Kirsten Ruigrok-Ritstier

Association of an Extracellular Matrix Gene Cluster with Breast Cancer Prognosis and Endocrine Therapy Response

Purpose: We previously discovered an extracellular matrix (ECM) gene cluster associated with resistance to first-line tamoxifen therapy of patients with metastatic breast cancer. In this study, we determined whether the six individual ECM genes [collagen 1A1 (COL1A1), fibronectin 1 (FN1), lysyl oxidase (LOX), secreted protein acidic cysteine-rich (SPARC), tissue inhibitor of metalloproteinase 3 (TIMP3), and tenascin C (TNC)] were associated with treatment response, prognosis, or both. Experimental Design: In 1,286 primary breast tumors, mRNA expression (quantitative real-time PCR) was related to clinicopathologic factors and disease outcome in univariate and multivariate analysis including traditional factors. Results:TIMP3, FN1, LOX, and SPARC expression levels (continuous variables) were significantly associated with distant metastasis-free survival (MFS) in 680 lymph node–negative untreated patients (P < 0.03). Using a calculated linear prognostic score, these patients were evenly divided into five prognostic groups with a significant difference in 10-year MFS of ∼40% between the two extreme prognostic groups. Furthermore, high TNC expression as continuous variable was associated with (a) shorter MFS in 139 estrogen receptor–positive and lymph node–positive patients who received adjuvant tamoxifen therapy (hazard ratio, 1.53; P = 0.001), and (b) no clinical benefit (odds ratio, 0.81; P = 0.035) and shorter progression-free survival (hazard ratio, 1.19; P = 0.002) in 240 patients in whom recurrence was treated with tamoxifen as first-line monotherapy. These results were also significant in multivariate analyses. Conclusion:FN1, LOX, SPARC, and TIMP3 expression levels are associated with the prognosis of patients with breast cancers, whereas TNC is associated with resistance to tamoxifen therapy. Further validation and functional studies are necessary to determine the use of these ECM genes in decisions regarding treatment and whether they can serve as targets for therapy.

Ovarian Cancer Cell Line Panel (OCCP): Clinical Importance of In Vitro Morphological Subtypes

Epithelial ovarian cancer is a highly heterogeneous disease and remains the most lethal gynaecological malignancy in the Western world. Therapeutic approaches need to account for inter-patient and intra-tumoural heterogeneity and detailed characterization of in vitro models representing the different histological and molecular ovarian cancer subtypes is critical to enable reliable preclinical testing. There are approximately 100 publicly available ovarian cancer cell lines but their cellular and molecular characteristics are largely undescribed. We have characterized 39 ovarian cancer cell lines under uniform conditions for growth characteristics, mRNA/microRNA expression, exon sequencing, drug response for clinically-relevant therapeutics and collated all available information on the original clinical features and site of origin. We tested for statistical associations between the cellular and molecular features of the lines and clinical features. Of the 39 ovarian cancer cell lines, 14 were assigned as high-grade serous, four serous-type, one low-grade serous and 20 non-serous type. Three morphological subtypes: Epithelial (n = 21), Round (n = 7) and Spindle (n = 12) were identified that showed distinct biological and molecular characteristics, including overexpression of cell movement and migration-associated genes in the Spindle subtype. Comparison with the original clinical data showed association of the spindle-like tumours with metastasis, advanced stage, suboptimal debulking and poor prognosis. In addition, the expression profiles of Spindle, Round and Epithelial morphologies clustered with the previously described C1-stromal, C5-mesenchymal and C4 ovarian subtype expression profiles respectively. Comprehensive profiling of 39 ovarian cancer cell lines under controlled, uniform conditions demonstrates clinically relevant cellular and genomic characteristics. This data provides a rational basis for selecting models to develop specific treatment approaches for histological and molecular subtypes of ovarian cancer.

