11q13 Amplification Research Articles

Sec62 Bridges the Gap from 3q Amplification to Molecular Cell Biology in Non–Small Cell Lung Cancer

The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non-small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro. Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62-overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non-small cell lung cancer and harbors various functions in cancer cell biology.

Gene Amplifications Are Rare Events in AML and MDS and Are Associated with Complex Karyotype, TP53 Deletions and Very Poor Survival

Abstract 2524 Background:Gene amplifications are usually defined as the presence of more than 6 copies of a gene per cell. These supernumerary copies are located either extrachromosomally in double minutes (small acentric chromosome structures) or intrachromosomally in homogeneously staining regions. Such gene amplifications are rare but recurrent phenomenons in AML and MDS. So far, only small case studies have been reported. Aims: 1) to determine the frequency of gene amplifications in a large AML and MDS cohort, 2) to characterize the amplified regions and accompanying abnormalities, 3) to analyze the impact of specific amplifications on outcome. Patients and Methods: Out of 4,248 AML and 3,689 MDS studied by chromosome banding analysis (CBA) we identified 105 AML patients (2.5%) with gene amplifications (80/3,478 (2.3%) de novo AML, 7/478 (1.5%) s-AML, 18/292 (6.2%) t-AML) and 46 (1.2%) MDS. All cases with gene amplification were studied by 24-color FISH in addition to CBA in order to characterize the amplified regions and the accompanying abnormalities in detail. Further, interphase (IP)-FISH was performed with probes for TP73, HOXD cluster, EVI1, CMYC, JAK2, NUP214, MLL, ZNF4, GLTSCR1, ERG, RUNX1, BCR and CLRF, if 24-color FISH suggested amplification of these genes. In a subcohort of 12 patients genomic arrays (Human CGH Whole-Genome Array, NimbleGen, Madison, WI; Genome-Wide Human SNP Array 6.0, Affymetrix, Santa Clara, CA) were performed to characterize the amplified region in more detail. Results: In 28/151 pts (18.5%) the amplification was located in double minutes and in the remaining 123 cases intrachromosomally (81.5%). The following regions were found to be amplified: 1p (n=1, containing TP73), 2q (n=1, containing HOXD cluster), 3q (n=1, containing EVI1), 7p (n=1), 8q (n=29, containing CMYC in 28/29 pts), 9p (n=2, containing JAK2), 9q (n=1, containing NUP214), 11q (n=81, containing MLL in 80/81 cases), 13q (n=2), chromosome 19 (n=10, containing ZNF4 in 5 cases and GLTSCR1 in 2 cases), 21q (n=19, containing ERG in 16 and RUNX1 in 6 cases), 22q (n=2, containing BCR) and Xp (n=1, containing CLRF). In median, 8 accompanying chromosomal aberrations per cases were observed (range 0–21). 124/151 (82.1%) cases had a complex aberrant karyotype, defined as 4 or more abnormalities. However, in 2 cases the double minutes were the sole abnormalities. Gene amplifications were not observed in patients with disease defining aberration like t(8;21), inv(16), t(15;17) or those carrying NPM1 or CEPBA mutations (mutation status available in 89 and 37 patients, respectively). However, 2 cases with t(6;11)(q27;q23)/MLL-AF6 harbored an amplification of CMYC. In 88 cases the copy number status of TP53 was determined by IP-FISH. A TP53 deletion was detected in 49 (55.7%) pts. Interestingly, 14/16 (87.5%) cases with double minutes compared to 35/72 (48.6%) patients with intrachromosomal gene amplifications showed a TP53 deletion (p=0.004). Only 3 chromosomal regions were amplified in double minutes: 8q24/CMYC (n=14), 11q23/MLL (n=12) and 13q (n=2). In 6 cases with 8q amplification, 2 cases with 11q amplification and 4 cases with 21q amplification genomic arrays were performed. While the amplified region was quite homogeneous in cases with 8q amplification and contained in all cases the CMYC gene, amplified regions on 11q and 21q were heterogeneous and amplified regions were interspersed with regions of deletions. Interestingly, MLL and CBL were amplified in all analyzed cases with 11q23 amplification. In all analyzed cases with 21q22 amplification ERG was located within the amplified region while RUNX1 was amplified in 3/4 cases and deleted in the remaining case. In AML, overall survival was short in cases with gene amplification (median OS 11.3 months) and was particularly short in cases accompanied by complex karyotype (6.3 mo vs 18.6 mo in cases with non-complex karyotype, p=0.049). Conclusions: 1) MLL is the most frequently amplified gene in AML and MDS. 2) Gene amplifications occur predominantly in complex aberrant karyotypes. 3) Prognosis is poor in this subset of cases, and even more dismal if these amplifications are accompanied by complex karyotype. 4) The association of gene amplifications, complex karyotypes and TP53 deletions suggests that the unfavorable prognosis is due to chromosome instability facilitating the occurrence of additional genetic aberrations triggering resistance to chemotherapy. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Jumping Translocation of Homogeneously Staining Region Hsr(11q) in An Erythroid Leukemia: Identification of Amplified Regions by Fluorescence Hybridization, M-FISH and M-Banding.

