The elucidation of the human genome sequence has made it possible to identify genetic alterations in cancers in unprecedented detail. To begin a systematic analysis of such alterations, we determined the sequence of well-annotated human protein-coding genes in two common tumor types. Analysis of 13,023 genes in 11 breast and 11 colorectal cancers revealed that individual tumors accumulate an average of approximately 90 mutant genes but that only a subset of these contribute to the neoplastic process. Using stringent criteria to delineate this subset, we identified 189 genes (average of 11 per tumor) that were mutated at significant frequency. The vast majority of these genes were not known to be genetically altered in tumors and are predicted to affect a wide range of cellular functions, including transcription, adhesion, and invasion. These data define the genetic landscape of two human cancer types, provide new targets for diagnostic and therapeutic intervention, and open fertile avenues for basic research in tumor biology.

Human cancer is caused by the accumulation of mutations in oncogenes and tumor suppressor genes. To catalog the genetic changes that occur during tumorigenesis, we isolated DNA from 11 breast and 11 colorectal tumors and determined the sequences of the genes in the Reference Sequence database in these samples. Based on analysis of exons representing 20,857 transcripts from 18,191 genes, we conclude that the genomic landscapes of breast and colorectal cancers are composed of a handful of commonly mutated gene "mountains" and a much larger number of gene "hills" that are mutated at low frequency. We describe statistical and bioinformatic tools that may help identify mutations with a role in tumorigenesis. These results have implications for understanding the nature and heterogeneity of human cancers and for using personal genomics for tumor diagnosis and therapy.

Mounting evidence indicates that Smad proteins are required for TGF beta signaling, but the way(s) in which Smad proteins propagate this signal is unclear. We found that two human Smad proteins (Smad3 and Smad4) could specifically recognize an identical 8 bp palindromic sequences (GTCTAGAC). Tandem repeats of this palindrome conferred striking TGF beta responsiveness to a minimal promoter. This responsiveness was abrogated by targeted deletion of the cellular Smad4 gene. These results define a novel biochemical property of Smad proteins that is likely to play a direct role in the biologic responses to TGF beta and related ligands.

To gain insights into the molecular basis for metastasis, we compared the global gene expression profile of metastatic colorectal cancer with that of primary cancers, benign colorectal tumors, and normal colorectal epithelium. Among the genes identified, the PRL-3 protein tyrosine phosphatase gene was of particular interest. It was expressed at high levels in each of 18 cancer metastases studied but at lower levels in nonmetastatic tumors and normal colorectal epithelium. In 3 of 12 metastases examined, multiple copies of the PRL-3 gene were found within a small amplicon located at chromosome 8q24.3. These data suggest that the PRL-3 gene is important for colorectal cancer metastasis and provide a new therapeutic target for these intractable lesions.

Purpose: Patients with pancreatic ductal adenocarcinoma usually present with advanced-stage disease and a dismal prognosis. One effective strategy likely to improve the morbidity and mortality from pancreatic cancer would be the identification of accurate, noninvasive diagnostic markers that would enable earlier diagnosis of symptomatic patients and earlier detection of cancer in asymptomatic individuals at high risk for developing pancreatic cancer. In this study, we evaluated serum macrophage inhibitory cytokine-1 (MIC-1) as a marker of pancreatic cancer.Experimental Design: MIC-1 expression in primary pancreatic cancers, intraductal papillary mucinous neoplasms, and pancreatic cancer cell lines was determined using the National Center for Biotechnology Information serial analysis of gene expression database, oligonucleotide microarrays analysis, in situ hybridization, and immunohistochemistry. Serum MIC-1 levels were determined by ELISA in 80 patients with pancreatic adenocarcinomas, in 30 patients with ampullary and cholangiocellular carcinomas, in 42 patients with benign pancreatic tumors, in 76 patients with chronic pancreatitis, and in 97 healthy control subjects. The diagnostic performance of serum MIC-1 as a marker of pancreatic cancer was compared with that of serum CA19 -9.Results: Oligonucleotide microarray and serial analysis of gene expression data demonstrated that MIC-1 RNA levels were higher in primary pancreatic cancers, intraductal papillary mucinous neoplasms, and pancreatic cancer cell lines than in nonneoplastic pancreatic ductal epithelium. MIC-1 expression was localized to the malignant epithelium in pancreatic adenocarcinomas by in situ hybridization. MIC-1 protein was expressed in 14 of 16 primary pancreatic adenocarcinomas (88%) by immunohistochemistry and was also expressed in some pancreata affected by pancreatitis but not in normal pancreas. Serum MIC-1 levels were significantly higher in patients with pancreatic ductal adenocarcinoma (mean ؎ SD, 2428 ؎ 2324 pg/ml) and in patients with ampullary and cholangiocellular carcinomas (2123 ؎ 2387 pg/ml) than in those with benign pancreatic neoplasms (940 ؎ 469 pg/ml), chronic pancreatitis (1364 ؎ 1236 pg/ml), or in healthy controls (546 ؎ 262 pg/ml). An elevated serum MIC-1 (defined as 2 SD above the mean for healthy controls) performed as well as CA19 -9 (area under the receiver operating characteristic curve, 0.81 and 0.77, respectively), and the combination of MIC-1 and CA19 -9 significantly improved diagnostic accuracy (P < 0.05; area under the receiver operating characteristic curve, 0.87; sensitivity, 70%; specificity, 85%).Conclusion: Serum MIC-1 measurement can aid in the diagnosis of pancreatic adenocarcinoma.

