Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are linked to metastasis via their ability to increase invasiveness and enhance tumor-initiating capacity. Growth factors, cytokines, and chemotherapies present in the tumor microenvironment (TME) are capable of inducing EMT, but the role of the extracellular matrix (ECM) in this process remains poorly understood. Here, a novel tessellated three-dimensional (3D) polymer scaffolding is used to produce a fibrillar fibronectin matrix that induces an EMT-like event that includes phosphorylation of STAT3 and requires expression of β1 integrin. Consistent with these findings, analysis of the METABRIC dataset strongly links high-level fibronectin (FN) expression to decreased patient survival. In contrast, analysis of the MCF-10A progression series indicated that intracellular FN expression was associated with nonmetastatic cells. Therefore, differential bioluminescent imaging was used to track the metastasis of isogenic epithelial and mesenchymal cells within heterogeneous tumors. Interestingly, mesenchymal tumor cells do not produce a FN matrix and cannot complete the metastatic process, even when grown within a tumor containing epithelial cells. However, mesenchymal tumor cells form FN-containing cellular fibrils capable of supporting the growth and migration of metastatic-competent tumor cells. Importantly, depletion of FN allows mesenchymal tumor cells to regain epithelial characteristics and initiate tumor growth within a metastatic microenvironment. In contrast to the tumor-promoting functions of fibronectin within the ECM, these data suggest that autocrine fibronectin production inhibits the metastatic potential of mesenchymal tumor cells. .

Mortalin (mot-2) induces inactivation of the tumor suppressor p53's transcriptional and apoptotic functions by cytoplasmic sequestration of p53 in select cancers. The mot-2-dependent cytoprotective function enables cancer cells to support malignant transformation. Abrogating the p53-mot-2 interaction can control or slow down the growth of cancer cells. In this study, we report the discovery of a ubiquitin-like (UBX)-domain-containing protein, UBXN2A, which binds to mot-2 and consequently inhibits the binding between mot-2 and p53. Genetic analysis showed that UBXN2A binds to mot-2's substrate binding domain, and it partly overlaps p53's binding site indicating UBXN2A and p53 likely bind to mot-2 competitively. By binding to mot-2, UBXN2A releases p53 from cytosolic sequestration, rescuing the tumor suppressor functions of p53. Biochemical analysis and functional assays showed that the overexpression of UBXN2A and the functional consequences of unsequestered p53 trigger p53-dependent apoptosis. Cells expressing shRNA against UBXN2A showed the opposite effect of that seen with UBXN2A overexpression. The expression of UBXN2A and its apoptotic effects were not observed in normal colonic epithelial cells and p53−/− colon cancer cells. Finally, significant reduction in tumor volume in a xenograft mouse model in response to UBXN2A expression was verified in vivo. Our results introduce UBXN2A as a home defense response protein, which can reconstitute inactive p53-dependent apoptotic pathways. Inhibition of mot-2-p53 interaction by UBXN2A is an attractive therapeutic strategy in mot-2-elevated tumors.

In breast cancer (BC), tissue stiffening via fibronectin (FN) and collagen accumulation is associated with advanced disease progression at both the primary tumor and metastatic sites. Here, we evaluate FN production in 15 BC cell lines, representing a variety of subtypes, phenotypes, metastatic potentials, and chemotherapeutic sensitivities. We demonstrate that intracellular and soluble FN is initially lost during tumorigenic transformation but is rescued in all lines with epithelial-mesenchymal plasticity (EMP). Importantly, we establish that no BC cell line was able to independently organize a robust FN matrix. Non-transformed mammary epithelial cells were also unable to deposit FN matrices unless transglutaminase 2, a FN crosslinking enzyme, was overexpressed. Instead, BC cells manipulated the FN matrix production of fibroblasts in a phenotypic-dependent manner. In addition, varied accumulation levels were seen depending if the fibroblasts were conditioned to model paracrine signaling or endocrine signaling of the metastatic niche. In the former, fibroblasts conditioned by BC cultures with high EMP resulted in the largest FN matrix accumulation. In contrast, mesenchymal BC cells produced extracellular vesicles (EV) that resulted in the highest levels of matrix formation by conditioned fibroblasts. Overall, we demonstrate a dynamic relationship between tumor and stromal cells within the tumor microenvironment, in which the levels and fibrillarization of FN in the extracellular matrix are modulated during the particular stages of disease progression.

The deubiquitinase (DUB) ubiquitin C‐terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small‐molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine‐based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.

Veratridine (VTD), an alkaloid derived from the Liliaceae plant shows anti-tumor effects; however, its molecular targets have not been thoroughly studied. Using a high-throughput drug screen, we found that VTD enhances transactivation of UBXN2A, resulting in upregulation of UBXN2A in the cytoplasm, where UBXN2A binds and inhibits the oncoprotein mortalin-2 (mot-2). VTD-treated cancer cells undergo cell death in UBXN2A- and mot-2-dependent manners. The cytotoxic function of VTD is grade-dependent, and the combined treatment with a sub-optimal dose of the standard chemotherapy, 5-Fluorouracil (5-FU) and etoposide, demonstrated a synergistic effect, resulting in higher therapeutic efficacy. VTD influences the CD44+ stem cells, possibly through UBXN2A-dependent inhibition of mot-2. The VTD-dependent expression of UBXN2A is a potential candidate for designing novel strategies for colon cancer treatment because: 1) In 50% of colon cancer patients, UBXN2A protein levels in tumor tissues are significantly lower than those in the adjacent normal tissues. 2) Cytoplasmic expression of the mot-2 protein is very low in non-cancerous cells; thus, VTD can produce tumor-specific toxicity while normal cells remain intact. 3) Finally, VTD or its modified analogs offer a valuable adjuvant chemotherapy strategy to improve the efficacy of 5-FU-based chemotherapy for colon cancer patients harboring WT-p53.

