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Obesity is associated with increased incidence and metastasis of triple-negative breast cancer, an aggressive breast cancer subtype. The extracellular matrix (ECM) is a major component of the tumor microenvironment that drives metastasis. To characterize the temporal effects of age and high-fat diet (HFD)-driven weight gain on the ECM, we injected allograft tumor cells at 4-week intervals into mammary fat pads of mice fed a control or HFD, assessing tumor growth and metastasis and evaluating the ECM composition of the mammary fat pads, lungs, and livers. Tumor growth was increased in obese mice after 12 weeks on HFD. Liver metastasis increased in obese mice only at 4 weeks, and elevated body weight correlated with increased metastasis to the lungs but not the liver. Whole decellularized ECM coupled with proteomics indicated that early stages of obesity were sufficient to induce changes in the ECM composition. Obesity led to an increased abundance of the proinvasive ECM proteins collagen IV and collagen VI in the mammary glands and enhanced the invasive capacity of cancer cells. Cells of stromal vascular fraction and adipose stem and progenitor cells were primarily responsible for secreting collagen IV and collagen VI, not adipocytes. Longer exposure to HFD increased the invasive potential of ECM isolated from the lungs and liver, with significant changes in ECM composition found in the liver with short-term HFD exposure. Together, these data suggest that changes in the breast, lungs, and liver ECM underlie some of the effects of obesity on triple-negative breast cancer incidence and metastasis. Significance: Organ-specific extracellular matrix changes in the primary tumor and metastatic microenvironment are mechanisms by which obesity contributes to breast cancer progression., (©2024 American Association for Cancer Research.)

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

The extracellular matrix (ECM) has always been studied in the context of the structural support it provides tissues. However, more recently, it has become clear that ECM proteins do more to regulate biological processes relevant to cancer progression: from activating complex signaling pathways to presenting soluble growth factors. In 2009, Ulrich and colleagues provided evidence that the physical properties of the ECM could also contribute to glioblastoma tumor cell proliferation and invasion using tunable hydrogels, emphasizing a role for tumor rigidity in central nervous system cancer progression. Here, we will discuss the results of this landmark article, as well as highlight other work that has shown the importance of tissue stiffness in glioblastoma and other tumor types in the tumor microenvironment. Finally, we will discuss how this research has led to the development of novel treatments for cancer that target tumor rigidity. See related article by Ulrich and colleagues, Cancer Res 2009;69:4167-74., (©2024 American Association for Cancer Research.)

Background: Metastasis is the leading cause of death in breast cancer patients. For metastasis to occur, tumor cells must invade locally, intravasate, and colonize distant tissues and organs, all steps that require tumor cell migration. The majority of studies on invasion and metastasis rely on human breast cancer cell lines. While it is known that these cells have different properties and abilities for growth and metastasis, the in vitro morphological, proliferative, migratory, and invasive behavior of these cell lines and their correlation to in vivo behavior is poorly understood. Thus, we sought to classify each cell line as poorly or highly metastatic by characterizing tumor growth and metastasis in a murine model of six commonly used human triple-negative breast cancer xenografts, as well as determine which in vitro assays commonly used to study cell motility best predict in vivo metastasis., Methods: We evaluated the liver and lung metastasis of human TNBC cell lines MDA-MB-231, MDA-MB-468, BT549, Hs578T, BT20, and SUM159 in immunocompromised mice. We characterized each cell line's cell morphology, proliferation, and motility in 2D and 3D to determine the variation in these parameters between cell lines., Results: We identified MDA-MB-231, MDA-MB-468, and BT549 cells as highly tumorigenic and metastatic, Hs578T as poorly tumorigenic and metastatic, BT20 as intermediate tumorigenic with poor metastasis to the lungs but highly metastatic to the livers, and SUM159 as intermediate tumorigenic but poorly metastatic to the lungs and livers. We showed that metrics that characterize cell morphology are the most predictive of tumor growth and metastatic potential to the lungs and liver. Further, we found that no single in vitro motility assay in 2D or 3D significantly correlated with metastasis in vivo., Conclusions: Our results provide an important resource for the TNBC research community, identifying the metastatic potential of 6 commonly used cell lines. Our findings also support the use of cell morphological analysis to investigate the metastatic potential and emphasize the need for multiple in vitro motility metrics using multiple cell lines to represent the heterogeneity of metastasis in vivo., (© 2024. The Author(s).)

