Jeanine Pignatelli

Invasive breast carcinoma cells from patients exhibit MenaINV- and macrophage-dependent transendothelial migration

Analysis of cells in fine-needle aspirate biopsies reveals signals between breast cancer cells and macrophages that promote metastasis. Retaining a Barrier to Metastasis Metastatic disease results from the migration of cancer cells out of the primary tumor and invasion into neighboring tissue or vasculature and establishment of secondary sites. Using an in vitro transendothelial migration assay and cells obtained from fine-needle aspiration (FNA) biopsies from patients with invasive breast cancer, Pignatelli et al. examined the molecular and cellular interactions that may enable the migration of cancer cells into blood vessels (see the Perspective by Kiersse et al.). Where cancer cells colocalized with macrophages in the assay, contacts between endothelial cells were degraded. Macrophages promoted cancer cell migration by secreting the growth factors EGF and CSF-1, and cancer cells in turn secreted CSF-1, which functioned as a paracrine signal to macrophages and for some cancer cell subtypes also as an autocrine signal. Blocking the interaction of CSF-1 with its receptor prevented the transendothelial migration of cancer cells in culture, suggesting that this may be one approach to prevent metastatic progression in cancer patients. Metastasis is a complex, multistep process of cancer progression that has few treatment options. A critical event is the invasion of cancer cells into blood vessels (intravasation), through which cancer cells disseminate to distant organs. Breast cancer cells with increased abundance of Mena [an epidermal growth factor (EGF)–responsive cell migration protein] are present with macrophages at sites of intravasation, called TMEM sites (for tumor microenvironment of metastasis), in patient tumor samples. Furthermore, the density of these intravasation sites correlates with metastatic risk in patients. We found that intravasation of breast cancer cells may be prevented by blocking the signaling between cancer cells and macrophages. We obtained invasive breast ductal carcinoma cells of various subtypes by fine-needle aspiration (FNA) biopsies from patients and found that, in an in vitro transendothelial migration assay, cells that migrated through a layer of human endothelial cells were enriched for the transcript encoding MenaINV, an invasive isoform of Mena. This enhanced transendothelial migration required macrophages and occurred with all of the breast cancer subtypes. Using mouse macrophages and the human cancer cells from the FNAs, we identified paracrine and autocrine activation of colony-stimulating factor-1 receptor (CSF-1R). The paracrine or autocrine nature of the signal depended on the breast cancer cell subtype. Knocking down MenaINV or adding an antibody that blocks CSF-1R function prevented transendothelial migration. Our findings indicate that MenaINV and TMEM frequency are correlated prognostic markers and CSF-1 and MenaINV may be therapeutic targets to prevent metastasis of multiple breast cancer subtypes.

Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism

Chemotherapy induces prometastatic changes in breast cancer, reversible by TIE2 or MENA inhibition. Closing the door to cancer cells Breast cancer is one of the most common tumor types, and metastasis greatly increases the risk of death from this disease. By studying the process of intravasation or entry of cells into the vasculature, Karagiannis et al. discovered that, in addition to killing tumor cells, chemotherapy treatment can also increase intravasation. Groups of cells collectively known as tumor microenvironment of metastasis (TMEM) can serve as gateways for tumor cells entering the vasculature, and the authors discovered that several types of chemotherapy can increase the amounts of TMEM complexes and circulating tumor cells in the bloodstream. The researchers also determined that a drug called rebastinib can interfere with TMEM activity and help overcome the increased risk of cancer cell dissemination. Breast cancer cells disseminate through TIE2/MENACalc/MENAINV-dependent cancer cell intravasation sites, called tumor microenvironment of metastasis (TMEM), which are clinically validated as prognostic markers of metastasis in breast cancer patients. Using fixed tissue and intravital imaging of a PyMT murine model and patient-derived xenografts, we show that chemotherapy increases the density and activity of TMEM sites and Mena expression and promotes distant metastasis. Moreover, in the residual breast cancers of patients treated with neoadjuvant paclitaxel after doxorubicin plus cyclophosphamide, TMEM score and its mechanistically connected MENAINV isoform expression pattern were both increased, suggesting that chemotherapy, despite decreasing tumor size, increases the risk of metastatic dissemination. Chemotherapy-induced TMEM activity and cancer cell dissemination were reversed by either administration of the TIE2 inhibitor rebastinib or knockdown of the MENA gene. Our results indicate that TMEM score increases and MENA isoform expression pattern changes with chemotherapy and can be used in predicting prometastatic changes in response to chemotherapy. Furthermore, inhibitors of TMEM function may improve clinical benefits of chemotherapy in the neoadjuvant setting or in metastatic disease.

