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author:("wei, Kevin A.")
1.  Identification of recurrent SMO and BRAF mutations in ameloblastomas 
Nature genetics  2014;46(7):722-725.
Here we report the discovery of oncogenic mutations in the Hedgehog and mitogen-activated protein kinase (MAPK) pathways in over 80% of ameloblastomas, locally destructive odontogenic tumors of the jaw, by genomic analysis of archival material. Mutations in SMO (encoding Smoothened, SMO) are common in ameloblastomas of the maxilla, whereas BRAF mutations are predominant in tumors of the mandible. We show that a frequently occurring SMO alteration encoding p.Leu412Phe is an activating mutation and that its effect on Hedgehog-pathway activity can be inhibited by arsenic trioxide (ATO), an anti-leukemia drug approved by the US Food and Drug Administration (FDA) that is currently in clinical trials for its Hedgehog-inhibitory activity. In a similar manner, ameloblastoma cells harboring an activating BRAF mutation encoding p.Val600Glu are sensitive to the BRAF inhibitor vemurafenib. Our findings establish a new paradigm for the diagnostic classification and treatment of ameloblastomas.
PMCID: PMC4418232  PMID: 24859340
2.  Modulators of Sensitivity and Resistance to Inhibition of PI3K Identified in a Pharmacogenomic Screen of the NCI-60 Human Tumor Cell Line Collection 
PLoS ONE  2012;7(9):e46518.
The phosphoinositide 3-kinase (PI3K) signaling pathway is significantly altered in a wide variety of human cancers, driving cancer cell growth and survival. Consequently, a large number of PI3K inhibitors are now in clinical development. To begin to improve the selection of patients for treatment with PI3K inhibitors and to identify de novo determinants of patient response, we sought to identify and characterize candidate genomic and phosphoproteomic biomarkers predictive of response to the selective PI3K inhibitor, GDC-0941, using the NCI-60 human tumor cell line collection. In this study, sixty diverse tumor cell lines were exposed to GDC-0941 and classified by GI50 value as sensitive or resistant. The most sensitive and resistant cell lines were analyzed for their baseline levels of gene expression and phosphorylation of key signaling nodes. Phosphorylation or activation status of both the PI3K-Akt signaling axis and PARP were correlated with in vitro response to GDC-0941. A gene expression signature associated with in vitro sensitivity to GDC-0941 was also identified. Furthermore, in vitro siRNA-mediated silencing of two genes in this signature, OGT and DDN, validated their role in modulating sensitivity to GDC-0941 in numerous cell lines and begins to provide biological insights into their role as chemosensitizers. These candidate biomarkers will offer useful tools to begin a more thorough understanding of determinants of patient response to PI3K inhibitors and merit exploration in human cancer patients treated with PI3K inhibitors.
PMCID: PMC3460918  PMID: 23029544
3.  Genomic and functional analysis identifies CRKL as an oncogene amplified in lung cancer 
Oncogene  2009;29(10):1421-1430.
DNA amplifications, leading to the overexpression of oncogenes, are a cardinal feature of lung cancer and directly contribute to its pathogenesis. To uncover novel such alterations, we performed an array-based comparative genomic hybridization survey of 128 non-small cell lung cancer cell lines and tumors. Prominent among our findings, we identified recurrent high-level amplification at cytoband 22q11.21 in 3% of lung cancer specimens, with another 11% of specimens exhibiting low-level gain spanning that locus. The 22q11.21 amplicon core contained eight named genes, only four of which were overexpressed (by transcript profiling) when amplified. Among these, CRKL encodes an adaptor protein functioning in signal transduction, best known as a substrate of the BCR-ABL kinase in chronic myelogenous leukemia. RNA interference-mediated knockdown of CRKL in lung cancer cell lines with (but not without) amplification led to significantly decreased cell proliferation, cell-cycle progression, cell survival, and cell motility and invasion. In addition, overexpression of CRKL in immortalized human bronchial epithelial cells led to EGF-independent cell growth. Our findings indicate that amplification and resultant overexpression of CRKL contributes to diverse oncogenic phenotypes in lung cancer, with implications for targeted therapy, and highlighting a role of adapter proteins as primary genetic drivers of tumorigenesis.
