Anna K. Unruh

Clonal Evolution in Breast Cancer Revealed by Single Nucleus Genome Sequencing

Sequencing studies of breast tumour cohorts have identified many prevalent mutations, but provide limited insight into the genomic diversity within tumours. Here we developed a whole-genome and exome single cell sequencing approach called nuc-seq that uses G2/M nuclei to achieve 91% mean coverage breadth. We applied this method to sequence single normal and tumour nuclei from an oestrogen-receptor-positive (ER+) breast cancer and a triple-negative ductal carcinoma. In parallel, we performed single nuclei copy number profiling. Our data show that aneuploid rearrangements occurred early in tumour evolution and remained highly stable as the tumour masses clonally expanded. In contrast, point mutations evolved gradually, generating extensive clonal diversity. Using targeted single-molecule sequencing, many of the diverse mutations were shown to occur at low frequencies (<10%) in the tumour mass. Using mathematical modelling we found that the triple-negative tumour cells had an increased mutation rate (13.3×), whereas the ER+ tumour cells did not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer.

Tumour angiogenesis regulation by the miR-200 family

The miR-200 family is well known to inhibit the epithelial-mesenchymal transition, suggesting it may therapeutically inhibit metastatic biology. However, conflicting reports regarding the role of miR-200 in suppressing or promoting metastasis in different cancer types have left unanswered questions. Here we demonstrate a difference in clinical outcome based on miR-200s role in blocking tumour angiogenesis. We demonstrate that miR-200 inhibits angiogenesis through direct and indirect mechanisms by targeting interleukin-8 and CXCL1 secreted by the tumour endothelial and cancer cells. Using several experimental models, we demonstrate the therapeutic potential of miR-200 delivery in ovarian, lung, renal and basal-like breast cancers by inhibiting angiogenesis. Delivery of miR-200 members into the tumour endothelium resulted in marked reductions in metastasis and angiogenesis, and induced vascular normalization. The role of miR-200 in blocking cancer angiogenesis in a cancer-dependent context defines its utility as a potential therapeutic agent.

Clinically relevant microRNAs in ovarian cancer.

microRNAs (miRNAs/miRs) belong to a class of small noncoding RNAs that can negatively regulate messenger RNA (mRNA) expression of target genes. miRNAs are involved in multiple aspects of ovarian cancer cell dysfunction and the phenotype of ovarian cancer cells can be modified by targeting miRNA expression. miRNA profiling has detected a number of candidate miRNAs with the potential to regulate many important biologic functions in ovarian cancer, but their role still needs to be clarified, given the remarkable heterogeneity among ovarian cancers and the context-dependent role of miRNAs. This review summarizes the data collected from The Cancer Genome Atlas (TCGA) and several other genome-wide projects to identify dysregulated miRNAs in ovarian cancers. Copy number variations (CNVs), epigenetic alterations, and oncogenic mutations are also discussed that affect miRNA levels in ovarian disease. Emphasis is given to the role of particular miRNAs in altering expression of genes in human ovarian cancers with the potential to provide diagnostic, prognostic, and therapeutic targets. Particular attention has been given to TP53, BRCA1/2, CA125 (MUC16), HE4 (WFDC2), and imprinted genes such as ARHI (DIRAS3). A better understanding of the abnormalities in miRNA expression and downstream transcriptional and biologic consequences will provide leads for more effective biomarkers and translational approaches in the management of ovarian cancer. Mol Cancer Res; 13(3); 393–401. ©2014 AACR.

Molecular Biomarkers of Residual Disease after Surgical Debulking of High-Grade Serous Ovarian Cancer

Purpose: Residual disease following primary cytoreduction is associated with adverse overall survival in patients with epithelial ovarian cancer. Accurate identification of patients at high risk of residual disease has been elusive, lacking external validity and prompting many to undergo unnecessary surgical exploration. Our goal was to identify and validate molecular markers associated with high rates of residual disease. Methods: We interrogated two publicly available datasets from chemonaïve primary high-grade serous ovarian tumors for genes overexpressed in patients with residual disease and significant at a 10% false discovery rate (FDR) in both datasets. We selected genes with wide dynamic range for validation in an independent cohort using quantitative RT-PCR to assay gene expression, followed by blinded prediction of a patient subset at high risk for residual disease. Predictive success was evaluated using a one-sided Fisher exact test. Results: Forty-seven probe sets met the 10% FDR criterion in both datasets. These included FABP4 and ADH1B, which tracked tightly, showed dynamic ranges >16-fold and had high expression levels associated with increased incidence of residual disease. In the validation cohort (n = 139), FABP4 and ADH1B were again highly correlated. Using the top quartile of FABP4 PCR values as a prespecified threshold, we found 30 of 35 cases of residual disease in the predicted high-risk group (positive predictive value = 86%) and 54 of 104 among the remaining patients (P = 0.0002; OR, 5.5). Conclusion: High FABP4 and ADH1B expression is associated with significantly higher risk of residual disease in high-grade serous ovarian cancer. Patients with high tumoral levels of these genes may be candidates for neoadjuvant chemotherapy. Clin Cancer Res; 20(12); 3280–8. ©2014 AACR.