Abstract 4890Gene amplification is a mechanism whereby a tumor cell can increase the copy number of specific gene sequences and gain a proliferative advantage. Although amplifications are common in solid tumors, they are relatively rare in hematological neoplasms. We report here on a patient with an acute erythroid leukemia and a jumping translocation of a hsr(11q) in a complex karyotype, without MLL amplification.The patient was 40 years old when he was admitted for asthenia, peripheral blood showing pancytopenia (Hb=111G/L; neutrophils= 0.23 G/L; platelets 92 G/L), without circulating blast cells. Bone marrow was hypercellular with the presence of more than 50% of erythroid precursors and more than 20% of myeloid blast cells in non-erythroid cell population, leading to the diagnosis of erythroblastic acute leukemia. A marked dysgranulopoiesis was also detected.Conventional cytogenetics showed a complex karyotype with a del(5q), a unbalanced t(7;17) leading to partial 7q deletion and homogeneously staining regions (hsr). Fluorescence in situ hybridization confirmed the del(5q), and showed that TP53 was not deleted in the t(7;17). Multi-FISH (M-FISH) pointed out that the hsr consisted of chromosome 11 and was implicated in 3 different translocations with 3 different partner chromosomes in 3 different clones. To further characterize the amplicon and determine which bands were implicated, we used a chromosome 11 m-band probe. It revealed that the bands implicated are the same on the der(3), der(12) and der(20) and are localized between 11q23.3 and 11q25. A series of BAC probes showed that different genes, present in these regions, were amplified: ETS1, FLI1, KCNJ5, NFRKB, SNX19, HNT, OPCM, but not MLL.Amplifications of chromosome 11q usually includes MLL, but more telomeric amplicons have also been reported in AML and myelodysplatic syndromes (MDS). A very close 11q amplification was identified in 3 AML/MDS cases (Crossen et al, 1999; Tyybäkinoja et al, 2006). Also, the ETS1 oncogene was found to be rearranged and 30-fold amplified in a case of acute myelomonocytic leukaemia, in which an hsr occurred on 11q23 (Rovigatti et al, 1986).The role of chromosomal amplifications in leukemia is unclear, but it has been suggested that they are associated with rapid disease progression and short survival. Jumping translocations are an unusual phenomenon and have been rarely reported in hematological malignancies. Whether jumping translocations play a role in tumor genesis or confer a selective growth advantage on tumor cells is unknown. The combination of hsr and jumping translocations as described here are very rare in hematological malignancies. The cases of the literature (Yoshida et al, 1999) and the one presented here suggest that the 11q24-q25 region may harbor new candidate oncogenes, together with unusual chromosomal mechanisms. Disclosures:No relevant conflicts of interest to declare.

Characterisation of amplification patterns and target genes at chromosome 11q13 in CCND1-amplified sporadic and familial breast tumours

