Signaling events at TMEM doorways provide potential targets for inhibiting breast cancer dissemination.

Tumor cell intravasation is essential for metastatic dissemination, but its exact mechanism is incompletely understood. We have previously shown that in breast cancer, the direct and stable association of a tumor cell expressing Mena, a Tie2hi/VEGFhi macrophage, and a vascular endothelial cell, creates an intravasation portal, called a "tumor microenvironment of metastasis" (TMEM) doorway, for tumor cell intravasation, leading to dissemination to distant sites. The density of TMEM doorways, also called TMEM doorway score, is a clinically validated prognostic marker of distant metastasis in breast cancer patients. Although we know that tumor cells utilize TMEM doorway-associated transient vascular openings to intravasate, the precise signaling mechanisms involved in TMEM doorway function are only partially understood. Using two mouse models of breast cancer and an in vitro assay of intravasation, we report that CSF-1 secreted by the TMEM doorway tumor cell stimulates local secretion of VEGF-A from the Tie2hi TMEM doorway macrophage, leading to the dissociation of endothelial junctions between TMEM doorway associated endothelial cells, supporting tumor cell intravasation. Acute blockade of CSF-1R signaling decreases macrophage VEGF-A secretion as well as TMEM doorway-associated vascular opening, tumor cell trans-endothelial migration, and dissemination. These new insights into signaling events regulating TMEM doorway function should be explored further as treatment strategies for metastatic disease.

Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However, for scientists wishing to publish obtained images and image-analysis results, there are currently no unified guidelines for best practices. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here, we present community-developed checklists for preparing light microscopy images and describing image analyses for publications. These checklists offer authors, readers and publishers key recommendations for image formatting and annotation, color selection, data availability and reporting image-analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby to heighten the quality and explanatory power of microscopy data.

Racial disparity in tumor microenvironment and distant recurrence in residual breast cancer after neoadjuvant chemotherapy.

Black, compared to white, women with residual estrogen receptor-positive (ER+) breast cancer after neoadjuvant chemotherapy (NAC) have worse distant recurrence-free survival (DRFS). Such racial disparity may be due to difference in density of portals for systemic cancer cell dissemination, called TMEM doorways, and pro-metastatic tumor microenvironment (TME). Here, we evaluate residual cancer specimens after NAC from 96 Black and 87 white women. TMEM doorways are visualized by triple immunohistochemistry, and cancer stem cells by immunofluorescence for SOX9. The correlation between TMEM doorway score and pro-metastatic TME parameters with DRFS is examined using log-rank and multivariate Cox regression. Black, compared to white, patients are more likely to develop distant recurrence (49% vs 34.5%, p = 0.07), receive mastectomy (69.8% vs 54%, p = 0.04), and have higher grade tumors (p = 0.002). Tumors from Black patients have higher TMEM doorway and macrophages density overall (p = 0.002; p = 0.002, respectively) and in the ER+/HER2- (p = 0.02; p = 0.02, respectively), but not in the triple negative disease. Furthermore, high TMEM doorway score is associated with worse DRFS. TMEM doorway score is an independent prognostic factor in the entire study population (HR, 2.02; 95%CI, 1.18-3.46; p = 0.01), with a strong trend in ER+/HER2- disease (HR, 2.38; 95%CI, 0.96-5.95; p = 0.06). SOX9 expression is not associated with racial disparity in TME or outcome. In conclusion, higher TMEM doorway density in residual breast cancer after NAC is associated with higher distant recurrence risk, and Black patients are associated with higher TMEM doorway density, suggesting that TMEM doorway density may contribute to racial disparities in breast cancer.

Cooperative NF-κB and Notch1 signaling promotes macrophage-mediated MenaINV expression in breast cancer.