Most expression and splicing changes in myotonic dystrophy type 1 and type 2 skeletal muscle are shared with other muscular dystrophies

The prevailing pathomechanistic paradigm for myotonic dystrophy (DM) is that aberrant expression of embryonic/fetal mRNA/protein isoforms accounts for most aspects of the pleiotropic phenotype. To identify aberrant isoforms in skeletal muscle of DM1 and DM2 patients, we performed exon-array profiling and RT-PCR validation on the largest DM sample set to date, including Duchenne, Becker and tibial muscular dystrophy (NMD) patients as disease controls, and non-disease controls. Strikingly, most expression and splicing changes in DM patients were shared with NMD controls. Comparison between DM and NMD identified almost no significant differences. We conclude that DM1 and DM2 are essentially identical for dysregulation of gene expression, and DM expression changes represent a subset of broader spectrum dystrophic changes. We found no evidence for qualitative splicing differences between DM1 and DM2. While some DM-specific splicing differences exist, most of the DM splicing differences were also seen in NMD controls. SSBP3 exon 6 missplicing was observed in all diseased muscle and led to reduced protein. We conclude there is no widespread DM-specific spliceopathy in skeletal muscle and suggest that missplicing in DM (and NMD) may not be the driving mechanism for the muscle pathology, since the same pathways show expression changes unrelated to splicing.

Abstract 2273: Mechanistic and functional implications of FABP4 in ovarian cancer metastasis

Purpose:The purpose of this study was to identify molecular predictors of residual disease (RD) in high grade serous ovarian cancer (HGSC) and further understand their role in promoting cancer metastasis. Method:The current study analyzed Affymetrix gene expression data of 504 HGSC cases from The Cancer Genome Atlas (TCGA) data to identify differentially expressed genes in tumors from patients with no gross residual disease after surgery (NRD) or presence of RD following initial debulking surgery. It was followed by qRT-PCR analysis of tumor samples for validation purposes. RPPA data of 354 patients from TCGA were analyzed. Immunohistochemical analysis was performed on the patient samples to determine the expression at the protein level (cancer versus stromal cells). Gene array was carried out after overexpressing the selected gene in ovarian cancer cells and the data was analyzed by Ingenuity Pathway Analysis (IPA). In vitro (migration and invasion) and in vivo (orthotopic mouse models) assays were used to determine the biological roles of gene(s) identified from the above analyses. Results: In TCGA data set, 97/107 (90.6%) of the patients with high expression of FABP4 gene had residual disease. In the validation cohort, among the 35 patients predicted to be at high risk for residual disease, 30 (86%) did have residual disease. In contrast, only 54 of the 104 patients with FABP4 values below the decision threshold (52%) had incomplete resection (p = 0.0002). RPPA analysis indicated that expression of FABP4 was positively correlated (Spearman correlation analysis) with expression of several other proteins known to increase tumor cell infiltration and metastasis such as JNK2 (p = 0.194), transglutaminase (p = 0.199), c-kit (p = 0.173), fibronectin (p = 0.364), PKC-A (p = 0.178), collagen-6 (p = 0.197) and paxillin (p = 0.239). It was negatively correlated with E-cadherin (p = -0.246) and claudin-7 (p = -0.201) expression. Immunohistochemical analysis confirmed that apart from endothelial cells and adipocytes, cancer cells also express significant amount of FABP4. In vitro assays showed significant reduction in invasion and migration after silencing FABP4 in HGSC cell lines (p Conclusion:These findings provide a new understanding of ovarian cancer metastasis and identify a potentially important target for therapeutic intervention. Citation Format: Kshipra M. Gharpure, Susan L. Tucker, Shelley M. Herbrich, Anna K. Unruh, Alpa M. Nick, Erin K. Crane, Robert L. Coleman, Jamie Guenthoer, Heather J. Dalton, Sherry Y. Wu, Rajesha Rupaimoole, Gabriel Lopez-Berestein, Bulent Ozpolat, Cristina Ivan, Wei Hu, Keith Baggerly, Anil Sood. Mechanistic and functional implications of FABP4 in ovarian cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2273. doi:10.1158/1538-7445.AM2015-2273