Amplification of chromosomal region 11q13, containing the cell cycle regulatory gene CCND1, is frequently found in breast cancer and other malignancies. It is associated with the favourable oestrogen receptor (ER)-positive breast tumour phenotype, but also with poor prognosis and treatment failure. 11q13 spans almost 14 Mb and contains more than 200 genes and is affected by various patterns of copy number gains, suggesting complex mechanisms and selective pressure during tumour progression. In this study, we used 32 k tiling BAC array CGH to analyse 94 CCND1-amplified breast tumours from sporadic, hereditary, and familial breast cancers to fine map chromosome 11q13. A set containing 281 CCND1-non-amplified breast tumours was used for comparisons. We used gene expression data to further validate the functional effect of gene amplification. We identified six core regions covering 11q13.1-q14.1 that were amplified in different combinations. The major core contained CCND1, whereas two cores were found proximal of CCND1 and three distal. The majority of the CCND1-amplified tumours were ER-positive and classified as luminal B. Furthermore, we found that CCND1 amplification is associated with a more aggressive phenotype within histological grade 2 tumours and luminal A subtype tumours. Amplification was equally prevalent in familial and sporadic tumours, but strikingly rare in BRCA1- and BRCA2-mutated tumours. We conclude that 11q13 includes many potential target genes in addition to CCND1.

DeltaN TP63 reactivation, epithelial phenotype maintenance, and survival in lung squamous cell carcinoma

Genes, active during normal development, are frequently reactivated during neoplastic transformation and may be related to progression. One of them, the transcription factor TP63, is crucial for pulmonary epithelial development and a possible target of the recurrent 3q amplifications in lung squamous cell carcinoma (SCC). Here, we explored whether TP63 reactivation could be associated to cancer progression in lung SCC through an epithelial to mesenchymal transition. We studied TP63 amplification and TP63 expression at RNA and protein levels and we analyzed the ΔNTP63/TATP63 ratio that quantifies the proportion of the isoform lacking the transactivation domain/the isoform containing the transactivation domain. We correlated TP63 status to survival and to the expression of epithelial (E-cadherin and plakoglobin) and mesenchymal (N-cadherin, vimentin, TWIST1, and SNAIL) markers. We found that high ΔN/TA TP63 ratio was related to high E-cadherin and plakoglobin mRNA levels (P < 0.05) and that E-cadherin mRNA level was the only marker related to survival. Kaplan-Meier survival curves stratified according to the expression level of E-cadherin showed, as already reported in breast cancer, that patients with low (first quartile) or high (last quartile) E-cadherin expression had a worse survival with respect to patients with intermediate E-cadherin expression. Altogether, our results indicate that a reactivation of ΔNTP63 is linked to the maintenance of epithelial markers and suggest that E-cadherin has a dual role in lung SCC.

NUAK2: an emerging acral melanoma oncogene.

Recent technological advances in cancer genomics make it possible to dissect complicated genomic aberrations of melanomas. In particular, several specific genomic aberrations including 11q13 amplification and KIT aberrations have been identified in acral melanomas. We recently identified NUAK2 at 1q32 as a promising oncogene in acral melanomas and reported its significant roles in tumorigenesis in melanoma cells using both in vitro and in vivo analyses. NUAK2 as a member of the AMPK family has several intriguing aspects both as an oncogene and as a tumor suppressor gene. Here we review genomic aberrations of melanomas focusing on acral melanomas to emphasize the possible roles of NUAK2 in tumorigenesis in general and suggest that NUAK2 has pivotal roles in acral melanomagenesis.

High‐resolution genomic analysis of the 11q13 amplicon in breast cancers identifies synergy with 8p12 amplification, involving the mTOR targets S6K2 and 4EBP1

The chromosomal region 11q13 is amplified in 15-20% of breast cancers; an event not only associated with estrogen receptor (ER) expression but also implicated in resistance to endocrine therapy. Coamplifications of the 11q13 and 8p12 regions are common, suggesting synergy between the amplicons. The aim was to identify candidate oncogenes in the 11q13 region based on recurrent amplification patterns and correlations to mRNA expression levels. Furthermore, the 11q13/8p12 coamplification and its prognostic value, was evaluated at the DNA and the mRNA levels. Affymetrix 250K NspI arrays were used for whole-genome screening of DNA copy number changes in 29 breast tumors. To identify amplicon cores at 11q13 and 8p12, genomic identification of significant targets in cancer (GISTIC) was applied. The mRNA expression levels of candidate oncogenes in the amplicons [RAD9A, RPS6KB2 (S6K2), CCND1, FGF19, FGF4, FGF3, PAK1, GAB2 (11q13); EIF4EBP1 (4EBP1), PPAPDC1B, and FGFR1 (8p12)] were evaluated using real-time PCR. Resulting data revealed three main amplification cores at 11q13. ER expression was associated with the central 11q13 amplification core, encompassing CCND1, whereas 8p12 amplification/gene expression correlated to S6K2 in a proximal 11q13 core. Amplification of 8p12 and high expression of 4EBP1 or FGFR1 was associated with a poor outcome in the group. In conclusion, single nucleotide polymorphism arrays have enabled mapping of the 11q13 amplicon in breast tumors with high resolution. A proximal 11q13 core including S6K2 was identified as involved in the coamplification/coexpression with 8p12, suggesting synergy between the mTOR targets S6K2 and 4EBP1 in breast cancer development and progression.