Metastasis is a multistep process that leads to the formation of clinically detectable tumor foci at distant organs and frequently to patient demise. Only a subpopulation of breast cancer cells within the primary tumor can disseminate systemically and cause metastasis. To disseminate, cancer cells must express MenaINV, an isoform of the actin regulatory protein Mena, encoded by the ENAH gene, that endows tumor cells with transendothelial migration activity, allowing them to enter and exit the blood circulation. We have previously demonstrated that MenaINV mRNA and protein expression is induced in cancer cells by macrophage contact. In this study, we discovered the precise mechanism by which macrophages induce MenaINV expression in tumor cells. We examined the promoter of the human and mouse ENAH gene and discovered a conserved NF-κB transcription factor binding site. Using live imaging of an NF-κB activity reporter and staining of fixed tissues from mouse and human breast cancer, we further determined that for maximal induction of MenaINV in cancer cells, NF-κB needs to cooperate with the Notch1 signaling pathway. Mechanistically, Notch1 signaling does not directly increase MenaINV expression, but it enhances and sustains NF-κB signaling through retention of p65, an NF-κB transcription factor, in the nucleus of tumor cells, leading to increased MenaINV expression. In mice, these signals are augmented following chemotherapy treatment and abrogated upon macrophage depletion. Targeting Notch1 signaling in vivo decreased NF-κB signaling activation and MenaINV expression in the primary tumor and decreased metastasis. Altogether, these data uncover mechanistic targets for blocking MenaINV induction that should be explored clinically to decrease cancer cell dissemination and improve survival of patients with metastatic disease.

Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality and explanatory power of microscopy data is in publications.

Cooperative NF-κB and Notch1 signaling promotes macrophage-mediated MenaINV expression in breast cancer.

Metastasis is a multistep process that leads to the formation of clinically detectable tumor foci at distant organs and frequently patient demise. Only a subpopulation of breast cancer cells within the primary tumor can disseminate systemically and cause metastasis. To disseminate, cancer cells must express MenaINV, an isoform of the actin-regulatory protein Mena encoded by the ENAH gene that endows tumor cells with transendothelial migration activity allowing them to enter and exit the blood circulation. We have previously demonstrated that MenaINV mRNA and protein expression is induced in cancer cells by macrophage contact. In this study, we discovered the precise mechanism by which macrophages induce MenaINV expression in tumor cells. We examined the promoter of the human and mouse ENAH gene and discovered a conserved NF-κB transcription factor binding site. Using live imaging of an NF-κB activity reporter and staining of fixed tissues from mouse and human breast cancer we further determined that for maximal induction of MenaINV in cancer cell NF-κB needs to cooperate with the Notch1 signaling pathway. Mechanistically, Notch1 signaling does not directly increase MenaINV expression, but it enhances and sustains NF-κB signaling through retention of p65, an NF-κB transcription factor, in the nucleus of tumor cells, leading to increased MenaINV expression. In mice, these signals are augmented following chemotherapy treatment and abrogated upon macrophage depletion. Targeting Notch1 signaling in vivo decreased NF-κB signaling and MenaINV expression in the primary tumor and decreased metastasis. Altogether, these data uncover mechanistic targets for blocking MenaINV induction that should be explored clinically to decrease cancer cell dissemination and improve survival of patients with metastatic disease.

Primary tumor associated macrophages activate programs of invasion and dormancy in disseminating tumor cells.

Metastases are initiated by disseminated tumor cells (DTCs) that colonize distant organs. Growing evidence suggests that the microenvironment of the primary tumor primes DTCs for dormant or proliferative fates. However, the manner in which this occurs remains poorly understood. Here, using the Window for High-Resolution Intravital Imaging of the Lung (WHRIL), we study the live lung longitudinally and follow the fate of individual DTCs that spontaneously disseminate from orthotopic breast tumors. We find that spontaneously DTCs have increased levels of retention, increased speed of extravasation, and greater survival after extravasation, compared to experimentally metastasized tumor cells. Detailed analysis reveals that a subset of macrophages within the primary tumor induces a pro-dissemination and pro-dormancy DTC phenotype. Our work provides insight into how specific primary tumor microenvironments prime a subpopulation of cells for expression of proteins associated with dissemination and dormancy.

Live tumor imaging shows macrophage induction and TMEM-mediated enrichment of cancer stem cells during metastatic dissemination.

