Carrie D. House

Voltage-Gated Na + Channel SCN5A Is a Key Regulator of a Gene Transcriptional Network That Controls Colon Cancer Invasion

Voltage-gated Na(+) channels (VGSC) have been implicated in the metastatic potential of human breast, prostate, and lung cancer cells. Specifically, the SCN5A gene encoding the VGSC isotype Na(v)1.5 has been defined as a key driver of human cancer cell invasion. In this study, we examined the expression and function of VGSCs in a panel of colon cancer cell lines by electrophysiologic recordings. Na(+) channel activity and invasive potential were inhibited pharmacologically by tetrodotoxin or genetically by small interfering RNAs (siRNA) specifically targeting SCN5A. Clinical relevance was established by immunohistochemistry of patient biopsies, with strong Na(v)1.5 protein staining found in colon cancer specimens but little to no staining in matched-paired normal colon tissues. We explored the mechanism of VGSC-mediated invasive potential on the basis of reported links between VGSC activity and gene expression in excitable cells. Probabilistic modeling of loss-of-function screens and microarray data established an unequivocal role of VGSC SCN5A as a high level regulator of a colon cancer invasion network, involving genes that encompass Wnt signaling, cell migration, ectoderm development, response to biotic stimulus, steroid metabolic process, and cell cycle control. siRNA-mediated knockdown of predicted downstream network components caused a loss of invasive behavior, demonstrating network connectivity and its function in driving colon cancer invasion.

A factor graph nested effects model to identify networks from genetic perturbations.

Complex phenotypes such as the transformation of a normal population of cells into cancerous tissue result from a series of molecular triggers gone awry. We describe a method that searches for a genetic network consistent with expression changes observed under the knock-down of a set of genes that share a common role in the cell, such as a disease phenotype. The method extends the Nested Effects Model of Markowetz et al. (2005) by using a probabilistic factor graph to search for a network representing interactions among these silenced genes. The method also expands the network by attaching new genes at specific downstream points, providing candidates for subsequent perturbations to further characterize the pathway. We investigated an extension provided by the factor graph approach in which the model distinguishes between inhibitory and stimulatory interactions. We found that the extension yielded significant improvements in recovering the structure of simulated and Saccharomyces cerevisae networks. We applied the approach to discover a signaling network among genes involved in a human colon cancer cell invasiveness pathway. The method predicts several genes with new roles in the invasiveness process. We knocked down two genes identified by our approach and found that both knock-downs produce loss of invasive potential in a colon cancer cell line. Nested effects models may be a powerful tool for inferring regulatory connections and genes that operate in normal and disease-related processes.

Identification of Candidate Genes and Gene Networks Specifically Associated with Analgesic Tolerance to Morphine

Chronic morphine administration may alter the expression of hundreds to thousands of genes. However, only a subset of these genes is likely involved in analgesic tolerance. In this report, we used a behavior genetics strategy to identify candidate genes specifically linked to the development of morphine tolerance. Two inbred genotypes [C57BL/6J (B6), DBA2/J (D2)] and two reciprocal congenic genotypes (B6D2, D2B6) with the proximal region of chromosome 10 (Chr10) introgressed into opposing backgrounds served as the behavior genetic filter. Tolerance after therapeutically relevant doses of morphine developed most rapidly in the B6 followed by the B6D2 genotype and did not develop in the D2 mice and only slightly in the D2B6 animals indicating a strong influence of the proximal region of Chr10 in the development of tolerance. Gene expression profiling and pattern matching identified 64, 53, 86, and 123 predisposition genes and 81, 96, 106, and 82 tolerance genes in the periaqueductal gray (PAG), prefrontal cortex, temporal lobe, and ventral striatum, respectively. A potential gene network was identified in the PAG in which 19 of the 34 genes were strongly associated with tolerance. Eleven of the network genes were found to reside in quantitative trait loci previously associated with morphine-related behaviors, whereas seven were predictive of tolerance (morphine-naive condition). Overall, the genes modified by chronic morphine administration show a strong presence in canonical pathways representative of neuroadaptation. A potentially significant role for the micro-RNA and epigenetic mechanisms in response to chronic administration of pharmacologically relevant doses of morphine was highlighted by candidate genes Dicer and H19.

