From chromosomal alterations to target genes for therapy: integrating cytogenetic and functional genomic views of the breast cancer genome

A vast number of recurrent chromosomal alterations have been implicated in cancer development and progression. However, most of the genes involved in recurrent chromosomal alterations in solid tumors remain unknown, despite the recent substantial progress in genomic research and availability of high-throughput technologies. For example, it is now possible to quickly identify large numbers of differentially expressed genes in cancer specimens using cDNA microarrays. Integration of this "functional genomic view" of the cancer genome with the "cytogenetic view" could lead to the identification of genes playing a critical role in cancer development and progression. In this review, we illustrate how the combination of three different microarray technologies, cDNA, CGH, and tissue microarrays, makes it possible to directly identify genes involved in chromosomal rearrangements in cell line model systems and then rapidly explore their significance as potential diagnostic and therapeutic targets in human primary breast cancer progression.     

Identification of molecular targets for immunotherapy of patients with head and neck squamous cell carcinoma

To identify molecular targets for immunotherapy of head and neck squamous cell carcinoma (HNSCC) patients, we analyzed gene expression profile in matched tumor (HN) and normal fibroblast (FB) cell lines established from a HNSCC patient using microarray technique followed by real-time RT-PCR. Screening against a series of established normal and malignant cell lines followed by screening against a panel of normal human tissues led to the identification of 7 genes (AREG, CDH3, KLK10, NmU, SLPI, ANAX3 and MAL2), which were over-expressed at least 10-fold in tumors over any of the normal tissues. We determined the expression of mRNA encoding these genes against a panel of 15 HNSCC primary tumor samples. Relative expression of these genes was at least 20-fold. Expression of AREG, CDH3, KLK10, NmU and SLPI at the protein level was determined by immunohistochemistry in seven supraglottic laryngeal cancer specimens. All five proteins were expressed in these tumor samples with high intensity. We conclude that these molecules are potential targets for immunotherapy of HNSCC patients.     

Large-scale molecular comparison of human schwann cells to malignant peripheral nerve sheath tumor cell lines and tissues

Malignant peripheral nerve sheath tumors (MPNST) are highly invasive soft tissue sarcomas that arise within the peripheral nerve and frequently metastasize. To identify molecular events contributing to malignant transformation in peripheral nerve, we compared eight cell lines derived from MPNSTs and seven normal human Schwann cell samples. We found that MPNST lines are heterogeneous in their in vitro growth rates and exhibit diverse alterations in expression of pRb, p53, p14(Arf), and p16(INK4a) proteins. All MPNST cell lines express the epidermal growth factor receptor and lack S100beta protein. Global gene expression profiling using Affymetrix oligonucleotide microarrays identified a 159-gene molecular signature distinguishing MPNST cell lines from normal Schwann cells, which was validated in Affymetrix microarray data generated from 45 primary MPNSTs. Expression of Schwann cell differentiation markers (SOX10, CNP, PMP22, and NGFR) was down-regulated in MPNSTs whereas neural crest stem cell markers, SOX9 and TWIST1, were overexpressed in MPNSTs. Previous studies have implicated TWIST1 in apoptosis inhibition, resistance to chemotherapy, and metastasis. Reducing TWIST1 expression in MPNST cells using small interfering RNA did not affect apoptosis or chemoresistance but inhibited cell chemotaxis. Our results highlight the use of gene expression profiling in identifying genes and molecular pathways that are potential biomarkers and/or therapeutic targets for treatment of MPNST and support the use of the MPNST cell lines as a primary analytic tool.     

Combining serial analysis of gene expression and array technologies to identify genes differentially expressed in breast cancer.

Several methods have been used recently to determine gene expression profiles of cell populations. Here we demonstrate the strength of combining two approaches, serial analysis of gene expression (SAGE) and DNA arrays, to help elucidate pathways in breast cancer progression by finding genes consistently expressed at different levels in primary breast cancers, metastatic breast cancers, and normal mammary epithelial cells. SAGE profiles of 21PT and 21MT, two well-characterized breast tumor cell lines, were compared with SAGE profiles of normal breast epithelial cells to identify differentially expressed genes. A subset of these candidates was then placed on an array and screened with clinical breast tumor samples to find genes and expressed sequence tags that are consistently expressed at different levels in diseased and normal tissues. In addition to finding the predicted overexpression of known breast cancer markers HER-2/neu and MUC-1, the powerful coupling of SAGE and DNA arrays resulted in the identification of genes and potential pathways not implicated previously in breast cancer. Moreover, these techniques also generated information about the differences and similarities of expression profiles in primary and metastatic breast tumors. Thus, combining SAGE and custom array technology allowed for the rapid identification and validation of the clinical relevance of many genes potentially involved in breast cancer progression. These differentially expressed genes may be useful as tumor markers and prognostic indicators and may be suitable targets for various forms of therapeutic intervention.     

