SKOR1 mediates FER kinase-dependent invasive growth of breast cancer cells.

High expression of the non-receptor tyrosine kinase FER is an independent prognostic factor that correlates with poor survival in breast cancer patients. To investigate whether the kinase activity of FER is essential for its oncogenic properties, we developed an ATP analogue-sensitive knock-in allele (FERASKI). Specific FER kinase inhibition in MDA-MB-231 cells reduces migration and invasion, as well as metastasis when xenografted into a mouse model of breast cancer. Using the FERASKI system, we identified Ski family transcriptional corepressor 1 (SKOR1) as a direct FER kinase substrate. SKOR1 loss phenocopies FER inhibition, leading to impaired proliferation, migration and invasion, and inhibition of breast cancer growth and metastasis formation in mice. We show that SKOR1 Y234, a candidate FER phosphorylation site, is essential for FER-dependent tumor progression. Finally, our work suggests that the SKOR1 Y234 residue promotes Smad2/3 signaling through SKOR1 binding to Smad3. Our study thus identifies SKOR1 as a mediator of FER-dependent progression of high-risk breast cancers.

Integration of Pathological Criteria and Immunohistochemical Evaluation for Invasive Lobular Carcinoma Diagnosis: Recommendations From the European Lobular Breast Cancer Consortium.

Invasive lobular carcinoma (ILC) is the second most frequent type of breast cancer (BC) and its peculiar morphology is mainly driven by inactivation of CDH1, the gene coding for E-cadherin cell adhesion protein. ILC-specific therapeutic and disease-monitoring approaches are gaining momentum in the clinic, increasing the importance of accurate ILC diagnosis. Several essential and desirable morphologic diagnostic criteria are currently defined by the World Health Organization, the routine use of immunohistochemistry (IHC) for E-cadherin is not recommended. Disagreement in the diagnosis of ILC has been repeatedly reported, but interpathologist agreement increases with the use of E-cadherin IHC. In this study, we aimed to harmonize the pathological diagnosis of ILC by comparing 5 commonly used E-cadherin antibody clones (NCH-38, EP700Y, Clone 36, NCL-L-E-cad [Clone 36B5], and ECH-6). We determined their biochemical specificity for the E-cadherin protein and IHC staining performance according to type and location of mutation on the CDH1 gene. Western blot analysis on mouse cell lines with conditional E-cadherin expression revealed a reduced specificity of EP700Y and NCL-L-E-cad for E-cadherin, with cross-reactivity of Clone 36 to P-cadherin. The use of IHC improved interpathologist agreement for ILC, lobular carcinoma in situ, and atypical lobular hyperplasia. The E-cadherin IHC staining pattern was associated with variant allele frequency and likelihood of nonsense-mediated RNA decay but not with the type or position of CDH1 mutations. Based on these results, we recommend the indication for E-cadherin staining, choice of antibodies, and their interpretation to standardize ILC diagnosis in current pathology practice.

The laminin-keratin link shields the nucleus from mechanical deformation and signalling.

The mechanical properties of the extracellular matrix dictate tissue behaviour. In epithelial tissues, laminin is a very abundant extracellular matrix component and a key supporting element. Here we show that laminin hinders the mechanoresponses of breast epithelial cells by shielding the nucleus from mechanical deformation. Coating substrates with laminin-111-unlike fibronectin or collagen I-impairs cell response to substrate rigidity and YAP nuclear localization. Blocking the laminin-specific integrin ß4 increases nuclear YAP ratios in a rigidity-dependent manner without affecting the cell forces or focal adhesions. By combining mechanical perturbations and mathematical modelling, we show that ß4 integrins establish a mechanical linkage between the substrate and keratin cytoskeleton, which stiffens the network and shields the nucleus from actomyosin-mediated mechanical deformation. In turn, this affects the nuclear YAP mechanoresponses, chromatin methylation and cell invasion in three dimensions. Our results demonstrate a mechanism by which tissues can regulate their sensitivity to mechanical signals.

Results of a worldwide survey on the currently used histopathological diagnostic criteria for invasive lobular breast cancer.

