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Invasive lobular carcinoma (ILC) accounts for 8%–14% of all breast cancer cases. The main hallmark of ILCs is the functional loss of the cell–cell adhesion protein E-cadherin. Nonetheless, loss of E-cadherin alone does not predispose mice to mammary tumor development, indicating that additional perturbations are required for ILC formation. Previously, we identified an N-terminal truncation variant of ASPP2 (t-ASPP2) as a driver of ILC in mice with mammary-specific loss of E-cadherin. Here we showed that expression of t-ASPP2 induced actomyosin relaxation, enabling adhesion and survival of E-cadherin–deficient murine mammary epithelial cells on stiff matrices like fibrillar collagen. The induction of actomyosin relaxation by t-ASPP2 was dependent on its interaction with protein phosphatase 1, but not on t-ASPP2–induced YAP activation. Truncated ASPP2 collaborated with both E-cadherin loss and PI3K pathway activation via PTEN loss in ILC development. t-ASPP2–induced actomyosin relaxation was required for ILC initiation, but not progression. Conversely, YAP activation induced by t-ASPP2 contributed to tumor growth and progression while being dispensable for tumor initiation. Together, these findings highlight two distinct mechanisms through which t-ASPP2 promotes ILC initiation and progression. Significance: Truncated ASPP2 cooperates with E-cadherin and PTEN loss to drive breast cancer initiation and progression via two distinct mechanisms. ASPP2-induced actomyosin relaxation drives tumor initiation, while ASPP2-mediated YAP activation enhances tumor progression.

E-cadherin (CDH1) is a master regulator of epithelial cell adherence junctions and a well-established tumor suppressor in Invasive Lobular Carcinoma (ILC). Intriguingly, somatic inactivation of E-cadherin alone in mouse mammary epithelial cells (MMECs) is insufficient to induce tumor formation. Here we show that E-cadherin loss induces extrusion of luminal MMECs to the basal lamina. Remarkably, E-cadherin-deficient MMECs can breach the basal lamina but do not disseminate into the surrounding fat pad. Basal lamina components laminin and collagen IV supported adhesion and survival of E-cadherin-deficient MMECs while collagen I, the principle component of the mammary stromal micro-environment did not. We uncovered that relaxation of actomyosin contractility mediates adhesion and survival of E-cadherin-deficient MMECs on collagen I, thereby allowing ILC development. Together, these findings unmask the direct consequences of E-cadherin inactivation in the mammary gland and identify aberrant actomyosin contractility as a critical barrier to ILC formation., E-cadherin is a well-known tumor suppressor albeit loss of E-cadherin alone is insufficient to induce mammary tumorigenesis. Here the authors report that epithelial cells that have lost E-cadherin can survive by extruding to the basal lamina but require rebalancing of actomyosin contractility to drive tumor development.

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. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell–cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice.

Post-translational modifications are used by cells to link additional information to proteins. Most modifications are subtle and concern small moieties such as a phosphate group or a lipid. In contrast, protein ubiquitylation entails the covalent attachment of a full-length protein such as ubiquitin. The protein ubiquitylation machinery is remarkably complex, comprising more than 15 Ubls (ubiquitin-like proteins) and several hundreds of ubiquitin-conjugating enzymes. Ubiquitin is best known for its role as a tag that induces protein destruction either by the proteasome or through targeting to lysosomes. However, addition of one or more Ubls also affects vesicular traffic, protein–protein interactions and signal transduction. It is by now well established that ubiquitylation is a component of most, if not all, cellular signalling pathways. Owing to its abundance in controlling cellular functions, ubiquitylation is also of key relevance to human pathologies, including cancer and inflammation. In the present review, we focus on its role in the control of cell adhesion, polarity and directional migration. It will become clear that protein modification by Ubls occurs at every level from the receptors at the plasma membrane down to cytoskeletal components such as actin, with differential consequences for the pathway's final output. Since ubiquitylation is fast as well as reversible, it represents a bona fide signalling event, which is used to fine-tune a cell's responses to receptor agonists.