Identification of genes involved in squamous cell carcinoma of the lung using synchronized data from DNA copy number and transcript expression profiling analysis

Lung cancer is the leading cause of cancer deaths in the world and squamous cell carcinoma (SqCC) is the second most common in this group. Genomic DNA copy number alterations are fundamental genetic events in the development and progression of SqCC as well as other epithelial-derived cancers. The ability to identify tumor suppressor genes (TSGs) and oncogenes that are affected during tumor initiation and progression could facilitate the identification of novel molecular targets for therapeutic intervention and provide diagnostic biomarkers. Despite the association of many genetic alterations in lung cancer the molecular mechanisms of tumor progression remain ambiguous since often too many candidates are revealed using conventional genetic microarray analysis. To overcome this limitation, we have identified genes in SqCC which show concordant gene expression changes defined using microarray analysis with DNA copy number alterations defined by BAC-array comparative genomic hybridization (aCGH) in the same tumors. An in-house overlay algorithm was used to synchronize the data resulting from the two analyses. Although the expression levels of many genes were altered when compared to normal controls, those which correlated with copy number changes were far fewer, providing a manageable number for biological studies. We identified over 2000 genes which displayed both gene expression alterations and mapped to BACs which demonstrated a corresponding loss or gain. A further stringent statistical analysis identified minimal regions of overlap for losses or gains which displayed a coincident decrease or increase in the expression of genes mapping to those regions. Consistent gains involved 3q23-q29, 5p15.1-q11.1 and chromosomes 18 and 20, while consistent losses involved 3p26.3-p12.3, 9p24.3-q34.3, and chromosomes 17 and 19. The concordance finding between these two approaches suggests that DNA copy number alterations can directly influence gene expression patterns that impact on tumorigenesis in SqCC of the lung.     

Epigenomic profiling reveals novel and frequent targets of aberrant DNA methylation-mediated silencing in malignant glioma

Malignant glioma is the most common central nervous system tumor of adults and is associated with a significant degree of morbidity and mortality. Gliomas are highly invasive and respond poorly to conventional treatments. Gliomas, like other tumor types, arise from a complex and poorly understood sequence of genetic and epigenetic alterations. Epigenetic alterations leading to gene silencing, in the form of aberrant CpG island promoter hypermethylation and histone deacetylation, have not been thoroughly investigated in brain tumors, and elucidating such changes is likely to enhance our understanding of their etiology and provide new treatment options. We used a combined approach of pharmacologic inhibition of DNA methylation and histone deacetylation, coupled with expression microarrays, to identify novel targets of epigenetic silencing in glioma cell lines. From this analysis, we identified >160 genes up-regulated by 5-aza-2'-deoxycytidine and trichostatin A treatment. Further characterization of 10 of these genes, including the putative metastasis suppressor CST6, the apoptosis-inducer BIK, and TSPYL5, whose function is unknown, revealed that they are frequent targets of epigenetic silencing in glioma cell lines and primary tumors and suppress glioma cell growth in culture. Furthermore, we show that other members of the TSPYL gene family are epigenetically silenced in gliomas and dissect the contribution of individual DNA methyltransferases to the aberrant promoter hypermethylation events. These studies, therefore, lay the foundation for a comprehensive understanding of the full extent of epigenetic changes in gliomas and how they may be exploited for therapeutic purposes.     

