New Ameloblastoma Cell Lines Enable Preclinical Study of Targeted Therapies.

Ameloblastoma (AB) is an odontogenic tumor that arises from ameloblast-lineage cells. Although relatively uncommon and rarely metastatic, AB tumors are locally invasive and destructive to the jawbone and surrounding structures. Standard-of-care surgical resection often leads to disfigurement, and many tumors will locally recur, necessitating increasingly challenging surgeries. Recent genomic studies of AB have uncovered oncogenic driver mutations, including in the mitogen-activated protein kinase (MAPK) and Hedgehog signaling pathways. Medical therapies targeting those drivers would be a highly desirable alternative or addition to surgery; however, a paucity of existing AB cell lines has stymied clinical translation. To bridge this gap, here we report the establishment of 6 new AB cell lines-generated by "conditional reprogramming"-and their genomic characterization that reveals driver mutations in FGFR2, KRAS, NRAS, BRAF, PIK3CA, and SMO. Furthermore, in proof-of-principle studies, we use the new cell lines to investigate AB oncogene dependency and drug sensitivity. Among our findings, AB cells with KRAS or NRAS mutation (MAPK pathway) are exquisitely sensitive to MEK inhibition, which propels ameloblast differentiation. AB cells with activating SMO-L412F mutation (Hedgehog pathway) are insensitive to vismodegib; however, a distinct small-molecule SMO inhibitor, BMS-833923, significantly reduces both downstream Hedgehog signaling and tumor cell viability. The novel cell line resource enables preclinical studies and promises to speed the translation of new molecularly targeted therapies for the management of ameloblastoma and related odontogenic neoplasms.

ARID1B, a member of the human SWI/SNF chromatin remodeling complex, exhibits tumour-suppressor activities in pancreatic cancer cell lines.

BACKGROUND: The human ATP-dependent SWItch/sucrose nonfermentable (SWI/SNF) complex functions as a primary chromatin remodeler during ontogeny, as well as in adult life. Several components of the complex have been suggested to function as important regulators of tumorigenesis in various cancers. In the current study, we have characterised a possible tumour suppressor role for the largest subunit of the complex, namely the AT-rich interaction domain 1B (ARID1B). METHODS: We performed Azacytidine and Trichostatin A treatments, followed by bisulphite sequencing to determine the possible DNA methylation-induced transcription repression of the gene in pancreatic cancer (PaCa) cell lines. Functional characterisation of effect of ARID1B ectopic expression in MiaPaCa2 PaCa cell line, which harboured ARID1B homozygous deletion, was carried out. Finally, we evaluated ARID1B protein expression in pancreatic tumour samples using immunohistochemistry on a tissue microarray. RESULTS: ARID1B was transcriptionally repressed due to promoter hypermethylation, and ectopic expression severely compromised the ability of MiaPaCa2 cells to form colonies in liquid culture and soft agar. In addition, ARID1B exhibited significantly reduced/loss of expression in PaCa tissue, especially in samples from advanced-stage tumours, when compared with normal pancreas. CONCLUSION: The results therefore suggest a possible tumour-suppressor function for ARID1B in PaCa, thus adding to the growing list of SWI/SNF components with a similar function. Given the urgent need to design efficient targeted therapies for PaCa, our study assumes significance.

EGFRvIII gene rearrangement is an early event in glioblastoma tumorigenesis and expression defines a hierarchy modulated by epigenetic mechanisms.