The peripheral nervous system plays an important role in cancer progression. Studies in multiple cancer types have shown that higher intratumoral nerve density is associated with poor outcomes. Peripheral nerves have been shown to directly regulate tumor cell properties, such as growth and metastasis, as well as affect the local environment by modulating angiogenesis and the immune system. In this Review, we discuss the identity of nerves in organs in the periphery where solid tumors grow, the known mechanisms by which nerve density increases in tumors, and the effects these nerves have on cancer progression. We also discuss the strengths and weaknesses of current in vitro and in vivo models used to study nerve-cancer interactions. Increased understanding of the mechanisms by which nerves impact tumor progression and the development of new approaches to study nerve-cancer interactions will facilitate the discovery of novel treatment strategies to treat cancer by targeting nerves., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

In breast cancer, nerve presence has been correlated with more invasive disease and worse prognosis, yet the mechanisms by which different types of peripheral nerves drive tumor progression remain poorly understood. In this study, we identified sensory nerves as more abundant in human triple-negative breast cancer (TNBC) tumors. Co-injection of sensory neurons isolated from the dorsal root ganglia (DRG) of adult female mice with human TNBC cells in immunocompromised mice increased the number of lung metastases. Direct in vitro co-culture of human TNBC cells with the dorsal root ganglia (DRG) of adult female mice revealed that TNBC cells adhere to sensory neuron fibers leading to an increase in migration speed. Species-specific RNA sequencing revealed that co-culture of TNBC cells with sensory nerves upregulates the expression of genes associated with cell migration and adhesion in cancer cells. We demonstrated that lack of the semaphorin receptor PlexinB3 in cancer cells attenuate their adhesion to and migration on sensory nerves. Together, our results identify a mechanism by which nerves contribute to breast cancer migration and metastasis by inducing a shift in TNBC cell gene expression and support the rationale for disrupting neuron-cancer cell interactions to target metastasis., (© 2022. The Author(s).)

Metastasis accounts for 90% of cancer-related deaths, yet the mechanisms by which cancer cells colonize secondary organs remain poorly understood. For breast cancer patients, metastasis to the liver is associated with poor prognosis and a median survival of 6 months. Standard of care is chemotherapy, but recurrence occurs in 30% of patients. Systemic chemotherapy has been shown to induce hepatotoxicity and fibrosis, but how chemotherapy impacts the composition of the liver extracellular matrix (ECM) remains unknown. Individual ECM proteins drive tumor cell proliferation and invasion, features that are essential for metastatic outgrowth in the liver. First, we find that the ECM of livers isolated from chemotherapy-treated MMTV-PyMT mice increases the invasion, but not proliferation, of metastatic breast cancer cells. Proteomic analysis of the liver ECM identified Collagen V to be more abundant in paclitaxel-treated livers. We show that Collagen V increases cancer cell invasion via α1β1 integrins and MAPK signaling, while also increasing the alignment of Collagen I, which has been associated with increased invasion. Treatment with obtustatin, an inhibitor specific to α1β1 integrins, inhibits tumor cell invasion in decellularized ECM from paclitaxel-treated livers. Overall, we show chemotherapy treatment alters the liver microenvironment, priming it as a pro-metastatic niche for cancer metastasis., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Triple-negative breast cancer (TNBC) is the most aggressive and deadly subtype of breast cancer, accounting for 30,000 cases annually in the United States. While there are several clinical trials ongoing to identify new agents to treat TNBC, the majority of patients with TNBC are treated with anthracycline- or taxane-based chemotherapies in the neoadjuvant setting, followed by surgical resection and adjuvant chemotherapy. While many patients respond well to this approach, as many as 25% will suffer local or metastatic recurrence within 5 years. Understanding the mechanisms that drive recurrence after chemotherapy treatment is critical to improving survival for patients with TNBC. It is well established that the extracellular matrix (ECM), which provides structure and support to tissues, is a major driver of tumor growth, local invasion, and dissemination of cancer cells to distant metastatic sites. In the present study, we show that decellularized ECM (dECM) obtained from chemotherapy-treated mice increases motility of treatment-naïve breast cancer cells compared with vehicle-treated dECM. Tandem-mass-tag proteomics revealed that anthracycline- and taxane-based chemotherapies induce drug-specific changes in tumor ECM composition. The basement membrane protein collagen IV was significantly upregulated in the ECM of chemotherapy-treated mice and patients treated with neoadjuvant chemotherapy. Collagen IV drove invasion via activation of Src and focal adhesion kinase signaling downstream of integrin α1 and α2, and inhibition of collagen IV-driven signaling decreased motility in chemotherapy-treated dECM. These studies provide a novel mechanism by which chemotherapy may induce metastasis via its effects on ECM composition., Significance: Cytotoxic chemotherapy induces significant changes in the composition of tumor ECM, inducing a more invasive and aggressive phenotype in residual tumor cells following chemotherapy., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: There is a critical need to better understand the mechanisms that drive local cell invasion and metastasis to develop new therapeutics targeting metastatic disease. Bioelectricity is an important mediator of cellular processes and changes in the resting membrane potential (RMP) are associated with increased cancer cell invasion. However, whether the RMP can be used to target invading cancer cells is unknown., Methods: We employed both genetic and pharmacological manipulation of potassium channel activity and characterized the effects on breast cancer cell migration and invasion in vitro, and metastasis in an animal model of breast cancer., Findings: Our data demonstrate that altering the RMP of triple-negative breast cancer (TNBC) cells by manipulating potassium channel expression increases in vitro invasion, in vivo tumour growth and metastasis, and is accompanied by changes in gene expression associated with cell adhesion., Interpretation: We describe a novel mechanism for RMP-mediated cell migration involving cadherin-11 and the MAPK pathway. Importantly, we identify a new strategy to target metastatic TNBC in vivo by repurposing an FDA-approved potassium channel blocker. Our results demonstrate that bioelectricity regulates cancer cell invasion and metastasis which could lead to a new class of therapeutics for patients with metastatic disease., Funding: This work was supported by the National Institutes of Health (R00-CA207866 to M.J.O.), Tufts University (Start-up funds from the School of Engineering to M.J.O., Tufts Collaborates Award to M.J.O. and M.L.), Allen Discovery centre program (Paul G. Allen Frontiers Group (12,171) to M.L.), and Breast Cancer Alliance Young Investigator Grant to M.J.O, Laidlaw Scholar funding to D.S. M.L. also gratefully acknowledges support of the Barton Family Foundation., Competing Interests: Declaration of interests ML receives funding from Augmanity Inc. for a separate project. There are no other conflicts., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cardiac fibroblasts are responsible for extracellular matrix turnover and repair in the cardiac environment and serve to help facilitate immune responses. However, it is well established that they have a significant phenotypic heterogeneity with respect to location, physiological conditions, and developmental age. The goal of this study was to provide an in-depth transcriptomic profile of cardiac fibroblasts derived from rat hearts at fetal, neonatal, and adult developmental ages to ascertain variations in gene expression that may drive functional differences in these cells at these specific stages of development. We performed RNA sequencing (RNA-seq) of cardiac fibroblasts isolated from fetal, neonatal, and adult rats and compared with the rat genome. Principal component analysis of RNA-seq data suggested that data variance was predominantly due to developmental age. Differential expression and gene set enrichment analysis against Gene Ontology and Kyoto Encyclopedia of Genes and Genomes datasets indicated an array of differences across developmental ages, including significant decreases in cardiac development and cardiac function-associated genes with age and a significant increase in immune- and inflammatory-associated functions, particularly immune cell signaling and cytokine and chemokine production, with respect to increasing developmental age. These results reinforce established evidence of diverse phenotypic heterogeneity of fibroblasts with respect to developmental age. Furthermore, based on our analysis of gene expression, age-specific alterations in cardiac fibroblasts may play a crucial role in observed differences in cardiac inflammation and immune response observed across developmental ages.

