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PDF file - 114K, Uncropped immunoblots and autoradiograms

PDF file - 643K, Rho-kinase inhibition rescues contractile and migratory phenotype of TRPM7 shRNA cells

PDF file - 54K, TRPM7 shRNA#2 treatment reduces malignant phenotype of MDA-MB-231 cells

PDF file - 119K, TRPM7 expression measurements on TRPM7 knockdown MDA-MB-231 and MCF7 cells and TRPM7 rescued cells

PDF file - 92K, Patient- and tumor characteristics in discovery and validation cohort

PDF file - 3.9MB, TRPM7 knockdown does not affect MDA-MB-231 cell proliferation but impairs metastasis formation in vivo

TRPM7 encodes a Ca2+-permeable nonselective cation channel with kinase activity. TRPM7 has been implicated in control of cell adhesion and migration, but whether TRPM7 activity contributes to cancer progression has not been established. Here we report that high levels of TRPM7 expression independently predict poor outcome in breast cancer patients and that it is functionally required for metastasis formation in a mouse xenograft model of human breast cancer. Mechanistic investigation revealed that TRPM7 regulated myosin II–based cellular tension, thereby modifying focal adhesion number, cell–cell adhesion and polarized cell movement. Our findings therefore suggest that TRPM7 is part of a mechanosensory complex adopted by cancer cells to drive metastasis formation. Cancer Res; 72(16); 4250–61. ©2012 AACR.

PDF file - 96K, TRPM7 expression levels and clinical parameters

The detyrosination-tyrosination cycle involves the removal and religation of the C-terminal tyrosine of α-tubulin and is implicated in cognitive, cardiac, and mitotic defects. The vasohibin–small vasohibin-binding protein (SVBP) complex underlies much, but not all, detyrosination. We used haploid genetic screens to identify an unannotated protein, microtubule associated tyrosine carboxypeptidase (MATCAP), as a remaining detyrosinating enzyme. X-ray crystallography and cryo–electron microscopy structures established MATCAP’s cleaving mechanism, substrate specificity, and microtubule recognition. Paradoxically, whereas abrogation of tyrosine religation is lethal in mice, codeletion of MATCAP and SVBP is not. Although viable, defective detyrosination caused microcephaly, associated with proliferative defects during neurogenesis, and abnormal behavior. Thus, MATCAP is a missing component of the detyrosination-tyrosination cycle, revealing the importance of this modification in brain formation.

DCIS has been virtually unknown before the introduction of breast cancer screening programs in the 1980s. Since then, its incidence has been rising steeply and it now accounts for about 25% of all newly diagnosed ‘breast cancer’ cases. Despite the increase in occurrence, the molecular underpinnings regulating DCIS initiation and progression remain elusive. In addition, almost all DCIS cases are hormone dependent, a characteristic which remains one of the biggest challenges to recapitulate in breast cancer research. We could successfully establish models for DCIS as well as estrogen receptor positive disease subtypes by somatically engineering the rat mammary gland via intraductal injection of lentiviral vectors encoding for the most commonly mutated genes (Ccnd1, Myc, Pik3ca and p53) in (pre-) breast cancer. Models with one perturbed gene remained mostly mammary tumor-free after an observation period of 18 months. However, when perturbed in two loci, animals showed mammary lesion formation within 5 and after up to 40 weeks. Histopathological analysis of these lesions characterized a broad spectrum of tumor types including ductal and lobular carcinomas, with both in situ and invasive phenotypes. Among these tumor types, DCIS lesions as well as invasive ductal carcinoma showed estrogen receptor positivity after immunohistochemical staining. Two distinct growth patterns were observed: a flat growing tumor, shaped by, and replacing the host rat mammary duct, and a round growing lesion, disregarding ductal structures. Interestingly, the flat growing tumor pattern evolved mostly in models expressing luminal drivers (Ccnd1 and Pik3ca) and strongly correlated with in situ growth. To increase luminal subtypes and to narrow down the broad tumor spectrum observed in these models, we generated a luminal cell type-specific promoter expressing our genes of interest. Pathological analysis of the arisen tumors indeed revealed a more homogeneous tumor subtype for all models, with an average tumor latency of 28 to 40 weeks. These genetically engineered models for DCIS and estrogen receptor positive breast cancer may aid in an improved treatment stratification by distinguishment between indolent and aggressive DCIS, a persisting challenge as of today. Additionally, these models broaden the scope of available hormone dependent breast cancer models, benefitting the search for novel treatment regiments for patients suffering from this breast cancer subtype. Citation Format: Catrin Lutz, Madelon Badoux, Timo Eijkman, Bim de Klein, Ji-Ying Song, Linda Henneman, Colinda Scheele, Jelle Wesseling, Jos Jonkers. Pioneering genetic rat models of Ductal Carcinoma in situ (DCIS) [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr PR003.