Invasive lobular carcinoma (ILC) represents the second most common subtype of breast cancer (BC), accounting for up to 15% of all invasive BC. Loss of cell adhesion due to functional inactivation of E-cadherin is the hallmark of ILC. Although the current world health organization (WHO) classification for diagnosing ILC requires the recognition of the dispersed or linear non-cohesive growth pattern, it is not mandatory to demonstrate E-cadherin loss by immunohistochemistry (IHC). Recent results of central pathology review of two large randomized clinical trials have demonstrated relative overdiagnosis of ILC, as only ~60% of the locally diagnosed ILCs were confirmed by central pathology. To understand the possible underlying reasons of this discrepancy, we undertook a worldwide survey on the current practice of diagnosing BC as ILC. A survey was drafted by a panel of pathologists and researchers from the European lobular breast cancer consortium (ELBCC) using the online tool SurveyMonkey®. Various parameters such as indications for IHC staining, IHC clones, and IHC staining procedures were questioned. Finally, systematic reporting of non-classical ILC variants were also interrogated. This survey was sent out to pathologists worldwide and circulated from December 14, 2020 until July, 1 2021. The results demonstrate that approximately half of the institutions use E-cadherin expression loss by IHC as an ancillary test to diagnose ILC and that there is a great variability in immunostaining protocols. This might cause different staining results and discordant interpretations. As ILC-specific therapeutic and diagnostic avenues are currently explored in the context of clinical trials, it is of importance to improve standardization of histopathologic diagnosis of ILC diagnosis.

Re-inforcing the cell death army in the fight against breast cancer.

Metastatic breast cancer is responsible for most breast cancer-related deaths. Disseminated cancer cells have developed an intrinsic ability to resist anchorage-dependent apoptosis (anoikis). Anoikis is caused by the absence of cellular adhesion, a process that underpins lumen formation and maintenance during mammary gland development and homeostasis. In healthy cells, anoikis is mostly governed by B-cell lymphoma-2 (BCL2) protein family members. Metastatic cancer cells, however, have often developed autocrine BCL2-dependent resistance mechanisms to counteract anoikis. In this Review, we discuss how a pro-apoptotic subgroup of the BCL2 protein family, known as the BH3-only proteins, controls apoptosis and anoikis during mammary gland homeostasis and to what extent their inhibition confers tumor suppressive functions in metastatic breast cancer. Specifically, the role of the two pro-apoptotic BH3-only proteins BCL2-modifying factor (BMF) and BCL2-interacting mediator of cell death (BIM) will be discussed here. We assess current developments in treatment that focus on mimicking the function of the BH3-only proteins to induce apoptosis, and consider their applicability to restore normal apoptotic responses in anchorage-independent disseminating tumor cells.

E-cadherin to P-cadherin switching in lobular breast cancer with tubular elements.

Loss of E-cadherin expression due to mutation of the CDH1 gene is a characteristic feature of invasive lobular breast cancer (ILBC). Beta-catenin, which binds to the cytoplasmic domain of E-cadherin, is simultaneously downregulated, reflecting disassembly of adherens junctions (AJs) and loss of cell adhesion. E-cadherin to P-cadherin expression switching can rescue AJs and cell adhesion. However, P-cadherin has not been implicated in ILBC, so far. We aimed to characterize 13 ILBCs with exceptional histomorphology, which we termed ILBCs with tubular elements. The CDH1 mutational status was determined by next generation sequencing and whole-genome copy number (CN) profiling. Expression of cadherins was assessed by immunohistochemistry. ILBCs with tubular elements were ER-positive (13/13) and HER2-negative (13/13) and harbored deleterious CDH1 mutations (11/13) accompanied by loss of heterozygosity due to deletion of chromosome 16q22.1 (9/11). E-cadherin expression was lost or reduced in noncohesive tumor cells and in admixed tubular elements (13/13). Beta-catenin expression was lost in noncohesive tumor cells, but was retained in tubular elements (11/13), indicating focal rescue of AJ formation. N-cadherin and R-cadherin were always negative (0/13). Strikingly, P-cadherin was commonly positive (12/13) and immunoreactivity was accentuated in tubular elements. Adjacent lobular carcinoma in situ (LCIS) was always P-cadherin-negative (0/7). In a reference cohort of LCIS specimens, P-cadherin was constantly not expressed (0/25). In a reference cohort of invasive mammary carcinomas, P-cadherin-positive cases (36/268, 13%) were associated with triple-negative nonlobular breast cancer (P < 0.001). Compared with ILBCs from the reference cohort, P-cadherin expression was more common in ILBCs with tubular elements (12/13 versus 7/84, P < 0.001). In summary, E-cadherin to P-cadherin switching occurs in a subset of ILBCs. P-cadherin is the molecular determinant of a mixed-appearing histomorphology in ILBCs with tubular elements.