Comprehensive exon array data processing method for quantitative analysis of alternative spliced variants

Alternative splicing of pre-mRNA generates protein diversity. Dysfunction of splicing machinery and expression of specific transcripts has been linked to cancer progression and drug response. Exon microarray technology enables genome-wide quantification of expression levels of the majority of exons and facilitates the discovery of alternative splicing events. Analysis of exon array data is more challenging than the analysis of gene expression data and there is a need for reliable quantification of exons and alternatively spliced variants. We introduce a novel, computationally efficient methodology, Multiple Exon Array Preprocessing (MEAP), for exon array data pre-processing, analysis and visualization. We compared MEAP with existing pre-processing methods, and validation of six exons and two alternatively spliced variants with qPCR corroborated MEAP expression estimates. Analysis of exon array data from head and neck squamous cell carcinoma (HNSCC) cell lines revealed several transcripts associated with 11q13 amplification, which is related with decreased survival and metastasis in HNSCC patients. Our results demonstrate that MEAP produces reliable expression values at exon, alternatively spliced variant and gene levels, which allows generating novel experimentally testable predictions.

Clinical and molecular approaches to well differentiated and dedifferentiated liposarcoma

Liposarcoma, a rare disease, is classified into five histologic subtypes. These include well differentiated liposarcoma (WDLS) and dedifferentiated liposarcoma (DDLS), both characterized by chromosome 12q13-15 amplification. This review will focus on the clinical management of WDLS and DDLS and examine recent molecular studies that have the potential to affect clinical management. Outcome of patients with WDLS and DDLS depends on completeness of surgical resection as well as tumor location and histologic subtype. Risk of recurrence is high for patients with dedifferentiated histology or retroperitoneal location. We now understand that surgical outcomes are poor for patients with rapidly growing or incompletely resectable tumors, so these patients should be managed nonoperatively. Radiation and chemotherapy have low response rates in WDLS and DDLS, but novel agents targeted at chromosome 12 gene products MDM2 and CDK4 have shown promise in preclinical studies and are being tested in clinical trials. Cell line, tissue microarray, and genomic analyses have identified additional targets including ZIC1, TOP2A, AURKA, and IGF-1R, which could form the basis of future therapies. Although complete surgical resection is currently the most effective treatment for WDLS and DDLS, the majority of patients with retroperitoneal liposarcoma will eventually have recurrence and die of disease. It is hoped that a multimodality approach, which incorporates targeted therapies and complete surgical resection, will significantly improve patient outcomes.

Combination effects of distinct cores in 11q13 amplification region on cervical lymph node metastasis of oral squamous cell carcinoma

Lymph node metastasis (LNM) in oral squamous cell carcinoma (OSCC) is known to associate with a significant decrease of 5-year survival. Genetic factors related to the difference of the LNM status in the OSCC have been not fully elucidated. Array-based comparative genomic hybridization (CGH) with individual gene-level resolution and real-time quantitative polymerase chain reaction (QPCR) were conducted using primary tumor materials resected from 54 OSCC patients with (n=22) or without (n=32) cervical LNM. Frequent gain was observed at the 11q13 region exclusively in patients with cervical LNM, which was confirmed by real-time QPCR experiments using 11 genes (TPCN2, MYEOV, CCND1, ORAOV1, FGF4, TMEM16A, FADD, PPFIA1, CTTN, SHANK2 and DHCR7) in this region. It was revealed that two distinct amplification cores existed, which were separated by a breakpoint between MYEOV and CCND1 in the 11q13 region. The combination of copy number amplification at CTTN (core 2) and/or TPCN2/MYEOV (core 1), selected from each core, was most significantly associated with cervical LNM (P=0.0035). Two amplification cores at the 11q13 region may have biological impacts on OSCC cells to spread from the primary site to local lymph nodes. Further study of a larger patient series should be conducted to validate these results.