Cellular communication is important in all aspects of tissue and organism functioning, from the level of single cells, two discreet populations, and distant tissues of the body. Long distance communication networks integrate individual cells into tissues to maintain a complex organism during development, but when communication between cells goes awry, disease states such as cancer emerge. Herein we discuss the growing body of evidence suggesting that communication methods known to be employed by neurons, also exist in other cell types. We identify three major areas of long-distance communication: bioelectric signaling, tunneling nanotubes (TNTs), and macrophage modulation of networks, and draw comparisons about how these systems operate in the context of development and cancer. Bioelectric signaling occurs between cells through exchange of ions and tissue-level electric fields, leading to changes in biochemical gradients and molecular signaling pathways to control normal development and tumor growth and invasion in cancer. TNTs transport key morphogens and other cargo long distances, mediating electrical coupling, tissue patterning, and malignancy of cancer cells. Lastly macrophages maintain long distance signaling networks through trafficking of vesicles during development, providing communication relays and priming favorable microenvironments for cancer metastasis. By drawing comparisons between non-neural long distance signaling in the context of development and cancer we aim to encourage crosstalk between the two fields to cultivate new hypotheses and potential therapeutic strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MZ declared a shared consortium with one of the author ML to the handling editor., (Copyright © 2021 McMillen, Oudin, Levin and Payne.)

High-quality developmental environments often improve individual performance into adulthood, but allocating toward early life traits, such as growth, development rate and reproduction, may lead to trade-offs with late-life performance. It is, therefore, uncertain how a rich developmental environment will affect the ageing process (senescence), particularly in wild insects. To investigate the effects of early life environmental quality on insect life-history traits, including senescence, we reared larval antler flies ( Protopiophila litigata ) on four diets of varying nutrient concentration, then recorded survival and mating success of adult males released in the wild. Declining diet quality was associated with slower development, but had no effect on other life-history traits once development time was accounted for. Fast-developing males were larger and lived longer, but experienced more rapid senescence in survival and lower average mating rate compared to slow developers. Ultimately, larval diet, development time and body size did not predict lifetime mating success. Thus, a rich environment led to a mixture of apparent benefits and costs, mediated by development time. Our results indicate that 'silver spoon' effects can be complex and that development time mediates the response of adult life-history traits to early life environmental quality.