Identification and gene expression profiling of tumor-initiating cells isolated from human osteosarcoma cell lines in an orthotopic mouse model

In the cancer stem cell model a cell hierarchy has been suggested as an explanation for intratumoral heterogeneity and tumor formation is thought to be driven by this tumor cell subpopulation. The identification of cancer stem cells in osteosarcoma (OS) and the biological processes dysregulated in this cell subpopulation, also known as tumor-initiating cells (TICs), may provide new therapeutic targets. The goal of this study, therefore, was to identify and characterize the gene expression profiles of TICs isolated from human OS cell lines. We analyzed the self-renewal capacity of OS cell lines and primary OS tumors based upon their ability to form sphere-like structures (sarcospheres) under serum-starving conditions. TICs were identify from OS cell lines using the long-term label retention dye PKH26. OS TICs and the bulk of tumor cells were isolated and used to assess their ability to initiate tumors in NOD/SCID mice. Gene expression profiles of OS TICs were obtained from fresh orthotopic tumor samples. We observed that increased sarcosphere efficiency correlated with an enhanced tumorigenic potential in OS. PKH26Hi cells were shown to constitute OS TICs based upon their capacity to form more sarcospheres, as well as to generate both primary bone tumors and lung metastases efficiently in NOD/SCID mice. Genomic profiling of OS TICs revealed that both bone development and cell migration processes were dysregulated in this tumor cell subpopulation. PKH26 labeling represents a valuable tool to identify OS TICs and gene expression analysis of this tumor cell compartment may identify potential therapeutic targets.     

Identification of SMURF1 as a possible target for 7q21.3-22.1 amplification detected in a pancreatic cancer cell line by in-house array-based comparative genomic hybridization

Pancreatic cancer (PC) cell lines provide a useful starting point for the discovery and functional analysis of genes driving the genesis and progression of this lethal cancer. To increase our understanding of the gene copy number changes in pancreatic carcinomas and to identify key amplification and deletion targets, we applied genome-wide array-based comparative genomic hybridization using in-house array (MCG Cancer Array-800) to 24 PC cell lines. Overall, the analyses revealed high genomic complexity, with several copy number changes detected in each line. Homozygous deletions (log₂ratio < –2) of eight genes (clones) were seen in 14 of the 24 cell lines, whereas high-level amplifications (log₂ratio > 2) of 10 genes (clones) were detected in seven lines. Among them, we focused on high-level amplification at 7q22.1, because target genes for this alteration remain unknown. Through precise mapping of the altered region by fluorescence in situ hybridization, determination of the expression status of genes located within those regions, and functional analysis using knockdown of the gene expression or the ectopic overexpression approach in PC cell lines, as well as immunohistochemical analyses of candidates in primary tumors of PC, we successfully identified SMURF1 as having the greatest potential as a 7q21.3-22.1 amplification target. SMURF1 may work as a growth-promoting gene in PC through overexpression and might be a good candidate as a therapeutic target. Our results suggest that array-based comparative genomic hybridization analysis combined with further genetic and functional examinations is a useful approach for identifying novel tumor-associated genes involved in the pathogenesis of this lethal disease. ( Cancer Sci 2008; 99: 986–994)     

Molecular typing of breast cancer: transcriptomics and DNA microarrays

Breast cancer is the most frequent and deadly cancer of women. Its great heterogeneity makes prognosis and response to current treatments highly variable and difficult to predict. Mammary oncogenesis remains poorly understood. These issues should benefit from recent development of techniques capable of large-scale molecular analyses. The use of cDNA array techniques allows for the simultaneous analysis of the mRNA expression levels of thousands of genes in mammary tumor cell lines and breast tumors. Expression profiles will help classify tumors and provide new prognostic tools and potential therapeutic targets. They will also boost our knowledge of the molecular events responsible for the development and progression of this cancer.     

DLC1 suppresses distant dissemination of human hepatocellular carcinoma cells in nude mice through reduction of RhoA GTPase activity, actin cytoskeletal disruption and down-regulation of genes involved in metastasis

The process of cell dissemination from the primary tumors to distant sites is the most harmful event during cancer progression, and the leading cause of cancer death. We have previously demonstrated that restoration of DLC1 tumor suppressor gene expression in the DLC1-negative Focus and 7703K human hepatocellular carcinoma (HCC) cell lines induced caspase-3 mediated apoptosis, reduced cell growth in vitro and tumorigenicity in vivo and diminished the ability to migrate through Matrigel, a property suggestive of metastatic potential in vivo. We now show that subcutaneous tumors developing after inoculation of Focus and 7703K cells into nude mice disseminate cells to liver and lung, and this process is markedly suppressed by restoration of DLC1 expression. Inhibition of tumor cell dissemination was associated with lower levels of RhoA activity, an increase in rounded cells and a reduction in actin stress fibers and focal adhesion molecules that are of critical importance in cancer cell invasion and metastasis. In addition, DLC1 down-regulated the expression of osteopontin and matrix metalloproteinase-9, which are highly up-regulated in most primary HCC with associated metastases. These observations implicate the DLC1 gene in suppression of HCC cell dissemination and identify novel cellular and genetic alterations that contribute to prevention of metastasis, a life-threatening event in cancer progression.     

