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Metastasis is the major cause of mortality in patients with breast cancer. Many signaling pathways have been linked to cancer invasiveness, but blockade of few protein components has succeeded in reducing metastasis. Thus, identification of proteins contributing to invasion that are manipulable by small molecules may be valuable in inhibiting spread of the disease. The protein kinase with no lysine (K) 1 (WNK1) has been suggested to induce migration of cells representing a range of cancer types. Analyses of mouse models and patient data have implicated WNK1 as one of a handful of genes uniquely linked to invasive breast cancer. Here, we present evidence that inhibition of WNK1 slows breast cancer metastasis. We show that depletion or inhibition of WNK1 reduces migration of several breast cancer cell lines in wound healing assays and decreases invasion in collagen matrices. Furthermore, WNK1 depletion suppresses expression of AXL, a tyrosine kinase implicated in metastasis. Finally, we demonstrate that WNK inhibition in mice attenuates tumor progression and metastatic burden. These data showing reduced migration, invasion, and metastasis upon WNK1 depletion in multiple breast cancer models suggest that WNK1 contributes to the metastatic phenotype, and that WNK1 inhibition may offer a therapeutic avenue for attenuating progression of invasive breast cancers.

Defining how interactions between tumor subpopulations contribute to invasion is essential for understanding how tumors metastasize. Here, we find that the heterogeneous expression of the transcription factor ΔNp63 confers distinct proliferative and invasive epithelial-to-mesenchymal transition (EMT) states in subpopulations that establish a leader–follower relationship to collectively invade. A ΔNp63-high EMT program coupled the ability to proliferate with an IL1α- and miR-205–dependent suppression of cellular protrusions that are required to initiate collective invasion. An alternative ΔNp63-low EMT program conferred cells with the ability to initiate and lead collective invasion. However, this ΔNp63-low EMT state triggered a collateral loss of fitness. Importantly, rare growth-suppressed ΔNp63-low EMT cells influenced tumor progression by leading the invasion of proliferative ΔNp63-high EMT cells in heterogeneous primary tumors. Thus, heterogeneous activation of distinct EMT programs promotes a mode of collective invasion that overcomes cell intrinsic phenotypic deficiencies to induce the dissemination of proliferative tumor cells. Significance: These findings reveal how an interaction between cells in different EMT states confers properties that are not induced by either EMT program alone.

Using a data driven analysis of a high-content screen, we have uncovered new regulators of epithelial-to-mesenchymal transition (EMT) induced cell migration. Our results suggest that increased expression of miR614 can alter cell intrinsic gene expression to enhance single cell and collective migration in multiple contexts. Interestingly, miR614 specifically increased the expression of the EMT transcription factor Slug while not altering existing epithelial character or inducing other canonical EMT regulatory factors. Analysis of two different cell lines identified a set of genes whose expression is altered by the miR614 through direct and indirect mechanisms. Prioritization driven by functional testing of 25 of the miR614 suppressed genes uncovered the mitochondrial small GTPase Miro1 and the transmembrane protein TAPT1 as miR614 suppressed genes that inhibit migration. Notably, the suppression of either Miro1 or TAPT1 was sufficient to increase Slug expression and the rate of cell migration. Importantly, reduced TAPT1 expression correlated with an increased risk of relapse in breast cancer patients. Together, our results reveal how increased miR614 expression and the suppression of TAPT1 and Miro1 modulate the EMT state and migratory properties of breast cancer cells.