PMCID: PMC3320568  PMID: 19966867
CRKL; lung cancer; DNA amplification; genomic profiling; adapter protein
4.  SMURF1 Amplification Promotes Invasiveness in Pancreatic Cancer 
PLoS ONE  2011;6(8):e23924.
Pancreatic cancer is a deadly disease, and new therapeutic targets are urgently needed. We previously identified DNA amplification at 7q21-q22 in pancreatic cancer cell lines. Now, by high-resolution genomic profiling of human pancreatic cancer cell lines and human tumors (engrafted in immunodeficient mice to enrich the cancer epithelial fraction), we define a 325 Kb minimal amplicon spanning SMURF1, an E3 ubiquitin ligase and known negative regulator of transforming growth factor β (TGFβ) growth inhibitory signaling. SMURF1 amplification was confirmed in primary human pancreatic cancers by fluorescence in situ hybridization (FISH), where 4 of 95 cases (4.2%) exhibited amplification. By RNA interference (RNAi), knockdown of SMURF1 in a human pancreatic cancer line with focal amplification (AsPC-1) did not alter cell growth, but led to reduced cell invasion and anchorage-independent growth. Interestingly, this effect was not mediated through altered TGFβ signaling, assayed by transcriptional reporter. Finally, overexpression of SMURF1 (but not a catalytic mutant) led to loss of contact inhibition in NIH-3T3 mouse embryo fibroblast cells. Together, these findings identify SMURF1 as an amplified oncogene driving multiple tumorigenic phenotypes in pancreatic cancer, and provide a new druggable target for molecularly directed therapy.
PMCID: PMC3161761  PMID: 21887346
5.  Genomic instability in breast cancer: pathogenesis and clinical implications 
Molecular oncology  2010;4(3):255-266.
Breast cancer is a heterogeneous disease, appreciable by molecular markers, gene expression profiles, and most recently, patterns of genomic alteration. In particular, genomic profiling has revealed three distinct patterns of DNA copy number alteration: a “simple” type with few gains or losses of whole chromosome arms, an “amplifier” type with focal high-level DNA amplifications, and a “complex” type marked by numerous low-amplitude changes and copy-number transitions. The three patterns are associated with distinct gene-expression subtypes, and preferentially target different loci in the genome (implicating distinct cancer genes). Moreover, the different patterns of alteration imply distinct underlying mechanisms of genomic instability. The amplifier pattern may arise from transient telomere dysfunction, although new data suggest ongoing “amplifier” instability. The complex pattern shows similarity to breast cancers with germline BRCA1 mutation, which also exhibit “basal-like” expression profiles and complex-pattern genomes, implicating a possible defect in BRCA1-associated repair of DNA double-strand breaks. As such, targeting presumptive DNA repair defects represents a promising area of clinical investigation. Future studies should clarify the pathogenesis of breast cancers with amplifier and complex pattern genomes, and will likely identify new therapeutic opportunities.