The extracellular matrix (ECM), a major component of the tumor microenvironment, promotes local invasion to drive metastasis. Here, we describe a method to study whole-tissue ECM effects from disease states associated with metastasis on tumor cell phenotypes and identify the individual ECM proteins and signaling pathways that are driving these effects. We show that decellularized ECM from tumor-bearing and obese mammary glands drives TNBC cell invasion. Proteomics of the ECM from the obese mammary gland led us to identify full-length collagen VI as a novel driver of TNBC cell invasion whose abundance in tumor stroma increases with body mass index in human TNBC patients. Last, we describe the mechanism by which collagen VI contributes to TNBC cell invasion via NG2-EGFR cross-talk and MAPK signaling. Overall, these studies demonstrate the value of decellularized ECM scaffolds obtained from tissues to identify novel functions of the ECM., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Interpreting how multicellular interactions in the tumor affect resistance pathways to BRAF and MEK1/2 MAPK inhibitors (MAPKi) remains a challenge. To investigate this, we profiled global ligand-receptor interactions among tumor and stromal/immune cells from biopsies of MAPK-driven disease. MAPKi increased tumor-associated macrophages (TAMs) in some patients, which correlated with poor clinical response, and MAPKi coamplified bidirectional tumor-TAM signaling via receptor tyrosine kinases (RTKs) including AXL, MERTK, and their ligand GAS6. In xenograft tumors, intravital microscopy simultaneously monitored in situ single-cell activities of multiple kinases downstream of RTKs, revealing MAPKi increased TAMs and enhanced bypass signaling in TAM-proximal tumor cells. As a proof-of-principle strategy to block this signaling, we developed a multi-RTK kinase inhibitor nanoformulation that accumulated in TAMs and delayed disease progression. Thus, bypass signaling can reciprocally amplify across nearby cell types, offering new opportunities for therapeutic design., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Metastasis, the leading cause of death in cancer patients, requires the invasion of tumor cells through the stroma in response to migratory cues, in part provided by the extracellular matrix (ECM). Recent advances in proteomics have led to the identification of hundreds of ECM proteins, which are more abundant in tumors relative to healthy tissue. Our goal was to develop a pipeline to easily predict which ECM proteins are more likely to have an effect on cancer invasion and metastasis. We evaluated the effect of four ECM proteins upregulated in breast tumor tissue in multiple human breast cancer cell lines in three assays. There was no linear relationship between cell adhesion to ECM proteins and ECM-driven 2D cell migration speed, persistence, or 3D invasion. We then used classifiers and partial-least squares regression analysis to identify which metrics best predicted ECM-driven 2D migration and 3D invasion responses. We find that ECM-driven 2D cell migration speed or persistence did not predict 3D invasion in response to the same cue. However, cell adhesion, and in particular cell elongation and shape irregularity, accurately predicted the magnitude of ECM-driven 2D migration and 3D invasion. Our models successfully predicted the effect of novel ECM proteins in a cell-line specific manner. Overall, our studies identify the cell morphological features that determine 3D invasion responses to individual ECM proteins. This platform will help provide insight into the functional role of ECM proteins abundant in tumor tissue and help prioritize strategies for targeting tumor-ECM interactions to treat metastasis., (© Author(s).)

All cells possess an electric potential across their plasma membranes and can generate and receive bioelectric signals. The cellular resting membrane potential (RMP) can regulate cell proliferation, differentiation and apoptosis. Current approaches to measure the RMP rely on patch clamping, which is technically challenging, low-throughput and not widely available. It is therefore critical to develop simple strategies to measure, manipulate and characterize the RMP. Here, we present a simple methodology to study the RMP of non-excitable cells and characterize the contribution of individual ions to the RMP using a voltage-sensitive dye. We define protocols using extracellular solutions in which permeable ions (Na + , Cl - and K + ) are substituted with non-permeable ions [N-Methyl-D-glucamine (NMDG), gluconate, choline, SO 4 2- ]. The resulting RMP modifications were assessed with both patch clamp and a voltage sensitive dye. Using an epithelial and cancer cell line, we demonstrate that the proposed ionic solutions can selectively modify the RMP and help determine the relative contribution of ionic species in setting the RMP. The proposed method is simple and reproducible and will make the study of bioelectricity more readily available to the cell biology community.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Metastasis is the main cause of death in cancer patients but remains a poorly understood process. Small cell lung cancer (SCLC) is one of the most lethal and most metastatic cancer types. SCLC cells normally express neuroendocrine and neuronal gene programs but accumulating evidence indicates that these cancer cells become relatively more neuronal and less neuroendocrine as they gain the ability to metastasize. Here we show that mouse and human SCLC cells in culture and in vivo can grow cellular protrusions that resemble axons. The formation of these protrusions is controlled by multiple neuronal factors implicated in axonogenesis, axon guidance, and neuroblast migration. Disruption of these axon-like protrusions impairs cell migration in culture and inhibits metastatic ability in vivo. The co-option of developmental neuronal programs is a novel molecular and cellular mechanism that contributes to the high metastatic ability of SCLC., Competing Interests: DY, FQ, HC, CC, JL, NJ, BG, CS, CK, MO No competing interests declared, MW has equity in, and is an advisor for, D2GOncology, JS Receives research funding from Stemcentrx/Abbvie, Pfizer, and Revolution Medicines and owns stock in Forty Seven Inc, (© 2019, Yang et al.)