Downregulation of SIAH2, an ubiquitin E3 ligase, is associated with resistance to endocrine therapy in breast cancer

Purpose In our microarray analysis we observed that Seven-in-Absentia Homolog 2 (SIAH2) levels were low in estrogen receptor (ER) positive breast tumors of patients resistant to first-line tamoxifen therapy. The aim of this study was to evaluate SIAH2 for its (a) predictive/prognostic value, and (b) functional role in endocrine therapy resistance. Patients and methods SIAH2 expression was measured with quantitative Real-Time-PCR (qRT-PCR) in 1205 primary breast tumor specimens and related to disease outcome. The functional role of SIAH2 was determined in human breast cancer cell lines ZR-75-1, ZR/HERc, and MCF7. Cell lines were treated with estrogen (E2), anti-estrogen ICI164.384 or epidermal growth factor (EGF). Moreover, MCF7 was treated with ICI164.384 after silencing SIAH2 expression. Results SIAH2 was not prognostic in 603 lymph node negative patients who had not received adjuvant systemic therapy. In multivariate analysis of ER-positive tumors of 235 patients with recurrent disease, SIAH2 as continuous variable, significantly predicted first-line tamoxifen treatment failure (OR = 1.48; P = 0.05) and progression-free survival (PFS) (HR = 0.79; P = 0.007). Furthermore, in primary breast cancer patients treated with adjuvant tamoxifen, SIAH2 predicted metastasis-free survival (MFS) (HR = 0.73; P = 0.005). In vitro experiments showed that SIAH2 silencing in MCF7 cells resulted in resistance to ICI164.384-treatment when compared with mock silenced cells (P = 0.008). Interestingly, in ZR cells transfected with EGFR (ZR/HERc), SIAH2 expression was induced by E2 but downregulated by EGF. Conclusion In primary breast tumor specimens as well as in vitro low SIAH2 levels associated with resistance to endocrine therapy. Moreover, SIAH2 expression showed an opposite regulation by E2 and EGF.

LH receptor gene expression is essentially absent in breast tumor tissue: implications for treatment

Worldwide, breast cancer is the most frequently occurring malignancy in women. Early age at full term pregnancy has a protective effect against breast cancer. Evidence coming from a rat breast cancer model suggests a possible role for the pregnancy hormone hCG, a ligand of the LH receptor, as a mediator for this effect. In a previous study, we found that a common polymorphism in the LH receptor associates with tumor progression in premenopausal breast cancer patients, as carriers of the variant receptor showed a shorter disease free survival compared to non-carriers. How hCG and its receptor exert their effects on breast cancer, however, is unclear. One possibility is that these effects take place through LH receptors present in the ovaries, thereby influencing steroid hormone production. Another possibility is that the effects take place through LH receptors present in breast tumor cells themselves, as some studies have detected the receptor in both normal and neoplastic breast tissues and in breast cancer cell lines. To investigate whether a direct effect of LH signaling in breast cancer is likely, we measured LH receptor mRNA expression levels in 1551 breast tumors and 42 different human breast cancer cell lines using a qRT-PCR with a wide dynamic range. In addition, associations between LH receptor expression and clinico-pathologic factors were investigated. Assay validation showed that as little as ?10 copies per reaction volume of LH receptor cDNA could still be detected by our assay. We show that LH receptors are undetectable in 62% of breast tumor samples and 41 of 42 breast cancer cell lines. For the remaining samples we found expression levels to be very low. Although low, expression of the LH receptor appears to be associated with normal breast cells, favorable tumor characteristics and low tumor percentage. Since expression of the LH receptor in breast cancer cells is very low, it almost excludes the possibility of direct signaling effects. We therefore conclude that signaling effects of the LH receptor on breast cancer most likely take place by an indirect pathway through the ovaries.

LRG1 mRNA expression in breast cancer associates with PIK3CA genotype and with aromatase inhibitor therapy outcome

PIK3CA is the most frequent somatic mutated oncogene in estrogen receptor (ER) positive breast cancer. We previously observed an association between PIK3CA genotype and aromatase inhibitors (AI) treatment outcome. This study now evaluates whether expression of mRNAs and miRs are linked to PIK3CA genotype and are independently related to AI therapy response in order to define potential expressed biomarkers for treatment outcome.

