High-throughput and targeted drug screens identify pharmacological candidates against MiT-translocation renal cell carcinoma.

BACKGROUND: MiT-Renal Cell Carcinoma (RCC) is characterized by genomic translocations involving microphthalmia-associated transcription factor (MiT) family members TFE3, TFEB, or MITF. MiT-RCC represents a specific subtype of sporadic RCC that is predominantly seen in young patients and can present with heterogeneous histological features making diagnosis challenging. Moreover, the disease biology of this aggressive cancer is poorly understood and there is no accepted standard of care therapy for patients with advanced disease. Tumor-derived cell lines have been established from human TFE3-RCC providing useful models for preclinical studies. METHODS: TFE3-RCC tumor derived cell lines and their tissues of origin were characterized by IHC and gene expression analyses. An unbiased high-throughput drug screen was performed to identify novel therapeutic agents for treatment of MiT-RCC. Potential therapeutic candidates were validated in in vitro and in vivo preclinical studies. Mechanistic assays were conducted to confirm the on-target effects of drugs. RESULTS: The results of a high-throughput small molecule drug screen utilizing three TFE3-RCC tumor-derived cell lines identified five classes of agents with potential pharmacological efficacy, including inhibitors of phosphoinositide-3-kinase (PI3K) and mechanistic target of rapamycin (mTOR), and several additional agents, including the transcription inhibitor Mithramycin A. Upregulation of the cell surface marker GPNMB, a specific MiT transcriptional target, was confirmed in TFE3-RCC and evaluated as a therapeutic target using the GPNMB-targeted antibody-drug conjugate CDX-011. In vitro and in vivo preclinical studies demonstrated efficacy of the PI3K/mTOR inhibitor NVP-BGT226, Mithramycin A, and CDX-011 as potential therapeutic options for treating advanced MiT-RCC as single agents or in combination. CONCLUSIONS: The results of the high-throughput drug screen and validation studies in TFE3-RCC tumor-derived cell lines have provided in vitro and in vivo preclinical data supporting the efficacy of the PI3K/mTOR inhibitor NVP-BGT226, the transcription inhibitor Mithramycin A, and GPNMB-targeted antibody-drug conjugate CDX-011 as potential therapeutic options for treating advanced MiT-RCC. The findings presented here should provide the basis for designing future clinical trials for patients with MiT-driven RCC.

Hard wiring of normal tissue-specific chromosome-wide gene expression levels is an additional factor driving cancer type-specific aneuploidies.

BACKGROUND: Many carcinomas have recurrent chromosomal aneuploidies specific to the tissue of tumor origin. The reason for this specificity is not completely understood. METHODS: In this study, we looked at the frequency of chromosomal arm gains and losses in different cancer types from the The Cancer Genome Atlas (TCGA) and compared them to the mean gene expression of each chromosome arm in corresponding normal tissues of origin from the Genotype-Tissue Expression (GTEx) database, in addition to the distribution of tissue-specific oncogenes and tumor suppressors on different chromosome arms. RESULTS: This analysis revealed a complex picture of factors driving tumor karyotype evolution in which some recurrent chromosomal copy number reflect the chromosome arm-wide gene expression levels of the their normal tissue of tumor origin. CONCLUSIONS: We conclude that the cancer type-specific distribution of chromosomal arm gains and losses is potentially "hardwiring" gene expression levels characteristic of the normal tissue of tumor origin, in addition to broadly modulating the expression of tissue-specific tumor driver genes.

Novel renal medullary carcinoma cell lines, UOK353 and UOK360, provide preclinical tools to identify new therapeutic treatments.