Amplification and rearrangements of the epidermal growth factor receptor (EGFR) gene are frequently found in glioblastoma multiforme (GBM). The most common variant is EGFR variant III (EGFRvIII). Research suggests that EGFRvIII could be a marker for a cancer stem cell or tumor-initiating population. If amplification and rearrangement are early events in tumorigenesis, this implies that they should be preserved throughout the tumor. However, in primary GBM, EGFRvIII expression is focal and sporadic. Unexpectedly, we found EGFR amplification and rearrangement throughout the tumor, including regions with no EGFRvIII expression, suggesting that mechanisms exist to modulate EGFRvIII expression even in the presence of high gene amplification. To study this phenomenon, we characterized three GBM cell lines with endogenous EGFRvIII. EGFRvIII expression was heterogeneous, with both positive and negative populations maintaining the genetic alterations, akin to primary tumors. Furthermore, EGFRvIII defined a hierarchy where EGFRvIII-positive cells gave rise to additional positive and negative cells. Only cells that had recently lost EGFRvIII expression could re-express EGFRvIII, providing an important buffer for maintaining EGFRvIII-positive cell numbers. Epigenetic mechanisms had a role in maintaining heterogeneous EGFRvIII expression. Demethylation induced a 20-60% increase in the percentage of EGFRvIII-positive cells, indicating that some cells could re-express EGFRvIII. Surprisingly, inhibition of histone deacetylation resulted in a 50-80% reduction in EGFRvIII expression. Collectively, this data demonstrates that EGFR amplification and rearrangement are early events in tumorigenesis and EGFRvIII follows a model of hierarchical expression. Furthermore, EGFRvIII expression is restricted by epigenetic mechanisms, suggesting that drugs that modulate the epigenome might be used successfully in glioblastoma tumors.

Recurrent deletion of CHD1 in prostate cancer with relevance to cell invasiveness.

Though prostate cancer is often indolent, it is nonetheless a leading cause of cancer death. Defining the underlying molecular genetic alterations may lead to new strategies for prevention or treatment. Towards this goal, we performed array-based comparative genomic hybridization (CGH) on 86 primary prostate tumors. Among the most frequent alterations not associated with a known cancer gene, we identified focal deletions within 5q21 in 15 out of 86 (17%) cases. By high-resolution tiling array CGH, the smallest common deletion targeted just one gene, the chromatin remodeler chromodomain helicase DNA-binding protein 1 (CHD1). Expression of CHD1 was significantly reduced in tumors with deletion (P=0.03), and compared with normal prostate (P=0.04). Exon sequencing analysis also uncovered nonsynonymous mutations in 1 out of 7 (14%) cell lines (LAPC4) and in 1 out of 24 (4%) prostate tumors surveyed. RNA interference-mediated knockdown of CHD1 in two nontumorigenic prostate epithelial cell lines, OPCN2 and RWPE-1, did not alter cell growth, but promoted cell invasiveness, and in OPCN2-enhanced cell clonogenicity. Taken together, our findings suggest that CHD1 deletion may underlie cell invasiveness in a subset of prostate cancers, and indicate a possible novel role of altered chromatin remodeling in prostate tumorigenesis.

Focal amplification and oncogene dependency of GAB2 in breast cancer.

DNA amplifications in breast cancer are frequent on chromosome 11q, in which multiple driver oncogenes likely reside in addition to cyclin D1 (CCND1). One such candidate, the scaffolding adapter protein, GRB2-associated binding protein 2 (GAB2), functions in ErbB signaling and was recently shown to enhance mammary epithelial cell proliferation, and metastasis of ERBB2 (HER2/neu)-driven murine breast cancer. However, the amplification status and function of GAB2 in the context of amplification remain undefined. In this study, by genomic profiling of 172 breast tumors, and fluorescence in situ hybridization validation in an independent set of 210 scorable cases, we observed focal amplification spanning GAB2 (11q14.1) independent of CCND1 (11q13.2) amplification, consistent with a driver role. Further, small interfering RNA (siRNA)-mediated knockdown of GAB2 in breast cancer lines (SUM52, SUM44PE and MDA468) with GAB2 amplification revealed a dependency on GAB2 for cell proliferation, cell-cycle progression, survival and invasion, likely mediated through altered phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling. GAB2 knockdown also reduced proliferation and survival in a cell line (BT474) with ERBB2 amplification, consistent with the possibility that GAB2 can function downstream of ERBB2. Our studies implicate focal amplification of GAB2 in breast carcinogenesis, and underscore an oncogenic role of scaffolding adapter proteins, and a potential new point of therapeutic intervention.