Approximately 75% of all breast cancers express the nuclear hormone receptor estrogen receptor α (ERα). However, the majority of mammary tumors from genetically engineered mouse models (GEMMs) are ERα-negative. To model ERα-positive breast cancer in mice, we exogenously introduced expression of mouse and human ERα in an existing GEMM of p53-deficient breast cancer. After initial ERα expression during mammary gland development, expression was reduced or lost in adult glands and p53-deficient mammary tumors. Chromatin immunoprecipitation (ChIP)-sequencing analysis of primary mouse mammary epithelial cells (MMECs) derived from these models, in which expression of the ERα constructs was induced in vitro, confirmed interaction of ERα with the DNA. In human breast and endometrial cancer, and also in healthy breast tissue, DNA binding of ERα is facilitated by the pioneer factor FOXA1. Surprisingly, the ERα binding sites identified in primary MMECs, but also in mouse mammary gland and uterus, showed an high enrichment of ERE motifs, but were devoid of Forkhead motifs. Furthermore, exogenous introduction of FOXA1 and GATA3 in ERα-expressing MMECs was not sufficient to promote ERα-responsiveness of these cells. Together, this suggests that species-specific differences in pioneer factor usage between mouse and human are dictated by the DNA sequence, resulting in ERα-dependencies in mice that are not FOXA1 driven. These species-specific differences in ERα-biology may limit the utility of mice for in vivo modeling of ERα-positive breast cancer.

Cancer-associated systemic inflammation is strongly linked to poor disease outcome in patients with cancer1,2. For most human epithelial tumour types, high systemic neutrophil-to-lymphocyte ratios are associated with poor overall survival3, and experimental studies have demonstrated a causal relationship between neutrophils and metastasis4,5. However, the cancer-cell-intrinsic mechanisms that dictate the substantial heterogeneity in systemic neutrophilic inflammation between tumour-bearing hosts are largely unresolved. Here, using a panel of 16 distinct genetically engineered mouse models for breast cancer, we uncover a role for cancer-cell-intrinsic p53 as a key regulator of pro-metastatic neutrophils. Mechanistically, loss of p53 in cancer cells induced the secretion of WNT ligands that stimulate tumour-associated macrophages to produce IL-1β, thus driving systemic inflammation. Pharmacological and genetic blockade of WNT secretion in p53-null cancer cells reverses macrophage production of IL-1β and subsequent neutrophilic inflammation, resulting in reduced metastasis formation. Collectively, we demonstrate a mechanistic link between the loss of p53 in cancer cells, secretion of WNT ligands and systemic neutrophilia that potentiates metastatic progression. These insights illustrate the importance of the genetic makeup of breast tumours in dictating pro-metastatic systemic inflammation, and set the stage for personalized immune intervention strategies for patients with cancer.