Renal medullary carcinoma (RMC) is a rare, aggressive disease that predominantly afflicts individuals of African or Mediterranean descent with sickle cell trait. RMC comprises 1% of all renal cell carcinoma diagnoses with a median overall survival of 13 months. Patients are typically young (median age-22) and male (male:female ratio of 2:1) and tumors are characterized by complete loss of expression of the SMARCB1 tumor suppressor protein. Due to the low incidence of RMC and the disease's aggressiveness, treatment decisions are often based on case reports. Thus, it is critical to develop preclinical models of RMC to better understand the pathogenesis of this disease and to identify effective forms of therapy. Two novel cell line models, UOK353 and UOK360, were derived from primary RMCs that both demonstrated the characteristic SMARCB1 loss. Both cell lines overexpressed EZH2 and other members of the polycomb repressive complex and EZH2 inhibition in RMC tumor spheroids resulted in decreased viability. High throughput drug screening of both cell lines revealed several additional candidate compounds, including bortezomib that had both in vitro and in vivo antitumor activity. The activity of bortezomib was shown to be partially dependent on increased oxidative stress as addition of the N-acetyl cysteine antioxidant reduced the effect on cell proliferation. Combining bortezomib and cisplatin further decreased cell viability both in vitro and in vivo that single agent bortezomib treatment. The UOK353 and UOK360 cell lines represent novel preclinical models for the development of effective forms of therapy for RMC patients.

Single-Cell-Derived Primary Rectal Carcinoma Cell Lines Reflect Intratumor Heterogeneity Associated with Treatment Response.

PURPOSE: The standard treatment of patients with locally advanced rectal cancer consists of preoperative chemoradiotherapy (CRT) followed by surgery. However, the response of individual tumors to CRT is extremely diverse, presenting a clinical dilemma. This broad variability in treatment response is likely attributable to intratumor heterogeneity (ITH). EXPERIMENTAL DESIGN: We addressed the impact of ITH on response to CRT by establishing single-cell-derived cell lines (SCDCL) from a treatment-naïve rectal cancer biopsy after xenografting. RESULTS: Individual SCDCLs derived from the same tumor responded profoundly different to CRT in vitro. Clonal reconstruction of the tumor and derived cell lines based on whole-exome sequencing revealed nine separate clusters with distinct proportions in the SCDCLs. Missense mutations in SV2A and ZWINT were clonal in the resistant SCDCL, but not detected in the sensitive SCDCL. Single-cell genetic analysis by multiplex FISH revealed the expansion of a clone with a loss of PIK3CA in the resistant SCDCL. Gene expression profiling by tRNA-sequencing identified the activation of the Wnt, Akt, and Hedgehog signaling pathways in the resistant SCDCLs. Wnt pathway activation in the resistant SCDCLs was confirmed using a reporter assay. CONCLUSIONS: Our model system of patient-derived SCDCLs provides evidence for the critical role of ITH for treatment response in patients with rectal cancer and shows that distinct genetic aberration profiles are associated with treatment response. We identified specific pathways as the molecular basis of treatment response of individual clones, which could be targeted in resistant subclones of a heterogenous tumor.

Bile acid-induced "Minority MOMP" promotes esophageal carcinogenesis while maintaining apoptotic resistance via Mcl-1.

Barrett's esophagus (BE) is associated with reflux and is implicated the development of esophageal adenocarcinoma (EAC). Apoptosis induces cell death through mitochondrial outer membrane permeabilization (MOMP), which is considered an irreversible step in apoptosis. Activation of MOMP to levels that fail to reach the apoptotic threshold may paradoxically promote cancer-a phenomenon called "Minority MOMP." We asked whether reflux-induced esophageal carcinogenesis occurred via minority MOMP and whether compensatory resistance mechanisms prevented cell death during this process. We exposed preneoplastic, hTERT-immortalized Barrett's cell, CP-C and CP-A, to the oncogenic bile acid, deoxycholic acid (DCA), for 1 year. Induction of minority MOMP was tested via comet assay, CyQuant, annexin V, JC-1, cytochrome C subcellular localization, caspase 3 activation, and immunoblots. We used bcl-2 homology domain-3 (BH3) profiling to test the mitochondrial apoptotic threshold. One-year exposure of Barrett's cells to DCA induced a malignant phenotype noted by clone and tumor formation. DCA promoted minority MOMP noted by minimal release of cytochrome C and limited caspase 3 activation, which resulted in DNA damage without apoptosis. Upregulation of the antiapoptotic protein, Mcl-1, ROS generation, and NF-κB activation occurred in conjunction with minority MOMP. Inhibition of ROS blocked minority MOMP and Mcl-1 upregulation. Knockdown of Mcl-1 shifted minority MOMP to complete MOMP as noted by dynamic BH3 profiling and increased apoptosis. Minority MOMP contributes to DCA induced carcinogenesis in preneoplastic BE. Mcl-1 provided a balance within the mitochondria that induced resistance complete MOMP and cell death. Targeting Mcl-1 may be a therapeutic strategy in EAC.