Cross-Talk of Expression Quantitative Trait Loci Within 2 Interacting Blood Pressure Quantitative Trait Loci

Genetic dissection of the S rat genome has provided strong evidence for the presence of 2 interacting blood pressure quantitative trait loci (QTLs), termed QTL1 and QTL2, on rat chromosome 5. However, the identities of the underlying interacting genetic factors remain unknown. Further experiments targeted to identify the interacting genetic factors by the substitution mapping approach alone are difficult because of the interdependency of natural recombinations to occur at the 2 QTLs. We hypothesized that the interacting genetic factors underlying these 2 QTLs may interact at the level of gene transcription and thereby represent expression QTLs or eQTLs. To detect these interacting expression QTLs, a custom QTL chip containing the annotated genes within QTL1 and QTL2 was developed and used to conduct a transcriptional profiling study of S and 2 congenic strains that retain either 1 or both of the QTLs. The results uncovered an interaction between 2 transcription factor genes, Dmrta2 and Nfia. Furthermore, the “biological signature” elicited by these 2 transcription factors was differential between the congenic strain that retained Lewis alleles at both QTL1 and QTL2 compared with the congenic strain that retained Lewis alleles at QTL1 alone. A network of transcription factors potentially affecting blood pressure could be traced, lending support to our hypothesis.

NFκB Promotes Ovarian Tumorigenesis via Classical Pathways That Support Proliferative Cancer Cells and Alternative Pathways That Support ALDH+ Cancer Stem–like Cells

Understanding the mechanisms supporting tumor-initiating cells (TIC) is vital to combat advanced-stage recurrent cancers. Here, we show that in advanced ovarian cancers NFκB signaling via the RelB transcription factor supports TIC populations by directly regulating the cancer stem-like associated enzyme aldehyde dehydrogenase (ALDH). Loss of RelB significantly inhibited spheroid formation, ALDH expression and activity, chemoresistance, and tumorigenesis in subcutaneous and intrabursal mouse xenograft models of human ovarian cancer. RelB also affected expression of the ALDH gene ALDH1A2 Interestingly, classical NFκB signaling through the RelA transcription factor was equally important for tumorigenesis in the intrabursal model, but had no effect on ALDH. In this case, classical signaling via RelA was essential for proliferating cells, whereas the alternative signaling pathway was not. Our results show how NFκB sustains diverse cancer phenotypes via distinct classical and alternative signaling pathways, with implications for improved understanding of disease recurrence and therapeutic response. Cancer Res; 77(24); 6927-40. ©2017 AACR.

Characterization of ovarian cancer cell lines as in vivo models for preclinical studies

OBJECTIVE The value of cell lines for pre-clinical work lies in choosing those with similar characteristics. Selection of cell lines is typically based on patient history, histological subtype at diagnosis, mutation patterns, or signaling pathways. Although recent studies established consensus regarding molecular characteristics of ovarian cancer cell lines, data on in vivo tumorigenicity remains only sporadically available, impeding translation of in vitro work to xenograft models. METHODS We introduced 18 ovarian cancer cell lines into athymic nude mice through subcutaneous, intraperitoneal, and ovary intrabursal routes, and observed tumor development over 6weeks. We also profiled cell line gene expression and identified differentially expressed gene sets based on their ability to form tumors in the subcutaneous or intraperitoneal locations. Representative cell lines were further subjected to proteomic analyses. RESULTS Ovarian cancer cell lines showed variable ability to grow in mice when implanted subcutaneous, intraperitoneal, or intrabursal. While some cell lines grew well in both SC and IP locations, others showed a strong propensity to grow in one location only. Gene expression profiles suggested that cell lines showing preference for IP growth had gene expression patterns more similar to primary tumors. CONCLUSIONS We report the tumorigenicity of 17 human ovarian cancer cell lines and one mouse cell line in three distinct anatomical locations, and associated gene networks. Growth patterns and histopathology, linked to molecular characteristics, provide a valuable resource to the research community, and better guide the choice of cell lines for in vitro studies to translate efficiently into xenograft testing.