AIB1Δ4 is an N-terminally truncated isoform of the oncogene amplified in breast cancer 1 (AIB1) with increased expression in high-grade human ductal carcinoma in situ (DCIS). However, the role of AIB1Δ4 in DCIS malignant progression has not been defined. Here we CRISPR-engineered RNA splice junctions to produce normal and early-stage DCIS breast epithelial cells that expressed only AIB1Δ4. These cells showed enhanced motility and invasion in 3D cell culture. In zebrafish, AIB1Δ4-expressing cells enabled invasion of parental cells when present in a mixed population. In mouse xenografts, a subpopulation of AIB1Δ4 cells mixed with parental cells enhanced tumor growth, recurrence, and lung metastasis. AIB1Δ4 chromatin immunoprecipitation sequencing revealed enhanced binding to regions including peroxisome proliferator-activated receptor (PPAR) and glucocorticoid receptor (GR) genomic recognition sites. H3K27ac and H3K4me1 genomic engagement patterns revealed selective activation of breast cancer-specific enhancer sites by AIB1Δ4. AIB1Δ4 cells displayed upregulated inflammatory response genes and downregulated PPAR signaling gene expression patterns. In the presence of AIB1Δ4 enabler cells, parental cells increased NF-κB and WNT signaling. Cellular cross-talk was inhibited by the PPARγ agonist efatutazone but was enhanced by treatment with the GR agonist dexamethasone. In conclusion, expression of the AIB1Δ4-selective cistrome in a small subpopulation of cells triggers an “enabler” phenotype hallmarked by an invasive transcriptional program and collective malignant progression in a heterogeneous tumor population. Significance: A minor subset of early-stage breast cancer cells expressing AIB1Δ4 enables bulk tumor cells to become invasive, suggesting that selective eradication of this population could impair breast cancer metastasis.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells. Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

Genes that are normally biased towards expression in the testis are often induced in tumor cells. These gametogenic genes, known as cancer-testis antigens (CTAs), have been extenstively investigated as targets for immunotherapy. However, despite their frequent detection, the degree to which CTAs support neoplastic invasion is poorly understood. Here, we find that the CTA genes SPANX-A/C/D and CTAG2 are coordinately induced in breast cancer cells and regulate distinct features of invasive behavior. Our functional analysis revealed that CTAG2 interacts with Pericentrin at the centrosome and is necessary for directional migration. Conversely, SPANX-A/C/D interacts with Lamin A/C at the inner nuclear membrane and is required for the formation of actin-rich cellular protrusions that reorganize the extracellular matrix. Importantly, SPANX-A/C/D was required for breast cancer cells to spontaneously metastasize to the lung, demonstrating that CTA reactivation can be critical for invasion dependent phenotypes in vivo. Moreover, elevated SPANX-A/C/D expression in breast cancer patient tumors correlated with poor outcome. Together, our results suggest that distinct CTAs promote tumor progression by regulating complementary cellular functions that are integrated together to induce invasive behavior.

Tumor cells can engage in a process called collective invasion, in which cohesive groups of cells invade through interstitial tissue. Here, we identified an epigenetically distinct subpopulation of breast tumor cells that have an enhanced capacity to collectively invade. Analysis of spheroid invasion in an organotypic culture system revealed that these “trailblazer” cells are capable of initiating collective invasion and promote non-trailblazer cell invasion, indicating a commensal relationship among subpopulations within heterogenous tumors. Canonical mesenchymal markers were not sufficient to distinguish trailblazer cells from non-trailblazer cells, suggesting that defining the molecular underpinnings of the trailblazer phenotype could reveal collective invasion-specific mechanisms. Functional analysis determined that DOCK10, ITGA11, DAB2, PDFGRA, VASN, PPAP2B, and LPAR1 are highly expressed in trailblazer cells and required to initiate collective invasion, with DOCK10 essential for metastasis. In patients with triple-negative breast cancer, expression of these 7 genes correlated with poor outcome. Together, our results indicate that spontaneous conversion of the epigenetic state in a subpopulation of cells can promote a transition from in situ to invasive growth through induction of a cooperative form of collective invasion and suggest that therapeutic inhibition of trailblazer cell invasion may help prevent metastasis.

