BackgroundCHEK2 c.1100delC was the first moderate-risk breast cancer (BC) susceptibility allele discovered. Despite several genomic, transcriptomic and functional studies, however, it is still... Show moreBackgroundCHEK2 c.1100delC was the first moderate-risk breast cancer (BC) susceptibility allele discovered. Despite several genomic, transcriptomic and functional studies, however, it is still unclear how exactly CHEK2 c.1100delC promotes tumorigenesis. Since the mutational landscape of a tumor reflects the processes that have operated on its development, the aim of this study was to uncover the somatic genomic landscape of CHEK2-associated BC.MethodsWe sequenced primary BC (pBC) and normal genomes of 20 CHEK2 c.1100delC mutation carriers as well as their pBC transcriptomes. Including pre-existing cohorts, we exhaustively compared CHEK2 pBC genomes to those from BRCA1/2 mutation carriers, those that displayed homologous recombination deficiency (HRD) and ER- and ER+ pBCs, totaling to 574 pBC genomes. Findings were validated in 517 metastatic BC genomes subdivided into the same subgroups. Transcriptome data from 168 ER+ pBCs were used to derive a TP53-mutant gene expression signature and perform cluster analysis with CHEK2 BC transcriptomes. Finally, clinical outcome of CHEK2 c.1100delC carriers was compared with BC patients displaying somatic TP53 mutations in two well-described retrospective cohorts totaling to 942 independent pBC cases.ResultsBC genomes from CHEK2 mutation carriers were most similar to ER+ BC genomes and least similar to those of BRCA1/2 mutation carriers in terms of tumor mutational burden as well as mutational signatures. Moreover, CHEK2 BC genomes did not show any evidence of HRD. Somatic TP53 mutation frequency and the size distribution of structural variants (SVs), however, were different compared to ER+ BC. Interestingly, BC genomes with bi-allelic CHEK2 inactivation lacked somatic TP53 mutations and transcriptomic analysis indicated a shared biology with TP53 mutant BC. Moreover, CHEK2 BC genomes had an increased frequency of > 1 Mb deletions, inversions and tandem duplications with peaks at specific sizes. The high chromothripsis frequency among CHEK2 BC genomes appeared, however, not associated with this unique SV size distribution profile.ConclusionsCHEK2 BC genomes are most similar to ER+ BC genomes, but display unique features that may further unravel CHEK2-driven tumorigenesis. Increased insight into this mechanism could explain the shorter survival of CHEK2 mutation carriers that is likely driven by intrinsic tumor aggressiveness rather than endocrine resistance. Show less
Grainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing... Show moreGrainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing epithelial to mesenchymal transitions (EMT). GRHL2 cooperates with androgen and estrogen receptors (ER) to regulate gene expression. We explore genome wide GRHL2 binding sites conserved in three ER⍺/GRHL2 positive luminal breast cancer cell lines by ChIP-Seq. Interaction with the ER⍺/FOXA1/GATA3 complex is observed, however, only for a minor fraction of conserved GRHL2 peaks. We determine genome wide transcriptional dynamics in response to loss of GRHL2 by nascent RNA Bru-seq using an MCF7 conditional knockout model. Integration of ChIP- and Bru-seq pinpoints candidate direct GRHL2 target genes in luminal breast cancer. Multiple connections between GRHL2 and proliferation are uncovered, including transcriptional activation of ETS and E2F transcription factors. Among EMT-related genes, direct regulation of CLDN4 is corroborated but several targets identified in other cells (including CDH1 and ZEB1) are ruled out by both ChIP- and Bru-seq as being directly controlled by GRHL2 in luminal breast cancer cells. Gene clusters correlating positively (including known GRHL2 targets such as ErbB3, CLDN4/7) or negatively (including TGFB1 and TGFBR2) with GRHL2 in the MCF7 knockout model, display similar correlation with GRHL2 in ER positive as well as ER negative breast cancer patients. Altogether, this study uncovers gene sets regulated directly or indirectly by GRHL2 in luminal breast cancer, identifies novel GRHL2-regulated genes, and points to distinct GRHL2 regulation of EMT in luminal breast cancer cells. Video Abstract. Show less
Grainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing... Show moreGrainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing epithelial to mesenchymal transitions (EMT). GRHL2 cooperates with androgen and estrogen receptors (ER) to regulate gene expression. We explore genome wide GRHL2 binding sites conserved in three ER?/GRHL2 positive luminal breast cancer cell lines by ChIP-Seq. Interaction with the ER?/FOXA1/GATA3 complex is observed, however, only for a minor fraction of conserved GRHL2 peaks. We determine genome wide transcriptional dynamics in response to loss of GRHL2 by nascent RNA Bru-seq using an MCF7 conditional knockout model. Integration of ChIP- and Bru-seq pinpoints candidate direct GRHL2 target genes in luminal breast cancer. Multiple connections between GRHL2 and proliferation are uncovered, including transcriptional activation of ETS and E2F transcription factors. Among EMT-related genes, direct regulation of CLDN4 is corroborated but several targets identified in other cells (including CDH1 and ZEB1) are ruled out by both ChIP- and Bru-seq as being directly controlled by GRHL2 in luminal breast cancer cells. Gene clusters correlating positively (including known GRHL2 targets such as ErbB3, CLDN4/7) or negatively (including TGFB1 and TGFBR2) with GRHL2 in the MCF7 knockout model, display similar correlation with GRHL2 in ER positive as well as ER negative breast cancer patients. Altogether, this study uncovers gene sets regulated directly or indirectly by GRHL2 in luminal breast cancer, identifies novel GRHL2-regulated genes, and points to distinct GRHL2 regulation of EMT in luminal breast cancer cells. Show less
Background Pre-eclampsia has a major impact on renal function as shown by the development of proteinuria and podocyturia. How the systemic, soluble Fms-like tyrosine kinase-1 (sFlt-1)-driven... Show moreBackground Pre-eclampsia has a major impact on renal function as shown by the development of proteinuria and podocyturia. How the systemic, soluble Fms-like tyrosine kinase-1 (sFlt-1)-driven inhibition of vascular endothelial growth factor (VEGF) activity detected in pre-eclampsia directly affects renal function remains unknown. The aim of the study was to clarify whether a non-canonical, renal-centred escape from VEGF inhibition in the case of pre-eclamptic pregnancy might have a direct impact on renal function. Methods We evaluated plasma and urinary VEGF and placental growth factor (PlGF), plasma sFlt-1 and carbonic anhydrase IX (CAIX), albuminuria and podocyturia in 18 women with uncomplicated pregnancy, 21 with pre-eclampsia and 18 non-pregnant. The three groups were matched for age and the pregnant groups also for gestational age at enrolment. Results Plasma VEGF was reduced in uncomplicated (P = 0.001) and pre-eclamptic (P = 0.0003) pregnancies when compared with controls. In uncomplicated pregnancy, the dysfunction was balanced by an increase (P = 0.009) of plasma PlGF. Increased (P = 0.0001) plasma CAIX in pre-eclampsia was in line with hypoxia. Pre-eclampsia resulted in a paradoxical increase (P = 0.0004) of urinary excretion of VEGF. Urinary concentrations of VEGF and podocytes were correlated to each other (r(2) = 0.48, P < 0.0005) but also to plasma sFlt-1 (r(2) = 0.56, P < 0.0001 and r(2) = 0.23, P = 0.03, respectively). Conclusions In the case of pre-eclampsia, the systemic VEGF inhibition leads the kidney, possibly the podocyte, to increase the VEGF synthesis. The mechanisms leading to local VEGF overproduction or the overproduced VEGF itself are reasonably involved in the pathogenesis of podocyturia and, as a consequence, renal dysfunction in pre-eclampsia. Show less
Koedoot, E.; Wolters, L.; Smid, M.; Stoilov, P.; Burger, G.A.; Herpers, B.H.A.; ... ; Water, B. van de 2021
Screening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these... Show moreScreening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these different culture conditions by RNAseq of 14 BC cell lines cultured in both 2D and 3D conditions. All 3D BC cell cultures demonstrated increased mitochondrial metabolism and downregulated cell cycle programs. Luminal BC cells in 3D demonstrated overall limited reprogramming. 3D basal B BC cells showed increased expression of extracellular matrix (ECM) interaction genes, which coincides with an invasive phenotype not observed in other BC cells. Genes downregulated in 3D were associated with metastatic disease progression in BC patients, including cyclin dependent kinases and aurora kinases. Furthermore, the overall correlation of the cell line transcriptome to the BC patient transcriptome was increased in 3D cultures for all TNBC cell lines. To define the most optimal culture conditions to study the oncogenic pathway of interest, an open source bioinformatics strategy was established. Show less
He, J.; McLaughlin, R.P.; Van der Beek, L.; Canisius, S.; Wessels, L.; Smid, M.; ... ; Water, B. van de 2020