As the leading cause of death in cancer, there is an urgent need to develop treatments to target the dissemination of primary tumor cells to secondary organs, known as metastasis. Bioelectric signaling has emerged in the last century as an important controller of cell growth, and with the development of current molecular tools we are now beginning to identify its role in driving cell migration and metastasis in a variety of cancer types. This review summarizes the currently available research for bioelectric signaling in solid tumor metastasis. We review the steps of metastasis and discuss how these can be controlled by bioelectric cues at the level of a cell, a population of cells, and the tissue. The role of ion channel, pump, and exchanger activity and ion flux is discussed, along with the importance of the membrane potential and the relationship between ion flux and membrane potential. We also provide an overview of the evidence for control of metastasis by external electric fields (EFs) and draw from examples in embryogenesis and regeneration to discuss the implications for endogenous EFs. By increasing our understanding of the dynamic properties of bioelectric signaling, we can develop new strategies that target metastasis to be translated into the clinic., Competing Interests: No competing financial interests exist., (Copyright 2019, Mary Ann Liebert, Inc., publishers.)

Taxane therapy remains the standard of care for triple-negative breast cancer. However, high frequencies of recurrence and progression in treated patients indicate that metastatic breast cancer cells can acquire resistance to this drug. The actin regulatory protein MENA and particularly its invasive isoform, MENA INV , are established drivers of metastasis. MENA INV expression is significantly correlated with metastasis and poor outcome in human patients with breast cancer. We investigated whether MENA isoforms might play a role in driving resistance to chemotherapeutics. We find that both MENA and MENA INV confer resistance to the taxane paclitaxel, but not to the widely used DNA-damaging agents doxorubicin or cisplatin. Furthermore, paclitaxel treatment does not attenuate growth of MENA INV -driven metastatic lesions. Mechanistically, MENA isoform expression alters the ratio of dynamic and stable microtubule populations in paclitaxel-treated cells. MENA expression also increases MAPK signaling in response to paclitaxel treatment. Decreasing ERK phosphorylation by co-treatment with MEK inhibitor restored paclitaxel sensitivity by driving microtubule stabilization in MENA isoform-expressing cells. Our results reveal a novel mechanism of taxane resistance in highly metastatic breast cancer cells and identify a combination therapy to overcome such resistance. Mol Cancer Ther; 16(1); 143-55. ©2016 AACR., (©2016 American Association for Cancer Research.)

Mena INV , an isoform of the motility regulator protein Mena, contributes to prometastatic phenotypes. Tumor microenvironment of metastasis (TMEM), a three-cell structure associated with intravasation, contains a stationary Mena-expressing tumor cell. TMEM density and Mena INV expression both correlate with poor clinical outcome in breast cancer patients. However, is Mena INV involved in TMEM assembly and function?, (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Treatment of BRAF-mutated melanoma tumors with BRAF inhibitor-based therapy produces high response rates, but of limited duration in the vast majority of patients. Published investigations of resistance mechanisms suggest numerous examples of tumor adaptation and signal transduction bypass mechanisms, but without insight into biomarkers that would predict which mechanism will predominate. Monitoring phenotypic response of multiple adaptive mechanisms simultaneously within the same tumor as it adapts during treatment has been elusive., Experimental Design: This study reports on a method to provide a more complete understanding of adaptive tumor responses. We simultaneously measured in vivo antitumor activity of 12 classes of inhibitors, which are suspected of enabling adaptive escape mechanisms, at various time points during systemic BRAF inhibition. We used implantable microdevices to release multiple compounds into distinct regions of a tumor to measure the efficacy of each compound independently and repeated these measurements as tumors progressed on systemic BRAF treatment., Results: We observed varying phenotypic responses to specific inhibitors before, during, and after prolonged systemic treatment with BRAF inhibitors. Our results specifically identify PI3K, PDGFR, EGFR, and HDAC inhibitors as becoming significantly more efficacious during systemic BRAF inhibition. The sensitivity to other targeted inhibitors remained mostly unchanged, whereas local incremental sensitivity to PLX4720 declined sharply., Conclusions: These findings suggest redundancy of several resistance mechanisms and may help identify optimal constituents of more effective combination therapy in BRAF-mutant melanoma. They also represent a new paradigm for dynamic measurement of adaptive signaling mechanisms within the same tumor during therapy. Clin Cancer Res; 22(24); 6031-8. ©2016 AACR., (©2016 American Association for Cancer Research.)