Molecular alterations associated with liver metastases development in colorectal cancer patients

Background:Understanding the molecular biology of colorectal cancer (CRC) provides opportunities for effective personalised patient management. We evaluated whether chromosomal aberrations, mutations in the PI(3)K signalling pathway and the CpG-island methylator phenotype (CIMP) in primary colorectal tumours can predict liver metastases.Methods:Formalin-fixed paraffin-embedded material from primary colorectal tumours of three different groups were investigated: patients with CRC without metastases (M0, n=39), patients who were treated with hyperthermal intraperitoneal chemotherapy for CRC metastases confined to the peritoneum (PM, n=46) and those who had isolated hepatic perfusion for CRC metastases confined to the liver (LM, n=48).Results:All samples were analysed for DNA copy number changes, PIK3CA, KRAS, BRAF mutations, CIMP and microsatellite instability. The primary CRCs of the LM group had significantly higher frequency of amplified chromosome 20q (P=0.003), significantly fewer mutations in the PI(3)K signalling pathway (P=0.003) and fewer CIMP high tumours (P=0.05). There was a strong inverse correlation between 20q and the PI(3)K pathway mutations.Conclusion:The development of CRC liver metastases is associated with amplification of chromosome 20q and not driven by mutations in the PI(3)K signalling pathway.

Overexpression of eIF5A‐2 is an adverse prognostic marker of survival in stage I non–small cell lung cancer patients

We have previously isolated an oncogene EIF5A2 (eukaryotic initiation factor 5A2) from a frequently amplified region at 3q of a primary ovarian cancer cell line, and demonstrated its impact on prognosis in human ovarian cancer. Amplification of chromosome 3q has also been detected frequently in non-small cell lung cancer (NSCLC), however, abnormalities of EIF5A2 and its clinicopathologic significance in NSCLC haven't been studied. In our study, the methods of immunohistochemistry and fluorescence in situ hybridization were utilized to examine protein expression and amplification of EIF5A2 in 248 surgically resected NSCLCs (learning cohort) and another validation cohort of 120 stage I NSCLC patients. Overexpression and amplification of EIF5A2 was detected informatively in 48.7% and 13.7% of NSCLCs in learning cohort, 33.3% and 6.0% of NSCLCs in validation cohort. Overexpression of eIF5A-2 was found to correlate with gene amplification, increased cell proliferation and advanced T stage. In learning cohort, eIF5A-2 expression was evaluated as a strong prognostic factor on disease-specific survival, but in subgroup analyses, it only retained its stratified significance in stage I set (Hazards ratio = 2.799, p = 0.001). In validation cohort, the impact of eIF5A-2 expression on survival in stage I NSCLC patients was also observed (Hazard ratio = 2.097, p = 0.014). Our findings suggested that overexpression of eIF5A-2 correlates with local invasion of NSCLC, and might serve as an adverse prognostic marker of survival for stage I NSCLC patients.

Genomic evidence of pre‐invasive clonal expansion, dispersal and progression in bronchial dysplasia

The term ‘field cancerization’ is used to describe an epithelial surface that has a propensity to develop cancerous lesions, and in the case of the aerodigestive tract this is often as a result of chronic exposure to carcinogens in cigarette smoke 1, 2. The clinical endpoint is the development of multiple tumours, either simultaneously or sequentially in the same epithelial surface. The mechanisms underlying this process remain unclear; one possible explanation is that the epithelium is colonized by a clonal population of cells that are at increased risk of progression to cancer. We now address this possibility in a short case series, using individual genomic events as molecular biomarkers of clonality. In squamous lung cancer the most common genomic aberration is 3q amplification. We use a digital PCR technique to assess the clonal relationships between multiple biopsies in a longitudinal bronchoscopic study, using amplicon boundaries as markers of clonality. We demonstrate that clonality can readily be defined by these analyses and confirm that field cancerization occurs at a pre-invasive stage and that pre-invasive lesions and subsequent cancers are clonally related. We show that while the amplicon boundaries can be shared between different biopsies, the degree of 3q amplification and the internal structure of the 3q amplicon varies from lesion to lesion. Finally, in this small cohort, the degree of 3q amplification corresponds to clinical progression. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 4963: An integrated approach for functional analysis of 11q13 amplification target genes in HNSCC