p120-Catenin Is Critical for the Development of Invasive Lobular Carcinoma in Mice.

Loss of E-cadherin expression is causal to the development of invasive lobular breast carcinoma (ILC). E-cadherin loss leads to dismantling of the adherens junction and subsequent translocation of p120-catenin (p120) to the cytosol and nucleus. Although p120 is critical for the metastatic potential of ILC through the regulation of Rock-dependent anoikis resistance, it remains unknown whether p120 also contributes to ILC development. Using genetically engineered mouse models with mammary gland-specific inactivation of E-cadherin, p120 and p53, we demonstrate that ILC formation induced by E-cadherin and p53 loss is severely impaired upon concomitant inactivation of p120. Tumors that developed in the triple-knockout mice were mostly basal sarcomatoid carcinomas that displayed overt nuclear atypia and multinucleation. In line with the strong reduction in ILC incidence in triple-knockout mice compared to E-cadherin and p53 double-knockout mice, no functional redundancy of p120 family members was observed in mouse ILC development, as expression and localization of ARVCF, p0071 or δ-catenin was unaltered in ILCs from triple-knockout mice. In conclusion, we show that loss of p120 in the context of the p53-deficient mouse models is dominant over E-cadherin inactivation and its inactivation promotes the development of basal, epithelial-to-mesenchymal-transition (EMT)-type invasive mammary tumors.

Lobular breast cancer: molecular basis, mouse and cellular models.

Infiltrating lobular breast cancer (ILC) is the most common special breast cancer subtype. With mutational or epigenetic inactivation of the cell adhesion molecule E-cadherin (CDH1) being confined almost exclusively to ILC, this tumor entity stands out from all other types of breast cancers. The molecular basis of ILC is linked to loss of E-cadherin, as evidenced by human CDH1 germline mutations and conditional knockout mouse models. A better understanding of ILC beyond the level of descriptive studies depends on physiologically relevant and functional tools. This review provides a detailed overview on ILC models, including well-characterized cell lines, xenograft tumors and genetically engineered mouse models. We consider advantages and limitations of these models and evaluate their representativeness for human ILC. The still incompletely defined mechanisms by which loss of E-cadherin drives malignant transformation are discussed based on recent findings in these models. Moreover, candidate genes and signaling pathways potentially involved in ILC development and progression as well as anticancer drug and endocrine resistance are highlighted.

p120-catenin in cancer - mechanisms, models and opportunities for intervention.

The epithelial adherens junction is an E-cadherin-based complex that controls tissue integrity and is stabilized at the plasma membrane by p120-catenin (p120, also known as CTNND1). Mutational and epigenetic inactivation of E-cadherin has been strongly implicated in the development and progression of cancer. In this setting, p120 translocates to the cytosol where it exerts oncogenic properties through aberrant regulation of Rho GTPases, growth factor receptor signaling and derepression of Kaiso (also known as ZBTB33) target genes. In contrast, indirect inactivation of the adherens junction through conditional knockout of p120 in mice was recently linked to tumor formation, indicating that p120 can also function as a tumor suppressor. Supporting these opposing functions are findings in human cancer, which show that either loss or cytoplasmic localization of p120 is a common feature in the progression of several types of carcinoma. Underlying this dual biological phenomenon might be the context-dependent regulation of Rho GTPases in the cytosol and the derepression of Kaiso target genes. Here, we discuss past and present findings that implicate p120 in the regulation of cancer progression and highlight opportunities for clinical intervention.