Single Chromosome Aneuploidy Induces Genome-Wide Perturbation of Nuclear Organization and Gene Expression.

Chromosomal aneuploidy is a defining feature of carcinomas and results in tumor-entity specific genomic imbalances. For instance, most sporadic colorectal carcinomas carry extra copies of chromosome 7, an aneuploidy that emerges already in premalignant adenomas, and is maintained throughout tumor progression and in derived cell lines. A comprehensive understanding on how chromosomal aneuploidy affects nuclear organization and gene expression, i.e., the nucleome, remains elusive. We now analyzed a cell line established from healthy colon mucosa with a normal karyotype (46,XY) and its isogenic derived cell line that acquired an extra copy of chromosome 7 as its sole anomaly (47,XY,+7). We studied structure/function relationships consequent to aneuploidization using genome-wide chromosome conformation capture (Hi-C), RNA sequencing and protein profiling. The gain of chromosome 7 resulted in an increase of transcript levels of resident genes as well as genome-wide gene and protein expression changes. The Hi-C analysis showed that the extra copy of chromosome 7 is reflected in more interchromosomal contacts between the triploid chromosomes. Chromatin organization changes are observed genome-wide, as determined by changes in A/B compartmentalization and topologically associating domain (TAD) boundaries. Most notably, chromosome 4 shows a profound loss of chromatin organization, and chromosome 14 contains a large A/B compartment switch region, concurrent with resident gene expression changes. No changes to the nuclear position of the additional chromosome 7 territory were observed when measuring distances of chromosome painting probes by interphase FISH. Genome and protein data showed enrichment in signaling pathways crucial for malignant transformation, such as the HGF/MET-axis. We conclude that a specific chromosomal aneuploidy has profound impact on nuclear structure and function, both locally and genome-wide. Our study provides a benchmark for the analysis of cancer nucleomes with complex karyotypes.

Sex-chromosome dosage effects on gene expression in humans.

A fundamental question in the biology of sex differences has eluded direct study in humans: How does sex-chromosome dosage (SCD) shape genome function? To address this, we developed a systematic map of SCD effects on gene function by analyzing genome-wide expression data in humans with diverse sex-chromosome aneuploidies (XO, XXX, XXY, XYY, and XXYY). For sex chromosomes, we demonstrate a pattern of obligate dosage sensitivity among evolutionarily preserved X-Y homologs and update prevailing theoretical models for SCD compensation by detecting X-linked genes that increase expression with decreasing X- and/or Y-chromosome dosage. We further show that SCD-sensitive sex-chromosome genes regulate specific coexpression networks of SCD-sensitive autosomal genes with critical cellular functions and a demonstrable potential to mediate previously documented SCD effects on disease. These gene coexpression results converge with analysis of transcription factor binding site enrichment and measures of gene expression in murine knockout models to spotlight the dosage-sensitive X-linked transcription factor ZFX as a key mediator of SCD effects on wider genome expression. Our findings characterize the effects of SCD broadly across the genome, with potential implications for human phenotypic variation.

A multiplex preclinical model for adenoid cystic carcinoma of the salivary gland identifies regorafenib as a potential therapeutic drug.

Adenoid cystic carcinomas (ACC) are rare salivary gland cancers with a high incidence of metastases. In order to study this tumor type, a reliable model system exhibiting the molecular features of this tumor is critical, but none exists, thereby inhibiting in-vitro studies and the analysis of metastatic behavior. To address this deficiency, we have coupled an efficient method to establish tumor cell cultures, conditional reprogramming (CR), with a rapid, reproducible and robust in-vivo zebrafish model. We have established cell cultures from two individual ACC PDX tumors that maintain the characteristic MYB translocation. Additional mutations found in one ACC culture also seen in the PDX tumor. Finally, the CR/zebrafish model mirrors the PDX mouse model and identifies regorafenib as a potential therapeutic drug to treat this cancer type that mimic the drug sensitivity profile in PDX model, further confirming the unique advantages of multiplex system.

ASXL3 Is a Novel Pluripotency Factor in Human Respiratory Epithelial Cells and a Potential Therapeutic Target in Small Cell Lung Cancer.