Autocrine CSF1R signaling mediates switching between invasion and proliferation downstream of TGFβ in claudin-low breast tumor cells

Patient data suggest that colony-stimulating factor-1 (CSF1) and its receptor (CSF1R) have critical roles during breast cancer progression. We have previously shown that in human breast tumors expressing both CSF1 and CSF1R, invasion in vivo is dependent both on a paracrine interaction with tumor-associated macrophages and an autocrine regulation of CSF1R in the tumor cells themselves. Although the role of the paracrine interaction between tumor cells and macrophages has been extensively studied, very little is known about the mechanism by which the autocrine CSF1R signaling contributes to tumor progression. We show here that breast cancer patients of the claudin-low subtype have significantly increased expression of CSF1R. Using a panel of breast cancer cell lines, we confirm that CSF1R expression is elevated and regulated by TGFβ specifically in claudin-low cell lines. Abrogation of autocrine CSF1R signaling in MDA-MB-231 xenografts (a claudin-low cell line) leads to increased tumor size by enhanced proliferation, but significantly reduced invasion, dissemination and metastasis. Indeed, we show that proliferation and invasion are oppositely regulated by CSF1R downstream of TGFβ only in claudin-low cell lines. Intravital multiphoton imaging revealed that inhibition of CSF1R in the tumor cells leads to decreased in vivo motility and a more cohesive morphology. We show that, both in vitro and in vivo, CSF1R inhibition results in a reversal of claudin-low marker expression by significant upregulation of luminal keratins and tight-junction proteins such as claudins. Finally, we show that artificial overexpression of claudins in MDA-MB-231 cells is sufficient to tip the cells from an invasive state to a proliferative state. Our results suggest that autocrine CSF1R signaling is essential in maintaining low claudin expression and that it mediates a switch between the proliferative and the invasive state in claudin-low tumor cells downstream of TGFβ.

The Selective Tie2 Inhibitor Rebastinib Blocks Recruitment and Function of Tie2Hi Macrophages in Breast Cancer and Pancreatic Neuroendocrine Tumors

Tumor-infiltrating myeloid cells promote tumor progression by mediating angiogenesis, tumor cell intravasation, and metastasis, which can offset the effects of chemotherapy, radiation, and antiangiogenic therapy. Here, we show that the kinase switch control inhibitor rebastinib inhibits Tie2, a tyrosine kinase receptor expressed on endothelial cells and protumoral Tie2-expressing macrophages in mouse models of metastatic cancer. Rebastinib reduces tumor growth and metastasis in an orthotopic mouse model of metastatic mammary carcinoma through reduction of Tie2+ myeloid cell infiltration, antiangiogenic effects, and blockade of tumor cell intravasation mediated by perivascular Tie2Hi/Vegf-AHi macrophages in the tumor microenvironment of metastasis (TMEM). The antitumor effects of rebastinib enhance the efficacy of microtubule inhibiting chemotherapeutic agents, either eribulin or paclitaxel, by reducing tumor volume, metastasis, and improving overall survival. Rebastinib inhibition of angiopoietin/Tie2 signaling impairs multiple pathways in tumor progression mediated by protumoral Tie2+ macrophages, including TMEM-dependent dissemination and angiopoietin/Tie2-dependent angiogenesis. Rebastinib is a promising therapy for achieving Tie2 inhibition in cancer patients. Mol Cancer Ther; 16(11); 2486–501. ©2017 AACR.