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Chemoresistance is a major obstacle in triple negative breast cancer (TNBC), the most aggressive breast cancer subtype. Here we identify hypoxia-induced ECM re-modeler, lysyl oxidase (LOX) as a key inducer of chemoresistance by developing chemoresistant TNBC tumors in vivo and characterizing their transcriptomes by RNA-sequencing. Inhibiting LOX reduces collagen cross-linking and fibronectin assembly, increases drug penetration, and downregulates ITGA5/FN1 expression, resulting in inhibition of FAK/Src signaling, induction of apoptosis and re-sensitization to chemotherapy. Similarly, inhibiting FAK/Src results in chemosensitization. These effects are observed in 3D-cultured cell lines, tumor organoids, chemoresistant xenografts, syngeneic tumors and PDX models. Re-expressing the hypoxiarepressed miR-142-3p, which targets HIF1A, LOX and ITGA5, causes further suppression of the HIF-1α/LOX/ITGA5/FN1 axis. Notably, higher LOX, ITGA5, or FN1, or lower miR-142-3p levels are associated with shorter survival in chemotherapy-treated TNBC patients. These results provide strong pre-clinical rationale for developing and testing LOX inhibitors to overcome chemoresistance in TNBC patients.

A mutational analysis of the matrix metalloproteinase (MMP) gene family in human melanoma identified somatic mutations in 23% of melanomas. Five mutations in one of the most commonly mutated genes, MMP8, reduced MMP enzyme activity. Expression of wild-type but not mutant MMP8 in human melanoma cells inhibited growth on soft agar in vitro and tumor formation in vivo, suggesting that wild-type MMP-8 has the ability to inhibit melanoma progression.MMPs are proteolytic enzymes that degrade components of extracellular matrix and basement membranes 1 . MMPs have been associated with cancer metastasis 2,3 , and small molecule inhibitors of MMPs were tested as potential anticancer agents. However, clinical trials using these inhibitors showed no effect and, occasionally, accelerated tumor growth 4,5 . In contrast to the idea that MMP activity promotes melanoma progression, mouse models suggested that MMPs can have an antitumor role 6-8 . In particular, an increase in skin tumor incidence was seen in MMP-8-deficient mice 6 . These findings suggest that an in-depth analysis of the specific role of individual MMPs in particular cancer types is warranted. We systematically addressed © 2009 Nature America, Inc. All rights reserved.Correspondence should be addressed to Y.S. (samuelsy@mail.nih.gov).. AUTHOR CONTRIBUTIONS L.H.P. and Y.S. designed the study; J.R.W., P.F., A.C.F. and S.A.R. collected and analyzed the melanoma samples, A.S.B., J.C.C., N.S.A., P.B., P.P.-G., S.D., C.W., C.E.B., J. Table 2 online) and sequenced with dye terminator chemistry. To determine whether a given mutation was somatic, we sequenced the gene in genomic DNA from matched normal tissue. From the ∼5.5 Mb of sequence information obtained, we identified eight MMP genes containing somatic mutations (Table 1). Genes found to have one or more nonsynonymous mutations were then screened for mutations in an additional 47 melanomas. Through this approach, we identified 28 somatic mutations in eight genes, affecting 23% of the melanoma tumors analyzed (Table 1 and Supplementary Fig. 1 online).In seven tumors, both alleles of the MMP gene were affected, a characteristic associated with tumor suppressor genes. In addition, 6 of the 28 mutations were nonsense or splice-site alterations, which were predicted to result in aberrant or truncated proteins. Most tumors with MMP gene mutations also contained mutations in NRAS or BRAF. The clinical information associated with melanoma tumors containing MMP mutations is described in Supplementary Table 3 online.The observed somatic mutations could be either 'driver' mutations that have a functional role underlying neoplasia or nonfunctional 'passenger' changes. In the eight genes found to be mutated, 28 nonsynonymous (N) and 5 synonymous (S) somatic mutations were identified, yielding a N:S ratio of 28:5, significantly higher than the N:S ratio of 2:1 predicted for nonselected passenger mutations 9 (P < 0.026), suggesting that these are driver mutations. The ratio of C>T mutations compared to other nucleotide s...