In this study, we generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) to investigate epigenetic mechanisms of stemness and pluripotency in lung cancers. We documented key hallmarks of reprogramming in lung iPSCs (Lu-iPSC) that coincided with modulation of more than 15,000 genes relative to parental SAECs. Of particular novelty, we identified the PRC2-associated protein, ASXL3, which was markedly upregulated in Lu-iPSCs and small cell lung cancer (SCLC) lines and clinical specimens. ASXL3 overexpression correlated with increased genomic copy number in SCLC lines. ASXL3 silencing inhibited proliferation, clonogenicity, and teratoma formation by Lu-iPSCs, and diminished clonogenicity and malignant growth of SCLC cells in vivo Collectively, our studies validate the utility of the Lu-iPSC model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and highlight ASXL3 as a novel candidate target for SCLC therapy. Cancer Res; 77(22); 6267-81. ©2017 AACR.

HPV positive neuroendocrine cervical cancer cells are dependent on Myc but not E6/E7 viral oncogenes.

Using conditional cell reprogramming, we generated a stable cell culture of an extremely rare and aggressive neuroendocrine cervical cancer. The cultured cells contained HPV-16, formed colonies in soft agar and rapidly produced tumors in immunodeficient mice. The HPV-16 genome was integrated adjacent to the Myc gene, both of which were amplified 40-fold. Analysis of RNA transcripts detected fusion of the HPV/Myc genes, arising from apparent microhomologous recombination. Spectral karyotyping (SKY) and fluorescent-in-situ hybridization (FISH) demonstrated coordinate localization and translocation of the amplified Myc and HPV genes on chromosomes 8 and 21. Similar to the primary tumor, tumor cell cultures expressed very high levels of the Myc protein and, in contrast to all other HPV-positive cervical cancer cell lines, they harbored a gain-of-function mutation in p53 (R273C). Unexpectedly, viral oncogene knockdown had no effect on the growth of the cells, but it did inhibit the proliferation of a conventional HPV-16 positive cervical cancer cell line. Knockdown of Myc, but not the mutant p53, significantly inhibited tumor cell proliferation. On the basis of these data, we propose that the primary driver of transformation in this aggressive cervical cancer is not HPV oncogene expression but rather the overexpression of Myc.

A ten-year retrospective analysis of the distribution, use and phenotypic characteristics of the LAD2 human mast cell line.

BACKGROUND: In 2003, this laboratory published an account of the human mast cell line LAD2 (Laboratory of Allergic Diseases 2) that expressed FcεRI, responded to recombinant human stem cell factor (rhSCF) and resembled CD34+-derived human mast cells. LAD2 cells have now been distributed worldwide. To study the impact of this transfer, we analyzed the number of investigators receiving LAD2 cells and resulting publications. METHODS: Records maintained in our laboratory, the Technology Transfer and Intellectual Property Office and Office of Technology Transfer, were reviewed for material transfer agreements (MTAs) and licensing agreements (LAs). Journals and impact factors were obtained from PubMed.gov by cross-referencing LAD2 and human mast cells from 2003 through November 2013. RESULTS: Over 300 MTAs and 40 LAs were approved. LAD2 cells were shipped to over 30 countries. More than 80 papers have been published in journals with impact factors from 1.31 to 13.21. Intended uses include the study of receptors, degranulation, and cell signaling. LAD2 cells continue to express described markers and have consistent FcεR1-mediated degranulation. CONCLUSIONS: Success of the LAD2 line reflects the demand for a human mast cell line in research, the uniqueness of this cell line, and that it continues to exhibit minimal variation from its original description. We hope that the awareness of the impact of this cell line on mast cell research will encourage others to develop and distribute other similar cell lines with additional characteristics so as to address the limitations of depending on the study of cultured human mast cells from tissues.

Telomerase variant A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal carcinomas.

BACKGROUND: Although implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas. METHODS: Sequencing techniques were used to evaluate mutational status of telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and ß-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability. RESULTS: Sequencing analysis revealed one deletion involving TERC (TERC del 341-360), and two non-synonymous TERT variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p<0.01). Relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-ß-catenin complex, markedly depleted ß-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin™ exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage. CONCLUSIONS: A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal cancer cells. These findings warrant further analysis of A279T expression in esophageal cancers and premalignant esophageal lesions.

Genome-wide analysis of HPV integration in human cancers reveals recurrent, focal genomic instability.