Abstract 4238: Unique ovarian cancer cell line panel: Molecular subtypes and therapy response

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Epithelial ovarian cancer is the most lethal gynecological malignancy in the Western world. Up to 70% of the patients do not survive the first 5 years after diagnosis, mostly due to acquired chemotherapy resistance. With the goal to stratify a heterogeneous group of patients, recently, a number of studies have identified molecular subtypes within ovarian carcinomas. However, the relation with response to chemotherapeutics is unknown. An extensive model system in which the response to different chemotherapeutics can be related to molecular subtypes is therefore imperative to know how to individualize treatment based on these molecular subtypes. We have an (increasing) collection of so far 40 ovarian cancer cell lines. The large number and extensive characterization of the cell lines makes this a unique model. We have data of the growth and morphological properties, exon-based gene expression (22.000 genes), microRNA expression (400 miRNAs), mutation analysis (PIK3CA, RAF & RAS genes), microsatellite instability status (MSI) and response curves for eight chemotherapeutics used in first- and second-line treatment. Unsupervised absolute clustering using the most variable expressed genes and microRNAs revealed two even clusters of cell lines with significantly different characteristics. The cell lines in the first cluster compared to the second: -show more often a round and less often an epithelial morphology (cell lines with spindle morphology are divided over both clusters), -show more often no Epithelial Cell Adhesion Molecule (EpCAM) and high CD44 membrane expression -are highly proliferative -are more often PI3K/Ras mutated and MSI Interestingly, the cell lines within the first cluster are significantly more sensitive to six of the eight tested chemotherapeutics compared to the second cluster BRB class comparison analysis revealed that 287 genes were differentially expressed between the two clusters (permutation p<0.01, FDR<5%, 10.000 permutations). Pathway analysis of these genes revealed the significantly overrepresented canonical pathways and molecular & cellular functions including Interferon Signaling (p=0.007), Arachidonic Acid Metabolism (p=0.030) and Cell-To-Cell Signaling and Interaction (p<0.03). We are currently identifying the published clinical molecular subtypes in our cell lines and relating it with response to treatment. This will give information on which of the commonly used therapeutics are expected to be most effective in the different molecular subtype. In the future, the response to targeted therapeutics can be tested in our cell line panel which may give additional information on how to treat each subtype. In conclusion, this large panel of extremely well characterized ovarian cancer cell lines is a unique model system that can be used in many future translational and functional studies and we are happy to collaborate (j.helleman@erasmusmc.nl). 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 4238. doi:1538-7445.AM2012-4238

Abstract P6-04-08: FOXA1 expression: regulated by EZH2 and associated with favorable outcome to tamoxifen in advanced breast cancer

Background: Enhancer of Zeste Homolog 2 (EZH2) is a member of the polycomb complex 2 and serves as a histone methyltransferase. Through trimethylation of histone 3 of lysine 27, EZH2 regulates ‘genome wide’ gene transcription silencing. Downregulation of EZH2 is associated with increased expression of the estrogen receptor (ER) and favorable outcome to tamoxifen in metastatic breast cancer. The binding of ER to its target genes is enhanced by Forkhead Box A1 (FOXA1), a pioneer factor that binds to and opens chromatized DNA. The aim of this study is to investigate the potential association between EZH2 and FOXA1 and their relation with treatment outcome in patients with advanced breast cancer. Experimental design: EZH2 and FOXA1 mRNA levels were analysed using available gene expression profile-data of 344 primary breast cancer specimens of lymph-node-negative (LNN) patients and in 109 ER-positive (ER+) tumors of patients with metastatic disease treated with first-line tamoxifen. To functionally analyze EZH2, cell lines were generated with different knockdown-constructs for EZH2, followed by evaluation of mRNA and protein expression levels and chromatin immunoprecipitation (ChIP) experiments combined with qPCR and/or next generation sequencing (NGS; ChIPseq). Results: In the 344 LNN breast tumors, EZH2 was highly expressed in breast tumors of the basal subtype. In contrast, FOXA1 was hardly expressed in this subtype, but highly in the luminal A and B subtypes. FOXA1 was related to a prolonged time to progression (TTP) after tamoxifen (HR = 0.51 [0.35–0.74], P We obtained 50–70% EZH2 knockdown in the breast cancer cell lines MCF7 (ER+/PR+/FOXA1+), SUM52PE (ER+/PR−/FOXA1+), and MM231 (ER−/PR−/FOXA1−). ChIP followed by qPCR demonstrated higher levels of H3K27 trimethylation at the ER-locus in the parental cell lines compared to the EZH2-knockdowns for MM231 and at the PR-locus for SUM52PE and MM231. ChIPseq evaluation in the EZH2 knockdown of MM231 identified 200 loci with a significant decrease in read numbers for H3K27me3 compared to the parental. Interestingly, amongst these 200 loci the FOXA1-locus was identified. These results suggest FOXA1 as an EZH2-target since its expression was not seen in the parental MM231. Conclusion: High EZH2 and low FOXA1 mRNA levels are associated with poor clinical outcome for tamoxifen therapy of advanced breast cancer. Functional studies indicate that EZH2 might regulate the expression of FOXA1. These results suggest that EZH2 and/or FOXA1 could be used as a predictive marker to identify patients at risk for tamoxifen therapy failure and possibly as a target for therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-08.