Abstract 5466: Integrative TCGA analyses identify Basonuclin1 (BNC1) as a key mediator for platinum resistance

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Objective. We have previously shown that platinum-resistant (disease progression within 6 months of diagnosis) high-grade serous ovarian cancer (HGSOC) expresses higher levels of basonuclin1 (BNC1) compared to platinum-sensitive tumors (no disease progression for more than 2 years). Here, we aim to systematically examine the functional effects of BNC1 silencing on tumor sensitivity to platinum and other commonly used chemotherapeutic agents in multiple cancer models. Methods. An integrative analysis was performed using The Cancer Genome Atlas (TCGA) dataset to systematically identify genes associated with platinum-resistance in patients with HGSOC. The biological effects of BNC1, one of the lead candidates identified, were studied in HGSOC (HeyA8-MDR, PEO4), endometrioid ovarian (A2780-CP20), clear cell ovarian (ES2), and squamous cell lung (H1299) cancer models both in vitro and in vivo. The effects of BNC1 silencing or overexpression on the sensitivity of cancer cells to paclitaxel, gemcitabine, doxorubicin, and cisplatin treatments were assessed. Results. BNC1 expression was increased by more than two-fold in platinum-resistant tumors in the TCGA ovarian cancer dataset. BNC1 silencing increased the sensitivity of A2780-CP20, HeyA8-MDR, PEO4, ES2, and H1299 cells to cisplatin and doxorubicin treatments, but not to gemcitabine or taxane-based therapies. BNC1-silenced cells showed a 3-fold average increase in the percentage of apoptotic cells following cisplatin treatment, when compared to cells treated with control siRNA. Importantly, combination treatment of siBNC1 and cisplatin resulted in a 90% reduction in tumor burden in platinum-resistant A2780-CP20 and HeyA8-MDR orthotopic ovarian cancer mouse models when compared to control siRNA plus cisplatin treatment group. Overexpression of BNC1 in platinum-sensitive A2780 cells led to a 2.5-fold reduction in cisplatin sensitivity in vivo as measured by total tumor burden 4 weeks after therapy. Conclusion. Collectively, BNC1 represents an important target for enhancing platinum-sensitivity in ovarian and lung cancer. Targeting BNC1 in platinum-resistant malignancies may improve patient survival. Note: This abstract was not presented at the meeting. Citation Format: Sherry Y. Wu, Justyna Filant, Michael McGuire, Rajesha Rupaimoole, Sunila Pradeep, Anna Unruh, Herbrich Shelley, Cristina Ivan, Ruder Dennis, Cristian Rodriguez-Aguayo, Vasudha Sehgal, Takahito Miyake, Archana Nagaraja, Kshipra Gharpure, Guillermo Armaiz, Rebecca Previs, Gabriel Lopez-Berestein, Prahlad Ram, Keith Baggerly, Anil Sood. Integrative TCGA analyses identify Basonuclin1 (BNC1) as a key mediator for platinum resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5466. doi:10.1158/1538-7445.AM2015-5466

Abstract LB-310: Single cell genome sequencing reveals clonal stability and diversity in breast cancer

Human breast cancers often display intratumor genomic heterogeneity. This clonal diversity confounds the clinical diagnosis and basic research of cancer, because single samples may not represent that tumor as a whole. Sequencing breast tumor cohorts en masse has identified many prevalent mutations, but has limited ability for resolving subclonal diversity. Here, we developed a whole-genome and exome single-cell sequencing approach (Nuc-Seq) using G2/M cells. To validate our method, we applied Nuc-Seq to sequence the whole genomes of two single cells from a genetically monoclonal breast cancer cell line (SK-BR-3) at high coverage depth (61X ± 5 sem, n=2) and breadth (83.70% ± 3.40 sem, n=2) to detect somatic mutations. Our analysis suggests that Nuc-Seq generates low allelic dropout rates (9.73% ± 2.19%) and low false positive error rates for point mutations (FPR = 1.24e-6). We then applied this method to sequence single normal and tumor cells from an estrogen-receptor positive breast cancer and a triple-negative ductal carcinoma at base-pair resolution. In parallel, we performed single cell copy number profiling. In both tumors, we observed a large number of rare variants that were not detected by sequencing the bulk tumor en masse. In contrast, we find that single cell copy number profiles are highly similar. Our data suggest that aneuploid rearrangements occurred early in tumor evolution and remained highly stable as the tumor mass expanded. In contrast we find that point mutations evolved gradually, generating extensive clonal diversity. Many of the diverse mutations were shown to occur at low frequencies (0.03 -10%) in the tumor mass by targeted duplex sequencing. Mathematical modeling suggests that the triple-negative tumor cells have an increased mutation rate (13.3X), while the ER+ tumor cells do not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer. Citation Format: Yong Wang, Nicholas Navin, Jill Waters, Marco Leung, Anna Unruh, Xiuqing Shi, Whijae Roh, Ken Chen, Paul Scheet, Selina Vattathil, Han Liang, Asha Multani, Hong Zhang, Funda Meric-Bernstam, Franziska Michor, Rui Zhao. Single cell genome sequencing reveals clonal stability and diversity in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-310. doi:10.1158/1538-7445.AM2014-LB-310