PMCID: PMC2904860  PMID: 20434415
Breast cancer; genomic instability; DNA amplification; DNA repair defect; basal-like; BRCA1
6.  Integrin A6 Cleavage in Mouse Skin Tumors 
We have previously identified a structural variant of the α6 integrin (Laminin receptor) called α6p. The α6p variant is a 70 kDa form of the full-length α6 integrin (140 kDa) that remains paired with either the β1 or β4 subunit on the cell surface. α6p is produced by urokinase-type plasminogen activator (uPA), which removes the extracellular β-barrel domain while the receptor is on the cell surface. The α6p integrin was present in human prostate cancer tissue but not in normal tissue and the cleavage of the α6 integrin extracellular domain promotes tumor cell invasion and migration on laminin. The objective of the present study was to determine whether the α6p integrin is observed in other models of carcinogenesis. Our results indicate detectable low levels of α6p in normal mouse skin, and comparatively elevated levels in mouse papillomas and squamous cell carcinomas induced by DMBA, TPA and MNNG treatments. Furthermore, we have found that α6p was present at high levels in skin melanomas of transgenic mice that over express activated Ha-ras under the control of the tyrosinase promoter. Finally, subcutaneous injection into athymic nude mice of a malignant mouse keratinocyte derived cell line (6M90) that is α6p negative, results in the development of tumors that contain α6p integrin. The latter results indicate that α6p is induced in vivo suggesting that the tumor microenvironment plays a major role in the production of α6p. Taken together, these data suggest that the cell surface cleavage of the α6 integrin may be a novel mechanism of integrin regulation and might be an important step during skin tissue remodeling and during carcinogenesis.
PMCID: PMC2906811  PMID: 20664806
7.  CAMK1D amplification implicated in epithelial-mesenchymal transition in basal-like breast cancer 
Molecular oncology  2008;2(4):327-339.
Breast cancer exhibits clinical and molecular heterogeneity, where expression-profiling studies have identified five major molecular subtypes. The basal-like subtype, expressing basal epithelial markers and negative for estrogen receptor (ER), progesterone receptor (PR) and HER2, is associated with higher overall levels of DNA copy number alteration (CNA), specific CNAs (like gain on chromosome 10p), and poor prognosis. Discovering the molecular genetic basis of tumor subtypes may provide new opportunities for therapy. To identify the driver oncogene on 10p associated with basal-like tumors, we analyzed genomic profiles of 172 breast carcinomas. The smallest shared region of gain spanned just seven genes at 10p13, including calcium/calmodulin-dependent protein kinase ID (CAMK1D), functioning in intracellular signaling but not previously linked to cancer. By microarray, CAMK1D was overexpressed when amplified, and by immunohistochemistry exhibited elevated expression in invasive carcinomas compared to carcinoma in situ. Engineered overexpression of CAMK1D in non-tumorigenic breast epithelial cells led to increased cell proliferation, and molecular and phenotypic alterations indicative of epithelial-mesenchymal transition (EMT), including loss of cell-cell adhesions and increased cell migration and invasion. Our findings identify CAMK1D as a novel amplified oncogene linked to EMT in breast cancer, and as a potential therapeutic target with particular relevance to clinically unfavorable basal-like tumors.
PMCID: PMC2653212  PMID: 19383354
Breast cancer; genomic profiling; DNA amplification; oncogene; epithelial-mesenchymal transition; EMT
8.  Molecular Profiling of Breast Cancer Cell Lines Defines Relevant Tumor Models and Provides a Resource for Cancer Gene Discovery 
PLoS ONE  2009;4(7):e6146.
Breast cancer cell lines have been used widely to investigate breast cancer pathobiology and new therapies. Breast cancer is a molecularly heterogeneous disease, and it is important to understand how well and which cell lines best model that diversity. In particular, microarray studies have identified molecular subtypes–luminal A, luminal B, ERBB2-associated, basal-like and normal-like–with characteristic gene-expression patterns and underlying DNA copy number alterations (CNAs). Here, we studied a collection of breast cancer cell lines to catalog molecular profiles and to assess their relation to breast cancer subtypes.
Whole-genome DNA microarrays were used to profile gene expression and CNAs in a collection of 52 widely-used breast cancer cell lines, and comparisons were made to existing profiles of primary breast tumors. Hierarchical clustering was used to identify gene-expression subtypes, and Gene Set Enrichment Analysis (GSEA) to discover biological features of those subtypes. Genomic and transcriptional profiles were integrated to discover within high-amplitude CNAs candidate cancer genes with coordinately altered gene copy number and expression.