Immune checkpoint inhibitor (ICI) treatment has thus far shown limited efficacy in triple-negative breast cancer (TNBC) patients, presumably due to sparse or unresponsive tumor-infiltrating lymphocytes. We reveal a strong correlation between MYC expression and loss of immune signatures in human TNBC. In mouse models of BRCA1-proficient and -deficient TNBC, MYC overexpression dramatically decreased lymphocyte infiltration in tumors, along with immune signature loss. Likewise, MYC overexpression suppressed inflammatory signaling induced by BRCA1/2 inactivation in human TNBC cell lines. Moreover, MYC overexpression prevented the recruitment and activation of lymphocytes in co-cultures with human and mouse TNBC models. Chromatin immunoprecipitation (ChIP)-sequencing revealed that MYC directly binds promoters of multiple interferon-signaling genes, which were downregulated upon MYC expression. Finally, MYC overexpression suppressed induction of interferon signaling and tumor growth inhibition by a Stimulator of Interferon Genes (STING) agonist. Together, our data reveal that MYC suppresses innate immunity and facilitates immune escape, explaining the poor immunogenicity of MYC-overexpressing TNBCs.Statement of SignificanceMYC suppresses recruitment and activation of immune cells in TNBC by repressing the transcription of interferon genes. These findings provide a mechanistic rationale for the association of high MYC expression levels with immune exclusion in human TNBCs, which might underlie the relatively poor response of many TNBCs to ICI.

Somatic hotspot mutations and structural amplifications and fusions that affect fibroblast growth factor receptor 2 (encoded by FGFR2) occur in multiple types of cancer1. However, clinical responses to FGFR inhibitors have remained variable1–9, emphasizing the need to better understand which FGFR2 alterations are oncogenic and therapeutically targetable. Here we apply transposon-based screening10,11 and tumour modelling in mice12,13, and find that the truncation of exon 18 (E18) of Fgfr2 is a potent driver mutation. Human oncogenomic datasets revealed a diverse set of FGFR2 alterations, including rearrangements, E1–E17 partial amplifications, and E18 nonsense and frameshift mutations, each causing the transcription of E18-truncated FGFR2 (FGFR2ΔE18). Functional in vitro and in vivo examination of a compendium of FGFR2ΔE18 and full-length variants pinpointed FGFR2-E18 truncation as single-driver alteration in cancer. By contrast, the oncogenic competence of FGFR2 full-length amplifications depended on a distinct landscape of cooperating driver genes. This suggests that genomic alterations that generate stable FGFR2ΔE18 variants are actionable therapeutic targets, which we confirmed in preclinical mouse and human tumour models, and in a clinical trial. We propose that cancers containing any FGFR2 variant with a truncated E18 should be considered for FGFR-targeted therapies.

Approximately 75% of all breast cancers express the nuclear hormone receptor estrogen receptor α (ERα). However, the majority of mammary tumors from genetically engineered mouse models (GEMMs) are ERα-negative. To model ERα-positive breast cancer in mice, we exogenously introduced expression of mouse and human ERα in an existing GEMM of p53-deficient breast cancer. After initial ERα expression during mammary gland development, expression was reduced or lost in adult glands and p53-deficient mammary tumors. Chromatin immunoprecipitation (ChIP)-sequencing analysis of primary mouse mammary epithelial cells (MMECs) derived from these models, in which expression of the ERα constructs was induced in vitro, confirmed interaction of ERα with the DNA. In human breast and endometrial cancer, and also in healthy breast tissue, DNA binding of ERα is facilitated by the pioneer factor FOXA1. Surprisingly, the ERα binding sites identified in primary MMECs, but also in mouse mammary gland and uterus, showed an high enrichment of ERE motifs, but were devoid of Forkhead motifs. Furthermore, exogenous introduction of FOXA1 and GATA3 in ERα-expressing MMECs was not sufficient to promote ERα-responsiveness of these cells. Together, this suggests that species-specific differences in pioneer factor usage between mouse and human are dictated by the DNA sequence, resulting in ERα-dependencies in mice that are not FOXA1 driven. These species-specific differences in ERα-biology may limit the utility of mice for in vivo modeling of ERα-positive breast cancer.