Cancer stem cells (CSCs) play an important role during metastasis, but the dynamic behavior and induction mechanisms of CSCs are not well understood. Here, we employ high-resolution intravital microscopy using a CSC biosensor to directly observe CSCs in live mice with mammary tumors. CSCs display the slow-migratory, invadopod-rich phenotype that is the hallmark of disseminating tumor cells. CSCs are enriched near macrophages, particularly near macrophage-containing intravasation sites called Tumor Microenvironment of Metastasis (TMEM) doorways. Substantial enrichment of CSCs occurs on association with TMEM doorways, contributing to the finding that CSCs represent >60% of circulating tumor cells. Mechanistically, stemness is induced in non-stem cancer cells upon their direct contact with macrophages via Notch-Jagged signaling. In breast cancers from patients, the density of TMEM doorways correlates with the proportion of cancer cells expressing stem cell markers, indicating that in human breast cancer TMEM doorways are not only cancer cell intravasation portals but also CSC programming sites.

SUN-MKL1 Crosstalk Regulates Nuclear Deformation and Fast Motility of Breast Carcinoma Cells in Fibrillar ECM Microenvironment.

Aligned collagen fibers provide topography for the rapid migration of single tumor cells (streaming migration) to invade the surrounding stroma, move within tumor nests towards blood vessels to intravasate and form distant metastases. Mechanisms of tumor cell motility have been studied extensively in the 2D context, but the mechanistic understanding of rapid single tumor cell motility in the in vivo context is still lacking. Here, we show that streaming tumor cells in vivo use collagen fibers with diameters below 3 µm. Employing 1D migration assays with matching in vivo fiber dimensions, we found a dependence of tumor cell motility on 1D substrate width, with cells moving the fastest and the most persistently on the narrowest 1D fibers (700 nm-2.5 µm). Interestingly, we also observed nuclear deformation in the absence of restricting extracellular matrix pores during high speed carcinoma cell migration in 1D, similar to the nuclear deformation observed in tumor cells in vivo. Further, we found that actomyosin machinery is aligned along the 1D axis and actomyosin contractility synchronously regulates cell motility and nuclear deformation. To further investigate the link between cell speed and nuclear deformation, we focused on the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex proteins and SRF-MKL1 signaling, key regulators of mechanotransduction, actomyosin contractility and actin-based cell motility. Analysis of The Cancer Genome Atlas dataset showed a dramatic decrease in the LINC complex proteins SUN1 and SUN2 in primary tumor compared to the normal tissue. Disruption of LINC complex by SUN1 + 2 KD led to multi-lobular elongated nuclei, increased tumor cell motility and concomitant increase in F-actin, without affecting Lamin proteins. Mechanistically, we found that MKL1, an effector of changes in cellular G-actin to F-actin ratio, is required for increased 1D motility seen in SUN1 + 2 KD cells. Thus, we demonstrate a previously unrecognized crosstalk between SUN proteins and MKL1 transcription factor in modulating nuclear shape and carcinoma cell motility in an in vivo relevant 1D microenvironment.

Author Correction: Optogenetic control of cofilin and αTAT in living cells using Z-lock.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: Septin 9 isoforms promote tumorigenesis in mammary epithelial cells by increasing migration and ECM degradation through metalloproteinase secretion at focal adhesions.

The original version of this Article contained an error in the author affiliations. Vladislav V. Verkhusha was incorrectly associated with the School of Mathematics, Statistics & Applied Mathematics, National University of Ireland Galway, Galway, Ireland. The correct affiliation is Anatomy and Structural Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY, USA.

Optogenetic control of cofilin and αTAT in living cells using Z-lock.

Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.

Septin 9 isoforms promote tumorigenesis in mammary epithelial cells by increasing migration and ECM degradation through metalloproteinase secretion at focal adhesions.