Abstract Gene amplification is one of the most common mechanisms for oncogenic activation in human cancer. Some genomic regions are more frequently amplified than others. For example, amplification of 11q13 occurs at high frequencies in several human cancers, such as lung, bladder, breast and ovarian carcinomas, as well as head and neck squamous cell carcinoma (HNSCC). Approximately 40% of HNSCC are affected by this specific amplification, which is associated with the presence of lymph node metastasis and poor prognosis. Distinct core regions of amplified areas within the 11q13 locus have been identified, and these can be amplified independently or concurrently in various combinations. A number of oncogenes or potential cancer-related genes have been mapped to the 11q13 chromosomal region, however it is still not known whether these genes, that undergo co-amplification and over-expression, cooperate in promoting tumorigenesis. We have used a complementary approach to comprehensively characterize the functional basis for selection and maintenance of 11q13 amplification in HNSCC. To facilitate this approach, each of the 15 HNSCC cell lines used in this study has undergone molecular characterization for genomic and transcriptomic alterations. We performed a loss-of-function screen on 11q13 positive HNSCC cell lines to identify the genes essential for growth and survival. Next we applied gain-of-function analysis using ORFs for CTTN, FADD, PPFIA1, ORAOV1, CCND1 on 11q13 negative HNSCC cell lines. A single or multiple genes mapping to the amplicon were stably co-overexpressed in different combinations and related phenotypes were analysed in several in vitro and in vivo assays for growth, invasion and sensitivity/resistance to drugs. In addition, changes associated with overexpression of those genes were analysed by exon arrays in order to identify deregulated signaling pathways associated with 11q13 amplification. Our preliminary data suggests that this integrated approach is an efficient method at identifying genes that co-operate due to their location in the same amplicon. Such information is particularly valuable for elucidating oncogenic drivers and addressing the relevance of 11q13 amplification to drug sensitivity/resistance in 11q13 positive HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4963. doi:10.1158/1538-7445.AM2011-4963

Abstract 4752: Cortactin and coronin 1B cooperate to promote tumor cell invasion in head and neck squamous cell carcinoma

Abstract Amplification of chromosome 11q13 in head and neck squamous cell carcinoma (HNSCC) is a common occurrence associated with poor patient prognosis. Within the core amplified region is the gene that encodes cortactin (CTTN), a filamentous (F)- actin binding protein and Src kinase substrate that stabilizes Arp2/3-F-actin branchpoints in dynamic cortical F-actin structures. CTTN amplification results in increased lymph node metastases and decreased survival rates in HNSCC patients. CTTN amplification in HNSCC cell lines enhances tumor cell motility and invasion. Chromosomal regions of varying length flanking the core 11q13 amplicon are often amplified in HNSCC, and contain several genes with the potential to cooperate with CTTN in regulating actin dynamics. One of these genes within the 11q13 region outside of the core amplicon is CORO1B (coronin 1B), an actin-binding protein that antagonizes cortactin function by promoting the breakdown of cortactin-stabilized Arp2/3-F-actin branches, resulting in impaired cell motility. Here we show that coronin 1B is a potential mediator of HNSCC invasion. CORO1B amplification and overexpression occurs in a subset of HNSCC patients with CTTN amplification. Coronin 1B localizes with cortactin within HNSCC invadopodia, ventral membranous protrusions responsible for degrading extracellular matrix to facilitate loco-regional invasion and extracapsular spread. RNAi-mediated coronin 1B knockdown decreases the number of invadopodia per cell, enhances cell spreading and reduces extracellular matrix degradation. These results collectively point to a potential role for coronin 1B in regulating HNSCC tumor cell invasion through modulating cortactin activity and invadopodia function. Our data also support a potential diagnostic function for CORO1B amplification in denoting a previously unidentified HNSCC subset that may display increased invasiveness in cases containing 11q13 amplification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4752. doi:10.1158/1538-7445.AM2011-4752

Abstract 4906: A systems approach into studying the changes in genetic regulation during prostate cancer progression