Genomic and functional analysis identifies CRKL as an oncogene amplified in lung cancer.

DNA amplifications, leading to the overexpression of oncogenes, are a cardinal feature of lung cancer and directly contribute to its pathogenesis. To uncover such novel 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 adapter 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 enhanced growth factor-independent cell growth. Our findings indicate that amplification and resultant overexpression of CRKL contribute to diverse oncogenic phenotypes in lung cancer, with implications for targeted therapy, and highlight a role of adapter proteins as primary genetic drivers of tumorigenesis.

Genomic profiling identifies TITF1 as a lineage-specific oncogene amplified in lung cancer.

Lung cancer is a leading cause of cancer death, where the amplification of oncogenes contributes to tumorigenesis. Genomic profiling of 128 lung cancer cell lines and tumors revealed frequent focal DNA amplification at cytoband 14q13.3, a locus not amplified in other tumor types. The smallest region of recurrent amplification spanned the homeobox transcription factor TITF1 (thyroid transcription factor 1; also called NKX2-1), previously linked to normal lung development and function. When amplified, TITF1 exhibited increased expression at both the RNA and protein levels. Small interfering RNA (siRNA)-mediated knockdown of TITF1 in lung cancer cell lines with amplification led to reduced cell proliferation, manifested by both decreased cell-cycle progression and increased apoptosis. Our findings indicate that TITF1 amplification and overexpression contribute to lung cancer cell proliferation rates and survival and implicate TITF1 as a lineage-specific oncogene in lung cancer.

c-Jun N-terminal kinase is activated in non-small-cell lung cancer and promotes neoplastic transformation in human bronchial epithelial cells.

c-Jun N-terminal kinase (JNK) has been reported to either potentiate or inhibit oncogenesis, depending upon the cellular context, but its role in lung neoplasia is unclear. Here we sought to define the role of JNK in lung neoplasia by examining evidence of JNK phosphorylation in non-small-cell lung cancer (NSCLC) biopsy samples and by using genetic and pharmacologic approaches to modulate JNK expression and activity in cultured cells. Immunohistochemical staining for JNK phosphorylation was detected in 114 (45%) of 252 NSCLC biopsy samples and was predominantly nuclear, providing evidence of JNK activation in a subset of NSCLC cases. Introduction of a doxycycline-inducible, constitutively active, mutant mitogen-activated protein kinase kinase 4 (MKK4) into the human bronchial epithelial cell lines BEAS-2B and HB56B increased the cells' proliferation, migration, invasion and clonogenicity. Depletion of JNK in MKK4 mutant-transformed BEAS-2B cells by introduction of JNK1/2 short hairpin RNA reversed the transformed phenotype, indicating that JNK activation is oncogenic and MKK4 confers neoplastic properties in these cells. The proliferation of NSCLC cell lines HCC827 and H2009, in which JNK and its substrate c-Jun are constitutively phosphorylated, was inhibited by SP600125, a JNK kinase inhibitor. We conclude that JNK is activated in a subset of NSCLC biopsy samples and promotes oncogenesis in the bronchial epithelium, suggesting that strategies to inhibit the JNK pathway should be considered for the prevention and treatment of NSCLC.

Molecular profiling reveals myeloid leukemia cell lines to be faithful model systems characterized by distinct genomic aberrations.

To model and investigate different facets of leukemia pathogenesis, a widely accepted approach is to use immortalized leukemia cell lines. Although these provide powerful tools to our knowledge, few studies have addressed the question whether hematopoietic cell lines represent accurate and reliable model systems. To improve the molecular characterization of these model systems, we analyzed 17 myeloid leukemia cell lines using DNA microarray technology. By array-based comparative genomic hybridization, we identified recurrent genomic DNA gains and losses, as well as high-level amplifications. Parallel analysis of gene expression helped delineate potential candidate genes, and unsupervised analysis of gene expression data revealed cell lines to cluster in part based on underlying cytogenetic abnormalities. Comparison with clinical leukemia specimens showed that key signatures were retained, as myeloid cell lines with characteristic cytogenetic aberrations co-clustered with leukemia samples carrying the respective abnormality. Signatures were also quite robust, as expression data from cell lines correlated highly with published data. Thus, our analyses demonstrate myeloid cell lines to exhibit conserved and stable signatures reflecting the underlying primary cytogenetic aberrations. Our refined molecular characterization of myeloid cell lines supports the utility of cell lines as faithful and powerful model systems and provides additional insights into the molecular mechanisms of leukemogenesis.