Transcriptional profiling of breast cancer cell lines identified one luminal and two basal-like (A and B) subtypes. Luminal lines displayed an estrogen receptor (ER) signature and resembled luminal-A/B tumors, basal-A lines were associated with ETS-pathway and BRCA1 signatures and resembled basal-like tumors, and basal-B lines displayed mesenchymal and stem/progenitor-cell characteristics. Compared to tumors, cell lines exhibited similar patterns of CNA, but an overall higher complexity of CNA (genetically simple luminal-A tumors were not represented), and only partial conservation of subtype-specific CNAs. We identified 80 high-level DNA amplifications and 13 multi-copy deletions, and the resident genes with concomitantly altered gene-expression, highlighting known and novel candidate breast cancer genes.
Overall, breast cancer cell lines were genetically more complex than tumors, but retained expression patterns with relevance to the luminal-basal subtype distinction. The compendium of molecular profiles defines cell lines suitable for investigations of subtype-specific pathobiology, cancer stem cell biology, biomarkers and therapies, and provides a resource for discovery of new breast cancer genes.
PMCID: PMC2702084  PMID: 19582160
9.  Genomic Profiling Identifies GATA6 as a Candidate Oncogene Amplified in Pancreatobiliary Cancer 
PLoS Genetics  2008;4(5):e1000081.
Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46%) primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies.
Author Summary
Pancreatic cancer is a devastating disease, having among the lowest survival rates of any cancer. A better understanding of the molecular basis of pancreatic cancer may lead to improved rationale therapies. We report here the discovery of amplification (i.e. extra copies) of the GATA6 gene in many human pancreatic cancers. GATA6 is a regulator of gene expression and functions in the development of the normal pancreas. Our findings indicate that its amplification and aberrant overexpression contribute to pancreatic cancer development. GATA6 joins a growing list of cancer genes with key roles in normal human development but pathogenic roles in cancer when aberrantly expressed. Our discovery of GATA6 amplification provides a new foothold into understanding the pathogenic mechanisms underlying pancreatic cancer, and suggests new strategies for therapy by targeting GATA6 or the genes it regulates.
PMCID: PMC2413204  PMID: 18535672
10.  Transcriptional Repression of Catalase in Mouse Skin Tumor Progression1 
Neoplasia (New York, N.Y.)  2004;6(5):440-448.
Previous studies in our laboratory have shown that the elevation of reactive oxygen species levels and the repression of the antioxidant enzyme, catalase, played a critical role in the in vitro progression of benign papilloma cells to malignant carcinoma cells. Catalase message, protein levels, and activity levels were found to be downregulated in the malignantly progressed cells. The goal of this study is to further characterize the repression of catalase in malignant progression of mouse skin tumors. To validate the in vitro observations, we examined catalase expression in tumor samples generated by the multistep chemical carcinogenesis protocol. Higher levels of catalase mRNA and protein were observed in benign papillomas versus malignant carcinomas. Nuclear run-on analysis showed that catalase repression in the cultured malignant cells was transcription-dependent. Results from luciferase reporter assays indicated that malignant cells have lower catalase promoter activities than benign papilloma cells, in part through the Wilm's tumor suppressor 1 (WT1) binding site within the proximal promoter region. The WT1 protein levels were found to be inversely correlated with the observed catalase promoter activities, with higher levels observed in the malignant cells versus the benign cells. These results led us to conclude that WT1 is acting as a transcription repressor in catalase gene regulation during tumor progression.
PMCID: PMC1550329  PMID: 15548352
Catalase; Wilm's tumor suppressor 1; antioxidant; transcriptional regulation; tumor progression and mouse skin carcinogenesis; ROS, reactive oxygen species; SCC, squamous cell carcinoma; DMBA, 7,12-dimethylbenzanthracene; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; MNNG, N-methyl-N-Vnitro-N-nitrosoguanidine; WT1, Wilm's tumor suppressor 1

Results 1-10 (10)