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Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer (IBC). Due to a lack of biomarkers able to distinguish high- from low-risk cases, DCIS is treated similar to early IBC even though the minority of untreated cases eventually become invasive. Here, we characterized 115 patient-derived mouse-intraductal (MIND) DCIS models reflecting the full spectrum of DCIS observed in patients. Utilizing the possibility to follow the natural progression of DCIS combined with omics and imaging data, we reveal multiple prognostic factors for high-risk DCIS including high grade, HER2 amplification, expansive 3D growth, and high burden of copy number aberrations. In addition, sequential transplantation of xenografts showed minimal phenotypic and genotypic changes over time, indicating that invasive behavior is an intrinsic phenotype of DCIS and supporting a multiclonal evolution model. Moreover, this study provides a collection of 19 distributable DCIS-MIND models spanning all molecular subtypes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cancer-associated fibroblasts (CAFs) are abundantly present in the microenvironment of virtually all tumors and strongly impact tumor progression. Despite increasing insight into their function and heterogeneity, little is known regarding the origin of CAFs. Understanding the origin of CAF heterogeneity is needed to develop successful CAF-based targeted therapies. Through various transplantation studies in mice, we show that CAFs in both invasive lobular breast cancer and triple-negative breast cancer originate from mammary tissue-resident normal fibroblasts (NFs). Single-cell transcriptomics, in vivo and in vitro studies reveal the transition of CD26+ and CD26- NF populations into inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs), respectively. Functional co-culture experiments show that CD26+ NFs transition into pro-tumorigenic iCAFs which recruit myeloid cells in a CXCL12-dependent manner and enhance tumor cell invasion via matrix-metalloproteinase (MMP) activity. Together, our data suggest that CD26+ and CD26- NFs transform into distinct CAF subpopulations in mouse models of breast cancer., (© 2023. The Author(s).)

Background: The majority of BRCA1-mutant breast cancers are characterized by a triple-negative phenotype and a basal-like molecular subtype, associated with aggressive clinical behavior. Current treatment options are limited, highlighting the need for the development of novel targeted therapies for this tumor subtype., Methods: Our group previously showed that EZH2 is functionally relevant in BRCA1-deficient breast tumors and blocking EZH2 enzymatic activity could be a potent treatment strategy. To validate the role of EZH2 as a therapeutic target and to identify new synergistic drug combinations, we performed a high-throughput drug combination screen in various cell lines derived from BRCA1-deficient and -proficient mouse mammary tumors., Results: We identified the combined inhibition of EZH2 and the proximal DNA damage response kinase ATM as a novel synthetic lethality-based therapy for the treatment of BRCA1-deficient breast tumors. We show that the combined treatment with the EZH2 inhibitor GSK126 and the ATM inhibitor AZD1390 led to reduced colony formation, increased genotoxic stress, and apoptosis-mediated cell death in BRCA1-deficient mammary tumor cells in vitro. These findings were corroborated by in vivo experiments showing that simultaneous inhibition of EZH2 and ATM significantly increased anti-tumor activity in mice bearing BRCA1-deficient mammary tumors., Conclusion: Taken together, we identified a synthetic lethal interaction between EZH2 and ATM and propose this synergistic interaction as a novel molecular combination for the treatment of BRCA1-mutant breast cancer., (© 2022. The Author(s).)

Purpose: Because BRCA1 is a high-risk breast/ovarian cancer susceptibility gene, BRCA1 sequence variants of uncertain clinical significance (VUS) complicate genetic counseling. As most VUS are rare, reliable classification based on clinical and genetic data is often impossible. However, all pathogenic BRCA1 variants analyzed result in defective homologous recombination DNA repair (HRR). Thus, BRCA1 VUS may be categorized based on their functional impact on this pathway., Experimental Design: Two hundred thirty-eight BRCA1 VUS-comprising most BRCA1 VUS known in the Netherlands and Belgium-were tested for their ability to complement Brca1- deficient mouse embryonic stem cells in HRR, using cisplatin and olaparib sensitivity assays and a direct repeat GFP (DR-GFP) HRR assay. Assays were validated using 25 known benign and 25 known pathogenic BRCA1 variants. For assessment of pathogenicity by a multifactorial likelihood analysis method, we collected clinical and genetic data for functionally deleterious VUS and VUS occurring in three or more families., Results: All three assays showed 100% sensitivity and specificity (95% confidence interval, 83%-100%). Out of 238 VUS, 45 showed functional defects, 26 of which were deleterious in all three assays. For 13 of these 26 variants, we could calculate the probability of pathogenicity using clinical and genetic data, resulting in the identification of 7 (likely) pathogenic variants., Conclusions: We have functionally categorized 238 BRCA1 VUS using three different HRR-related assays. Classification based on clinical and genetic data alone for a subset of these variants confirmed the high sensitivity and specificity of our functional assays., (©2020 American Association for Cancer Research.)

Effective treatment of invasive lobular carcinoma (ILC) of the breast is hampered by late detection, invasive growth, distant metastasis, and poor response to chemotherapy. Phosphoinositide 3-kinase (PI3K) signaling, one of the major druggable oncogenic signaling networks, is frequently activated in ILC. We investigated treatment response and resistance to AZD8055, an inhibitor of mammalian target of rapamycin (mTOR), in the K14-cre;Cdh1 Flox/Flox ;Trp53 Flox/Flox (KEP) mouse model of metastatic ILC. Inhibition of mTOR signaling blocked the growth of primary KEP tumors as well as the progression of metastatic disease. However, primary tumors and distant metastases eventually acquired resistance after long-term AZD8055 treatment, despite continued effective suppression of mTOR signaling in cancer cells. Interestingly, therapeutic responses were associated with increased expression of genes related to antigen presentation. Consistent with this observation, increased numbers of tumor-infiltrating major histocompatibility complex class II-positive (MHCII+) immune cells were observed in treatment-responsive KEP tumors. Acquisition of treatment resistance was associated with loss of MHCII+ cells and reduced expression of genes related to the adaptive immune system. The therapeutic efficacy of mTOR inhibition was reduced in Rag1 -/- mice lacking mature T and B lymphocytes, compared to immunocompetent mice. Furthermore, therapy responsiveness could be partially rescued by transplanting AZD8055-resistant KEP tumors into treatment-naïve immunocompetent hosts. Collectively, these data indicate that the PI3K signaling pathway is an attractive therapeutic target in invasive lobular carcinoma, and that part of the therapeutic effect of mTOR inhibition is mediated by the adaptive immune system., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The GATA-type zinc finger transcription factor TRPS1 has been implicated in breast cancer. However, its precise role remains unclear, as both amplifications and inactivating mutations in TRPS1 have been reported. Here, we used in vitro and in vivo loss-of-function approaches to dissect the role of TRPS1 in mammary gland development and invasive lobular breast carcinoma, which is hallmarked by functional loss of E-cadherin. We show that TRPS1 is essential in mammary epithelial cells, since TRPS1-mediated suppression of interferon signaling promotes in vitro proliferation and lactogenic differentiation. Similarly, TRPS1 expression is indispensable for proliferation of mammary organoids and in vivo survival of luminal epithelial cells during mammary gland development. However, the consequences of TRPS1 loss are dependent on E-cadherin status, as combined inactivation of E-cadherin and TRPS1 causes persistent proliferation of mammary organoids and accelerated mammary tumor formation in mice. Together, our results demonstrate that TRPS1 can function as a context-dependent tumor suppressor in breast cancer, while being essential for growth and differentiation of normal mammary epithelial cells., (© 2020 Cornelissen et al.; Published by Cold Spring Harbor Laboratory Press.)

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.