Abstract: Readthrough fusions across adjacent genes in the genome, or transcription-induced chimeras (TICs), have been estimated using expressed sequence tag (EST) libraries to involve 4-6% of all genes. Deep transcriptional sequencing (RNA-Seq) now makes it possible to study the occurrence and expression levels of TICs in individual samples across the genome. Methods We performed single-end RNA-Seq on three human prostate adenocarcinoma samples and their corresponding normal tissues, as well as brain and universal reference samples. We developed two bioinformatics methods to specifically identify TIC events: a targeted alignment method using artificial exon-exon junctions within 200,000 bp from adjacent genes, and genomic alignment allowing splicing within individual reads. We performed further experimental verification and characterization of selected TIC and fusion events using quantitative RT-PCR and comparative genomic hybridization microarrays. Results Targeted alignment against artificial exon-exon junctions yielded 339 distinct TIC events, including 32 gene pairs with multiple isoforms. The false discovery rate was estimated to be 1.5%. Spliced alignment to the genome was less sensitive, finding only 18% of those found by targeted alignment in 33-nt reads and 59% of those in 50-nt reads. However, spliced alignment revealed 30 cases of TICs with intervening exons, in addition to distant inversions, scrambled genes, and translocations. Our findings increase the catalog of observed TIC gene pairs by 66%. We verified 6 of 6 predicted TICs in all prostate samples, and 2 of 5 predicted novel distant gene fusions, both private events among 54 prostate tumor samples tested. Expression of TICs correlates with that of the upstream gene, which can explain the prostate-specific pattern of some TIC events and the restriction of the SLC45A3-ELK4 e4-e2 TIC to ERG -negative prostate samples, as confirmed in 20 matched prostate tumor and normal samples and 9 lung cancer cell lines. Conclusions Deep transcriptional sequencing and analysis with targeted and spliced alignment methods can effectively identify TIC events across the genome in individual tissues. Prostate and reference samples exhibit a wide range of TIC events, involving more genes than estimated previously using ESTs. Tissue specificity of TIC events is correlated with expression patterns of the upstream gene. Some TIC events, such as MSMB-NCOA4 , may play functional roles in cancer.

Abstract: Breast cancer is a heterogeneous disease with distinct molecular subtypes characterized by differential response to targeted and chemotherapeutic agents. Enhanced understanding of the genetic alterations characteristic of different subtypes is needed to pave the way for more personalized administration of therapeutic agents. We have taken a functional genomics approach using a well-characterized panel of breast cancer cell lines to identify putative biomarkers of resistance to antimitotic agents such as paclitaxel and monomethyl-auristatin-E (MMAE). In vitro studies revealed a striking difference in sensitivity to these agents between cell lines from different subtypes, with basal-like cell lines being significantly more sensitive to both agents than luminal or HER2-amplified cell lines. Genome-wide association studies using copy number data from Affymetrix single nucleotide polymorphism arrays identified amplification of the chromosome 17q21 region as being highly associated with resistance to both paclitaxel and MMAE. An unbiased approach consisting of RNA interference and high content analysis was used to show that amplification and concomitant overexpression of the gene encoding the ABCC3 drug transporter is responsible for conferring in vitro resistance to paclitaxel and MMAE. We also show that amplification of ABCC3 is present in primary breast tumors and that it occurs predominantly in HER2-amplified and luminal tumors, and we report on development of a specific fluorescence in situ hybridization assay that may have utility as a predictive biomarker of taxane resistance in breast cancer. [Cancer Res 2008;68(13):5380–9]

Abstract: The echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK) fusion gene has been identified as an oncogene in a subset of non–small cell lung cancers (NSCLC). We used profiling of cancer genomes on an exon array to develop a novel computational method for the global search of gene rearrangements. This approach led to the detection of EML4-ALK fusion in breast and colorectal carcinomas in addition to NSCLC. Screening of a large collection of patient tumor samples showed the presence of EML4-ALK fusion in 2.4% of breast (5 of 209), 2.4% of colorectal (2 of 83), and in 11.3% of NSCLC (12 of 106). Besides previously known EML4-ALK variants 1 (E13; A20) and 2 (E20; A20), a novel variant E21; A20 was found in colorectal carcinoma. The presence of an EML-ALK rearrangement was verified by identifying genomic fusion points in tumor samples representative of breast, colon, and NSCLC. EML4-ALK translocation was also confirmed by fluorescence in situ hybridization assay, which revealed its substantial heterogeneity in both primary tumors and tumor-derived cell lines. To elucidate the functional significance of EML4-ALK, we examined the growth of cell lines harboring the fusion following EML4 and ALK silencing by small interfering RNA. Significant growth inhibition was observed in some but not all cell lines, suggesting their variable dependence on ALK-mediated cell survival signaling. Collectively, these findings show the recurrence of EML4-ALK fusion in multiple solid tumors and further substantiate its role in tumorigenesis. (Mol Cancer Res 2009;7(9):1466–76)