Somatic inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis.

Metastatic disease is the primary cause of death in breast cancer, the most common malignancy in Western women. Loss of E-cadherin is associated with tumor metastasis, as well as with invasive lobular carcinoma (ILC), which accounts for 10%-15% of all breast cancers. To study the role of E-cadherin in breast oncogenesis, we have introduced conditional E-cadherin mutations into a mouse tumor model based on epithelium-specific knockout of p53. Combined loss of E-cadherin and p53 resulted in accelerated development of invasive and metastatic mammary carcinomas, which show strong resemblance to human ILC. Moreover, loss of E-cadherin induced anoikis resistance and facilitated angiogenesis, thus promoting metastatic disease. Our results suggest that loss of E-cadherin contributes to both mammary tumor initiation and metastasis.

Differential transcriptional invasion signatures from patient derived organoid models define a functional prognostic tool for head and neck cancer.

Clinical outcome for patients suffering from HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. This is mostly due to highly invasive tumors that cause loco-regional relapses after initial therapeutic intervention and metastatic outgrowth. The molecular pathways governing the detrimental invasive growth modes in HNSCC remain however understudied. Here, we have established HNSCC patient derived organoid (PDO) models that recapitulate 3-dimensional invasion in vitro. Single cell mRNA sequencing was applied to study the differences between non-invasive and invasive conditions, and in a collective versus single cell invading PDO model. Differential expression analysis under invasive conditions in Collagen gels reveals an overall upregulation of a YAP-centered transcriptional program, irrespective of the invasion mode. However, we find that collectively invading HNSCC PDO cells show elevated levels of YAP transcription targets when compared to single cell invasion. Also, collectively invading cells are characterized by increased nuclear translocation of YAP within the invasive strands, which coincides with Collagen-I matrix alignment at the invasive front. Using gene set enrichment analysis, we identify immune cell-like migratory pathways in the single cell invading HNSCC PDO, while collective invasion is characterized by overt upregulation of adhesion and migratory pathways. Lastly, based on clinical head and neck cancer cohorts, we demonstrate that the identified collective invasion signature provides a candidate prognostic platform for survival in HNSCC. By uncoupling collective and single cell invasive programs, we have established invasion signatures that may guide new therapeutic options.

Reporting on invasive lobular breast cancer in clinical trials: a systematic review.

Invasive lobular breast cancer (ILC) differs from invasive breast cancer of no special type in many ways. Evidence on treatment efficacy for ILC is, however, lacking. We studied the degree of documentation and representation of ILC in phase III/IV clinical trials for novel breast cancer treatments. Trials were identified on Pubmed and clinicaltrials.gov. Inclusion/exclusion criteria were reviewed for requirements on histological subtype and tumor measurability. Documentation of ILC was assessed and ILC inclusion rate, central pathology and subgroup analyses were evaluated. Inclusion restrictions concerning tumor measurability were found in 39/93 manuscripts. Inclusion rates for ILC were documented in 13/93 manuscripts and varied between 2.0 and 26.0%. No central pathology for ILC was reported and 3/13 manuscripts had ILC sub-analyses. ILC is largely disregarded in most trials with poor representation and documentation. The current inclusion criteria using RECIST v1.1, fall short in recognizing the unique non-measurable metastatic infiltration of ILC.

Rapid autopsies to enhance metastatic research: the UPTIDER post-mortem tissue donation program.

Research on metastatic cancer has been hampered by limited sample availability. Here we present the breast cancer post-mortem tissue donation program UPTIDER and show how it enabled sampling of a median of 31 (range: 5-90) metastases and 5-8 liquids per patient from its first 20 patients. In a dedicated experiment, we show the mild impact of increasing time after death on RNA quality, transcriptional profiles and immunohistochemical staining in tumor tissue samples. We show that this impact can be counteracted by organ cooling. We successfully generated ex vivo models from tissue and liquid biopsies from distinct histological subtypes of breast cancer. We anticipate these and future findings of UPTIDER to elucidate mechanisms of disease progression and treatment resistance and to provide tools for the exploration of precision medicine strategies in the metastatic setting.

Intravital microscopy: new insights into metastasis of tumors.