Combined microarray analysis of small cell lung cancer reveals altered apoptotic balance and distinct expression signatures of MYC family gene amplification.

DNA amplifications and deletions frequently contribute to the development and progression of lung cancer. To identify such novel alterations in small cell lung cancer (SCLC), we performed comparative genomic hybridization on a set of 24 SCLC cell lines, using cDNA microarrays representing approximately 22,000 human genes (providing an average mapping resolution of <70 kb). We identified localized DNA amplifications corresponding to oncogenes known to be amplified in SCLC, including MYC (8q24), MYCN (2p24) and MYCL1 (1p34). Additional highly localized DNA amplifications suggested candidate oncogenes not previously identified as amplified in SCLC, including the antiapoptotic genes TNFRSF4 (1p36), DAD1 (14q11), BCL2L1 (20q11) and BCL2L2 (14q11). Likewise, newly discovered PCR-validated homozygous deletions suggested candidate tumor-suppressor genes, including the proapoptotic genes MAPK10 (4q21) and TNFRSF6 (10q23). To characterize the effect of DNA amplification on gene expression patterns, we performed expression profiling using the same microarray platform. Among our findings, we identified sets of genes whose expression correlated with MYC, MYCN or MYCL1 amplification, with surprisingly little overlap among gene sets. While both MYC and MYCN amplification were associated with increased and decreased expression of known MYC upregulated and downregulated targets, respectively, MYCL1 amplification was associated only with the latter. Our findings support a role of altered apoptotic balance in the pathogenesis of SCLC, and suggest that MYC family genes might affect oncogenesis through distinct sets of targets, in particular implicating the importance of transcriptional repression.

Molecular portraits of human breast tumours.

Human breast tumours are diverse in their natural history and in their responsiveness to treatments. Variation in transcriptional programs accounts for much of the biological diversity of human cells and tumours. In each cell, signal transduction and regulatory systems transduce information from the cell's identity to its environmental status, thereby controlling the level of expression of every gene in the genome. Here we have characterized variation in gene expression patterns in a set of 65 surgical specimens of human breast tumours from 42 different individuals, using complementary DNA microarrays representing 8,102 human genes. These patterns provided a distinctive molecular portrait of each tumour. Twenty of the tumours were sampled twice, before and after a 16-week course of doxorubicin chemotherapy, and two tumours were paired with a lymph node metastasis from the same patient. Gene expression patterns in two tumour samples from the same individual were almost always more similar to each other than either was to any other sample. Sets of co-expressed genes were identified for which variation in messenger RNA levels could be related to specific features of physiological variation. The tumours could be classified into subtypes distinguished by pervasive differences in their gene expression patterns.

Genome-wide analysis of DNA copy-number changes using cDNA microarrays.

Gene amplifications and deletions frequently contribute to tumorigenesis. Characterization of these DNA copy-number changes is important for both the basic understanding of cancer and its diagnosis. Comparative genomic hybridization (CGH) was developed to survey DNA copy-number variations across a whole genome. With CGH, differentially labelled test and reference genomic DNAs are co-hybridized to normal metaphase chromosomes, and fluorescence ratios along the length of chromosomes provide a cytogenetic representation of DNA copy-number variation. CGH, however, has a limited ( approximately 20 Mb) mapping resolution, and higher-resolution techniques, such as fluorescence in situ hybridization (FISH), are prohibitively labour-intensive on a genomic scale. Array-based CGH, in which fluorescence ratios at arrayed DNA elements provide a locus-by-locus measure of DNA copy-number variation, represents another means of achieving increased mapping resolution. Published array CGH methods have relied on large genomic clone (for example BAC) array targets and have covered only a small fraction of the human genome. cDNAs representing over 30,000 radiation-hybrid (RH)-mapped human genes provide an alternative and readily available genomic resource for mapping DNA copy-number changes. Although cDNA microarrays have been used extensively to characterize variation in human gene expression, human genomic DNA is a far more complex mixture than the mRNA representation of human cells. Therefore, analysis of DNA copy-number variation using cDNA microarrays would require a sensitivity of detection an order of magnitude greater than has been routinely reported. We describe here a cDNA microarray-based CGH method, and its application to DNA copy-number variation analysis in breast cancer cell lines and tumours. Using this assay, we were able to identify gene amplifications and deletions genome-wide and with high resolution, and compare alterations in DNA copy number and gene expression.