Genetically engineered mouse models (GEMMs) of cancer have proven to be of great value for basic and translational research. Although CRISPR-based gene disruption offers a fast-track approach for perturbing gene function and circumvents certain limitations of standard GEMM development, it does not provide a flexible platform for recapitulating clinically relevant missense mutations in vivo. To this end, we generated knock-in mice with Cre-conditional expression of a cytidine base editor and tested their utility for precise somatic engineering of missense mutations in key cancer drivers. Upon intraductal delivery of sgRNA-encoding vectors, we could install point mutations with high efficiency in one or multiple endogenous genes in situ and assess the effect of defined allelic variants on mammary tumorigenesis. While the system also produces bystander insertions and deletions that can stochastically be selected for when targeting a tumor suppressor gene, we could effectively recapitulate oncogenic nonsense mutations. We successfully applied this system in a model of triple-negative breast cancer, providing the proof of concept for extending this flexible somatic base editing platform to other tissues and tumor types.

Heterozygous mutations in the transcription factor GATA3 are identified in 10–15% of all breast cancer cases. Most of these are protein-truncating mutations, concentrated within or downstream of the second GATA-type zinc-finger domain. Here, we investigated the functional consequences of expression of two truncated GATA3 mutants, in vitro in breast cancer cell lines and in vivo in the mouse mammary gland. We found that the truncated GATA3 mutants display altered DNA binding activity caused by preferred tethering through FOXA1. In addition, expression of the truncated GATA3 mutants reduces E-cadherin expression and promotes anchorage-independent growth in vitro. However, we could not identify any effects of truncated GATA3 expression on mammary gland development or mammary tumor formation in mice. Together, our results demonstrate that both truncated GATA3 mutants promote cistromic re-programming of GATA3 in vitro, but these mutants are not sufficient to induce tumor formation in mice.

BRCA1-mutated breast cancer is primarily driven by DNA copy-number alterations (CNAs) containing large numbers of candidate driver genes. Validation of these candidates requires novel approaches for high-throughput in vivo perturbation of gene function. Here we develop genetically engineered mouse models (GEMMs) of BRCA1-deficient breast cancer that permit rapid introduction of putative drivers by either retargeting of GEMM-derived embryonic stem cells, lentivirus-mediated somatic overexpression or in situ CRISPR/Cas9-mediated gene disruption. We use these approaches to validate Myc, Met, Pten and Rb1 as bona fide drivers in BRCA1-associated mammary tumorigenesis. Iterative mouse modeling and comparative oncogenomics analysis show that MYC-overexpression strongly reshapes the CNA landscape of BRCA1-deficient mammary tumors and identify MCL1 as a collaborating driver in these tumors. Moreover, MCL1 inhibition potentiates the in vivo efficacy of PARP inhibition (PARPi), underscoring the therapeutic potential of this combination for treatment of BRCA1-mutated cancer patients with poor response to PARPi monotherapy., It is difficult to identify cancer driver genes in cancers, for instance BRCA1 mutated breast cancer, that are characterised by large scale genomic alterations. Here, the authors develop genetically engineered mouse models of BRCA1-deficient breast cancer that allow highthroughput in vivo perturbation of candidate driver genes, validating drivers Myc, Met, Pten and Rb1, and identifying MCL1 as a collaborating driver whose targeting can impact efficacy of PARP inhibition.

The isoprenoid biosynthesis pathway provides the cell with a variety of compounds which are involved in multiple cellular processes. Inhibition of this pathway with statins and bisphosphonates is widely applied in the treatment of hypercholesterolemia and metabolic bone disease, respectively. In addition, since isoprenylation of proteins is an important therapeutic target in cancer research there is interest in interfering with isoprenoid biosynthesis, for which new inhibitors to block farnesylation and geranylgeranylation of small GTPases are being developed. We recently developed a sensitive method using UPLC-MS/MS that allows the direct detection and quantification of all intermediates of the mevalonate pathway from MVA to GGPP which can be used to verify the specificity of inhibitors of the isoprenoid biosynthesis pathway. We here investigated the specificity of several inhibitors of the isoprenoid biosynthesis pathway in HepG2 cells, fibroblasts and lymphoblasts. The nitrogen-containing bisphosphonates pamidronate and zoledronate specifically inhibit farnesyl pyrophosphate synthase indicated by the accumulation of IPP/DMAPP. However, zaragozic acid A. a squalene synthase inhibitor, causes an increase of MVA in addition to the expected increase of FPP. Analysis of isoprenoid intermediate profiles after incubation with 6-fluoromevalonate showed a very nonspecific result with an increase in MVA, MVAP, MVAPP and IPP/DMAPP. These results show that inhibitors of a particular enzyme of the isoprenoid biosynthesis pathway can have additional effects another enzymes of the pathway either direct or indirect through accumulation of isoprenoid intermediates. Our method can be used to test new inhibitors and their effect on overall isoprenoid biosynthesis. (C) 2011 Elsevier B.V. All rights reserved

Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. For these reasons, new technologies are needed to expand the genetic toolbox of cancer biologists and allow a more rapid and systematic in vivo interrogation of gene perturbations. In this regard, the advent of CRISPR/Cas9 technologies for somatic genome editing has already paved the way for a new generation of non-germline animal tumor models. For example, liver-specific gene disruption was achieved by transient delivery of components of the CRISPR/Cas9 system in the tail veins of mice, leading to hepatocellular carcinoma (Xue et al., 2014; Weber et al., 2015). Similar approaches have been used to deliver targeted oncogenic mutations to the lung (Platt et al., 2014; Sánchez-Rivera et al., 2014), brain (Zuckermann et al., 2015), and pancreas (Chiou et al., 2015). We have previously shown that the mammary tissue is also amenable to somatic gene editing using intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in the mammary glands of female mice carrying conditional predisposing alleles (Annunziato et al., 2016). This approach was successfully applied to validate in vivo candidate tumor suppressors implicated in invasive lobular carcinoma (Kas et al., 2017), but it is conceivable that more breast cancer subtypes can be modeled via somatic engineering in mice with distinct predisposing mutations. In this study, we show how somatic engineering via intraductal injection of lentiviruses can be used to develop innovative mouse models of triple-negative breast cancer (TNBC), one of the subtypes with the poorest prognosis in the clinics. Using intraductal injection of Cre-encoding lentiviruses in mice with conditional Brca1 and Trp53 alleles (B1P), we were able to target TNBC-initiating cells from the basal compartment, and induce tumors that retained all the hallmark features of the transgenic Cre-driven tumor counterparts, including latency, histopathology, and DNA copy-number variation (CNV) pattern. Moreover, we could dramatically accelerate tumorigenesis when lentiviral Cre was injected in B1PM mice that harbored, in addition to conditional Brca1 and Trp53 alleles, a conditional knock-in allele overexpressing the Myc oncogene, a candidate driver in BRCA1-associated cancers. Alternatively, somatic Myc expression and loss of BRCA1 and p53 was induced in the mammary glands of B1P mice via intraductal injection of lentiviral vectors encoding Myc and Cre under a viral promoter. In both cases the resulting tumors displayed a reshaped CNV profile that strongly implicated a limited number of recurrent focal amplifications and deletions in the tumorigenic cascade. We reiterated the process by lentiviral overexpression of these candidate driver oncogenes and Cre in the mammary glands of B1PM mice, or by achieving in situ CRISPR/Cas9-mediated somatic gene disruption of one or multiple candidate tumor-suppressor genes in Cas9-transgenic WB1P-Cas9 mice. This versatile somatic platform allows for rapid, flexible, and multiplexable in vivo testing of putative oncogenic hits and deals with the need for increasingly complex mouse models while avoiding the bottlenecks of classical transgenesis. Notably, we show here that we can extend the applicability of non-germline breast cancer modeling in mice to produce genetically and histologically accurate TNBC, which allows in vivo investigation of the underlying biology as well as rapid generation of tailored preclinical models for this aggressive breast cancer subtype. Citation Format: Stefano Annunziato, Julian R. de Ruiter, Chiara S. Brambillasca, Sjors M. Kas, Federica Ferrante, Catrin Lutz, Bjorn Siteur, Bas van Gerwen, Marieke van de Ven, Martine H. van Miltenburg, Linda Henneman, Jos Jonkers. Somatic engineering of the mammary gland for the development of novel mouse models of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A01.