Abstract 2036: Overcoming platinum-resistance in ovarian cancer via targeting basonuclin1 (BNC1).

Purpose This study aims to interrogate if basonuclin1 (BNC1), a key gene identified through integrative TCGA-based functional genomic analysis, is a relevant target for overcoming chemo-resistance in high-grade serous ovarian cancer (HGS-OvCa). BNC1 is a transcription factor which can regulate ribosomal biogenesis and cell proliferation. Methods TCGA mRNA expression and clinical response data were used to systematically identify genes associated with chemo-resistance in patients with HGS-OvCa. Gene-specific effects were subsequently studied for selected targets in vitro. BNC1 targeted siRNA was used to study the functional role of BNC1 in chemo-resistance. Apoptosis and cell cycle analyses were carried out following BNC1 silencing in ovarian cancer cell lines with or without the presence of cisplatin. The in vivo effects of silencing BNC1 on the chemosensitivity were tested using an orthotopic mouse model (A2780CP20) of ovarian cancer. Results The expression of BNC1 was found to be increased by more than two-fold (Agilent platform; p Citation Format: Sherry Y. Wu, Anna K. Unruh, Rajesha Rupaimoole, Cristina Ivan, Dennis Ruder, Sunila Pradeep, Vasudha Sehgal, Cristian Rodriguez-Aguayo, Chad Pecot, Takahito Miyake, Gabriel Lopez-Berestein, Prahlad Ram, Keith A. Baggerly, Anil K. Sood. Overcoming platinum-resistance in ovarian cancer via targeting basonuclin1 (BNC1). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2036. doi:10.1158/1538-7445.AM2013-2036

Abstract 3157: Clinical relevance of miR-200 family members in various cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Low expression of miR-200 family members in some cancers has been linked to epithelial-mesenchymal transition and subsequent metastatic spread. Thus, low expression of miR-200 members would be expected to reduce survival. We investigated whether expression levels of miR-200 family members are associated with clinical outcome in several cancer types. Methods: We used clinically annotated data from The Cancer Genome Atlas (TCGA). Our preliminary analysis focused on nine different cancer types, subsequently reduced to four that had a large number of samples (>200). Expression data for all five miR-200 members (miR-141, -200a, -200b, -200c, and -429) were obtained from either Agilent miRNA microarrays (8x15K; glioblastoma multiforme and ovarian cancers) or miR-Seq (Illumina GA and HiSeq; breast and clear cell renal cancers). Approximately 2/3 of the samples for each tumor type were used as a training cohort to obtain the miRNA expression thresholds yielding the most significant log rank test p-values (one-tailed test), thus dividing the data into good and poor prognosis groups. These thresholds were then used to divide the data in the remaining 1/3 of the samples that functioned as independent validation cohorts, and p-values were computed for those cohorts. The same procedure was repeated again to look for significant differences in progression free survival. Kaplan-Meier curves were also plotted for all datasets, with a p-value 3500 days for low versus high miR-200a expression, respectively (p=0.005). However, in breast cancer (n=325) and glioblastoma multiforme (GBM, n=487), low expression of a miR-200 member was associated with an improved clinical outcome. In the breast cancer validation set (113 samples), median overall survival was > 7000 versus 3100 days for low versus high miR-141 expression, respectively (p=0.036). In the GBM validation set (174 samples), median progression free survival was 290 versus 150 days for low versus high miR-429 expression, respectively (p=0.009). Discussion: Here we show that altered expression levels of miR-200 members may have clinical relevance in common cancers. Interestingly, depending on the cancer type, low miR-200 member levels may either portend a better or worse prognosis. This study highlights the need to further elucidate novel mechanisms of the miR-200 family and perhaps exploit their diverse functions for therapeutic intent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3157. doi:1538-7445.AM2012-3157