Bladder cancer outcome and subtype classification by gene expression.

Models of bladder tumor progression have suggested that genetic alterations may determine both phenotype and clinical course. We have applied expression microarray analysis to a divergent set of bladder tumors to further elucidate the course of disease progression and to classify tumors into more homogeneous and clinically relevant subgroups. cDNA microarrays containing 10,368 human gene elements were used to characterize the global gene expression patterns in 80 bladder tumors, 9 bladder cancer cell lines, and 3 normal bladder samples. Robust statistical approaches accounting for the multiple testing problem were used to identify differentially expressed genes. Unsupervised hierarchical clustering successfully separated the samples into two subgroups containing superficial (pT(a) and pT(1)) versus muscle-invasive (pT(2)-pT(4)) tumors. Supervised classification had a 90.5% success rate separating superficial from muscle-invasive tumors based on a limited subset of genes. Tumors could also be classified into transitional versus squamous subtypes (89% success rate) and good versus bad prognosis (78% success rate). The performance of our stage classifiers was confirmed in silico using data from an independent tumor set. Validation of differential expression was done using immunohistochemistry on tissue microarrays for cathepsin E, cyclin A2, and parathyroid hormone-related protein. Genes driving the separation between tumor subsets may prove to be important biomarkers for bladder cancer development and progression and eventually candidates for therapeutic targeting.     

Identification of maspin and S100P as novel hypomethylation targets in pancreatic cancer using global gene expression profiling

DNA hypomethylation is one of the major epigenetic alterations in human cancers. We have previously shown that genes identified as hypomethylated in pancreatic cancer are expressed in pancreatic cancer cell lines, but not in normal pancreatic ductal epithelium and can be reexpressed in nonexpressing cells using 'epigenetic modifying agents' such as DNA methyltransferase inhibitors. To identify additional targets for aberrant hypomethylation in pancreatic cancer, we used oligonucleotide microarrays to screen for genes that displayed expression patterns associated with hypomethylation. This analysis identified a substantial number of candidates including previously reported hypomethylated genes. A subset of eight genes were selected for further methylation analysis, and two cancer-related genes, maspin and S100P, were found to be aberrantly hypomethylated in a large fraction of pancreatic cancer cell lines and primary pancreatic carcinomas. Combined treatment with 5-aza-2'-deoxycytidie and trichostatin A resulted in synergistic induction of maspin and S100P mRNA in MiaPaCa2 cells where both genes were methylated. Furthermore, there was an inverse correlation between methylation and mRNA expression level for maspin and S100P in a large panel of pancreatic cancer cell lines. We also found a significant difference in the methylation patterns of maspin and two previously identified hypomethylated genes (trefoil factor 2 and lipocalin 2) between pancreatic and breast cancer cell lines, suggesting cancer-type specificity for some hypomethylation patterns. Thus, our present results confirm that DNA hypomethylation is a frequent epigenetic event in pancreatic cancer, and suggest that gene expression profiling may help to identify potential targets affected by this epigenetic alteration.     

A mouse skin multistage carcinogenesis model reflects the aberrant DNA methylation patterns of human tumors

Whereas accepted models of tumorigenesis exist for genetic lesions, the timing of epigenetic alterations in cancer is not clearly understood. We have analyzed the profile of aberrations in DNA methylation occurring in cells lines and primary tumors of one of the best-characterized mouse carcinogenesis systems, the multistage skin cancer progression model. Initial analysis using high-performance capillary electrophoresis and immunolocalization revealed a loss of genomic 5-methylcytosine associated with the degree of tumor aggressiveness. Paradoxically, this occurs in the context of a growing number of hypermethylated CpG islands of tumor suppressor genes at the most malignant stages of carcinogenesis. We have observed this last phenomenon using two approaches, a candidate gene approach, studying genes with well-known methylation-associated silencing in human tumors, and a mouse cDNA microarray expression analysis after treatment with DNA demethylating drugs. The transition from epithelial to spindle cell morphology is particularly associated with major epigenetic alterations, such as E-cadherin methylation, demethylation of the Snail promoter, and a decrease of the global DNA methylation. Analysis of data obtained from the cDNA microarray strategy led to the identification of new genes that undergo methylation-associated silencing and have growth-inhibitory effects, such as the insulin-like growth factor binding protein-3. Most importantly, all of the above genes were also hypermethylated in human cancer cell lines and primary tumors, underlining the value of the mouse skin carcinogenesis model for the study of aberrant DNA methylation events in cancer cells.     