Breast cancer is the most common cancer among women in the Western world. Approximately 8% of all breast cancer cases may be caused by inheritance of mutations in breast cancer susceptibility genes. Of these, BRCA1 is the most important one, contributing to approximately half of all familial breast cancer cases. BRCA1-related breast tumors show a triple-negative (TBNC) basal-like phenotype, which correlates with aggressive characteristics and poor prognosis. MYC has been identified as a potential driver gene in BRCA1-associated breast cancer. In line with this, we found MYC to be frequently amplified in both mouse and human BRCA1-deficient breast cancers. Thus, evaluating the contribution of MYC to BRCA1-associated breast cancer might unravel new mechanistic insights and potentially open the way to new therapeutic approaches. To investigate a causal role of MYC in BRCA1-associated breast cancer, we generated WAPCre;Brca1F/F;Trp53F/F (WB1P) and WAPCre;Brca1F/F;Trp53F/F;invCAG-Myc (WB1P-Myc) mouse models, which were monitored for tumor development. Whereas WB1P female mice developed mammary tumors after 300 days, WB1P-Myc mice showed a dramatic acceleration in tumor development with a median survival of 100 days. Tumors of both models were histopathologically classified as poorly differentiated solid carcinomas resembling TNBC. To characterize the differences between the WB1P and WB1P-Myc tumors, we performed whole-transcriptome analysis using RNAseq. Unsupervised hierarchical cluster analysis showed that WB1P and WB1P-Myc tumors cluster separately. Interestingly, Gene Set Enrichment Analysis (GSEA) showed that the most significant deregulated pathway is the cytokine-cytokine interaction pathway. Compared to WB1P tumors, WB1P-Myc tumors showed downregulation of different molecules involved in immune cell chemoattraction and antigen presentation. Characterization of the immune infiltrate of WB1P and WB1P-Myc tumors showed that MYC overexpression leads to depletion of different tumor-infiltrating immune cells, including leukocytes, T cells, myeloid cells, and NK cells. To further investigate the role of MYC on the tumor microenvironment, we generated different Myc inducible platforms, including a “double-layered” system for Cre-conditional and doxycycline-regulatable overexpression of the MYC gene in WB1P mice. We also obtained tumor organoids from the WB1P and WB1P-Myc tumors which can be manipulated in vitro and orthotopically transplanted in vivo. We transduced the WB1P organoids with a lentiviral MycERT2 construct, enabling us to switch on and off MYC in organoids and study if its overexpression is the cause of the differences observed in the WB1P-Myc tumors. In addition, we are studying if MYC upregulation induces secretion of immune-suppressive factors and/or upregulation of molecules that can create an immunosuppressive microenvironment. Moreover, we are studying the secretome of WB1P and WB1P-Myc organoids in vitro. Of note, we discovered that WB1P-Myc tumors strongly downregulate the CIITA-CD74-MHCII axis, which is important for T-cell mediated antitumor activity. Taking advantage of the aforementioned Myc-inducible platforms, we are currently investigating if this axis is the underlying cause of the immune-depleted microenvironment we observed in WB1P-Myc tumors. In addition, we are somatically inducing CIITA expression in the mammary epithelium of WB1P-Myc mice to investigate if this leads to delayed mammary tumor formation and/or increased influx of immune cells in the resulting tumors. Citation Format: Chiara Svetlana Brambillasca, Linda Henneman, Julian de Ruiter, Max Wellenstein, Anne Paulien Drenth, Karin de Visser, Jos Jonkers. Dissecting MYC immunoregulatory activity in BRCA1-associated breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A21.

Preclinical in vivo validation of target genes for therapeutic intervention requires careful selection and characterization of the most suitable animal model in order to assess the role of these genes in a particular process or disease. To this end, genetically engineered mouse models (GEMMs) are typically used. However, the appropriate engineering of these models is often cumbersome and time consuming. Recently, we and others described a modular approach for fast-track modification of existing GEMMs by re-derivation of embryonic stem cells (ESCs) that can be modified by recombinase-mediated transgene insertion and subsequently used for the production of chimeric mice. This 'GEMM-ESC strategy' allows for rapid in vivo analysis of gene function in the chimeras and their offspring. Moreover, this strategy is compatible with CRISPR/Cas9-mediated genome editing. This protocol describes when and how to use the GEMM-ESC strategy effectively, and it provides a detailed procedure for re-deriving and manipulating GEMM-ESCs under feeder- and serum-free conditions. This strategy produces transgenic mice with the desired complex genotype faster than traditional methods: generation of validated GEMM-ESC clones for controlled transgene integration takes 9-12 months, and recombinase-mediated transgene integration and chimeric cohort production takes 2-3 months. The protocol requires skills in embryology, stem cell biology and molecular biology, and it is ideally performed within, or in close collaboration with, a transgenic facility.