BRD4 facilitates DNA damage response and represses CBX5/Heterochromatin protein 1 (HP1)

Ovarian cancer (OC) is a heterogeneous disease characterized by defective DNA repair. Very few targets are universally expressed in the high grade serous (HGS) subtype. We previously identified that CHK1 was overexpressed in most of HGSOC. Here, we sought to understand the DNA damage response (DDR) to CHK1 inhibition and increase the anti-tumor activity of this pathway. We found BRD4 suppression either by siRNA or BRD4 inhibitor JQ1 enhanced the cytotoxicity of CHK1 inhibition. Interestingly, BRD4 was amplified and/or upregulated in a subset of HGSOC with statistical correlation to overall survival. BRD4 inhibition increased CBX5 (HP1α) level. CHK1 inhibitor induced DDR marker, γ-H2AX, but BRD4 suppression did not. Furthermore, nuclear localization of CBX5 and γ-H2AX was mutually exclusive in BRD4-and CHK1-inhibited cells, suggesting BRD4 facilitates DDR by repressing CBX5. Our results provide a strong rationale for clinical investigation of CHK1 and BRD4 co-inhibition, especially for HGSOC patients with BRD4 overexpression.Ovarian cancer (OC) is a heterogeneous disease characterized by defective DNA repair. Very few targets are universally expressed in the high grade serous (HGS) subtype. We previously identified that CHK1 was overexpressed in most of HGSOC. Here, we sought to understand the DNA damage response (DDR) to CHK1 inhibition and increase the anti-tumor activity of this pathway. We found BRD4 suppression either by siRNA or BRD4 inhibitor JQ1 enhanced the cytotoxicity of CHK1 inhibition. Interestingly, BRD4 was amplified and/or upregulated in a subset of HGSOC with statistical correlation to overall survival. BRD4 inhibition increased CBX5 (HP1α) level. CHK1 inhibitor induced DDR marker, γ-H2AX, but BRD4 suppression did not. Furthermore, nuclear localization of CBX5 and γ-H2AX was mutually exclusive in BRD4-and CHK1-inhibited cells, suggesting BRD4 facilitates DDR by repressing CBX5. Our results provide a strong rationale for clinical investigation of CHK1 and BRD4 co-inhibition, especially for HGSOC patients with BRD4 overexpression.

IΚΚε cooperates with either MEK or non-canonical NF-kB driving growth of triple-negative breast cancer cells in different contexts

BackgroundMetastatic breast cancer carries a poor prognosis despite the success of newly targeted therapies. Treatment options remain especially limited for the subtype of triple negative breast cancer (TNBC). Several signaling pathways, including NF-κB, are altered in TNBC, and the complexity of this disease implies multi-faceted pathway interactions. Given that IKKε behaves as an oncogene in breast cancer, we hypothesized that IKKε regulates NF-κB signaling to control diverse oncogenic functions in TNBC.MethodsVector expression and RNA interference were used to investigate the functional role of IKKε in triple-negative breast cancer cells. Viability, protein expression, NF-κB binding activity, invasion, anoikis, and spheroid formation were examined in cells expressing high or low levels of IKKε, in conjunction with p52 RNA interference or MEK inhibition.ResultsThis study found that non-canonical NF-κB p52 levels are inversely proportional to ΙΚΚε, and growth of TNBC cells in anchorage supportive, high-attachment conditions requires IKKε and activated MEK. Growth of these cells in anchorage resistant conditions requires IKKε and activated MEK or p52. In this model, IKKε and MEK cooperate to support overall viability whereas the p52 transcription factor is only required for viability in low attachment conditions, underscoring the contrasting roles of these proteins.ConclusionsThis study illustrates the diverse functions of IKKε in TNBC and highlights the adaptability of NF-κB signaling in maintaining cancer cell survival under different growth conditions. A better understanding of the diversity of NF-κB signaling may ultimately improve the development of novel therapeutic regimens for TNBC.