Abstract Prostate cancer (PC) is a complex disease that is currently the most common malignancy in males, and the second most frequent cause of cancer-related mortality in developed countries. Despite its high prevalence, the molecular mechanisms that induce PC progression are not well understood. The most common alterations affecting genetic regulation in PC are TMPRSS2:ERG gene fusions, 8q24 amplifications (including MYC-oncogene), and androgen receptor (AR) mutations and amplifications in castration-resistant (late stage) tumors. In normal prostatic tissue, local androgen metabolism maintains a balance between the proliferation and apoptotic cell death of prostatic epithelial cells. However in PC, androgen metabolism drives malignant transformation. The exact mechanisms which cause the imbalance in proliferation and apoptosis are not known, but likely include dysregulation of AR/MYC/ERG-regulated transcription factors (TFs) and/or microRNAs. Using a systems biology approach, we jointly studied the accumulation of expression and copy number differences of TFs and microRNAs during PC progression. We first established a generic regulatory network consisting of transcription factors and microRNAs using three sets of sequence-based binding site predictions. First, TF binding sites were predicted by scanning the sequences upstream from gene transcription start sites for binding motifs from Transfac database, second, TF binding to microRNA promoters was predicted with ProbTF, and third, microRNA binding sites were obtained from Targetscan predictions. The inferred generic network was then adapted for studying PC by pruning it based on a set of publicly available gene expression, microRNA expression data. That is, we only considered those hypothetical regulatory connections that were supported by the data. By dividing these data into normal prostate tissue, primary PC's of different grade, and metastatic tumors, we were able to study the changes in regulation during different stages of the disease. We uncovered putative regulatory motifs consisting of differentially expressed TFs and microRNAs. Another level of regulatory information was added by integrating copy number information to the regulatory motifs. We concentrated on the motifs that could be associated to AR-, ERG-, or MYC-signaling, due to their relevance in prostate cancer, or to molecules that are known to be regulated by these TFs. We found previously unknown microRNAs and TFs that may directly bound to the promoters of the TFs and therefore may have a critical effect to the malignancy of prostate cancers. The experimental validations of the most promising new candidate TF/microRNA regulatory connections are currently underway. We demonstrated here an application of a systems biology based method to prostate cancer which was used to gain insights into the changes in genetic regulation during progression of the disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4906. doi:10.1158/1538-7445.AM2011-4906

Knockdown of ovarian cancer amplification target ADRM1 leads to downregulation of GIPC1 and upregulation of RECK

Approximately 25,000 ovarian cancers are diagnosed in the United States annually, and 75% of cases are in the advanced stage when they are largely incurable. There is a critical need for improved early detection tools and development of novel treatments. Recently, we showed that among 20q13-amplified genes in ovarian cancer, ADRM1 overexpression was the most highly correlated with amplification and was significantly upregulated with respect to stage, recurrence, and metastasis. In addition, overexpression of ADRM1 correlated significantly with shorter time to recurrence and overall survival. Herein, array-CGH and microarray expression of ovarian cancer cell lines provides evidence consistent with the primary tumor data that ADRM1 is a 20q13 amplification target. Knockdown of ADRM1 in amplified ovarian cell-line OAW42 results in downregulation of growth factor GIPC1 and upregulation of tumor-suppressor RECK RNA and protein. In our dataset of 141 ovarian primary tumors, ADRM1 overexpression significantly correlates with GIPC1 overexpression. In addition, there is a significant anticorrelation between ADRM1 overexpression and RECK expression. Further research is necessary to determine whether targeting knockdown of ADRM1 in 20q13-amplified ovarian cancers results in growth inhibition and tumor suppression via downstream targets GIPC1 and RECK.

Integrated mutation, copy number and expression profiling in resectable non-small cell lung cancer.

BackgroundThe aim of this study was to identify critical genes involved in non-small cell lung cancer (NSCLC) pathogenesis that may lead to a more complete understanding of this disease and identify novel molecular targets for use in the development of more effective therapies.MethodsBoth transcriptional and genomic profiling were performed on 69 resected NSCLC specimens and results correlated with mutational analyses and clinical data to identify genetic alterations associated with groups of interest.ResultsCombined analyses identified specific patterns of genetic alteration associated with adenocarcinoma vs. squamous differentiation; KRAS mutation; TP53 mutation, metastatic potential and disease recurrence and survival. Amplification of 3q was associated with mutations in TP53 in adenocarcinoma. A prognostic signature for disease recurrence, reflecting KRAS pathway activation, was validated in an independent test set.ConclusionsThese results may provide the first steps in identifying new predictive biomarkers and targets for novel therapies, thus improving outcomes for patients with this deadly disease.