The genetically heterogeneous triple-negative breast cancer (TNBC) continues to be an intractable disease, due to lack of effective targeted therapies. Gene amplification is a major event in... Show moreThe genetically heterogeneous triple-negative breast cancer (TNBC) continues to be an intractable disease, due to lack of effective targeted therapies. Gene amplification is a major event in tumorigenesis. Genes with amplification-dependent expression are being explored as therapeutic targets for cancer treatment. In this study, we have applied Analytical Multi-scale Identification of Recurring Events analysis and transcript quantification in the TNBC genome across 222 TNBC tumors and identified 138 candidate genes with positive correlation in copy number gain (CNG) and gene expression. siRNA-based loss-of-function screen of the candidate genes has validated EGFR, MYC, ASAP1, IRF2BP2, and CCT5 genes as drivers promoting proliferation in different TNBC cells. MYC, ASAP1, IRF2BP2, and CCT5 display frequent CNG and concurrent expression over 2173 breast cancer tumors (cBioPortal dataset). More frequently are MYC and ASAP1 amplified in TNBC tumors (>30%, n = 320). In particular, high expression of ASAP1, the ADP-ribosylation factor GTPase-activating protein, is significantly related to poor metastatic relapse-free survival of TNBC patients (n = 257, bc-GenExMiner). Furthermore, we have revealed that silencing of ASAP1 modulates numerous cytokine and apoptosis signaling components, such as IL1B, TRAF1, AIFM2, and MAP3K11 that are clinically relevant to survival outcomes of TNBC patients. ASAP1 has been reported to promote invasion and metastasis in various cancer cells. Our findings that ASAP1 is an amplification-dependent TNBC driver gene promoting TNBC cell proliferation, functioning upstream apoptosis components, and correlating to clinical outcomes of TNBC patients, support ASAP1 as a potential actionable target for TNBC treatment. Show less
Ren, J.; Smid, M.; Iaria, J.; Salvatori, D.C.F.; Dam, H. van; Zhu, H.J.; ... ; Dijke, P. ten 2019
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor clinical prognosis and limited targeted treatment strategies. Kinase inhibitor screening of a panel of 20... Show moreTriple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor clinical prognosis and limited targeted treatment strategies. Kinase inhibitor screening of a panel of 20 TNBC cell lines uncovered three critical TNBC subgroups: 1) sensitive to only MEK inhibitors; 2) sensitive to only Akt inhibitors; 3) resistant to both MEK/Akt inhibitors. Using genomic, transcriptomic and proteomic datasets of these TNBC cell lines we unravelled molecular features associated with the MEK and Akt drug resistance. MEK inhibitor-resistant TNBC cell lines were discriminated from Akt inhibitor-resistant lines by the presence of PIK3CA/PIK3R1/PTEN mutations, high p-Akt and low p-MEK levels, yet these features could not distinguish double-resistant cells. Gene set enrichment analyses of transcriptomic and proteomic data of the MEK and Akt inhibitor response groups revealed a set of cell cycle-related genes associated with the double-resistant phenotype; these genes were overexpressed in a subset of breast cancer patients. CDK inhibitors targeting the cell cycle programme could overcome the Akt and MEK inhibitor double-resistance. In conclusion, we uncovered molecular features and alternative treatment strategies for TNBC that are double-resistant to Akt and MEK inhibitors. Show less
McLaughlin, R.P.; He, J.; Noord, V.E. van der; Redel, J.; Foekens, J.A.; Martens, J.W.M.; ... ; Water, B. van de 2019
The effective treatment of triple-negative breast cancer (TNBC) remains a profound clinical challenge. Despite frequent epidermal growth factor receptor (EGFR) overexpression and reliance on... Show moreThe effective treatment of triple-negative breast cancer (TNBC) remains a profound clinical challenge. Despite frequent epidermal growth factor receptor (EGFR) overexpression and reliance on downstream signalling pathways in TNBC, resistance to EGFR-tyrosine kinase inhibitors (TKIs) remains endemic. Therefore, the identification of targeted agents, which synergise with current therapeutic options, is paramount.Compound-based, high-throughput, proliferation screening was used to profile the response of TNBC cell lines to EGFR-TKIs, western blotting and siRNA transfection being used to examine the effect of inhibitors on EGFR-mediated signal transduction and cellular dependence on such pathways, respectively. A kinase inhibitor combination screen was used to identify compounds that synergised with EGFR-TKIs in TNBC, utilising sulphorhodamine B (SRB) assay as read-out for proliferation. The impact of drug combinations on cell cycle arrest, apoptosis and