The cytoskeletal interacting protein Septin 9 (SEPT9), a member of the septin gene family, has been proposed to have oncogenic functions. It is a known hot spot of retroviral tagging insertion and a fusion partner of both de novo and therapy-induced mixed lineage leukemia (MLL). Of all septins, SEPT9 holds the strongest link to cancer, especially breast cancer. Murine models of breast cancer frequently exhibit SEPT9 amplification in the form of double minute chromosomes, and about 20% of human breast cancer display genomic amplification and protein over expression at the SEPT9 locus. Yet, a clear mechanism by which SEPT9 elicits tumor-promoting functions is lacking. To obtain unbiased insights on molecular signatures of SEPT9 upregulation in breast tumors, we overexpressed several of its isoforms in breast cancer cell lines. Global transcriptomic profiling supports a role of SEPT9 in invasion. Functional studies reveal that SEPT9 upregulation is sufficient to increase degradation of the extracellular matrix, while SEPT9 downregulation inhibits this process. The degradation pattern is peripheral and associated with focal adhesions (FAs), where it is coupled with increased expression of matrix metalloproteinases (MMPs). SEPT9 overexpression induces MMP upregulation in human tumors and in culture models and promotes MMP3 secretion to the media at FAs. Downregulation of SEPT9 or chemical inhibition of septin filament assembly impairs recruitment of MMP3 to FAs. Our results indicate that SEPT9 promotes upregulation and both trafficking and secretion of MMPs near FAs, thus enhancing migration and invasion of breast cancer cells.

Optimizing leading edge F-actin labeling using multiple actin probes, fixation methods and imaging modalities.

We systematically evaluated the performance and reliability of several widely used, commercially available actin-filament probes in a highly motile breast adenocarcinoma cell line to optimize the visualization of F-actin-rich dynamic lamellipodia. We evaluated four Phalloidin-fluorophores, two anti-actin antibodies, and three live-cell actin probes in five fixation conditions across three imaging platforms as a basis for the design of optimized protocols. Of the fluorescent phalloidin-dye conjugates tested, Alexa Fluor-488 Phalloidin ranked best in overall labeling of the actin cytoskeleton and maintenance of the fluorescence signal over time. Use of actin monoclonal antibodies revealed significant limitations under a variety of fixation-permeabilization conditions. Evaluation of commonly used live-cell probes provides evidence for actin filament bias, with TagRFP-Lifeact excluded from lamellipodia, but not mEGFP-Lifeact or F-tractin-EGFP.

Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors.

Metastatic dissemination of cancer cells from the primary tumor and their spread to distant sites in the body is the leading cause of mortality in breast cancer patients. While researchers have identified treatments that shrink or slow metastatic tumors, no treatment that permanently eradicates metastasis exists at present. Here, we show that the ABL kinase inhibitors imatinib, nilotinib, and GNF-5 impede invadopodium precursor formation and cortactin-phosphorylation dependent invadopodium maturation, leading to decreased actin polymerization in invadopodia, reduced extracellular matrix degradation, and impaired matrix proteolysis-dependent invasion. Using a mouse xenograft model we demonstrate that, while primary tumor size is not affected by ABL kinase inhibitors, the in vivo matrix metalloproteinase (MMP) activity, tumor cell invasion, and consequent spontaneous metastasis to lungs are significantly impaired in inhibitor-treated mice. Further proteogenomic analysis of breast cancer patient databases revealed co-expression of the Abl-related gene (Arg) and cortactin across all hormone- and human epidermal growth factor receptor 2 (HER2)-receptor status tumors, which correlates synergistically with distant metastasis and poor patient prognosis. Our findings establish a prognostic value for Arg and cortactin as predictors of metastatic dissemination and suggest that therapeutic inhibition of ABL kinases may be used for blocking breast cancer metastasis.

Pyk2 and FAK differentially regulate invadopodia formation and function in breast cancer cells.

The nonreceptor tyrosine kinase Pyk2 is highly expressed in invasive breast cancer, but the mechanism by which it potentiates tumor cell invasiveness is unclear at present. Using high-throughput protein array screening and bioinformatic analysis, we identified cortactin as a novel substrate and interactor of proline-rich tyrosine kinase 2 (Pyk2). Pyk2 colocalizes with cortactin to invadopodia of invasive breast cancer cells, where it mediates epidermal growth factor-induced cortactin tyrosine phosphorylation both directly and indirectly via Src-mediated Abl-related gene (Arg) activation, leading to actin polymerization in invadopodia, extracellular matrix degradation, and tumor cell invasion. Both Pyk2 and the closely related focal adhesion kinase (FAK) regulate tumor cell invasion, albeit via distinct mechanisms. Although Pyk2 regulates tumor cell invasion by controlling invadopodium-mediated functions, FAK controls invasiveness of tumor cells by regulating focal adhesion-mediated motility. Collectively, our findings identify Pyk2 as a unique mediator of invadopodium formation and function and also provide a novel insight into the mechanisms by which Pyk2 mediates tumor cell invasion.