Identification of therapeutic targets applicable to clinical strategies in ovarian cancer

BackgroundshRNA-mediated lethality screening is a useful tool to identify essential targets in cancer biology. Ovarian cancer (OC) is extremely heterogeneous and most cases are advanced stages at diagnosis. OC has a high response rate initially, but becomes resistant to standard chemotherapy. We previously employed high throughput global shRNA sensitization screens to identify NF-kB related pathways. Here, we re-analyzed our previous shRNA screens in an unbiased manner to identify clinically applicable molecular targets.MethodsWe proceeded with siRNA lethality screening using the top 55 genes in an expanded set of 6 OC cell lines. We investigated clinical relevance of candidate targets in The Cancer Genome Atlas OC dataset. To move these findings towards the clinic, we chose four pharmacological inhibitors to recapitulate the top siRNA effects: Oxozeaenol (for MAP3K7/TAK1), BI6727 (PLK1), MK1775 (WEE1), and Lapatinib (ERBB2). Cytotoxic effects were measured by cellular viability assay, as single agents and in 2-way combinations. Co-treatments were evaluated in either sequential or simultaneous exposure to drug for short term and extended periods to simulate different treatment strategies.ResultsLoss-of-function shRNA screens followed by short-term siRNA validation screens identified therapeutic targets in OC cells. Candidate genes were dysregulated in a subset of TCGA OCs although the alterations of these genes showed no statistical significance to overall survival. Pharmacological inhibitors such as Oxozeaenol, BI6727, and MK1775 showed cytotoxic effects in OC cells regardless of cisplatin responsiveness, while all OC cells tested were cytostatic to Lapatinib. Co-treatment with BI6727 and MK1775 at sub-lethal concentrations was equally potent to BI6727 alone at lethal concentrations without cellular re-growth after the drugs were washed off, suggesting the co-inhibition at reduced dosages may be more efficacious than maximal single-agent cytotoxic concentrations.ConclusionsLoss-of-function screen followed by in vitro target validation using chemical inhibitors identified clinically relevant targets. This approach has the potential to systematically refine therapeutic strategies in OC. These molecular target-driven strategies may provide additional therapeutic options for women whose tumors have become refractory to standard chemotherapy.

Abstract AP13: NF-KAPPAB CLASSICAL VERSUS ALTERNATIVE PATHWAYS SUPPORT DISTINCT POPULATIONS OF OVARIAN CANCER TUMOR-INITIATING CELLS

Ovarian cancer is the most lethal gynecological malignancy in the United States with high morbidity and mortality due to recurrence and chemoresistance. Our data suggest tumor-initiating cells (TICs) play an important role in disease biology. We previously showed that a subset of ovarian cancer cells depends on NF-kappaB signaling, and that expression of NF-kappaB proteins is associated with poor survival. Given that NF-kappaB expression correlates with a poor outcome in ovarian cancer, and NF-kappaB activity supports drug resistance and tumorigenicity, we hypothesize that NF-kappaB supports a TIC program responsible for ovarian cancer relapse. To investigate this pathway in TICs, we designed a novel method to enrich for TICs from cell lines and patient samples by culturing non-adherent, floating cells in stem cell conditions defined by low attachment flasks and serum free media. Preliminary data confirm that these cells have higher stem cell marker expression, are chemoresistant, and are more tumorigenic in nude mice compared to their adherent counterparts. These TIC-enriched culture conditions enhance NF-kappaB expression and activity. More specifically, alternative NF-kappaB signaling through the RelB transcription factor supports TIC populations by regulating aldehyde dehydrogenase (ALDH), an enzyme with high activity in TICs. Using an inducible shRNA targeting RelB we show that spheroid formation, ALDH expression and activity, chemoresistance, and tumorigenesis in both subcutaneous and intrabursal xenograft models, significantly decreased in the absence of RelB. Interestingly, loss of classical NF-kappaB signaling, through an shRNA targeting the RelA transcription factor, was less effective in targeting TICs, but more effective at targeting a proliferative subpopulation with high Ki67 staining. We conclude that classical and alternative NF-kappaB signaling support ovarian tumor cells with distinct phenotypes and the collaboration of these pathways may be critical for supporting tumor repopulation following chemotherapy. Current studies will measure system level changes induced by these shRNAs, and identify a gene signature specific to each NF-kappaB pathway in ovarian TICs. Clarifying the nuances of NF-kappaB signaling in TICs will increase our understanding of ovarian cancer recurrence and further focus therapeutic strategies to prevent relapse. Citation Format: Carrie D. House, Elizabeth A. Jordan, Christina M. Annunziata. NF-KAPPAB CLASSICAL VERSUS ALTERNATIVE PATHWAYS SUPPORT DISTINCT POPULATIONS OF OVARIAN CANCER TUMOR-INITIATING CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP13.