The regulation of Focal Adhesion (FA) dynamics is a key aspect of cellular motility. FAs concentrate integrins and associated cytoskeletal elements as well as a large number of regulatory proteins, including adapters, kinases and small GtPases of the rho Family. We have recently shown that activated rac1 can localize to FAs and can initiate the accumulation of the adapter protein Caveolin1 (Cav1) at FAs. As reported by several groups including ours, this translocation requires Cav1 phosphorylation at tyr14, presumably by Src. here we provide additional data regarding this process and briefly review recent literature. Finally, we incorporated the different pieces of available information into a mechanistic model. this model proposes that local rac1 activation initiates a series of events that involve endosomal traffic of Cav1 and Src, targeting these proteins to or near FAs. next, within specific membrane domains, Src can mediate the phosphorylation of Cav1 at tyr 14, which is important for the stable FA localization of Cav1. Finally, dephosphorylation of Cav1 may represent a key step required for internalization, FA turnover and cell motility.

Invasive lobular carcinoma (ILC) accounts for 10-15% of breast cancer cases in women. One of the characteristics of ILC is the loss of intercellular adhesion, which occurs as a consequence of inactivation of E-cadherin (CDH1). Inactivation of E-cadherin often occurs through truncating mutations followed by loss of heterozygosity. E-cadherin is a master regulator of cell-cell interactions and thereby functions as an important tumor suppressor. Nevertheless, loss of E-cadherin by itself does not induce mammary tumor formation in mice. Moreover, somatic inactivation of E-cadherin in mouse mammary epithelium resulted in outgrowth mammary ducts with only E-cadherin proficient mammary epithelial cells, suggesting that loss of E-cadherin by itself is not tolerated. Why the inactivation of E-cadherin is not tolerated in mammary epithelial cells remains unclear. To uncover the fate of mammary epithelial cells upon inactivation of E-cadherin, a WapCre;Cdh1F/F;mTmg mouse model was generated. The introduction of the mTmG reporter allows for the identification and tracking of Cre-driven E-cadherin inactivated mammary epithelial cells. Analysis of the mammary ducts of these mice revealed that at least a portion of E-cadherin inactivated mammary epithelial cells survive. At 12 weeks of age clusters of E-cadherin inactivated mammary epithelial cells were found nested between the myoepithelial cells and the basal membrane. Despite the fact that they survive, these cells appear dormant since these clusters do not increase in size over time. Using in vivo imaging of WapCre;Cdh1F/F;mTmg mice, it becomes clear that the E-cadherin inactivated cells are motile and show a high degree of membrane blebbing. The E-cadherin inactivated cells have increased cortical accumulation of MYH9 and myosin light chain phosphorylation, which indicate increased actin contraction. Recently we identified PPP1R12A/B, TP53BP2, and MYH9 as drivers of ILC in an in vivo insertional mutagenesis screen. The identified drivers did not have any effect on the PI3 kinase and MAP kinase pathways nor did they alter P53 activation. They did, however, allow survival of E-cadherin inactivated mammary cells in vitro and in vivo. PPP1R12A/B and MYH9 are primarily known for their critical role in actin contractility. Interestingly, overexpression of the TRP53BP2 truncation variant identified in the screen reduced myosin light chain phosphorylation in a similar manner as the PPP1R12A truncation variant. To provide more evidence that the reduction of actin contraction allows rescue of E-cadherin inactivated cells, we inhibited actin contraction pharmacologically. Both exposure to a Rho kinase inhibitor as well as blebbistatin (inhibits non-muscle myosin ATPase activity) allowed for the growth of E-cadherin inactivated primary epithelial cells. Overall our data suggest that the inactivation of E-cadherin in mammary epithelial cells leads to aberrant actin contraction preventing oncogenic transformation. By reducing actin contraction, it allows for the many benefits of losing E-cadherin to drive the development of ILC. We are currently investigating through which signaling routes the increase in actin contraction prevents growth. Citation Format: Koen Schipper, Micha Nethe, Sjors M. Kas, Julian R. de Ruiter, Anne Paulien Drenth, Eline van der Burg, Ute Boon, Eva Schut, Jos Jonkers. Reducing actin contraction in E-cadherin inactivated mammary epithelial cells leads to the development of invasive lobular carcinoma [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 A47.