Abstract 397: Rebastinib potently inhibits function of perivascular TIE2 expressing macrophagesin vitroandin vivo

Introduction Significant cross-talk between tumor cells and the surrounding stromal tissue are essential for tumor vascularization, survival, immunotolerance, invasion, and metastasis. The angiopoietin (ANG)/TIE2 kinase signaling pathway is a pivotal cross talk axis in the tumor microenvironment. It has been demonstrated that TIE2-expressing macrophages (TEMs) mediate invasion and metastasis in the PyMT syngeneic breast cancer model, and that TIE2 expression correlates with poor overall survival and high risk of metastasis in breast cancer patients. In this study, rebastinib was evaluated as a TIE2 inhibitor in in vitro and in vivo models, and was shown to have profound effects on the structure and function of perivascular TEMs. Procedures TIE2 kinase assays used a standard PK/LDH coupled spectrophotometric continuous assay. CHO cells were transiently transfected to express human TIE2 for cellular studies. CHOs, HUVECs and EA.hy926 cells were used to evaluate rebastinib for inhibition of ANG1-stimulated TIE2 phosphorylation. In vitro intravasation studies were performed in a transwell transendothelial migration chamber wherein TIE2HI macrophages interact with breast tumor cells to cause transendothelial migration of tumor cells across a sealed HUVEC endothelial monolayer. In vivo evaluations of rebastinib were performed using intravital high-resolution two-photon microscopy in the murine PyMT breast cancer model to evaluate effects on tumor vascular permeability and tumor cell intravasation. Results Rebastinib is a potent inhibitor of TIE2 kinase (IC50 = 0.63 nM). Rebastinib slowly dissociated from TIE2 (koff = 0.0012 minutes−1; T1/2 = 10 hr). In HUVECs or EA.hy926 cells, rebastinib inhibited ANG1-stimulated TIE2 kinase activity (IC50s of 0.018 and 0.091 nM, respectively). In TIE2 CHO cells, rebastinib inhibited TIE2 phosphorylation (IC50 2.0 nM), and demonstrated a prolonged off-rate (> 24 hr) against TIE2 after inhibitor washout. Rebastinib exhibited an IC50 Rebastinib was evaluated in vivo in the PyMT syngeneic breast cancer model. Rebastinib dosed at 10 mg/kg orally twice weekly impaired tumoral perivascular TEMs, resulting in a significant reduction in vascular permeability and in tumor cell intravasation as quantified by CTCs. Conclusion Rebastinib is a potent inhibitor of TIE2 kinase and exhibits durable cellular inhibition in endothelial cells and in TIE2 macrophages. Oral dosing of rebastinib resulted in a significant reduction in TIE2-macrophage mediated tumor vascular permeability and in the intravasation of tumor cells into the circulation. Rebastinib is currently in Phase 1 clinical evaluation in solid tumors. Citation Format: Allison Harney, Jeanine Pignatelli, Edison Leung, Maja Oktay, Yarong Wang, Bryan D. Smith, Daniel L. Flynn, John S. Condeelis. Rebastinib potently inhibits function of perivascular TIE2 expressing macrophages in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 397. doi:10.1158/1538-7445.AM2015-397

Abstract 898: Elucidation of the molecular mechanism of MenaINVexpression, invadopodium maturation and tumor cell intravasation during breast cancer dissemination by TMEM

Numerous clinical studies have identified Tumor MicroEnvironment of Metastasis (TMEM) and MenaCalc as distinct but functionally interrelated prognostic indicators of distant metastasis in breast cancer patients. TMEM sites are the only sites where tumor cells intravasate in mammary tumors. TMEM score is calculated histologically as the density of tripartite microanatomical structures involving a perivascular macrophage, a Mena-expressing tumor cell and an endothelial cell, all three in direct physical contact. On the other hand, MenaCalc represents the pattern of Mena splice-isoforms present in a tumor sample. MenaINV is the key metastasis-promoting Mena splice-isoform driving tumor cell migration toward blood vessels, intravasation and dissemination. However, the precise molecular mechanisms relating TMEM formation and function and MenaINV expression in these critical steps of the metastatic cascade have not been elucidated. Here we show that MenaINV promotes invadopodium-based proteolysis, which is required for tumor cell invasion and transendothelial migration, by preventing the localization of the phosphatase PTP1B to invadopodia. Interestingly, PTP1B regulates invadopodium maturation by limiting cortactin phosphorylation at a key residue (Y421) that is necessary for actin polymerization during invadopodium maturation. Additionally, we demonstrate that MenaINV expression, invadopodium activity, and subsequent transendothelial migration are induced in tumor cells via Notch1-mediated signaling induced by contact of tumor cells with macrophages. Knock-down of MenaINV expression in tumor cells leads to a proportional decrease in mature invadopodium formation. Complete knock-out of Mena in mouse mammary tumors (PyMT-MMTV) abolishes TMEM assembly and TMEM functions, including TMEM-dependent vascular permeability, circulating tumor cells and lung metastases. In summary, our work shows that macrophage contact of TMEM-associated tumor cells during TMEM assembly stimulates Notch1 to drive expression of MenaINV in tumor cells. MenaINV expression then inhibits PTP1B at invadopodia to promote cortactin phosphorylation at invadopodium precursors, driving invadopodium maturation and promoting transendothelial migration. These findings provide, for the first time, an integrative molecular mechanism for two clinically validated prognostic indicators of metastatic risk, TMEM and MenaCalc, and identify new drug targets for limiting the metastatic spread of breast cancer. Citation Format: Maxwell D. Weidmann, Chinmay R. Surve, Jeanine Pignatelli, Javier J. Bravo-Cordero, George S. Karagiannis, Maja H. Oktay, John S. Condeelis. Elucidation of the molecular mechanism of MenaINV expression, invadopodium maturation and tumor cell intravasation during breast cancer dissemination by TMEM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 898. doi:10.1158/1538-7445.AM2017-898