Directed cell migration, a key process in metastasis, arises from the combined influence of multiple processes, including chemotaxis-the directional movement of cells to soluble cues-and haptotaxis-the migration of cells on gradients of substrate-bound factors. However, it is unclear how chemotactic and haptotactic pathways integrate with each other to drive overall cell behavior. Mena INV has been implicated in metastasis by driving chemotaxis via dysregulation of phosphatase PTP1B and more recently in haptotaxis via interaction with integrin α5β1. Here we find that Mena INV -driven haptotaxis on fibronectin (FN) gradients requires intact signaling between α5β1 integrin and the epidermal growth factor receptor (EGFR), which is influenced by PTP1B. Furthermore, we show that Mena INV -driven haptotaxis and ECM reorganization both require the Rab-coupling protein RCP, which mediates α5β1 and EGFR recycling. Finally, Mena INV promotes synergistic migratory response to combined EGF and FN in vitro and in vivo, leading to hyperinvasive phenotypes. Together our data demonstrate that Mena INV is a shared component of multiple prometastatic pathways that amplifies their combined effects, promoting synergistic cross-talk between RTKs and integrins., (© 2016 Oudin et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Unlabelled: Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here, we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo Haptotaxis on FN gradients requires direct interaction between α5β1 integrin and MENA, an actin regulator, and involves increases in focal complex signaling and tumor cell-mediated extracellular matrix (ECM) remodeling. Compared with MENA, higher levels of the prometastatic MENA(INV) isoform associate with α5, which enables 3-D haptotaxis of tumor cells toward the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MENA(INV) and FN levels were correlated in two breast cancer cohorts, and high levels of MENA(INV) were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor cell-intrinsic mechanism that promotes metastasis through ECM remodeling and ECM-guided directional migration., Significance: Here, we provide new insight into how tumor cell:ECM interactions generate signals and structures that promote directed tumor cell migration, a critical component of metastasis. Our results identify a tumor cell-intrinsic mechanism driven by the actin regulatory protein MENA that promotes ECM remodeling and haptotaxis along FN gradients. Cancer Discov; 6(5); 516-31. ©2016 AACR.See related commentary by Santiago-Medina and Yang, p. 474This article is highlighted in the In This Issue feature, p. 461., (©2016 American Association for Cancer Research.)

Unlabelled: Kinase inhibitor resistance often involves upregulation of poorly understood "bypass" signaling pathways. Here, we show that extracellular proteomic adaptation is one path to bypass signaling and drug resistance. Proteolytic shedding of surface receptors, which can provide negative feedback on signaling activity, is blocked by kinase inhibitor treatment and enhances bypass signaling. In particular, MEK inhibition broadly decreases shedding of multiple receptor tyrosine kinases (RTK), including HER4, MET, and most prominently AXL, an ADAM10 and ADAM17 substrate, thus increasing surface RTK levels and mitogenic signaling. Progression-free survival of patients with melanoma treated with clinical BRAF/MEK inhibitors inversely correlates with RTK shedding reduction following treatment, as measured noninvasively in blood plasma. Disrupting protease inhibition by neutralizing TIMP1 improves MAPK inhibitor efficacy, and combined MAPK/AXL inhibition synergistically reduces tumor growth and metastasis in xenograft models. Altogether, extracellular proteomic rewiring through reduced RTK shedding represents a surprising mechanism for bypass signaling in cancer drug resistance., Significance: Genetic, epigenetic, and gene expression alterations often fail to explain adaptive drug resistance in cancer. This work presents a novel post-translational mechanism of such resistance: Kinase inhibitors, particularly targeting MAPK signaling, increase tumor cell surface receptor levels due to widely reduced proteolysis, allowing tumor signaling to circumvent intended drug action., Competing Interests: of Potential Conflicts of Interest: No potential conflicts of interest were disclosed., (©2016 American Association for Cancer Research.)