Genomic instability is a hallmark of human cancers, including the 5% caused by human papillomavirus (HPV). Here we report a striking association between HPV integration and adjacent host genomic structural variation in human cancer cell lines and primary tumors. Whole-genome sequencing revealed HPV integrants flanking and bridging extensive host genomic amplifications and rearrangements, including deletions, inversions, and chromosomal translocations. We present a model of "looping" by which HPV integrant-mediated DNA replication and recombination may result in viral-host DNA concatemers, frequently disrupting genes involved in oncogenesis and amplifying HPV oncogenes E6 and E7. Our high-resolution results shed new light on a catastrophic process, distinct from chromothripsis and other mutational processes, by which HPV directly promotes genomic instability.

CKAP2 ensures chromosomal stability by maintaining the integrity of microtubule nucleation sites.

Integrity of the microtubule spindle apparatus and intact cell division checkpoints are essential to ensure the fidelity of distributing chromosomes into daughter cells. Cytoskeleton-associated protein 2, CKAP2, is a microtubule-associated protein that localizes to spindle poles and aids in microtubule stabilization, but the exact function and mechanism of action are poorly understood. In the present study, we utilized RNA interference to determine the extent to which the expression of CKAP2 plays a role in chromosome segregation. CKAP2-depleted cells showed a significant increase of multipolar mitoses and other spindle pole defects. Notably, when interrogated for microtubule nucleation capacity, CKAP2-depleted cells showed a very unusual phenotype as early as two minutes after release from mitotic block, consisting of dispersal of newly polymerized microtubule filaments through the entire chromatin region, creating a cage-like structure. Nevertheless, spindle poles were formed after one hour of mitotic release suggesting that centrosome-mediated nucleation remained dominant. Finally, we showed that suppression of CKAP2 resulted in a higher incidence of merotelic attachments, anaphase lagging, and polyploidy. Based on these results, we conclude that CKAP2 is involved in the maintenance of microtubule nucleation sites, focusing microtubule minus ends to the spindle poles in early mitosis, and is implicated in maintaining genome stability.

Defective telomere elongation and hematopoiesis from telomerase-mutant aplastic anemia iPSCs.

Critically short telomeres activate p53-mediated apoptosis, resulting in organ failure and leading to malignant transformation. Mutations in genes responsible for telomere maintenance are linked to a number of human diseases. We derived induced pluripotent stem cells (iPSCs) from 4 patients with aplastic anemia or hypocellular bone marrow carrying heterozygous mutations in the telomerase reverse transcriptase (TERT) or the telomerase RNA component (TERC) telomerase genes. Both mutant and control iPSCs upregulated TERT and TERC expression compared with parental fibroblasts, but mutant iPSCs elongated telomeres at a lower rate compared with healthy iPSCs, and the deficit correlated with the mutations' impact on telomerase activity. There was no evidence for alternative lengthening of telomere (ALT) pathway activation. Elongation varied among iPSC clones derived from the same patient and among clones from siblings harboring identical mutations. Clonal heterogeneity was linked to genetic and environmental factors, but was not influenced by residual expression of reprogramming transgenes. Hypoxia increased telomere extension in both mutant and normal iPSCs. Additionally, telomerase-mutant iPSCs showed defective hematopoietic differentiation in vitro, mirroring the clinical phenotype observed in patients and demonstrating that human telomere diseases can be modeled utilizing iPSCs. Our data support the necessity of studying multiple clones when using iPSCs to model disease.

Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells.

Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research.

Spontaneous transformation of murine epithelial cells requires the early acquisition of specific chromosomal aneuploidies and genomic imbalances.