Abstract A15: High miRNA-26a and low EZH2 expression levels are associated with favorable outcome to tamoxifen in advanced breast cancer

Background: In breast cancer we have recently discovered that decreased EZH2 expression levels are associated with a favorable outcome to tamoxifen in advanced disease. Furthermore, EZH2 knockdown in the breast cancer cell line MCF7 resulted in estrogen receptor (ER) upregulation and increased sensitivity to anti-estrogens. Interestingly, EZH2 has been identified as target of miRNA-26a and miRNA-101. Objective: The main objective of this study is to investigate miRNA-26a and miRNA-101 for: A) their association with EZH2 and B) their predictive value in breast cancer patients treated with first-line tamoxifen monotherapy. Materials & Methods: Expression levels of miRNA-26a, miRNA-101, EZH2 and references were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in 235 ER-positive primary breast cancer specimens from patients with advanced disease. The levels of expression were related to clinicopathologic factors and disease outcome. Computations were performed with STATA and P-values Results: Expression levels of miRNA-26a and miRNA-101 were not correlated with age, tumor grade, tumor size, and nodal status. The miRNA-26a levels were significantly associated with progesterone (PgR) levels, HER2-status and EGFR levels, whereas miRNA-101 levels showed significant relations with PgR expression and menopausal status. The miRNA-26a and miRNA-101 levels showed an inverse relation with EZH2 mRNA levels (Spearman Rank Correlation of −0.21 and −0.15, respectively, P Conclusion & Discussion: The miRNA-26a and miRNA-101 levels have an inverse relation with EZH2, but only miRNA-26a has predictive value in advanced breast cancer. High levels of miRNA-26a and low EZH2 levels are associated with a favorable outcome to tamoxifen therapy. Interestingly, estrogens repress miRNA-26a expression suggesting that ER-positive tumors have less miRNA-26a than ER-negative tumors. This contradicts, however, with the lower EZH2 levels observed in ER-positive compared to ER-negative breast cancers. To clarify this paradox, future (functional) studies will focus on miRNA-26a and EZH2 in different breast cancer subtypes and determine their activated pathways in relation with response to anti-estrogens. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A15

Abstract P4-02-16: High miRNA26A1 and Low EZH2 Expression Levels Are Associated with Favorable Outcome to Tamoxifen in Advanced Breast Cancer through Similar Molecular Pathways