signal transduction was assessed using flow cytometry, automated live-cell imaging and western blotting, respectively. RNA sequencing was employed to unravel transcriptomic changes elicited by this synergistic combination and to permit identification of the signalling networks most sensitive to co-inhibition.We demonstrate that a dual cdc7/CDK9 inhibitor, PHA-767491, synergises with multiple EGFR-TKIs (lapatinib, erlotinib and gefitinib) to overcome resistance to EGFR-targeted therapy in various TNBC cell lines. Combined inhibition of EGFR and cdc7/CDK9 resulted in reduced cell proliferation, accompanied by induction of apoptosis, G2-M cell cycle arrest, inhibition of DNA replication and abrogation of CDK9-mediated transcriptional elongation, in contrast to mono-inhibition. Moreover, high expression of cdc7 and RNA polymerase II Subunit A (POLR2A), the direct target of CDK9, is significantly correlated with poor metastasis-free survival in a cohort of breast cancer patients. RNA sequencing revealed marked downregulation of pathways governing proliferation, transcription and cell survival in TNBC cells treated with the combination of an EGFR-TKI and a dual cdc7/CDK9 inhibitor. A number of genes enriched in these downregulated pathways are associated with poor metastasis-free survival in TNBC.Our results highlight that dual inhibition of cdc7 and CDK9 by PHA-767491 is a potential strategy for targeting TNBC resistant to EGFR-TKIs. Show less
Koedoot, E.; Fokkelman, M.; Rogkoti, V.M.; Smid, M.; Sandt, I. van de; Bont, H.J.G.M. de; ... ; Water, B. van de 2019
Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an... Show moreTtriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug development. Show less
Koedoot, E.; Smid, M.; Foekens, J.A.; Martens, J.W.M.; Le Dévédec, S.E.; Water, B. van de 2019
Splicing factors (SFs) act in dynamic macromolecular complexes to modulate RNA processing. To understand the complex role of SFs in cancer progression, we performed a systemic analysis of the co... Show moreSplicing factors (SFs) act in dynamic macromolecular complexes to modulate RNA processing. To understand the complex role of SFs in cancer progression, we performed a systemic analysis of the co-regulation of SFs using primary tumor RNA sequencing data. Co-regulated SFs were associated with aggressive breast cancer phenotypes and enhanced metastasis formation, resulting in the classification of Enhancer- (21 genes) and Suppressor-SFs (64 genes). High Enhancer-SF levels were related to distinct splicing patterns and expression of known oncogenic pathways such as respiratory electron transport, DNA damage and cell cycle regulation. Importantly, largely identical SF co-regulation was observed in almost all major cancer types, including lung, pancreas and prostate cancer. In conclusion, we identified cancer-associated co-regulated expression of SFs that are associated with aggressive phenotypes. This study increases the global understanding of the role of the spliceosome in cancer progression and also contributes to the development of strategies to cure cancer patients. Show less
Breast cancer has for long been recognized as a highly diverse tumor group, but the underlying genetic basis has been elusive. Here, we report an extensive molecular characterization of a... Show moreBreast cancer has for long been recognized as a highly diverse tumor group, but the underlying genetic basis has been elusive. Here, we report an extensive molecular characterization of a collection of 41 human breast cancer cell lines. Protein and gene expression analyses indicated that the collection of breast cancer cell lines has retained most, if not all, molecular characteristics that are typical for clinical breast cancers. Gene mutation analyses identified 146 oncogenic mutations among 27 well-known cancer genes, amounting to an average of 3.6 mutations per cell line. Mutations in genes from the p53, RB and PI3K tumor suppressor pathways were widespread among all breast cancer cell lines. Most important, we have identified two gene mutation profiles that are specifically associated with luminal-type and basal-type breast cancer cell lines. The luminal mutation profile involved E-cadherin and MAP2K4 gene mutations and amplifications of Cyclin D1, ERBB2 and HDM2, whereas the basal mutation profile involved BRCA1, RB1, RAS and BRAF gene mutations and deletions of p16 and p14ARF. These subtype-specific gene mutation profiles constitute a genetic basis for the heterogeneity observed among human breast cancers, providing clues for their underlying biology and providing guidance for targeted pharmacogenetic intervention in breast cancer patients. Show less