Human carcinomas are defined by recurrent chromosomal aneuploidies, which result in a tissue-specific distribution of genomic imbalances. In order to develop models for these genome mutations and to determine their role in tumorigenesis, we generated 45 spontaneously transformed murine cell lines from normal epithelial cells derived from bladder, cervix, colon, kidney, lung, and mammary gland. Phenotypic changes, chromosomal aberrations, centrosome number, and telomerase activity were assayed in control uncultured cells and in three subsequent stages of transformation. Supernumerary centrosomes, binucleate cells, and tetraploidy were observed as early as 48 hr after explantation. In addition, telomerase activity increased throughout progression. Live-cell imaging revealed that failure of cytokinesis, not cell fusion, promoted genome duplication. Spectral karyotyping demonstrated that aneuploidy preceded immortalization, consisting predominantly of whole chromosome losses (4, 9, 12, 13, 16, and Y) and gains (1, 10, 15, and 19). After transformation, focal amplifications of the oncogenes Myc and Mdm2 were frequently detected. Fifty percent of the transformed lines resulted in tumors on injection into immunocompromised mice. The phenotypic and genomic alterations observed in spontaneously transformed murine epithelial cells recapitulated the aberration pattern observed during human carcinogenesis. The dominant aberration of these cell lines was the presence of specific chromosomal aneuploidies. We propose that our newly derived cancer models will be useful tools to dissect the sequential steps of genome mutations during malignant transformation, and also to identify cancer-specific genes, signaling pathways, and the role of chromosomal instability in this process.

Genomic instability influences the transcriptome and proteome in endometrial cancer subtypes.

BACKGROUND: In addition to clinical characteristics, DNA aneuploidy has been identified as a prognostic factor in epithelial malignancies in general and in endometrial cancers in particular. We mapped ploidy-associated chromosomal aberrations and identified corresponding gene and protein expression changes in endometrial cancers of different prognostic subgroups. METHODS: DNA image cytometry classified 25 endometrioid cancers to be either diploid (n = 16) or aneuploid (n = 9), and all uterine papillary serous cancers (UPSC) to be aneuploid (n = 8). All samples were subjected to comparative genomic hybridization and gene expression profiling. Identified genes were subjected to Ingenuity pathway analysis (IPA) and were correlated to protein expression changes. RESULTS: Comparative genomic hybridization revealed ploidy-associated specific, recurrent genomic imbalances. Gene expression analysis identified 54 genes between diploid and aneuploid endometrioid carcinomas, 39 genes between aneuploid endometrioid cancer and UPSC, and 76 genes between diploid endometrioid and aneuploid UPSC to be differentially expressed. Protein profiling identified AKR7A2 and ANXA2 to show translational alterations consistent with the transcriptional changes. The majority of differentially expressed genes and proteins belonged to identical molecular functions, foremost Cancer, Cell Death, and Cellular Assembly and Organization. CONCLUSIONS: We conclude that the grade of genomic instability rather than the histopathological subtype correlates with specific gene and protein expression changes. The identified genes and proteins might be useful as molecular targets for improved diagnostic and therapeutic intervention and merit prospective validation.

Genome and transcriptome profiles of CD133-positive colorectal cancer cells.

Colorectal carcinomas (CRC) might be organized hierarchically and contain a subpopulation of tumorigenic, putative cancer stem cells that are CD133 positive. We studied the biological and genetic characteristics of such cells in CRC cell lines and primary tumors. Three CRC cell lines were sorted in CD133 positive and negative fractions. The respective genetic aberration profiles were studied using array comparative genomic hybridization (aCGH) and expression profiling. Tumorigenicity for each cellular population was tested by injection into nude mice. Additionally, we compared CD133+ and CD133- cells of 12 primary colorectal tumors using laser capture microdissection and aCGH. Three of five CRC cell lines displayed both CD133+ and CD133- cells, but tumorigenicity of these subfractions did not differ significantly and aCGH revealed essentially identical genomic imbalances. However, 96 genes were differentially expressed between the two populations. Array comparative genomic hybridization analysis after laser capture microdissection of CD133+ and CD133- areas in primary colorectal tumors revealed genetic differences in 7 of 12 cases. The use of cell lines for studying genomic alterations that define cancer stem cell characteristics, therefore, seems questionable. In contrast, CD133+ cells in primary cancer samples showed a unique genomic aberration profile. In conclusion, our data suggest that CD133 positivity defines a genetically distinct cellular compartment in primary CRC, which potentially includes tumor initiating cells.

Definitive molecular cytogenetic characterization of 15 colorectal cancer cell lines.

In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. Here, we present the results of a comprehensive investigation of 15 established colorectal cancer cell lines using spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI-H508, NCI-H716, and SK-CO-1) is described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines; isochromosomes were the most common recurrent abnormalities; and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities predominantly result in copy number changes rather than specific chromosome or gene fusions suggests that this may be the major mechanism leading to carcinogenesis in colorectal cancer.