Rho-like guanosine triphosphatases (RhoGTPases) control many aspects of cellular physiology through their effects on the actin cytoskeleton and on gene transcription. Signalling by RhoGTPases is tightly coordinated and requires a series of regulatory proteins, including guanine-nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanine-nucleotide dissociation inhibitors (GDIs). GEFs and GAPs regulate GTPase cycling between the active (GTP-bound) and inactive (GDP-bound) states, whereas GDI is a cytosolic chaperone that binds inactive RhoGTPases. Like many other proteins, RhoGTPases are subject to degradation following the covalent conjugation of ubiquitin. There have been increasing indications that ubiquitylation of small GTPases occurs in a regulated fashion, primarily upon activation, and is an important means to control signalling output. Recent work has identified cellular proteins that control RasGTPase and RhoGTPase ubiquitylation and degradation, allowing us to amend the canonical model for GTPase (in)activation. Moreover, accumulating evidence for indirect regulation of GTPase function through the ubiquitylation of GTPase regulators makes this post-translational modification a key feature of GTPase-dependent signalling pathways. Here, we will discuss these recent insights into the regulation of RhoGTPase ubiquitylation and their relevance for cell signalling.

Invasive lobular carcinoma (ILC) is the second most common breast cancer subtype and accounts for 8-14% of all cases. Although the majority of human ILCs are characterized by the functional loss of E-cadherin (encoded by CDH1), inactivation of Cdh1 does not predispose mice to develop mammary tumors, implying that mutations in additional genes are required for ILC formation in mice. To identify these genes, we performed an insertional mutagenesis screen using the Sleeping Beauty transposon system in mice with mammary-specific inactivation of Cdh1. These mice developed multiple independent mammary tumors of which the majority resembled human ILC in terms of morphology and gene expression. Recurrent and mutually exclusive transposon insertions were identified in Myh9, Ppp1r12a, Ppp1r12b and Trp53bp2, whose products have been implicated in the regulation of the actin cytoskeleton. Notably, MYH9, PPP1R12B and TP53BP2 were also frequently aberrated in human ILC, highlighting these genes as drivers of a novel oncogenic pathway underlying ILC development.

Invasive lobular carcinoma (ILC) is the second most common breast cancer subtype, accounting for 5% to 15% of breast tumors.The majority of ILCs are characterized by the complete loss of the cell adhesion protein E-cadherin encoded by the CDH1 gene. However, WAPcre;Cdh1F/F mice with mammary gland-specific E-cadherin loss do not develop ILC, unless coupled with the additional disruption of a tumor suppressor gene, like Pten or Trp53. Compound mutant mice develop lesions that closely resemble the human disease in terms of histology and invasivity. However, genome-wide sequencing projects and forward genetic screens identified a number of additional putative ILC-initiating loci. Hence, there is now an urgent need for validation and characterization of these candidate cancer genes. We sought to determine if it was possible to deploy CRISPR/Cas9 technology to somatically inactivate candidate tumor suppressor genes in mammary gland tissue of adult mice. For this purpose we performed in Cdh1F/F mice intraductal injections of high-titer lentiviral vectors carrying different combinations of Cre, Cas9 and sgRNAs targeting several candidate genes, including Pten, Tgfbr2, Myh9, Nf1 and Fbxw7. We found that Cas9-bearing vectors elicited strong immune responses against transduced cells, which resulted in small ILC lesions surrounded by immune cells. However, when Cas9 was expressed endogenously in the mammary tissue from a knock-in allele, intraductal injection of lentiviral vectors encoding the sgRNA moiety of the CRISPR system was sufficient to induce penetrant and multifocal ILC formation in WAPcre;Cdh1F/F;Cas9 female mice. Sequencing revealed specific mutations clustered in the CRISPR targeted gene, and positive selection for loss-of-function indels. Indeed, immunohistochemistry and immunofluorescence analysis confirmed that tumors were largely deficient for E-cadherin and the targeted gene. In sum, we have successfully established a new platform for in vivo CRISPR-Cas9 mediated somatic gene editing in the mouse mammary gland. This rapid and versatile platform can be used to identify novel tumor drivers (e.g.by employing forward genetic screens) and validate candidate cancer genes in ILC and other breast cancer types. Citation Format: Stefano Annunziato, Sjors Kas, Micha Nethe, Hatice Yucel, Jessica del Bravo, Colin Pritchard, Rahmen Bin Ali, Bas van Gerwen, Bjorn Siteur, Anne Paulien Drenth, Eva Schut-Kregel, Sjoerd Klarenbeek, Ivo Huijbers, Martine van Miltenburg, Jos Jonkers. Rapid in vivo testing of tumor suppressors in ILC by CRISPR-Cas9 mediated somatic gene editing of the mammary gland. [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 2687.

