Multiplex Zymography Captures Stage-specific Activity Profiles of Cathepsins K, L, and S in Human Breast, Lung, and Cervical Cancer

<p>Abstract</p> <p>Background</p> <p>Cathepsins K, L, and S are cysteine proteases upregulated in cancer and proteolyze extracellular matrix to facilitate metastasis, but difficulty distinguishing specific cathepsin activity in complex tissue extracts confounds scientific studies and employing them for use in clinical diagnoses. Here, we have developed multiplex cathepsin zymography to profile cathepsins K, L, and S activity in 10 μg human breast, lung, and cervical tumors by exploiting unique electrophoretic mobility and renaturation properties.</p> <p>Methods</p> <p>Frozen breast, lung, and cervix cancer tissue lysates and normal organ tissue lysates from the same human patients were obtained (28 breast tissues, 23 lung tissues, and 23 cervix tissues), minced and homogenized prior to loading for cathepsin gelatin zymography to determine enzymatic activity.</p> <p>Results</p> <p>Cleared bands of cathepsin activity were identified and validated in tumor extracts and detected organ- and stage-specific differences in activity. Cathepsin K was unique compared to cathepsins L and S. It was significantly higher for all cancers even at the earliest stage tested (stage I for lung and cervix (n = 6, p < .05), and stage II for breast; n = 6, p < .0001). Interestingly, cervical and breast tumor cathepsin activity was highest at the earliest stage we tested, stages I and II, respectively, and then were significantly lower at the latest stages tested (III and IV, respectively) (n = 6, p < 0.01 and p < 0.05), but lung cathepsin activity increased from one stage to the next (n = 6, p < .05). Using cathepsin K as a diagnostic biomarker for breast cancer detected with multiplex zymography, yielded 100% sensitivity and specificity for 20 breast tissue samples tested (10 normal; 10 tumor) in part due to the consistent absence of cathepsin K in normal breast tissue across all patients.</p> <p>Conclusions</p> <p>To summarize, this sensitive assay provides quantitative outputs of cathepsins K, L, and S activities from mere micrograms of tissue and has potential use as a supplement to histological methods of clinical diagnoses of biopsied human tissue.</p

Intratumoral macrophages contribute to epithelial-mesenchymal transition in solid tumors

<p>Abstract</p> <p>Background</p> <p>Several stromal cell subtypes including macrophages contribute to tumor progression by inducing epithelial-mesenchymal transition (EMT) at the invasive front, a mechanism also linked to metastasis. Tumor associated macrophages (TAM) reside mainly at the invasive front but they also infiltrate tumors and in this process they mainly assume a tumor promoting phenotype. In this study, we asked if TAMs also regulate EMT intratumorally. We found that TAMs through TGF-β signaling and activation of the β-catenin pathway can induce EMT in intratumoral cancer cells.</p> <p>Methods</p> <p>We depleted macrophages in F9-teratocarcinoma bearing mice using clodronate-liposomes and analyzed the tumors for correlations between gene and protein expression of EMT-associated and macrophage markers. The functional relationship between TAMs and EMT was characterized <it>in vitro </it>in the murine F9 and mammary gland NMuMG cells, using a conditioned medium culture approach. The clinical relevance of our findings was evaluated on a tissue microarray cohort representing 491 patients with non-small cell lung cancer (NSCLC).</p> <p>Results</p> <p>Gene expression analysis of F9-teratocarcinomas revealed a positive correlation between TAM-densities and mesenchymal marker expression. Moreover, immunohistochemistry showed that TAMs cluster with EMT phenotype cells in the tumors. <it>In vitro</it>, long term exposure of F9-and NMuMG-cells to macrophage-conditioned medium led to decreased expression of the epithelial adhesion protein E-cadherin, activation of the EMT-mediating β-catenin pathway, increased expression of mesenchymal markers and an invasive phenotype. In a candidate based screen, macrophage-derived TGF-β was identified as the main inducer of this EMT-associated phenotype. Lastly, immunohistochemical analysis of NSCLC patient samples identified a positive correlation between intratumoral macrophage densities, EMT markers, intraepithelial TGF-β levels and tumor grade.</p> <p>Conclusions</p> <p>Data presented here identify a novel role for macrophages in EMT-promoted tumor progression. The observation that TAMs cluster with intra-epithelial fibroblastoid cells suggests that the role of macrophages in tumor-EMT extends beyond the invasive front. As macrophage infiltration and pronounced EMT tumor phenotype correlate with increased grade in NSCLC patients, we propose that TAMs also promote tumor progression by inducing EMT locally in tumors.</p

Depletion of Kinesin 5B Affects Lysosomal Distribution and Stability and Induces Peri-Nuclear Accumulation of Autophagosomes in Cancer Cells

Background: Enhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from highly invasive MCF-7 cells that express an active form of the ErbB2 (DN-ErbB2). Methodology/Principal Findings: Mass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic DN-ErbB2 enhanced its lysosomal association. KIF5B associated with lysosomes also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells. Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase endocytosis functioned, however, normally in these cells. Both HeLa and MCF-7 cells appeared to express similar levels of the KIF5B isoform but the death phenotype was weaker in KIF5B-depleted MCF-7 cells. Surprisingly, KIF5B depletion inhibited the rapamycin-induced accumulation of autophagosomes in MCF-7 cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the cytoplasm. Conclusions/Significance: Our data identify KIF5B as a cancer relevant lysosomal motor protein with additional functions in autophagosome formatio

MAGUKs, scaffolding proteins at cell junctions, are substrates of different proteases during apoptosis