Metaplastic breast carcinoma (MBC) is a rare histological breast cancer subtype characterized by mesenchymal elements and poor clinical outcome. A large fraction of MBCs harbor defects in breast cancer 1 (BRCA1). As BRCA1 deficiency sensitizes tumors to DNA cross-linking agents and poly(ADP-ribose) polymerase (PARP) inhibitors, we sought to investigate the response of BRCA1-deficient MBCs to the PARP inhibitor olaparib. To this end, we established a genetically engineered mouse model (GEMM) for BRCA1-deficient MBC by introducing the MET proto-oncogene into a BRCA1-associated breast cancer model, using our novel female GEMM ES cell (ESC) pipeline. In contrast to carcinomas, BRCA1-deficient mouse carcinosarcomas resembling MBC show intrinsic resistance to olaparib caused by increased P-glycoprotein (Pgp) drug efflux transporter expression. Indeed, resistance could be circumvented by using another PARP inhibitor, AZD2461, which is a poor Pgp substrate. These preclinical findings suggest that patients with BRCA1-associated MBC may show poor response to olaparib and illustrate the value of GEMM-ESC models of human cancer for evaluation of novel therapeutics.

Neuroblastoma is an embryonal tumor derived from poorly differentiated neural crest cells. Current research is aimed at identifying the molecular mechanisms that maintain the progenitor state of neuroblastoma cells and to develop novel therapeutic strategies that induce neuroblastoma cell differentiation. Mechanisms controlling neural crest development are typically dysregulated during neuroblastoma progression, and provide an appealing starting point for drug target discovery. Transcriptional programs involved in neural crest development act as a context dependent gene regulatory network. In addition to BMP, Wnt and Notch signaling, activation of developmental gene expression programs depends on the physical characteristics of the tissue microenvironment. TRPM7, a mechanically regulated TRP channel with kinase activity, was previously found essential for embryogenesis and the maintenance of undifferentiated neural crest progenitors. Hence, we hypothesized that TRPM7 may preserve progenitor-like, metastatic features of neuroblastoma cells. Using multiple neuroblastoma cell models, we demonstrate that TRPM7 expression closely associates with the migratory and metastatic properties of neuroblastoma cells in vitro and in vivo. Moreover, microarray-based expression profiling on control and TRPM7 shRNA transduced neuroblastoma cells indicates that TRPM7 controls a developmental transcriptional program involving the transcription factor SNAI2. Overall, our data indicate that TRPM7 contributes to neuroblastoma progression by maintaining progenitor-like features.