17q23 amplifications in breast cancer involve the PAT1, RAD51C, PS6K, and SIGma1B genes

Amplification of the 17q23 region occurs frequently in breast tumors. To characterize the structure of 17q23 amplicons and to identify oncogene targets associated with this alteration, we performed a copy number analysis of 87 17q23 localized expressed sequence tags in seven breast cancer cell lines. Three major regions of amplification were detected in the MCF7 and BT474 cell lines. Amplification of at least one of four known genes (PAT1, PS6K, RAD51C, and SIGMA1B) was detected in the cell lines and in 28% of 94 breast tumors. In most cases, these four genes were overexpressed when amplified, but there was a particularly good association between amplification of the SIGMA1B gene and elevated expression in tumors, which suggested a possible role for this gene in tumor progression. Our data show that this region contains at least four independent targets of amplification, which suggests that there is considerable variability in the structure of the 17q23 amplicon.     

Connective tissue growth factor as a novel therapeutic target in high grade serous ovarian cancer

Ovarian cancer is the most common cause of death among women with gynecologic cancer. We examined molecular profiles of fibroblasts from normal ovary and high-grade serous ovarian tumors to identify novel therapeutic targets involved in tumor progression. We identified 2,300 genes that are significantly differentially expressed in tumor-associated fibroblasts. Fibroblast expression of one of these genes, connective tissue growth factor (CTGF), was confirmed by immunohistochemistry. CTGF protein expression in ovarian tumor fibroblasts significantly correlated with gene expression levels. CTGF is a secreted component of the tumor microenvironment and is being pursued as a therapeutic target in pancreatic cancer. We examined its effect in in vitro and ex vivo ovarian cancer models, and examined associations between CTGF expression and clinico-pathologic characteristics in patients. CTGF promotes migration and peritoneal adhesion of ovarian cancer cells. These effects are abrogated by FG-3019, a human monoclonal antibody against CTGF, currently under clinical investigation as a therapeutic agent. Immunohistochemical analyses of high-grade serous ovarian tumors reveal that the highest level of tumor stromal CTGF expression was correlated with the poorest prognosis. Our findings identify CTGF as a promoter of peritoneal adhesion, likely to mediate metastasis, and a potential therapeutic target in high-grade serous ovarian cancer. These results warrant further studies into the therapeutic efficacy of FG-3019 in high-grade serous ovarian cancer.     

Discovery of novel epigenetic markers in non-Hodgkin's lymphoma

Non-Hodgkin's lymphoma (NHL) is a group of malignancies with heterogeneous genetic and epigenetic alterations. Discovery of molecular markers that better define NHL should improve diagnosis, prognosis and understanding of the biology. We developed a CpG island DNA microarray for discovery of aberrant methylation targets in cancer, and now apply this method to examine NHL cell lines and primary tumors. This methylation profiling revealed differential patterns in six cell lines originating from different subtypes of NHL. We identified 30 hypermethylated genes in these cell lines and independently confirmed 10 of them. Methylation of 6 of these genes was then further examined in 75 primary NHL specimens composed of four subtypes representing different stages of maturation. Each gene (DLC-1, PCDHGB7, CYP27B1, EFNA5, CCND1 and RARbeta2) was frequently hypermethylated in these NHLs (87, 78, 61, 53, 40 and 38%, respectively), but not in benign follicular hyperplasia. Although some genes such as DLC-1 and PCDHGB7 were methylated in the vast majority of NHLs, others were differentially methylated in specific subtypes. The methylation of the candidate tumor suppressor gene DLC-1 was detected in a high proportion of primary tumor and plasma DNA samples by using quantitative methylation-specific PCR analysis. This promoter hypermethylation inversely correlated with DLC-1 gene expression in primary NHL samples. Thus, this CpG island microarray is a powerful discovery tool to identify novel methylated genes for further studies of their relevant molecular pathways in NHLs and identification of potential epigenetic biomarkers of disease.