Our results underscore the value of gene expression signatures to classify the interstitial lung diseases and to understand pathogenic mechanisms, and suggest new ways to improve the diagnosis and treatment of patients with these diseases.

Marked increased expression of cyclooxygenase 2 (COX-2), a prostaglandin-synthesizing enzyme that is pharmacologically inhibited by nonsteroid anti-inflammatory-type drugs, is a major early oncogenic event in the genesis of human colon neoplasia. We report that, in addition to inducing expression of COX-2, colon cancers further target the prostaglandin biogenesis pathway by ubiquitously abrogating expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme that physiologically antagonizes COX-2. We find that 15-PGDH transcript and protein are both highly expressed by normal colonic epithelia but are nearly undetectable in colon cancers. Using gene transfection to restore 15-PGDH expression in colon cancer cells strongly inhibits the ability of these cells to form tumors in immune-deficient mice and demonstrates 15-PGDH to have functional colon cancer tumor suppressor activity. In interrogating the mechanism for 15-PGDH expression loss in colon cancer, we determined that colonic 15-PGDH expression is directly controlled and strongly induced by activation of the TGF-␤ tumor suppressor pathway. These findings thus delineate an enzymatic pathway that induces colon cancer suppression, a pathway that is activated by TGF-␤ and mediated by 15-PGDH.colon ͉ gastric

A multidisciplinary method combining transcriptional data, specificity profiling, and biological characterization of an enzyme may be used to predict novel substrates. By integrating protease substrate profiling with microarray gene coexpression data from nearly 2,000 human normal and cancerous tissue samples, three fundamental components of a protease-activated signaling pathway were identified. We find that MT-SP1 mediates extracellular signaling by regulating the local activation of the prometastatic growth factor MSP-1. We demonstrate MT-SP1 expression in peritoneal macrophages, and biochemical methods confirm the ability of MT-SP1 to cleave and activate pro-MSP-1 in vitro and in a cellular context. MT-SP1 induced the ability of MSP-1 to inhibit nitric oxide production in bone marrow macrophages. Addition of HAI-1 or an MT-SP1-specific antibody inhibitor blocked the proteolytic activation of MSP-1 at the cell surface of peritoneal macrophages. Taken together, our work indicates that MT-SP1 is sufficient for MSP-1 activation and that MT-SP1, MSP-1, and the previously shown MSP-1 tyrosine kinase receptor RON are required for peritoneal macrophage activation. This work shows that this triad of growth factor, growth factor activator protease, and growth factor receptor is a protease-activated signaling pathway. Individually, MT-SP1 and RON overexpression have been implicated in cancer progression and metastasis. Transcriptional coexpression of these genes suggests that this signaling pathway may be involved in several human cancers.cancer ͉ macrophage activation ͉ protease substrate specificity ͉ proteomics D espite the successful physiological and biochemical characterization of many proteases, the vast majority of the Ͼ2% of the human genome that encodes proteases has yet to be functionally classified. Although many approaches demonstrate the sufficiency of a protease to cleave a given substrate, very few are able to address the physiological relevance of such in vitro findings. Cell-surface proteolysis is suggested to play a major role in cancer progression and metastasis through the processing of macromolecules important for regulating the extracellular environment. The cell-surface localization, high activity, and exquisite specificity of type II transmembrane serine proteases (TTSPs) suggest a role in outside-in signaling and interaction with the microenvironment. We elected to apply a multifaceted approach to identify physiologically relevant substrates of one prominent member of this family, membrane type serine protease 1 (MT-SP1/matriptase).Members of the TTSP family, such as hepsin and MT-SP1, are highly expressed in many cancers, including those of the prostate, breast, colon, and ovary (1-9). Both overexpression and inhibition studies have supported the role of MT-SP1 in tumorigenesis and tumor growth. Targeted overexpression of MT-SP1 in squamous epithelia in mice results in skin-limited nodules of squamous cell carcinoma that become metastatic in the presence of the chemical carcinogen DMBA (10). ...

We developed a method for deep mutational scanning of antibody complementarity-determining regions (CDRs) that can determine in parallel the effect of every possible single amino acid CDR substitution on antigen binding. The method uses libraries of full length IgGs containing more than 1000 CDR point mutations displayed on mammalian cells, sorted by flow cytometry into subpopulations based on antigen affinity and analyzed by massively parallel pyrosequencing. Higher, lower and neutral affinity mutations are identified by their enrichment or depletion in the FACS subpopulations. We applied this method to a humanized version of the anti-epidermal growth factor receptor antibody cetuximab, generated a near comprehensive data set for 1060 point mutations that recapitulates previously determined structural and mutational data for these CDRs and identified 67 point mutations that increase affinity. The large-scale, comprehensive sequence-function data sets generated by this method should have broad utility for engineering properties such as antibody affinity and specificity and may advance theoretical understanding of antibody-antigen recognition.