Several functionally distinct isoforms of the actin regulatory Mena are produced by alternative splicing during tumor progression. Forced expression of the Mena(INV) isoform drives invasion, intravasation and metastasis. However, the abundance and distribution of endogenously expressed Mena(INV) within primary tumors during progression remain unknown, as most studies to date have only assessed relative mRNA levels from dissociated tumor samples. We have developed a Mena(INV) isoform-specific monoclonal antibody and used it to examine Mena(INV) expression patterns in mouse mammary and human breast tumors. Mena(INV) expression increases during tumor progression and to examine the relationship between Mena(INV) expression and markers for epithelial or mesenchymal status, stemness, stromal cell types and hypoxic regions. Further, while Mena(INV) robustly expressed in vascularized areas of the tumor, it is not confined to cells adjacent to blood vessels. Altogether, these data demonstrate the specificity and utility of the anti-Mena(INV)-isoform specific antibody, and provide the first description of endogenous Mena(INV) protein expression in mouse and human tumors.

Cancer metastasis requires the invasion of tumor cells into the stroma and the directed migration of tumor cells through the stroma toward the vasculature and lymphatics where they can disseminate and colonize secondary organs. Physical and biochemical gradients that form within the primary tumor tissue promote tumor cell invasion and drive persistent migration toward blood vessels and the lymphatics to facilitate tumor cell dissemination. These microenvironment cues include hypoxia and pH gradients, gradients of soluble cues that induce chemotaxis, and ions that facilitate galvanotaxis, as well as modifications to the concentration, organization, and stiffness of the extracellular matrix that produce haptotactic, alignotactic, and durotactic gradients. These gradients form through dynamic interactions between the tumor cells and the resident fibroblasts, adipocytes, nerves, endothelial cells, infiltrating immune cells, and mesenchymal stem cells. Malignant progression results from the integrated response of the tumor to these extrinsic physical and chemical cues. Here, we first describe how these physical and chemical gradients develop, and we discuss their role in tumor progression. We then review assays to study these gradients. We conclude with a discussion of clinical strategies used to detect and inhibit these gradients in tumors and of new intervention opportunities. Clarifying the role of these gradients in tumor evolution offers a unique approach to target metastasis., (© 2016 Oudin and Weaver; Published by Cold Spring Harbor Laboratory Press.)

During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes., (© 2015 Hughes, Oudin, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Despite the fact that the majority of lung cancer deaths are due to metastasis, the molecular mechanisms driving metastatic progression are poorly understood. Here, we present evidence that loss of Foxa2 and Cdx2 synergizes with loss of Nkx2-1 to fully activate the metastatic program. These three lineage-specific transcription factors are consistently down-regulated in metastatic cells compared with nonmetastatic cells. Knockdown of these three factors acts synergistically and is sufficient to promote the metastatic potential of nonmetastatic cells to that of naturally arising metastatic cells in vivo. Furthermore, silencing of these three transcription factors is sufficient to account for a significant fraction of the gene expression differences between the nonmetastatic and metastatic states in lung adenocarcinoma, including up-regulated expression of the invadopodia component Tks5long, the embryonal proto-oncogene Hmga2, and the epithelial-to-mesenchymal mediator Snail. Finally, analyses of tumors from a genetically engineered mouse model and patients show that low expression of Nkx2-1, Foxa2, and Cdx2 strongly correlates with more advanced tumors and worse survival. Our findings reveal that a large part of the complex transcriptional network in metastasis can be controlled by a small number of regulatory nodes that function redundantly, and loss of multiple nodes is required to fully activate the metastatic program., (© 2015 Li et al.; Published by Cold Spring Harbor Laboratory Press.)

During development and after birth neural stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to populate the olfactory bulb (OB) with neurons. Multiple factors promote neuroblast migration, but the contribution that many of these make to guidance within the intact RMS is not known. In the present study we have characterised in detail how endocannabinoid (eCB), BDNF and FGF receptor (FGFR) signalling regulates motility and guidance, and also determined whether any of these receptors operate in a regionally restricted manner. We used in vivo electroporation in postnatal mice to fluorescently label neuroblasts, and live cell imaging to detail their migratory properties. Cannabinoid receptor antagonists rendered neuroblasts less mobile, and when they did move guidance was lost. Similar results were obtained when eCB synthesis was blocked with diacylglycerol lipase (DAGL) inhibitors, and importantly eCB function is required for directed migration at both ends of the RMS. Likewise, inhibition of BDNF signalling disrupted motility and guidance in a similar manner along the entire RMS. In contrast, altering FGFR signalling inhibits motility and perturbs guidance, but only at the beginning of the stream. Inhibition of FGFR signalling in vivo also reduces the length of the leading process on migratory neuroblasts in a graded manner along the RMS. These results provide evidence for a guidance function for all three of the above receptor systems in the intact RMS, but show that FGFR signalling is unique as it is required in a regionally specific manner., (Copyright © 2014. Published by Elsevier Inc.)