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

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.

Tumor Cell Invadopodia: Invasive Protrusions that Orchestrate Metastasis.

Invadopodia are a subset of invadosomes that are implicated in the integration of signals from the tumor microenvironment to support tumor cell invasion and dissemination. Recent progress has begun to define how tumor cells regulate the plasticity necessary for invadopodia to assemble and function efficiently in the different microenvironments encountered during dissemination in vivo. Exquisite mapping by many laboratories of the pathways involved in integrating diverse invadopodium initiation signals, from growth factors, to extracellular matrix (ECM) and cell-cell contact in the tumor microenvironment, has led to insight into the molecular basis of this plasticity. Here, we integrate this new information to discuss how the invadopodium is an important conductor that orchestrates tumor cell dissemination during metastasis.

MenaINV dysregulates cortactin phosphorylation to promote invadopodium maturation.

Invadopodia, actin-based protrusions of invasive carcinoma cells that focally activate extracellular matrix-degrading proteases, are essential for the migration and intravasation of tumor cells during dissemination from the primary tumor. We have previously shown that cortactin phosphorylation at tyrosine residues, in particular tyrosine 421, promotes actin polymerization at newly-forming invadopodia, promoting their maturation to matrix-degrading structures. However, the mechanism by which cells regulate the cortactin tyrosine phosphorylation-dephosphorylation cycle at invadopodia is unknown. Mena, an actin barbed-end capping protein antagonist, is expressed as various splice-isoforms. The MenaINV isoform is upregulated in migratory and invasive sub-populations of breast carcinoma cells, and is involved in tumor cell intravasation. Here we show that forced MenaINV expression increases invadopodium maturation to a far greater extent than equivalent expression of other Mena isoforms. MenaINV is recruited to invadopodium precursors just after their initial assembly at the plasma membrane, and promotes the phosphorylation of cortactin tyrosine 421 at invadopodia. In addition, we show that cortactin phosphorylation at tyrosine 421 is suppressed by the phosphatase PTP1B, and that PTP1B localization to the invadopodium is reduced by MenaINV expression. We conclude that MenaINV promotes invadopodium maturation by inhibiting normal dephosphorylation of cortactin at tyrosine 421 by the phosphatase PTP1B.

MicroRNA-375 Suppresses Extracellular Matrix Degradation and Invadopodial Activity in Head and Neck Squamous Cell Carcinoma.

CONTEXT: Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer with an association with locoregional recurrence and lymph node metastasis. We have previously reported that low microRNA-375 (miR-375) expression levels correlate with poor patient survival, increased locoregional recurrence, and distant metastasis. Increasing miR-375 expression in HNSCC cell lines to levels found in normal cells results in suppressed invasive properties. HNSCC invasion is mediated in part by invadopodia-associated degradation of the extracellular matrix. OBJECTIVE: To determine whether elevated miR-375 expression in HNSCC cell lines also affects invadopodia formation and activity. DESIGN: For evaluation of the matrix degradation properties of the HNSCC lines, an invadopodial matrix degradation assay was used. The total protein levels of invadopodia-associated proteins were measured by Western blot analyses. Immunoprecipitation experiments were conducted to evaluate the tyrosine phosphorylation state of cortactin. Human protease arrays were used for the detection of the secreted proteases. Quantitative real time-polymerase chain reaction measurements were used to evaluate the messenger RNA (mRNA) expression of the commonly regulated proteases. RESULTS: Increased miR-375 expression in HNSCC cells suppresses extracellular matrix degradation and reduces the number of mature invadopodia. Higher miR-375 expression does not reduce cellular levels of selected invadopodia-associated proteins, nor is tyrosine phosphorylation of cortactin altered. However, HNSCC cells with higher miR-375 expression had significant reductions in the mRNA expression levels and secreted levels of specific proteases. CONCLUSIONS: MicroRNA-375 regulates invadopodia maturation and function potentially by suppressing the expression and secretion of proteases.