The gut microbiota strongly impacts the development of sporadic colorectal cancer (CRC), but it is largely unknown how the microbiota affects the pathogenesis of mismatch-repair-deficient CRC in... Show moreThe gut microbiota strongly impacts the development of sporadic colorectal cancer (CRC), but it is largely unknown how the microbiota affects the pathogenesis of mismatch-repair-deficient CRC in the context of Lynch syndrome. In a mouse model for Lynch syndrome, we found a nearly complete loss of intestinal tumor development when animals were transferred from a conventional "open" animal facility to specific-pathogen-free (SPF) conditions. Using 16S sequencing we detected large changes in microbiota composition between the two facilities. Transcriptomic analyses of tumor-free intestinal tissues showed signs of strong intestinal inflammation in conventional mice. Whole exome sequencing of tumors developing in Msh2-Lynch mice revealed a much lower mutational load in the single SPF tumor than in tumors developing in conventional mice, suggesting reduced epithelial proliferation in SPF mice. Fecal microbiota transplantations with conventional feces altered the immune landscape and gut homeostasis, illustrated by increased gut length and elevated epithelial proliferation and migration. This was associated with drastic changes in microbiota composition, in particular increased relative abundances of different mucus-degrading taxa such as Desulfovibrio and Akkermansia, and increased bacterial-epithelial contact. Strikingly, transplantation of conventional microbiota increased microsatellite instability in untransformed intestinal epithelium of Msh2-Lynch mice, indicating that the composition of the microbiota influences the rate of mutagenesis in MSH2-deficient crypts. Show less
Pulver, E.M.; Mukherjee, C.; Kamp, G. van de; Roobol, S.J.; Rother, M.B.; Gulden, H. van der; ... ; Jonkers, J. 2021
The BRCA1 tumor suppressor gene encodes a multidomain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand... Show moreThe BRCA1 tumor suppressor gene encodes a multidomain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand breaks, which is shared with two other highrisk hereditary breast cancer suppressors, BRCA2 and PALB2. Although both BRCA1 and BRCA2 interact with PALB2, BRCA1 domain are considered variants of uncertain clinical significance (VUS). Using genetically engineered mice, we show here that a BRCA1 coiled-coil domain VUS, Brca1 p.L1363P, disrupts the interaction with PALB2 and leads to embryonic lethality. Brca1 p.L1363P led to a similar acceleration in the development of Trp53deficient mammary tumors as Brca1 loss, but the tumors showed distinct histopathologic features, with more stable DNA copy number profiles in Brca1 p.L1363P tumors. Nevertheless, Brca1 p.L1363P mammary tumors were HRR incompetent and responsive to cisplatin and PARP inhibition. Overall, these results provide the first direct evidence that a BRCA1 missense variant outside of the RING and BRCT domains increases the risk of Show less
Invasive lobular carcinoma (ILC) is the second most common type of breast cancer. Hallmarks of ILC include disruption of adherens junctions and hyperactivation of phosphoinositide 3-kinase (PI3K)... Show moreInvasive lobular carcinoma (ILC) is the second most common type of breast cancer. Hallmarks of ILC include disruption of adherens junctions and hyperactivation of phosphoinositide 3-kinase (PI3K)-mTOR signaling. The tumor suppressor PTEN regulates PI3K signaling. We present a preclinical mouse model of ILC metastasis, based on inactivation of the adherens junction protein E-cadherin and the tumor suppressor p53 and surgical excision of primary tumors. In this model, pharmacological mTOR inhibition blocks growth of primary tumors as well as metastatic disease, and this response is partially dependent on the adaptive immune system. Loss of E-cadherin mouse mammary epithelium leads to apoptosis, and PTEN activation alone results in squamous metaplastic mammary tumors, but a combination of these events leads to ILC formation, indicating a causal role of PI3K signaling together with E-cadherin loss in ILC. Combined somatic loss of the adherens junction molecule p120 and p53 in the mouse mammary gland leads to metaplastic mammary tumors, and loss of p120 in breast cancer cell lines promotes anoikis resistance through hypersensitization of growth factor receptor (GFR) signaling. Combined inactivation of E-cadherin, p120 and p53 induces basal-like tumors, with an epithelial-to- mesenchymal-transition (EMT) phenotype, and no ILC formation. Show less
Klarenbeek, S.; Doornebal, C.W.; Kas, S.M.; Bonzanni, N.; Bhin, J.; Braumuller, T.M.; ... ; Jonkers, J. 2020
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... Show moreEffective 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-naive 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. Show less
Miltenburg, M.H. van; Nimwegen, M.J. van; Tijdens, R.B.; Lalai, R.A.; Kuiper, R.; Klarenbeek, S.; ... ; Water, B. van de 2014
BACKGROUND Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary... Show moreBACKGROUND Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development. METHODS To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53(lox/lox)/FAK(+/+)/WapCre, p53(lox/lox)/FAK(flox/+)/WapCre and p53(lox/lox)/FAK(flox/-)/WapCre mice, and mice with WapCre-mediated conditional expression of p53(R270H), the mouse equivalent of human p53(R273H) hot spot mutation, together with conditional deletion of FAK, P53(R270H/+)/FAK(lox/+)/WapCre and p53(R270H/+)/FAK(flox/-)/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours. RESULTS Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53(R270H)-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures. CONCLUSIONS Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53(R270H) mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53(R270H)-induced mammary tumour development. Show less