Abstract: Our results point the way to novel strategies for sensitizing cancer cells to death receptor-mediated apoptosis. Mechanistically, we found that both EDD1 and GRHL2 inhibit death receptor-mediated apoptosis by repressing the expression of death receptors FAS and DR5 ( Fig. P1 D ). Furthermore, EDD1 expression is elevated in cancer cell lines classified as resistant to death receptor-mediated apoptosis and is consistent with our finding that silencing either EDD1 or GRHL2 sensitizes resistant breast cancer cells to ligand-induced apoptosis. Therefore, the identification of EDD1 and GRHL2 raises the possibility that targeting their activity or expression would provide a therapeutically relevant strategy in improving cancer cell responsiveness to death receptor-mediated apoptosis. These findings are significant because they describe a unique mechanism for regulating apoptosis and highlight potential therapeutic strategies for activating apoptosis in cancer. Given that our functional data implicate a role for EDD1 and GRHL2 in suppressing death receptor-mediated apoptosis, we hypothesized that amplification of either gene could contribute to a mechanism by which cancer cells evade therapeutic challenge with death receptor agonists. To address this question, we interrogated previously published gene expression profiles of cancer cells resistant to a proapoptotic receptor agonist, dulanermin ( 5 ). Studies on dulanermin-resistant pancreatic, breast, and lung cancer cell lines supported our hypothesis that these genes confer resistance to targeted apoptosis, particularly EDD1. The expression of EDD1 is elevated in these cancer cell lines, which was quantitatively confirmed. We did not find a statistically significant difference in GRHL2 expression between dulanermin-sensitive and -resistant cell lines. However, we were able to confirm elevated EDD1 and GRHL2 protein expression in several dulanermin-resistant breast cancer cell lines exhibiting copy number gains at the 8q22 locus. The functional relevance for the 8q22 gene cluster at contributing to resistance of cancer cells to death receptor agonists was tested directly by quantifying the effect of silencing EDD1 or GRHL2 in dulanermin-resistant breast cancer cell lines. We found that silencing EDD1 or GRHL2 sensitized four breast cancer cell lines with amplified 8q22 to dulanermin-induced apoptosis; this correlated with the degree of EDD1 and GRHL2 silencing as well as with a corresponding induction of DR5 expression. We next investigated the mechanism of how these top three hits regulate FASL-mediated apoptosis. For this purpose, we silenced each gene to determine if they modulate the expression of death receptors. Somewhat surprisingly, we found that silencing each gene induced a two- to fourfold increase in the mRNA levels of a receptor called FAS. A similar impact on the mRNA levels of a related death receptor, DR5, was also observed upon silencing EDD1 and GRHL2, whereas silencing PTK7 exhibited little or no effect on DR5 expression. In contrast, there was little or no induction of another death receptor, TNFR1, when any of the three genes were silenced. Thus, all the top three hits modulated the expression of the FAS receptor, which is the likely mechanism by which silencing of PTK7, EDD1, or GRHL2 sensitizes cells to FASL. Interestingly, PTK7 exhibits the greatest specificity for regulating the FAS receptor, whereas EDD1 and GRHL2 regulate the expression of both FAS and DR5 receptors. We focused further experiments on two genes with the most therapeutic potential by first inspecting their genomic and expression data in closer detail. Interestingly, we found that both genes localize to a genomic region on chromosome 8q22, one already implicated in multiple cancers. Using GISTIC, an algorithm for quantifying chromosomal copy number changes ( 4 ), we found that both GRHL2 and EDD1 localize within a large region on chromosome 8q frequently amplified in breast ( Fig. P1 B ), lung, melanoma, and ovarian cancers. Importantly, the genomic amplification correlated well with the expression level for both EDD1 and GRHL2. We then used FACS and Western blot analyses to measure the impact on death receptor expression following silencing of EDD1 and GRHL2. Upon silencing of either gene, FAS ( Fig. P1 C ) and DR5 protein levels were induced, indicating negative regulation of death receptor activity. To remove erroneous hits resulting from a common problem known as an off-target RNAi effect, we performed a series of validation experiments. First, we retested the top ∼250 hits as individual shRNAs. Second, we repeated the treatment with FASL for the top hits by using siRNAs, which also act as an RNAi trigger. For the tertiary-level validation, five hits, PTK7, UNC13D, TAOK2, EDD1, and GRHL2, were selected based on the finding that they exhibit frequent overexpression and/or copy number gain in cancer. We retested each of these hits with four different siRNAs for each gene and looked for increased FASL sensitivity with at least two independent siRNAs. This method identified the three top hits: PTK7, EDD1, and GRHL2. We screened the human genome by using shRNAs that can selectively turn off or silence targeted genes. We delivered these shRNAs into the cells using Lentivirus as the vector ( 3 ). We first divided the shRNA library into two viral pools, each consisting of ∼35,000 shRNAs. A common cell line known as fibrosarcoma HT1080 was treated with each shRNA pool. This was followed by treatment with one of the important ligands in death receptor-mediated apoptosis, the FAS ligand (FASL). Nine days following the initial FASL treatment, the shRNAs integrated into target cell DNA were recovered by PCR amplification. The change in shRNA representation between the FASL-treated and control cells was quantified using two different methods to overcome the bias introduced by each of these procedures ( Fig. P1 A ). Our screen focused on the apoptotic pathway activated via cell surface receptors commonly referred to as the extrinsic signaling pathway. This apoptotic pathway is induced by small signaling molecules called ligands that bind to the cell surface receptors, aptly named “death receptors.” The appropriate ligand binds to its corresponding death receptor, which initiates a signaling cascade that eventually activates the molecules that carry out apoptosis ( 1 , 2 ). With the aim of uncovering novel strategies to activate death receptor-mediated apoptosis in cancer cells selectively, we searched for genes that regulate the sensitivity to death receptor ligands. The potential to kill cancer cells selectively via apoptosis, or programmed cell death, is recognized as a highly valuable therapeutic strategy. However, because of the inherent signaling complexities of cancer cells, simultaneous activation of multiple components of apoptotic pathways is likely required to achieve effective treatment. We screened an entire genome using an RNAi approach and identified several novel regulators of death receptor-mediated apoptosis. Two of these regulators inhibit the expression of cell surface receptors involved in apoptosis and are frequently amplified in cancer. Collectively, our findings highlight a mechanism by which cancer cells may evade apoptosis and provide insights into the search for targets and functional biomarkers for the receptor-mediated apoptotic pathway.