Most ductal breast carcinoma cells are weakly invasive in vitro and in vivo, suggesting that components of their microenvironment may facilitate a transition from in situ to invasive stages during progression. Here, we report that coculture of mammary fibroblasts specifically triggers invasive behavior in basal-type breast cancer cells through a ligand independent mechanism. When cultured alone in organotypic culture, both basal- and luminal-type breast cancer cells formed noninvasive spheroids with characteristics of ductal carcinoma in situ (DCIS). In contrast, when cocultured with mammary fibroblasts, basal-type spheroids exhibited invasive character whereas the luminal-type spheroids retained a benign and noninvasive duct-like architecture. Real-time imaging and functional studies revealed that the specificity of invasion was linked to a unique capacity of basal-type breast cancer cells to move within spheroids. Mammary fibroblasts induced invasion by triggering basal-type breast cancer cells to convert from a noninvasive program of mammary epithelial morphogenesis to an invasive program of sprouting endothelial angiogenesis. Contrary to the existing invasion models, soluble ligands produced by the fibroblasts were not sufficient to trigger invasion. Instead, basal-type invasion relied upon a Cdc42-dependent reorganization of collagen fibers in the extracellular matrix by fibroblasts. Inhibiting basal-type cell movement with clinically relevant drugs blocked invasion both in organotypic culture and in animals, suggesting a new treatment strategy for early-stage patients. Together our findings establish that fibroblast recruitment by basal-type breast cancer cells into early-stage tumors is sufficient to trigger their conversion from a benign, noninvasive DCIS-like stage to a malignant invasive stage. Furthermore, our findings suggest that different subtypes of breast cancer may require distinct types of contributions from the microenvironment to undergo malignant progression. Cancer Res; 71(21); 6857–66. ©2011 AACR.

Cell identity signals influence the invasive capability of tumor cells, as demonstrated by the selection for programs of epithelial-to-mesenchymal transition (EMT) during malignant progression. Breast cancer cells retain canonical epithelial traits and invade collectively as cohesive groups of cells, but the signaling pathways critical to their invasive capabilities are still incompletely understood. Here we report that the transcription factor ΔNp63α drives the migration of basal-like breast cancer (BLBC) cells by inducing a hybrid mesenchymal/epithelial state. Through a combination of expression analysis and functional testing across multiple BLBC cell populations, we determined that ΔNp63α induces migration by elevating the expression of the EMT program components Slug and Axl. Interestingly, ΔNp63α also increased the expression of miR-205, which can silence ZEB1/2 to prevent the loss of epithelial character caused by EMT induction. In clinical specimens, co-expression of various elements of the ΔNp63α pathway confirmed its implication in motility signaling in BLBC. We observed that activation of the ΔNp63α pathway occurred during the transition from noninvasive ductal carcinoma in situ to invasive breast cancer. Notably, in an orthotopic tumor model, Slug expression was sufficient to induce collective invasion of E-cadherin–expressing BLBC cells. Together, our results illustrate how ΔNp63α can drive breast cancer cell invasion by selectively engaging promigratory components of the EMT program while, in parallel, still promoting the retention of epithelial character. Cancer Res; 75(18); 3925–35. ©2015 AACR.

Cells integrate signals to select the appropriate response from an array of possible outcomes. Signal integration causes the reorganization of signaling pathways by undescribed events. To analyze the molecular changes in signaling pathways that elicit different responses, we focused on the interaction between cyclic AMP (cAMP) and growth factors. We show that the activation of extracellular signal-regulated kinase 5 (ERK5), but not ERK1/2, by growth factors is disrupted by cAMP through cAMP-dependent protein kinase (PKA). Activation of MEKK2, a mitogen-activated protein (MAP) kinase kinase kinase upstream of ERK5 that is required for growth factor activation of ERK5, is also disrupted by PKA. Transcription of c-Jun is induced by ERK5, and like ERK5, c-Jun induction is also blocked by cAMP. Transcription from the serum response element, like activation of ERK1/2, is not blocked by cAMP. Collectively, these results support a model in which cAMP shapes the growth factor-induced cellular response through PKA-dependent uncoupling of selected MAP kinase cascades from activating signals.