Site-specific RNA binding by a hepatitis B virus reverse transcriptase initiates two distinct reactions: RNA packaging and DNA synthesis.

Hepatitis B viruses encode a polymerase (P) protein with key roles in both reverse transcription and genomic RNA encapsidation. Genetic analysis of cis-acting signals required for viral replication implicates an RNA stem-loop structure in both RNA packaging and the initiation of reverse transcription, a process in which P protein also serves as the primer. We now show that duck hepatitis B virus (DHBV) polymerase binds specifically and with high affinity to this RNA stem-loop structure. Mutational analysis indicates that all mutations in the RNA target that inhibit the P protein-RNA interaction inhibit both in vivo RNA packaging and in vitro DNA priming to comparable extents. However, certain mutations in the loop region of the RNA have minimal impact on P protein-RNA binding but are nonetheless severely defective for packaging and DNA synthesis. Thus, P protein-RNA complex formation is necessary but not sufficient to initiate these activities. In addition, examination of RNA binding by truncated P proteins indicates that the C terminus of the polymerase, although required for RNA encapsidation in vivo, is dispensable for RNA binding and DNA priming.

Hepatitis B virus reverse transcriptase and its many roles in hepadnaviral genomic replication.

The replication of hepatitis B virus DNA proceeds through reverse transcription of a pregenomic RNA intermediate, a reaction that takes place within viral nucleocapsids and is catalyzed by the viral P protein. P protein is involved in all phases of the reaction, serving as (a) a recognition factor for the selective encapsidation of the pregenomic RNA template; (b) the protein primer for the initiation of minus strand DNA synthesis; (c) the reverse transcriptase and DNA polymerase involved in strand elongation; and (d) the RNaseH activity required to remove RNA template prior to plus strand synthesis. P protein is capable of site-specific RNA recognition, specifically binding to a stem-loop structure at the 5' end of pregenomic RNA. This interaction is required for both RNA encapsidation and reverse transcription.

An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation.

Selective encapsidation of hepatitis B virus (HBV) genomic RNA within cytoplasmic core particles requires recognition of the cis-encapsidation signal, (termed epsilon) located at the 5' end of genomic RNA. By transfecting plasmids expressing chimeric RNAs bearing HBV sequences fused to lacZ, we have mapped the minimal region of epsilon to the 5' 94 nucleotides (nt) of genomic RNA. Enzymatic probing of the RNA secondary structure in this region (by using either in vitro transcripts or RNA extracted from HBV core particles) reveals a stem-loop structure containing a lower stem, a 6-nt bulge, an upper stem with a single unpaired U residue, and a 6-nt loop. The functional role of this structure in encapsidation was explored by examining the effects of mutations in epsilon on encapsidation of RNA in vivo. These studies reveal that (i) in the lower stem, base pairing but not specific primary sequence is required for function; (ii) there is no requirement for base pairing in the lower portion of the upper stem, but base pairing elsewhere in this stem contributes to packaging efficiency; (iii) the presence of the 6-nt bulge, but not its primary sequence, is important for function; and (iv) specific nucleotide sequences in the loop and in regions of the upper stem are critical for RNA encapsidation.

Influence of therapeutic plasmapheresis on elimination of ceftiaxone.