Hyaluronic acid (HA) is an extracellular matrix (ECM) component that has been shown to play a significant role in regulating muscle cell behavior during repair and regeneration. For instance, ECM remodeling after muscle injury involves an upregulation in HA expression that is coupled with skeletal muscle precursor cell recruitment. However, little is known about the role of HA during skeletal muscle development. To gain insight into the way in which HA mediates embryonic myogenesis, we first determined the spatial distribution and gene expression of CD44, RHAMM and other HA related proteins in embryonic day (E)10.5 to E12.5 murine forelimbs. While HA and CD44 expression remained high, RHAMM decreased at both the protein (via immunohistochemistry) and RNA (via qPCR) levels. Next, we determined that 4-methylumbelliferone-mediated knockdown of HA synthesis inhibited the migration and proliferation of E11.5/E12.5 forelimb-derived cells. Then, the influence of CD44 and RHAMM on myoblast and connective tissue cell behavior was investigated using antibodies against these receptors. Anti-RHAMM, but not anti-CD44, significantly decreased the total distance myogenic progenitors migrated over 24 h, whereas both inhibited connective tissue cell migration. In contrast, anti-CD44 inhibited the proliferation of connective tissue cells and muscle progenitors, but anti-RHAMM had no effect. However, when myoblasts and connective tissue cells were depleted of CD44 and RHAMM by shRNA, motility and proliferation were significantly inhibited in both cells indicating that blocking cell surface-localized CD44 and RHAMM does not have as pronounced effect as global shRNA-mediated depletion of these receptors. These results show, for the first time, the distribution and activity of RHAMM in the context of skeletal muscle. Furthermore, our data indicate that HA, through interactions with CD44 and RHAMM, promotes myogenic progenitor migration and proliferation. Confirmation of the role of HA and its receptors in directing myogenesis will be useful for the design of regenerative therapies that aim to promote the restoration of damaged or diseased muscle.

Human epidermal growth factor receptor 2 (HER2)-amplified breast cancers are treated using targeted antibodies and kinase inhibitors, but resistance to these therapies leads to systemic tumor recurrence of metastatic disease. Herein, we conducted gene expression analyses of HER2 kinase inhibitor-resistant cell lines as compared to their drug-sensitive counterparts. These data demonstrate the induction of epithelial–mesenchymal transition (EMT), which included enhanced expression of fibroblast growth factor receptor 1 (FGFR1) and axonal guidance molecules known as neuropilins (NRPs). Immunoprecipitation of FGFR1 coupled with mass spectroscopy indicated that FGFR1 forms a physical complex with NRPs, which is enhanced upon induction of EMT. Confocal imaging revealed that FGFR1 and NRP1 predominantly interact throughout the cytoplasm. Along these lines, short hairpin RNA-mediated depletion of NRP1, but not the use of NRP1-blocking antibodies, inhibited FGFR signaling and reduced tumor cell growth in vitro and in vivo. Our results further indicate that NRP1 upregulation during EMT is mediated via binding of the chromatin reader protein, bromodomain containing 4 (BRD4) in the NRP1 proximal promoter region. Pharmacological inhibition of BRD4 decreased NRP1 expression and ablated FGF-mediated tumor cell growth. Overall, our studies indicate that NRPs facilitate aberrant growth factor signaling during EMT-associated drug resistance and metastasis. Pharmacological combination of epigenetic modulators with FGFR-targeted kinase inhibitors may provide improved outcomes for breast cancer patients with drug-resistant metastatic disease.

The effectiveness of immunotherapy as a treatment for metastatic breast cancer is limited due to low numbers of infiltrating lymphocytes in metastatic lesions. Herein, we demonstrated that adjuvant therapy using FIIN4, a covalent inhibitor of fibroblast growth factor receptor (FGFR), dramatically delayed the growth of pulmonary metastases in syngeneic models of metastatic breast cancer. In addition, we demonstrated in a syngeneic model of systemic tumor dormancy that targeting of FGFR enhanced the immunogenicity of the pulmonary tumor microenvironment through increased infiltration of CD8+ lymphocytes and reduced presence of myeloid suppressor cells. Similar impacts on immune cell infiltration were observed upon genetic depletion of FGFR1 in tumor cells, which suggested a direct influence of FGFR signaling on lymphocyte trafficking. Suppression of CD8+ lymphocyte infiltration was consistent with FGFR-mediated inhibition of the T-cell chemoattractant CXCL16. Initial attempts to concomitantly administer FIIN4 with immune checkpoint blockade failed due to inhibition of immune-mediated tumor cell killing via blockade of T-cell receptor signaling by FIIN4. However, this was overcome by using a sequential dosing protocol that consisted of FIIN4 treatment followed by anti–PD-L1. These data illustrate the complexities of combining kinase inhibitors with immunotherapy and provide support for further assessment of FGFR targeting as an approach to enhance antitumor immunity and improve immunotherapy response rates in patients with metastatic breast cancer.