The with no lysine (K) (WNK) family of enzymes is best known for control of blood pressure through regulation of the function and membrane localization of ion cotransporters. In mice, global as well as endothelial-specific WNK1 gene disruption results in embryonic lethality due to angiogenic and cardiovascular defects. WNK1(-/-) embryos can be rescued by endothelial-specific expression of a constitutively active form of the WNK1 substrate protein kinase OSR1 (oxidative stress responsive 1). Using human umbilical vein endothelial cells (HUVECs), we explored mechanisms underlying the requirement of WNK1-OSR1 signaling for vascular development. WNK1 is required for cord formation in HUVECs, but the actions of the two major WNK1 effectors, OSR1 and its close relative SPAK (STE20/SPS1-related proline-, alanine-rich kinase), are distinct. SPAK is important for endothelial cell proliferation, whereas OSR1 is required for HUVEC chemotaxis and invasion. We also identified the zinc-finger transcription factor Slug in WNK1-mediated control of endothelial functions. Our study identifies a separation of functions for the WNK1-activated protein kinases OSR1 and SPAK in mediating proliferation, invasion, and gene expression in endothelial cells and an unanticipated link between WNK1 and Slug that is important for angiogenesis.

Mitogen-activated protein kinase (MAPK) pathways couple intrinsic and extrinsic signals to hypertrophic growth of cardiomyocytes. The MAPK kinase MEK5 activates the MAPK ERK5. To investigate the potential involvement of MEK5-ERK5 in cardiac hypertrophy, we expressed constitutively active and dominant-negative forms of MEK5 in cardiomyocytes in vitro. MEK5 induced a form of hypertrophy in which cardiomyocytes acquired an elongated morphology and sarcomeres were assembled in a serial manner. The cytokine leukemia inhibitory factor (LIF), which stimulates MEK5 activity, evoked a similar response. Moreover, a dominant-negative MEK5 mutant specifically blocked LIF-induced elongation of cardiomyocytes and reduced expression of fetal cardiac genes without blocking other aspects of LIF-induced hypertrophy. Consistent with the ability of MEK5 to induce serial assembly of sarcomeres in vitro, cardiac-specific expression of activated MEK5 in transgenic mice resulted in eccentric cardiac hypertrophy that progressed to dilated cardiomyopathy and sudden death. These findings reveal a specific role for MEK5-ERK5 in the induction of eccentric cardiac hypertrophy and in transduction of cytokine signals that regulate serial sarcomere assembly.

Mitogen-activated protein (MAP) kinases comprise a family of ubiquitous proline-directed, protein-serine/threonine kinases, which participate in signal transduction pathways that control intracellular events including acute responses to hormones and major developmental changes in organisms. MAP kinases lie in protein kinase cascades. This review discusses the regulation and functions of mammalian MAP kinases. Nonenzymatic mechanisms that impact MAP kinase functions and findings from gene disruption studies are highlighted. Particular emphasis is on ERK1/2.

Growth factor receptors of the epidermal growth factor (EGF) receptor family play pivotal roles in the regulation of cell proliferation and differentiation and are involved in the development of human cancers. It has been well documented that these receptors undergo growth factor-stimulated homo- and heterodimerization as a first step in the initiation of signaling cascades. Here we provide evidence for a new mechanism for growth factor-stimulated receptor dimer formation, designated secondary dimerization. The growth factor-induced dimerization and ensuing receptor trans-autophosphorylation results in the dissociation of the original (primary) receptor dimer. Each phosphorylated receptor monomer then interacts with a new (nonphosphorylated) receptor to form a secondary dimer. Treatment of cells with EGF yields Neu-ErbB3 secondary dimers, and heregulin treatment induces the formation of Neu-EGF receptor (secondary) dimers. The ability of EGF and heregulin to stimulate a cascade of dimerization events points to a novel mechanism by which multiple signaling activities and diverse biological responses are initiated by members of the EGF receptor family.

