Matriptase and its cognate, Kunitz-type serine protease inhibitor, HAI-1, comprise a newly characterized extracellular matrix-degrading protease system that may function as an epithelial membrane activator for other proteases and latent growth factors. Both enzyme and inhibitor have been detected in breast cancer cells, immortalized mammary epithelial cells, and human milk, but not in cultured fibroblasts nor in fibrosarcoma cells. To test the hypothesis that this system is expressed by normal breast epithelium, invasive breast cancers, and other cancers of an epithelial origin (carcinomas) but not in cancers of a mesenchymal origin, we have expanded our expression analysis of matriptase and HAI-1 in vitro and in vivo. Matriptase and HAI-1 were detected at the protein and mRNA levels both in hormone-dependent and hormone-independent cultured breast cancer cells, and this expression correlated with the expression of the epithelial markers E-cadherin or ZO-1. However, none of the breast cancer cell lines tested that express the mesenchymal marker vimentin express matriptase or HAI-1, consistent with an epithelial-selective expression of this system. Expression of matriptase, as determined by Western blot analysis, was observed in primary human breast, gynecological, and colon carcinomas, but not in stromal-derived ovarian tumors and human sarcomas of various origins and histological grades. It has long been proposed that metastasis is a multistep process. This includes the breakdown of the basement membrane, detachment of cancer cells from the primary tumor, invasion into the stroma, intravasation into blood vessels, survival in the blood stream, extravasation through target organ blood vessels, and the establishment and proliferation of cancer cells in remote tissues. To accomplish these events, cancer cells must acquire an enhanced ability to migrate through and degrade extracellular matrix components. An array of extracellular matrix-degrading proteases and cell motility factors have been characterized and implicated in cancer invasion and metastasis.1 Among the protease systems, the plasmin/urokinase-type plasminogen activator (uPA) system, 2-6 and the matrix metalloproteases 7-12 have received the most attention. Although these extracellular matrix-degrading proteases have been implicated in breast cancer invasion and metastasis, they are mainly expressed by stromal components of human breast tumors. 9,11,[13][14][15][16] The stromal origins of these extracellular matrix-degrading proteases in breast cancer suggests that malignant invasion is an event that depends at least in part on a stromal-epithelial interaction. 17 Furthermore, growth and motility factors secreted by stromal cells may also contribute to the ability of cancer cells to migrate through the extracellular matrix. Hepatocyte growth factor (HGF)/scattering factor (SF) is one of these mesenchymal cell-derived proteins. On binding to the c-Met receptor on the surfaces of epithelial cells, HGF can dissociate epithelial colonies and scatter cells. ...

Right heart failure is the cause of death of most patients with severe pulmonary arterial hypertensive (PAH) disorders, yet little is known about the cellular and molecular causes of right ventricular failure (RVF). We first showed a differential gene expression pattern between normal rat right and left ventricles, and postulated the existence of a molecular right heart failure program that distinguishes RVF from adaptive right ventricular hypertrophy (RVH), and that may differ in some respects from a left heart failure program. By means of microarrays and transcriptional sequencing strategies, we used two models of adaptive RVH to characterize a gene expression pattern reflective of growth and the maintenance of myocardial structure. Moreover, two models of RVF were associated with fibrosis, capillary rarefaction, the decreased expression of genes encoding the angiogenesis factors vascular endothelial growth factor, insulin-like growth factor 1, apelin, and angiopoeitin-1, and the increased expression of genes encoding a set of glycolytic enzymes. The treatment of established RVF with a β-adrenergic receptor blocker reversed RVF, and partly reversed the molecular RVF program. We conclude that normal right and left ventricles demonstrate clearly discernable differences in the expression of mRNA and microRNA, and that RVH and RVF are characterized by distinct patterns of gene expression that relate to cell growth, angiogenesis, and energy metabolism.

Regulatory proteins are often ubiquitinated, depending on their phosphorylation status as well as on their association with ancillary proteins that serve as adapters of the ubiquitination machinery. We previously demonstrated that c-Jun is targeted for ubiquitination by its association with inactive c-Jun NH 2 -terminal kinase (JNK). Phosphorylation by activated JNK protects cJun from ubiquitination, thus by prolonging its half-life. In the study reported here, we determined the ability of JNK to target ubiquitination of its other substrates (Elk1 and activating transcription factor 2 (ATF2)) and associated proteins (ATF2 and JunB). We demonstrate that phosphorylation by JNK protects ATF2, but not Elk1, from JNK-targeted ubiquitination. We also show that association of inactive JNK with JunB or ATF2 is necessary to target them for ubiquitination. Unlike its targeting of c-Jun, JNK requires additional cellular components, yet to be identified, to target the ubiquitination of ATF2. Elk1 is phosphorylated by JNK, but JNK neither associates with nor targets Elk1 for ubiquitination. The implications for the dual role of JNK in the regulation of ubiquitination and stability of c-Jun, ATF2, and JunB in normally growing versus stressed cells are discussed.

Tumor growth and metastasis are critically dependent on the formation of new blood vessels. The present study found that extracellular matrix protein 1 (ECM1), a newly described secretory glycoprotein, promotes angiogenesis. This was initially suggested by in situ hybridization studies of mouse embryos indicating that the ECM1 message was associated with blood vessels and its expression pattern was similar to that of flk-1, a recognized marker for endothelium. More direct evidence for the role of ECM1 in angiogenesis was provided by the fact that highly purified recombinant ECM1 stimulated the proliferation of cultured endothelial cells and promoted blood vessel formation in the chorioallantoic membrane of chicken embryos. Immunohistochemical staining with specific antibodies indicated that ECM1 was expressed by the human breast cancer cell lines MDA-435 and LCC15, both of which are highly tumorigenic. In addition, staining of tissue sections from patients with breast cancer revealed that ECM1 was present in a significant proportion of primary and secondary tumors. Collectively, the results of this study suggest that ECM1 possesses angiogenic properties that may promote tumor progression.

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Aberrant DNA hypermethylation of gene promoter regions has been increasingly recognized as a common molecular alteration in carcinogenesis. We evaluated the association between major clinicopathological features and hypermethylation of genes in tumors among 803 incidence breast cancer cases from a large population-based case-control study conducted in Western New York State. DNA samples were isolated from archive paraffin embedded tumor tissue and were analyzed for hypermethylation status of the E-cadherin, p16, and RAR-β 2 genes using real time methylation-specific polymerase chain reaction. The frequencies of hypermethylation were 20.0% for E-cadherin, 25.9% for p16, and 27.5% for RAR-β 2 genes. For postmenopausal women, hypermethylation of E-cadherin tended to be more likely in progesterone receptor (PR) negative than in PR-positive tumors (odds ratio (OR), 1.41; 95% confidence interval (CI), 0.91-2.18). Hypermethylation of p16 tended to be more frequent among estrogen receptor (ER) negative cases than ER-positive cases (OR, 1.51; 95% CI, 1.01-2.32). Hypermethylation of RAR-β 2 gene was inversely associated with histological and nuclear grade of breast cancer.

Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9–mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleotides to recombinant Cas9 protein containing a genetically encoded noncanonical amino acid with orthogonal chemical reactivity. The Cas9-oligonucleotide conjugates recruited an unmodified donor DNA template to the target site through base pairing, markedly increasing homology-directed repair efficiency in both human cell culture and mouse zygotes. These chemically modified Cas9 mutants provide an additional tool, one that is complementary to chemically modified nucleic acids, for improving the utility of CRISPR-Cas9–based genome-editing systems.