Breast cancer is the most common cancer among women in the Western world. Approximately 8% of all breast cancer cases may be caused by inheritance of mutations in breast cancer susceptibility genes. Of these, BRCA1 is the most important one, contributing to approximately half of all familial breast cancer cases. BRCA1-related breast tumors show a triple-negative (TBNC) basal like phenotype, that correlates with aggressive characteristics and bad prognosis. A number of BRCA1 associated genes have been identified; among those MYC overexpression seems to have an important role in aggressive TBNC. In line with this, we found MYC to be frequently amplified in both mouse and human BRCA1-deficient breast cancers. Thus, evaluating the contribution of MYC to BRCA1-associated breast cancer might unravel new mechanistic insights and potentially open the way to new therapeutic approaches. To pursue this, we generated a WAPCre;Brca1F/F;Trp53F/F;Myc (WB1P-MYC) mouse model to monitor tumor development. Whereas the WAPCre;Brca1F/F;Trp53F/F (WB1P) mouse model develops tumors after 300 days, Cre-conditional overexpression of the MYC transgene leads to a dramatic acceleration in tumor development with a median survival of 100 days. Tumors of both models were histopathologically classified as solid carcinomas. Further characterization revealed that WB1P-MYC tumors show higher levels of apoptosis and DNA damage. Moreover, we also observed a strong difference in immune infiltrate between WB1P and WB1P-MYC tumors. To better characterize these differences between WB1P and WB1P-MYC tumors we obtained tumor organoids. Importantly, these tumor organoids maintained characteristics of the original tumor like Brca1, P53 deletion status and MYC overexpression. Moreover, they can be orthotopically transplanted in vivo, leading to formation of solid carcinomas. Therefore, they represent a versatile platform to test new drug combinations both in vitro and in vivo. To test if MYC overexpression is also required for maintenance of established tumors, we are developing a “double-layered” system for Cre-conditional and doxycycline-regulatable overexpression of the MYC gene in the WAPCre;Brca1F/F;Trp53F/F model. By administering doxycycline we will induce MYC only in Cre-expressing mammary epithelial cells. After tumor formation MYC will be switched-off to assess its role in tumor maintenance and progression. Furthermore, we would like to test new therapeutic approaches, like combining Myc down-regulation with administration of PARP inhibitors. In addition, tumor organoids of this inducible WB1P-MYC model will be a powerful system to study the MYC-associated phenotype, drugs combinations and forward genetic screens. Citation Format: Chiara Svetlana Brambillasca, Linda Henneman, Julian de Ruiter, Anne Paulien Drenth, Jos Jonkers. Dissecting the role of MYC in BRCA1-associated breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 889.

Contains fulltext : 109674.pdf (Publisher’s version ) (Closed access) TRPM7 encodes a Ca(2+)-permeable nonselective cation channel with kinase activity. TRPM7 has been implicated in control of cell adhesion and migration, but whether TRPM7 activity contributes to cancer progression has not been established. Here we report that high levels of TRPM7 expression independently predict poor outcome in breast cancer patients and that it is functionally required for metastasis formation in a mouse xenograft model of human breast cancer. Mechanistic investigation revealed that TRPM7 regulated myosin II-based cellular tension, thereby modifying focal adhesion number, cell-cell adhesion and polarized cell movement. Our findings therefore suggest that TRPM7 is part of a mechanosensory complex adopted by cancer cells to drive metastasis formation. Cancer Res; 72(16); 4250-61. (c)2012 AACR.

Invasive lobular carcinoma (ILC) is a form of breast cancer that develops in the milk-producing glands (lobules) of the breast. ILC is an aggressive form of breast cancer due to its ability to spread to other parts of the body, both by bloodstream as well as lymphatic system. One of the key features found in ILC is loss of E-cadherin, encoding CDH1. Mammary-gland specific deletion of E-cadherin does not lead to tumour formation indicating that other factors are involved in the induction of ILC. Indeed, combined tissue-specific inactivation of E-cadherin and p53 induces development of invasive and metastatic mammary tumours resembling human ILC. However, a limitation of the current conditional mouse models is that human ILCs rarely contain p53 mutations. In contrast, PI3K pathway mutations are frequently found in human ILCs. Currently we are developing novel mouse ILC models to systematically examine the contribution of PI3K pathway components such as PIK3CA, AKT and PTEN to ILC development. To reduce time spent on generating a genetically engineered mouse (GEM) tumour cohort expressing three or more transgenes we set-up embryonic stem (ES) cell derivation and targeting of ES cells from validated GEM models, the so-called GEMM-ESC strategy. WapCre-Cdh1F/F ES cells targeted with PIK3CA or AKT mutants are now being used to generate the elaborate PI3K pathway tumour cohort. These advanced models will allow us to study the role of the PI3K pathway in the pathogenesis of ILC. Moreover, we can use these advanced mouse models which recapitulate key hallmarks of human ILC to perform refined and realistic preclinical prevention and intervention studies for ILC. 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 3295. doi:1538-7445.AM2012-3295