To study the plasma elimination rate of ceftriaxone given to patients undergoing plasmapheresis, a single 2-g intravenous dose of ceftriaxone was given to 11 patients who underwent plasmapheresis 3 h (early group) or 15 h (late group) later. Even though the early patient group had twofold more ceftriaxone removed than the late group, all individual patients had 24-h ceftriaxone levels of 9 micrograms/ml or higher. We conclude that patients undergoing plasmapheresis lasting 150 min or less may safely be treated with 2 g of ceftriaxone once daily without risking periods of subtherapeutic plasma ceftriaxone levels.

cis-acting sequences required for encapsidation of duck hepatitis B virus pregenomic RNA.

Hepadnavirus reverse transcription requires that pregenomic RNA first be selectively packaged into a cytoplasmic core particle. This process presumably requires the presence of specific recognition sequences on the pregenomic RNA. To define the cis-acting sequences required for pregenome encapsidation in the duck hepatitis B virus (DHBV), we assayed the packaging efficiency of a series of pregenomic RNA deletion mutants and hybrid DHBV/lacZ fusion transcripts. The 5' boundary of the packaging signal lies within the precore region, starting approximately 35 nucleotides from the cap site of pregenomic RNA; thus, the DR1 sequence required for proper viral DNA synthesis is not included in this signal. To define the 3' boundary of the encapsidation signal, fusion transcripts bearing foreign (lacZ) sequences fused to DHBV at different sites 3' to the pregenomic RNA start site were examined. A surprisingly large region of the DHBV genome proved to be required for packaging of such chimeras, which are efficiently encapsidated only when they contain the first 1,200 to 1,400 nucleotides of DHBV pregenomic RNA. However, mutant genomes bearing insertions within this region are packaged efficiently, making it likely that the actual recognition elements for encapsidation are smaller discontinuous sequences located within this region.

Iron uptake in Salmonella typhimurium: utilization of exogenous siderochromes as iron carriers.

Aerobic microorganisms have evolved a variety of siderochromes, special ligands which can dissolve insoluble ferric iron and facilitate its transport into the cell. We have found that enb mutants of Salmonella typhimurium blocked in the biosynthesis of enterobactin (its natural iron carrier) are able to utilize siderochromes of different types made by other microorganisms as iron carriers. The antibiotic albomycin delta(2) was used to select mutants defective in ferrichrome-mediated iron uptake. Twelve classes of albomycin-resistant mutants, named sid, were defined on the basis of their growth responses to other siderochromes. Most of these classes have genetic lesions in loci that are cotransduced with panC (represented at 9 min on the genetic map). The locus designated sidJ is cotransduced with enb, whereas sidK and sidL are linked with neither panC nor enb. Genetic and physiological data indicate that S. typhimurium has several transport systems of high specificity for a variety of siderochromes produced by other microorganisms.

Iron transport in Salmonella typhimurium: mutants blocked in the biosynthesis of enterobactin.

A number of mutants of Salmonella typhimurium were isolated which are blocked in the biosynthesis of enterobactin, an iron chelator that is secreted by the wild-type bacteria when they are grown on low iron media. One class of these enb mutants accumulates the enterobactin precursor 2,3-dihydroxybenzoic acid, and another class does not accumulate any detectable catechol precursor. The enb mutants grow very well on a glucose-mineral salts medium. Addition of citrate, itself an iron chelator, to the medium drastically inhibits growth unless the medium is supplemented with enterobactin or iron salts. Citrate inhibits iron uptake from the medium by enb mutants; enterobactin counteracts this inhibition and also, by itself, increases iron uptake. Thus, the apparent function of enterobactin is to promote the absorption of iron from the medium by the bacteria. Transduction experiments showed that the genes for enterobactin biosynthesis are closely linked on the S. typhimurium chromosome. It is suggested that they form an operon which is repressed by the presence of iron. S. typhimurium can utilize the iron chelate ferrichrome. (Deferriferrichrome is a cyclic hexapeptide that is produced by some fungi but not by S. typhimurium.) The enb mutants use ferrichrome as an effective growth factor.