Cell polarization is essential for neuronal development in both the embryonic and postnatal brain. Here, using primary cultures, in vivo postnatal electroporation and conditional genetic ablation, we show that the Ras-like small GTPase RalA and its effector, the exocyst, regulate the morphology and polarized migration of neural progenitors derived from the subventricular zone, a major neurogenic niche in the postnatal brain. Active RalA promotes the direct binding between the exocyst subunit Exo84 and the PDZ domain of Par6 through a non-canonical PDZ-binding motif. Blocking the Exo84-Par6 interaction impairs polarization in postnatal neural progenitors and cultured embryonic neurons. Our results provide the first in vivo characterization of RalA function in the mammalian brain and highlight a novel molecular mechanism for cell polarization. Given that the exocyst and the Par complex are conserved in many tissues, the functional significance of their interaction and its regulation by RalA are likely to be important in a wide range of polarization events.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration. In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

Until recently, endocannabinoid (eCB) signalling was largely studied in the context of synaptic plasticity in the postnatal brain in the absence of detailed knowledge of the nature of the enzyme(s) responsible for the synthesis of the eCBs. However, the identification of two diacylglycerol lipases (DAGLα and DAGLβ) responsible for the synthesis of 2-arachidonoylglycerol (2-AG) has increased the understanding of where this eCB is synthesised in relationship to the expression of cannabinoid receptor (CB)1 and CB2. Furthermore, the generation of knockout animals for each enzyme has allowed for the direct testing of their importance for established and emerging eCB functions. Based on this, we now know that DAGLα is enriched in dendritic spines that appose CB1-positive synaptic terminals, and that 2-AG functions as a retrograde signal controlling synaptic strength throughout the nervous system. Consequently, we have built on the principle that expression of eCB components dictates function to identify additional physiological functions of this signalling cassette. A number of studies have now provided support for DAGL-dependent eCB signalling playing important roles in brain development and in cellular plasticity in the adult nervous system. In this article, we will review evidence based on the localisation of the enzymes, as well as from genetic and pharmacological studies, that show DAGL-dependent eCB signalling to play an important role in axonal growth and guidance during development, in retrograde synaptic signalling at mature synapses, and in the control of adult neurogenesis in the hippocampus and subventricular zone., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

In the adult brain, neural stem cells proliferate within the subventricular zone before differentiating into migratory neuroblasts that travel along the rostral migratory stream (RMS) to populate the olfactory bulb with new neurons. Because neuroblasts have been shown to migrate to areas of brain injury, understanding the cues regulating this migration could be important for brain repair. Recent studies have highlighted an important role for endocannabinoid (eCB) signaling in the proliferation of the stem cell population, but it remained to be determined whether this pathway also played a role in cell migration. We now show that mouse migratory neuroblasts express cannabinoid receptors, diacylglycerol lipase α (DAGLα), the enzyme that synthesizes the endocannabinoid 2-arachidonoylglycerol (2-AG), and monoacylglycerol lipase, the enzyme responsible for its degradation. Using a scratch wound assay for a neural stem cell line and RMS explant cultures, we show that inhibition of DAGL activity or CB(1)/CB(2) receptors substantially decreases migration. In contrast, direct activation of cannabinoid receptors or preventing the breakdown of 2-AG increases migration. Detailed analysis of primary neuroblast migration by time-lapse imaging reveals that nucleokinesis, as well as the length and branching of the migratory processes are under dynamic control of the eCB system. Finally, similar effects are observed in vivo by analyzing the morphology of green fluorescent protein-labeled neuroblasts in brain slices from mice treated with CB(1) or CB(2) antagonists. These results describe a novel role for the endocannabinoid system in neuroblast migration in vivo, highlighting its importance in regulating an additional essential step in adult neurogenesis.

The subventricular zone (SVZ) is a major site of neurogenesis in the adult. We now show that ependymal and proliferating cells in the adult mouse SVZ express diacylglycerol lipases (DAGLs), enzymes that synthesise a CB1/CB2 cannabinoid receptor ligand. DAGL and CB2 antagonists inhibit the proliferation of cultured neural stem cells, and the proliferation of progenitor cells in young animals. Furthermore, CB2 agonists stimulate progenitor cell proliferation in vivo, with this effect being more pronounced in older animals. A similar response was seen with a fatty acid amide hydrolase (FAAH) inhibitor that limits degradation of endocannabinoids. The effects on proliferation were mirrored in changes in the number of neuroblasts migrating from the SVZ to the olfactory bulb (OB). In this context, CB2 antagonists reduced the number of newborn neurons appearing in the OB in the young adult animals while CB2 agonists stimulated this in older animals. These data identify CB2 receptor agonists and FAAH inhibitors as agents that can counteract the naturally observed decline in adult neurogenesis that is associated with ageing.