent prognostic marker for relapse in high-risk node-negative and node-positive BC. It is tempting to speculate that high-risk BC patients with elevated tumor ezrin levels could benefit from adjuvant ezrin-targeted therapy. Blocking cancer cell dissemination by ezrin-targeted therapy could also be beneficial in certain neoadjuvant settings, where therapies such as surgery, chemotherapy, or radiation have been shown to induce cancer cell motility leading to higher numbers of circulating tumor cells [
41]. Moreover, Karagiannis and colleagues have recently shown that neoadjuvant chemotherapy increases the risk of metastatic dissemination, despite decreasing the primary tumor size, in a mechanism driven by upregulation of actin-regulatory protein Mammalian-enabled (MENA) in cancer cells. Authors were able to reverse the chemotherapy-induced metastatic activity by knockdown of MENA [
42]. Interestingly, ezrin acts as a protein kinase A anchoring protein (AKAP) in regulating the phosphorylation of MENA, and silencing of ezrin has been shown to inhibit MENA’s function [
43]. Finally, as a low tumor ezrin level is associated with improved DFS in node-positive and high-risk node-negative BC, a less aggressive treatment regimen may be warranted in these patients to improve quality of life.
Acknowledgments
The authors thank Colleen Schick for assisting with technical aspects of this study. They would also like to thank Matt Gordon at Queen’s University Biomedical Imaging Centre (QUBIC) for technical assistance in flow cytometry and imaging. TMA construction and staining were performed at the Queen’s Laboratory of Molecular Pathology (QLMP) by Lee Boudreau and Shakeel Virk.