Directional cell migration is crucially dependent on the spatiotemporal control of intracellular signalling events. These events regulate polarized actin dynamics, resulting in protrusion at the front of the cell and contraction at the rear. The actin cytoskeleton is regulated through signalling by Rho-like GTPases, such as RhoA, which stimulates myosin-based contractility, and CDC42 and Rac1, which promote actin polymerization and protrusion. Here, we show that Rac1 binds the adapter protein caveolin-1 (Cav1) and that Rac1 activity promotes Cav1 accumulation at Rac1-positive peripheral adhesions. Using Cav1-deficient mouse fibroblasts and depletion of Cav1 expression in human epithelial and endothelial cells mediated by small interfering RNA and short hairpin RNA, we show that loss of Cav1 induces an increase in Rac1 protein and its activated, GTP-bound form. Cav1 controls Rac1 protein levels by regulating ubiquitylation and degradation of activated Rac1 in an adhesion-dependent fashion. Finally, we show that Rac1 ubiquitylation is not required for effector binding, but regulates the dynamics of Rac1 at the periphery of the cell. These data extend the canonical model of Rac1 inactivation and uncover Cav1-regulated polyubiquitylation as an additional mechanism to control Rac1 signalling.

Summary Invasive lobular carcinoma (ILC) is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary epithelium, we generated a mouse model of classical ILC (CLC), the main histological ILC subtype. While loss of E-cadherin induced cell dissemination and apoptosis, additional PTEN inactivation promoted cell survival and rapid formation of invasive mammary tumors that recapitulate the histological and molecular features, estrogen receptor (ER) status, growth kinetics, metastatic behavior, and tumor microenvironment of human CLC. Combined inactivation of E-cadherin and PTEN is sufficient to cause CLC development. These CLCs showed significant tumor regression upon BEZ235-mediated inhibition of PI3K signaling. In summary, this mouse model provides important insights into CLC development and suggests inhibition of phosphatidylinositol 3-kinase (PI3K) signaling as a potential therapeutic strategy for targeting CLC., Graphical Abstract, Highlights • PTEN loss rescues apoptosis induced by E-cadherin loss in mouse mammary epithelium • Combined loss of E-cadherin and PTEN is sufficient to cause mouse mammary tumors • These mouse mammary tumors closely resemble human classical lobular carcinoma (CLC) • Mouse CLCs induced by loss of E-cadherin and PTEN regress upon PI3K inhibition, Boelens et al. demonstrate that combined inactivation of E-cadherin and PTEN in mouse mammary epithelium results in rapid formation of classical invasive lobular carcinoma (CLC). This mouse model provides important insights into CLC development and suggests that inhibition of PI3K signaling is a potential strategy for targeting CLC.