To determine how extracellular signal–regulated kinases (ERK) 1/2 promote mammary tumorigenesis, we examined the real-time behavior of cells in an organotypic culture of the mammary glandular epithelium. Inducible activation of ERK1/2 in mature acini elicits cell motility and disrupts epithelial architecture in a manner that is reminiscent of ductal carcinoma in situ; however, motile cells do not invade through the basement membrane and branching morphogenesis does not take place. ERK1/2-induced motility causes cells to move both within the cell monolayer that contacts the basement membrane surrounding the acinus and through the luminal space of the acinus. E-cadherin expression is reduced after ERK1/2 activation, but motility does not involve an epithelial–mesenchymal transition. Cell motility and the disruption of epithelial architecture require a Rho kinase– and myosin light chain kinase–dependent increase in the phosphorylation of myosin light chain 2. Our results identify a new mechanism for the disruption of architecture in epithelial acini and suggest that ERK1/2 can promote noninvasive motility in preinvasive mammary tumors.

We showed previously that p90 RSK was activated in cells expressing an activated mutant of MEK5, the activator of the MAP kinase ERK5. Based on the following evidence, we suggest that ERK5 can directly activate RSK in cells. ERK5 binds to RSK in vitro and co-immunoprecipitates from cell extracts; activation of ERK5 weakens its binding to RSK, suggesting that RSK is released upon activation. Phosphorylation of RSK by ERK5 in vitro causes its activation, indicating that RSK is a substrate of ERK5. In cells activation of ERK5 but not p38 or the c-Jun N-terminal kinase is associated with RSK activation. The large C-terminal domain of ERK5 is not required for binding or activation of RSK by ERK5; however, the common docking or CD domain of ERK5 and the docking or D domain of RSK are important for their association.

Introduction Epithelial tumors, including breast cancer, are being identified and treated at earlier stages of tumor development because of technological advances in screening and detection methods. It is likely that early-stage epithelial tumors, such as mammary ductal carcinoma in situ (DCIS), will be amenable to new and more efficacious diagnostic tests and forms of therapy. However, our limited understanding of the underlying molecular mechanisms of early-stage epithelial tumor growth has hampered the development of new forms treatment and preventative therapy. Methods The Raf–MEK1/2–ERK1/2 mitogen-activated protein kinase module is activated by stimuli complicit in mammary neoplastic progression. We have recently demonstrated that the activation of ERK1/2 induces a non-invasive form of motility, where cells can track along the basement membrane and adjacent epithelial cells, but do not become invasive over time, using real-time imaging of a mammary epithelial organotypic culture model. Using this novel approach combined with traditional biochemical techniques, we have analyzed at the molecular level how ERK1/2 induces this new non-invasive form of motility as well as proliferation and cell survival. Results We find that the activation of Raf:ER in the differentiated epithelium of fully formed acini promotes proliferation and cell survival, which are characteristic features of pre-invasive DCIS lesions. The activation of ERK1/2 correlated with induction of c-Fos, a transcriptional regulator of proliferation and reduced expression of the pro-apoptotic BH3-only protein BIM. Both ERK1/2 and PI-3 kinase-dependent effector pathways were required for activated Raf:ER to reduce expression of p27 and promote proliferation. In addition, PI-3K activity was necessary for the induction of non-invasive motility induced by ERK1/2. Conclusions ERK1/2 activation is sufficient to induce cell behaviors in organotypic culture that could promote recurrent and invasive growth in DCIS patients. Interestingly, PI-3K activity is necessary for two of these behaviors, proliferation and cell motility. Collectively, our results suggest that the relationship between the activity state of the ERK1/2 and PI-3K signaling pathways and recurrent growth in DCIS patients should be investigated.