Abstract PD5-02: Paclitaxel induced mena- and TMEM-mediated pro-metastatic changes in the breast cancer microenvironment

Background: Breast cancer cell intravasation and dissemination occurs specifically at microanatomical structures that we call tumor-microenvironment of metastasis (TMEM), representing direct physical contact between a tumor cell expressing the actin-regulatory protein Mammalian-enabled (Mena), a perivascular Tie2 hi /Vegf hi -expressing macrophage, and an endothelial cell (Harney et al. Cancer Discovery 2015). TMEM sites have been identified in mouse and human mammary carcinomas, and both TMEM density (Rohan et al. JNCI 2014) and invasive Mena isoform expression (Agarwal et al. Breast Cancer Res, 2012; Forse et al. BMC Cancer, 2015]) correlates with metastasis in early stage breast cancer. Since cytotoxic agents such as PTX induce influx of bone marrow-derived progenitors that differentiate into Tie2 hi /VEGF hi macrophages in the primary tumor, we hypothesized that PTX may potentiate tumor cell invasion and metastasis by inducing the formation of TMEM sites and/or function. Methods and Results in humans: We analyzed the effect of chemotherapy on TMEM and invasive Mena isoforms in 10 patients with localized breast cancer who had residual disease after neoadjuvant chemotherapy (NAC: weekly paclitaxel followed by dose-dense doxorubicin-cyclophosphamide [AC]), of whom 7 had more than 2-fold increase in TMEM density in residual disease compared with pretreatment. In a separate cohort of 5 patients, NAC produced an acute increase of up to 150-fold in invasive Mena isoforms after 1-2 doses of NAC. Methods and Results in mice: After our preliminary data in humans, we evaluated effects of PTX in 4 different models, including 2 mouse models (PyMT-spontaneous & transplantation) and 2 patient-derived xenograft (PDX) triple negative models (HT17, HT33). Although PTX delayed primary tumor growth, tumors in PTX-treated mice had significantly more TMEM sites, circulating tumor cells (CTCs) and metastatic foci when compared to vehicle-treated animals. Using intravital imaging of MMTV-PyMT-Dendra2/Cfms-CFP mice, PTX induced influx of macrophages into primary tumors and intravasation of cancer cells at TMEM sites. Furthermore, PTX treatment significantly increased expression of Mena at the gene and protein levels, including invasive Mena isoforms. Deletion of the Mena gene completely abolished dissemination and metastasis in all cases, including those treated with PTX. Conclusions: We show in mammary carcinoma mouse models and PDX models that although PTX delays tumor growth, it induces invasive Mena isoform expression and significantly increases the density of TMEM sites that are responsible for cancer cell intravasation, dissemination and metastasis. Thus, our data indicate that PTX paradoxically induces dissemination of breast cancer cells by promoting invasive Mena isoforms and TMEM-mediated cancer cell intravasation, suggesting that blockade of TMEM assembly and/or function could enhance the effectiveness of PTX and possibly other cytotoxic agents commonly used to treat early and advanced stage breast cancer. Citation Format: Karagiannis GS, Pastoriza JM, Wang Y, Harney AS, Entenberg D, Pignatelli J, Jones JG, Anampa J, Sparano JA, Rohan TE, Condeelis JS, Oktay MH. Paclitaxel induced mena- and TMEM-mediated pro-metastatic changes in the breast cancer microenvironment [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD5-02.