Background: We showed that decreased expression levels of EZH2 are associated with a favorable outcome to tamoxifen in advanced breast cancer. Furthermore, EZH2 knockdown in MCF7 cells resulted in estrogen receptor (ER) upregulation and increased sensitivity to anti-estrogens. Recently, EZH2 has been identified as a target of miRNA26A1 and miRNA101. Objective: To associate miRNA26A1 and miRNA101 expression levels with: A) EZH2 and B) molecular pathways and C) outcome to first-line tamoxifen monotherapy for advanced disease. Materials & Methods: Expression levels of miRNA26A1, miRNA101, EZH2 and references (miRNA-132 and miRNA-374) were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in 235 ER-positive primary breast cancer specimens from patients with advanced disease. The levels of expression were related to clinicopathologic factors and disease outcome. Pathway analysis was performed in a subset of 65 ER-positive tumors with available gene expression microarray data available. Computations were performed with STATA and P-values Results: The miRNA26A1 levels were significantly associated with levels of ER, progesterone (PgR), HER2 and EGFR, whereas miRNA101 levels showed significant relations with PgR expression and menopausal status. The miRNA26A1 and miRNA101 levels showed an inverse relation with EZH2 mRNA levels (Spearman Rank Correlation of -0.21 and -0.15, respectively, P Conclusions: The miRNA26A1 and miRNA101 levels have an inverse relation with levels of EZH2, however, only miRNA26A1 has predictive value in advanced breast cancer. Pathways comparison between miRNA26A1 and EZH2 identified 2 overlapping cell cycle related pathways and the genes CCNE1 and CDC2. Low levels of EZH2, CCNE1 and CDC2 and high levels of miRNA26A1 are associated with a favorable outcome to tamoxifen therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-16.

Abstract P4-07-01: Correlation of Breast Cancer Susceptibility Loci with Patient Characteristics, Metastasis-Free Survival and Expression of the Nearest Genes

Background: Genome-wide association studies has identified single nucleotide polymorphisms (SNPs) in several loci being associated with breast cancer risk: fibroblast growth factor receptor 2 (FGFR2), trinucleotide repeat containing 9 (TNRC9 or TOX3), mitogen-activated protein kinase kinase kinase 1 (MAP3K1), lymphocyte-specific protein 1 (LSP1) and the imprinted maternally expressed H19, and loci in gene deserts at 8q24 and 2q35. However, the mechanism by which these loci confer breast cancer risk in patients is still largely unknown. Here, we addressed this question by associating these SNPs with the mRNA expression of the genes located at or nearest to the SNP in the same linkage disequilibrium region. In addition, we associated the SNPs with clinical, pathological and patient characteristics and prognosis. Material and Methods : SNPs tagging breast cancer loci were genotyped in genomic tumor DNA samples of 2,480 breast cancer patients. All samples were collected between 1978-2004. The mean age was 55.6 years and median follow-up up was 106 months. The SNPs were correlated with patients and tumor characteristics. Of the 1,262 patients with lymph-node negative disease and who did not receive any adjuvant systemic therapy, SNP status was associated with distant metastasis-free survival (MFS) using Cox regression analysis. Finally, in a subset of 1,400 of the 2,480 patients, the mRNA expression of FGFR2, TNRC9, MAP3K1, LSP1 and H19 genes was determined by quantitative RT-PCR and correlated with SNP genotypes. Results: The SNP rs2981582 in FGFR2 was significantly associated with ER and PgR status of the tumors (both p=0.001). Besides weak associations with tumor grade (FGFR2, p=0.01), ER (MAP3K1, p=0.03; LSP1, p=0.03), and PgR (LSP1, p=0.05), no other association with any clinical or pathological variable for any of the SNPs was observed. Of the SNPs analyzed, only rs2107425 near H19 was significantly linked in uni-and multivariable analysis with MFS (Hazard ratio [HR]=1.44, 95% Confidence interval [CI]: 1.04-1.98; p=0.026; HR=1.53; 95% CI: 1.09-2.14; p=0.013, respectively) with the more aggressive minor allele displaying a recessive trade. Interestingly, the minor allele of SNP rs3803662 was significantly associated with lower mRNA expression of the 8 kb downstream TNRC9 gene (p=0.0019). However, none of the other risk alleles, including the one in FGFR2, had an association with mRNA expression of the nearest located gene. Conclusions: In agreement with previous studies, a clear correlation of the SNPs in FGFR2 with ER and PR status of tumors was observed. The lower level of TNRC9 mRNA in tumors having the minor allele genotype suggests that TNRC9 may act as a tumor suppressor gene and its expression might have a protective effect. A significant association of the SNP near H19 with poor outcome without apparent effect on H19 mRNA expression suggests that this prognostic SNP in a well-known imprinted region is not linked to prognosis through altering H19 gene expression. This study indicates that most of the studied SNPs that confer breast cancer risk are not linked to prognosis nor do they effect, in primary tumors mRNA expression of the nearest gene. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-07-01.