Amplification of the 20q Chromosomal Arm Occurs Early in Tumorigenic Transformation and May Initiate Cancer

Duplication of chromosomal arm 20q occurs in prostate, cervical, colon, gastric, bladder, melanoma, pancreas and breast cancer, suggesting that 20q amplification may play a causal role in tumorigenesis. According to an alternative view, chromosomal imbalance is mainly a common side effect of cancer progression. To test whether a specific genomic aberration might serve as a cancer initiating event, we established an in vitro system that models the evolutionary process of early stages of prostate tumor formation; normal prostate cells were immortalized by the over-expression of human telomerase catalytic subunit hTERT, and cultured for 650 days till several transformation hallmarks were observed. Gene expression patterns were measured and chromosomal aberrations were monitored by spectral karyotype analysis at different times. Several chromosomal aberrations, in particular duplication of chromosomal arm 20q, occurred early in the process and were fixed in the cell populations, while other aberrations became extinct shortly after their appearance. A wide range of bioinformatic tools, applied to our data and to data from several cancer databases, revealed that spontaneous 20q amplification can promote cancer initiation. Our computational model suggests that 20q amplification induced deregulation of several specific cancer-related pathways including the MAPK pathway, the p53 pathway and Polycomb group factors. In addition, activation of Myc, AML, B-Catenin and the ETS family transcription factors was identified as an important step in cancer development driven by 20q amplification. Finally we identified 13 "cancer initiating genes", located on 20q13, which were significantly over-expressed in many tumors, with expression levels correlated with tumor grade and outcome suggesting that these genes induce the malignant process upon 20q amplification.

Abstract PD07-08: Integrated Genomic Analysis before and after Brief Exposure to Trastuzumab (T): The11q13 and 17q12 Amplicons Are Associated with Response to T+Chemotherapy in Early Stage HER2 Positive Breast Cancer

Abstract Background: The receptor tyrosine kinase HER2 is overexpressed in 18-20% of breast cancers and is the target of the highly effective therapy, trastuzumab. Coamplicons are frequent in HER2 amplified tumors, yet their role in response to T is not defined. To identify amplicons which might predict response to T in patients we performed gene expression and copy number analysis in tumor tissue from a preoperative trial with brief exposure to T, followed by T+C. Methods: Fresh tumor core biopsies were taken before at a 2 week timepoint after a single dose of T (8mg/m2) from 80 HER2-overpressing, early breast cancer patients enrolled on a clinical trial of T&amp;gt;T+C. Nucleic acids were extracted using Qiagen AllPrep and were analyzed with Illumina Ref8 and Illumina QUAD 610 SNP arrays. Eight commonly amplified regions (11q13, 14q32, 17q12, 20q13, 21q22, 8p22, 8q24) were identified by FISH probe coordinates and copy number calculated using the GPHMM algorithm. Amplification was defined as 4 or more copies of the amplicon. Gene expression was analyzed in Bioconductor using LIMMA analysis. Genomic biomarkers were compared pathologic complete response (pCR) vs objective response (CR+PR) and non-response (SD+PD) by RECIST criteria at the surgical timepoint. Results: There were 50 tumor pairs with adequate RNA for gene expression and 40 baseline tumors for copy number analysis with 28 paired samples. In the 40 baseline biopsies, the 11q13 amplicon was present in 14/40 (35%) of tumors but never seen in tumors with a pCR (t-test; p=0.004975). Of note, in the tumors with amplification of 11q13 there was no evidence of change in copy number after exposure to T, however numbers were small (7 pairs). Gene expression of the 11q13 region showed no significant change in common amplicon genes after one dose of T. The 17q12 amplicon, which harbors the HER2 locus, was amplified in 35/40 (87.5%) of cases - copy number was not associated with response, however, HER2 and GRB7 gene expression levels significantly declined in pCR tumors compared with &amp;lt;pCR (p=0.003). Conclusions: Integrated genomic analysis after brief exposure to T may provide useful predictors for response to T+C in early stage, HER2 positive breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD07-08.