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.
We present biochemical, structural and cellular characterization of a newly reported class of cyanopyrrolidine covalent inhibitors of the important oncotarget UCHL1. The insight into mechanism of action and structural analysis learned from this work provides information for the future design of potent and selective UCHL1 covalent inhibitors.
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.
The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest ...family of ubiquitin ligases Background: The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases. Supported primarily by the decrease of substrate receptor (SR) proteins of CRLs in cells deficient of a CSN subunit, CSN-mediated cullin deneddylation is believed to prevent autoubiquitination and self-destruction of the SR in active CRLs. However, it is unclear whether the decrease in SRs is solely due to protein destabilization. Moreover, our prior studies have demonstrated that cardiac specific knockout of
(Cops8-CKO) impairs autophagosome maturation and causes massive necrosis in cardiomyocytes but the underlying mechanism remains poorly understood. Given that Cops8 is nucleus-enriched and a prior report showed its binding to the promoter of several genes and association of its ablation with decreased mRNA levels of these genes, we sought to determine the dynamic changes of myocardial transcriptome in mice with perinatal Cops8-CKO and to explore their functional implications.
Myocardial transcriptomes of
,
, and
littermate mice at postnatal 2 and 3 weeks were analyzed. The data were imported into an in-house analysis pipeline using Bioconductor for quantile normalization and statistical analysis. Differentially expressed genes (DEGs) between groups at each time point or between time points within the group were revealed by
-test. Genes with
< 0.05 after Benjamini and Hochberg false discovery rate correction for multiple hypothesis testing were considered as significant DEGs. We found that (1) the Ingenuity Pathway Analysis (IPA) revealed significant enrichment of DEGs in multiple pathways, especially those responding to oxidative stress, in homozygous Cops8-CKO hearts at both 2 and 3 weeks, corroborating the occurrence of massive cardiomyocyte necrosis at 3 weeks; (2) the decreases in multiple CRL SR proteins were associated with decreased transcript levels; and (3) enrichment of DEGs in the chromatin remodeling pathway and the microtubule motility and vesicle trafficking pathways.
Our data are consistent with the notion that Cops8/CSN plays a role in the transcriptional regulation of CRL SRs and in the redox and vesicle trafficking pathways.
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.
•CD44, RHAMM and HA expression temporally varied during forelimb development.•shRNA-mediated depletion of CD44 and RHAMM inhibited proliferation and migration.•Antibody blocking of CD44 and RHAMM had a differential effect than shRNA depletion.
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.
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.
Overexpression of the oncoprotein mortalin in cancer cells and its protein partners enables mortalin to promote multiple oncogenic signaling pathways and effectively antagonize chemotherapy-induced ...cell death. A UBX-domain-containing protein, UBXN2A, acts as a potential mortalin inhibitor. This current study determines whether UBXN2A effectively binds to and occupies mortalin's binding pocket, resulting in a direct improvement in the tumor's sensitivity to chemotherapy. Molecular modeling of human mortalin's binding pocket and its binding to the SEP domain of UBXN2A followed by yeast two-hybrid and His-tag pulldown assays revealed that three amino acids (PRO442, ILE558, and LYS555) within the substrate-binding domain of mortalin are crucial for UBXN2A binding to mortalin. As revealed by chase experiments in the presence of cycloheximide, overexpression of UBXN2A seems to interfere with the mortalin-CHIP E3 ubiquitin ligase and consequently suppresses the C-terminus of the HSC70-interacting protein (CHIP)-mediated destabilization of p53, resulting in its stabilization in the cytoplasm and upregulation in the nucleus. Overexpression of UBXN2A causes a significant inhibition of cell proliferation and the migration of colon cancer cells. We silenced UBXN2A in the human osteosarcoma U2OS cell line, an enriched mortalin cancer cell, followed by a clinical dosage of the chemotherapeutic agent 5-fluorouracil (5-FU). The UBXN2A knockout U2OS cells revealed that UBXNA is essential for the cytotoxic effect achieved by 5-FU. UBXN2A overexpression markedly increased the apoptotic response of U2OS cells to the 5-FU. In addition, silencing of UBXN2A protein suppresses apoptosis enhanced by UBXN2A overexpression in U2OS. The knowledge gained from this study provides insights into the mechanistic role of UBXN2A as a potent mortalin inhibitor and as a potential chemotherapy sensitizer for clinical application.
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