A major feature of apoptotic cell death is gross structural changes, one of which is the loss of cell–cell contacts. The caspases, executioners of apoptosis, were shown to cleave several proteins involved in the formation of cell junctions. The membrane-associated guanylate kinases (MAGUKs), which are typically associated with cell junctions, have a major role in the organization of protein–protein complexes at plasma membranes and are therefore potentially important caspase targets during apoptosis. We report here that MAGUKs are cleaved and/or degraded by executioner caspases, granzyme B and several cysteine cathepsins in vitro. When apoptosis was induced by UV-irradiation and staurosporine in different epithelial cell lines, caspases were found to efficiently cleave MAGUKs in these cell models, as the cleavages could be prevented by a pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethylketone. Using a selective lysosomal disrupting agent -leucyl--leucine methyl ester, which induces apoptosis through the lysosomal pathway, it was further shown that MAGUKs are also cleaved by the cathepsins in HaCaT and CaCo-2 cells. Immunohistological data showed rapid loss of MAGUKs at the sites of cell–cell contacts, preceding actual cell detachment, suggesting that cleavage of MAGUKs is an important step in fast and efficient cell detachment

A Mouse Stromal Response to Tumor Invasion Predicts Prostate and Breast Cancer Patient Survival

Primary and metastatic tumor growth induces host tissue responses that are believed to support tumor progression. Understanding the molecular changes within the tumor microenvironment during tumor progression may therefore be relevant not only for discovering potential therapeutic targets, but also for identifying putative molecular signatures that may improve tumor classification and predict clinical outcome. To selectively address stromal gene expression changes during cancer progression, we performed cDNA microarray analysis of laser-microdissected stromal cells derived from prostate intraepithelial neoplasia (PIN) and invasive cancer in a multistage model of prostate carcinogenesis. Human orthologs of genes identified in the stromal reaction to tumor progression in this mouse model were observed to be expressed in several human cancers, and to cluster prostate and breast cancer patients into groups with statistically different clinical outcomes. Univariate Cox analysis showed that overexpression of these genes is associated with shorter survival and recurrence-free periods. Taken together, our observations provide evidence that the expression signature of the stromal response to tumor invasion in a mouse tumor model can be used to probe human cancer, and to provide a powerful prognostic indicator for some of the most frequent human malignancies

Unlocking the potential of weberite-type metal fluorides in electrochemical energy storage

Sodium-ion batteries (NIBs) are a front-runner among the alternative battery technologies suggested for substituting the state-of-the-art lithium-ion batteries (LIBs). The specific energy of Na-ion batteries is significantly lower than that of LIBs, which is mainly due to the lower operating potentials and higher molecular weight of sodium insertion cathode materials. To compete with the high energy density of LIBs, high voltage cathode materials are required for NIBs. Here we report a theoretical investigation on weberite-type sodium metal fluorides (SMFs), a new class of high voltage and high energy density materials which are so far unexplored as cathode materials for NIBs. The weberite structure type is highly favorable for sodium-containing transition metal fluorides, with a large variety of transition metal combinations (M, M’) adopting the corresponding Na2MM’F7 structure. A series of known and hypothetical compounds with weberite-type structure were computationally investigated to evaluate their potential as cathode materials for NIBs. Weberite-type SMFs show two-dimensional pathways for Na+ diffusion with surprisingly low activation barriers. The high energy density combined with low diffusion barriers for Na+ makes this type of compounds promising candidates for cathode materials in NIBs

BRCA1 loss activates cathepsin L–mediated degradation of 53BP1 in breast cancer cells

Loss of 53BP1 rescues BRCA1 deficiency and is associated with BRCA1-deficient and triple-negative breast cancers (TNBC) and with resistance to genotoxic drugs. The mechanisms responsible for decreased 53BP1 transcript and protein levels in tumors remain unknown. Here, we demonstrate that BRCA1 loss activates cathepsin L (CTSL)–mediated degradation of 53BP1. Activation of this pathway rescued homologous recombination repair and allowed BRCA1-deficient cells to bypass growth arrest. Importantly, depletion or inhibition of CTSL with vitamin D or specific inhibitors stabilized 53BP1 and increased genomic instability in response to radiation and poly(adenosine diphosphate–ribose) polymerase inhibitors, compromising proliferation. Analysis of human breast tumors identified nuclear CTSL as a positive biomarker for TNBC, which correlated inversely with 53BP1. Importantly, nuclear levels of CTSL, vitamin D receptor, and 53BP1 emerged as a novel triple biomarker signature for stratification of patients with BRCA1-mutated tumors and TNBC, with potential predictive value for drug response. We identify here a novel pathway with prospective relevance for diagnosis and customization of breast cancer therapy

Decidual-Secreted Factors Alter Invasive Trophoblast Membrane and Secreted Proteins Implying a Role for Decidual Cell Regulation of Placentation

Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the ‘extravillous trophoblast’ (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10−8 M), medroxyprogesterone acetate (10−7 M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN) before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition. Overall, we have demonstrated the potential of a proteomics approach to identify novel proteins expressed by EVT and to uncover the mechanisms leading to disease states

Topical Application of Activity-based Probes for Visualization of Brain Tumor Tissue

Several investigators have shown the utility of systemically delivered optical imaging probes to image tumors in small animal models of cancer. Here we demonstrate an innovative method for imaging tumors and tumor margins during surgery. Specifically, we show that optical imaging probes topically applied to tumors and surrounding normal tissue rapidly differentiate between tissues. In contrast to systemic delivery of optical imaging probes which label tumors uniformly over time, topical probe application results in rapid and robust probe activation that is detectable as early as 5 minutes following application. Importantly, labeling is primarily associated with peri-tumor spaces. This methodology provides a means for rapid visualization of tumor and potentially infiltrating tumor cells and has potential applications for directed surgical excision of tumor tissues. Furthermore, this technology could find use in surgical resections for any tumors having differential regulation of cysteine cathepsin activity