Abstract LB-006: Macrophage-dependent activation of Notch1 signaling regulates breast tumor cell intravasation

The process of intravasation, a key component to the metastatic cascade, remains poorly understood. How the multi-cell type tumor microenvironment facilitates tumor cell intravasation is largely unknown. Intravital imaging of rodent mammary tumors has shown that direct contact between a Mena expressing tumor cell, a perivascular macrophage and an endothelial cell forms a microanatomical structure named TMEM (Tumor Microenvironment of Metastasis) which is the site where intravasation occurs in mammary tumors. Clinical studies have shown that the number of TMEM is correlated with increased risk of developing distant metastasis in breast cancer patients. While TMEM is an excellent prognostic marker for predicting metastasis, the mechanisms of TMEM assembly and function are not well understood. Recently, we showed that heterotypic cell contact between tumor cells and macrophages induces the formation of invadopodia in tumor cells, invasive structures necessary for matrix degradation and required for tumor cell intravasation. In further work we found that Notch1, a known receptor involved in heterotypic cell contact signaling, is involved. In the absence of Notch1 signaling, macrophage-induced invadopodium formation and tumor cell intravasation are abolished. This heterotypic tumor cell - macrophage interaction regulates the expression profile of Mena in the tumor cell. Upon touching, the transcription of Mena shows a rapid kinetic response, with detectable changes in single transcriptome activity within 1 hour. Inhibition of Notch1 in vivo results in decreased intravasation of mammary tumor cells. Our findings indicate that Notch1 signaling regulates heterotypic cell contact mediated invadopodium formation, transendothelial migration and intravasation and reveals a novel Notch1/Mena pathway as a molecular target to prevent TMEM function and therefore metastasis. Citation Format: Jeanine Pignatelli, Jose Javier Bravo-Cordero, Minna Roh-Johnson, Saumil Gandhi, Yarong Wang, Robert Singer, Louis Hodgson, Maja Oktay, John Condeelis. Macrophage-dependent activation of Notch1 signaling regulates breast tumor cell intravasation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-006.

Abstract 4112: Mechanisms of transendothelial migration by invasive breast carcinoma cells from patients

The majority of breast cancer related deaths are not due to the primary tumor, but rather to the dissemination of metastatic tumor cells from it to distant sites. Our lab has previously identified the tumor microenvironment of metastasis (TMEM) in mouse and human mammary tumors, sites where transendothelial migration, intravasation and dissemination occur. The constituent cells of TMEM are an endothelial cell, a perivascular TIE2-expressing macrophage (TEM) and an invasive Mena-over expressing tumor cell in direct contact. TMEM are present in human invasive breast tumors and the density of TMEM is positively associated with the risk of developing metastases. Using invasive ductal carcinoma cells of the breast obtained from patients by fine needle aspiration (FNA), we demonstrated that intravasation-directed transendothelial migration (iTEM) of these cancer cells requires macrophages and, depending on clinical subtype, involves either paracrine (macrophage CSF1-R; tumor cell EGFR), or both paracrine and autocrine (tumor cell EGFR + CSF1-R) signaling. Compared to the total population of primary breast cancer cells assayed, cells capable of transendothelial migration expressed relatively high Mena INV and low Mena11a levels, independently of clinical subtype. Mena INV and Mena11a are functionally distinct isoforms of Mena, a key regulator of motility and invasion. Depletion of Mena INV using siRNA showed that Mena INV is required for efficient macrophage-dependent iTEM of tumor cells. Furthermore, relative Mena INV expression correlated with the density of TMEM, which were previously shown to correlate with risk of metastasis in patients. From these observations we have identified the signaling pathways involved in macrophage-dependent tumor cell iTEM. Two novel small molecule inhibitors, Rebastinib (Tie2 inhibitor) and Altiratinib (c-MET inhibitor), have been identified that show great promise in preventing key steps in iTEM. Our data reveal targetable signaling events required for transendothelial migration of human breast cancer cells, and indicate that relative Mena INV levels and TMEM frequency are correlated prognostic markers of metastasis and therapeutic targets for most human breast cancers. Citation Format: Jeanine Pignatelli, Joan Jones, Xiaoming Chen, Bryan Smith, Daniel Flynn, John Condeelis, Maja Oktay. Mechanisms of transendothelial migration by invasive breast carcinoma cells from patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4112. doi:10.1158/1538-7445.AM2015-4112