Intravital imaging reveals systemic ezrin inhibition impedes cancer cell migration and lymph node metastasis in breast cancer

Abstract

BackgroundLimited understanding of the cancer biology of metastatic sites is a major factor contributing to poor outcomes in cancer patients. The regional lymph nodes are the most common site of metastasis in most solid cancers and their involvement is a strong predictor of relapse in breast cancer (BC). We have previously shown that ezrin, a cytoskeletal–membrane linker protein, is associated with lymphovascular invasion and promotes metastatic progression in BC. However, the efficacy of pharmacological inhibition of ezrin in blocking cancer cell migration and metastasis remains unexplored in BC.MethodsWe quantified ezrin expression in a BC tissue microarray (n = 347) to assess its correlation with risk of relapse. Next, we developed a quantitative intravital microscopy (qIVM) approach, using a syngeneic lymphatic reporter mouse tumor model, to investigate the effect of systemic ezrin inhibition on cancer cell migration and metastasis.ResultsWe show that ezrin is expressed at significantly higher levels in lymph node metastases compared to matched primary tumors, and that a high tumor ezrin level is associated with increased risk of relapse in BC patients with regional disease. Using qIVM, we observe a subset of cancer cells that retain their invasive and migratory phenotype at the tumor-draining lymph node. We further show that systemic inhibition of ezrin, using a small molecule compound (NSC668394), impedes the migration of cancer cells in vivo. Furthermore, systemic ezrin inhibition leads to reductions in metastatic burden at the distal axillary lymph node and lungs.ConclusionsOur findings demonstrate that the tumor ezrin level act as an independent biomarker in predicting relapse and provide a rationale for therapeutic targeting of ezrin to reduce the metastatic capacity of cancer cells in high-risk BC patients with elevated ezrin expression.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-1079-7) contains supplementary material, which is available to authorized users.

Untitled

“…Preliminary evidence from our single-cell resolution intravital model shows increased tumor cell/T-cell engagement and number of active T cells (CD69 pos /CD62L neg ) in TDLN of mice treated with the ezrin inhibitor, suggesting that ezrin-targeted therapy, in addition to inhibiting metastasis, may also enhance host T cell antitumour activity (unpublished results).In summary, ezrin is involved in multiple aspects of metastasis [4,5]. Our recent clinical studies provide valuable information regarding the prognostic potential of ezrin in high risk LN-negative breast cancer patients [3] and possibly in other cancer subtypes. Furthermore, there is strong preclinical evidence demonstrating ezrin as a targetable biomarker of metastasis and convincing rationale for investigating the effect of combining ezrin-targeted therapy with chemotherapeutic or immunotherapeutic agents.…”

“…These multifaceted roles for ezrin in cancer makes it an attractive therapeutic target, where its inhibition may suppress metastasis, promote anti-tumor immunity and enhance cytotoxic drug sensitivity. Illustration was created by Designs That Cell.Oncotarget 6756 www.oncotarget.com of metastasis, within tumor-draining LNs (TDLNs) [3]. Furthermore, we demonstrate that reducing ezrin-mediated cell motility within TDLNs also impedes cancer cells from spreading to more distal axillary LNs and to the lungs [3].…”

“…This statistic highlights the limitations of LN status and the need for markers that will better stratify patients who are at risk of relapse. In a locally accrued cohort of breast cancer patients, we recently show that ezrin is an independent marker of recurrence in both LN positive and high risk LN-negative breast cancer patients [3], suggesting that the prognostic power of ezrin is not restricted to the LN positive patient population and may potentially be a more robust marker for disease recurrence. Ezrin expression is also shown to increase from primary tumors to LN metastasis in matched tissues from the same patient [3]; this finding in combination with results from our intravital model demonstrates that ezrin activity confers a metastatic advantage for tumor cells and remains biologically active at metastatic sites.Interestingly, targeting ezrin in our LN metastasis model principally affected metastatic dissemination with minimal changes to primary tumor growth [3], consistent with previous knockdown studies on ezrin [4].…”

“…It is therefore important to consider combination strategies that effectively target both the primary tumor and micrometastatic cell populations. Our preclinical data suggest that ezrin-targeted therapy is effective at reducing the burden of metastasis in the LN and lungs [3]. Furthermore, given ezrin's role in promoting survival of metastatic cells [4], targeting ezrin, or similarly acting molecules, may also provide additional anti-metastatic benefit in combination with anti-tumor/cytotoxic therapies [9].…”

“…Ezrin, a member of the ERM (Ezrin-Radixin-Moesin) family of cytoskeleton adaptor proteins, is frequently over-expressed in invasive cancers and is associated with poor overall survival [2]. Ezrin controls pro-metastatic phenotypes including cell migration and invasion [3,4], and is known to regulate multiple aspects of the metastatic process, such as angioand lymphangiogenesis [5] and distant organ seeding [4] ( Figure 1) -all of which make this molecule an intriguing anti-metastatic target and prognostic biomarker.Our group has recently demonstrated the efficacy of a small molecule inhibitor of ezrin in blocking metastatic progression, using a novel intravital imaging model of lymph node (LN) metastasis [3]. LNs are a common site of metastasis for many invasive epithelial cancers, including breast and prostate, both of which show a preference for lymphatic dissemination [6].…”