Metastasis, the process by which cells spread from the primary tumor to a distant site to form secondary tumors, is still not fully understood. Although histological techniques have provided important information, they give only a static image and thus compromise interpretation of this dynamic process. New advances in intravital microscopy (IVM), such as two-photon microscopy, imaging chambers, and multicolor and fluorescent resonance energy transfer imaging, have recently been used to visualize the behavior of single metastasizing cells at subcellular resolution over several days, yielding new and unexpected insights into this process. For example, IVM studies showed that tumor cells can switch between multiple invasion strategies in response to various densities of extracellular matrix. Moreover, other IVM studies showed that tumor cell migration and blood entry take place not only at the invasive front, but also within the tumor mass at tumor-associated vessels that lack an intact basement membrane. In this Commentary, we will give an overview of the recent advances in high-resolution IVM techniques and discuss some of the latest insights in the metastasis field obtained with IVM.

R-spondin-3 promotes proliferation and invasion of breast cancer cells independently of Wnt signaling.

We recently identified R-spondin-3 (RSPO3) as a novel driver of breast cancer associating with reduced patient survival, expanding its clinical value as potential therapeutic target that had been recognized mostly for colorectal cancer so far. (Pre)clinical studies exploring RSPO3 targeting in colorectal cancer approach this indirectly with Wnt inhibitors, or directly with anti-RSPO3 antibodies. Here, we address the clinical relevance of RSPO3 in breast cancer and provide insight in the oncogenic activities of RSPO3. Utilizing the RSPO3 breast cancer mouse model, we show that RSPO3 drives the aberrant expansion of luminal progenitor cells expressing cancer stem cell marker CD61, inducing proliferative, poorly differentiated and invasive tumors. Complementary studies with tumor organoids and human breast cancer cell lines demonstrate that RSPO3 consistently promotes the proliferation and invasion of breast cancer cells. Importantly, RSPO3 exerts these oncogenic effects independently of Wnt signaling, rejecting the therapeutic value of Wnt inhibitors in RSPO3-driven breast cancer. Instead, direct RSPO3 targeting effectively inhibited RSPO3-driven growth of breast cancer cells. Conclusively, our data indicate that RSPO3 exerts unfavorable oncogenic effects in breast cancer, enhancing proliferation and malignancy in a Wnt-independent fashion, proposing RSPO3 itself as a valuable therapeutic target in breast cancer.

LGR6-dependent conditional inactivation of E-cadherin and p53 leads to invasive skin and mammary carcinomas in mice.

Tissue-specific inactivation of E-cadherin combined with tumor suppressor loss leads to invasive and metastatic cancers in mice. While epidermal E-cadherin loss in mice induces squamous cell carcinomas, inactivation of E-cadherin in the mammary gland leads to invasive lobular carcinoma. To further explore the carcinogenic consequences of cell-cell adhesion loss in these compartments, we developed a new conditional mouse model inactivating E-cadherin (Cdh1) and p53 (Trp53) simultaneously in cells expressing the leucine-rich repeat-containing G-protein coupled receptor 6 (Lgr6), a putative epithelial stem cell marker in the skin and alveolar progenitor marker in the mammary gland. Compound Lgr6-CreERT2;Cdh1F;Trp53F female mice containing either heterozygous or homozygous Cdh1F alleles were bred, and Lgr6-driven Cre expression was activated in pre-puberal mice using tamoxifen. We observed that 41% of the mice (16/39) developed mostly invasive squamous-type skin carcinomas, but also a non-lobular mammary tumor was formed. In contrast to previous K14cre or WAPcre E-cadherin and p53 compound models, no significant differences were detected in the tumor-free survival of Lgr6-CreERT2 heterozygous Cdh1F/WT;Trp53F/F versus homozygous Cdh1F/F;Trp53F/F mice (778 versus 754 days, p=0.5). One Cdh1F homozygous mouse presented with lung metastasis that originated from a non-lobular and ERα negative invasive mammary gland carcinoma with squamous metaplasia. In total, 2/8 (25%) Cdh1F heterozygous and 3/12 (25%) Cdh1F homozygous mice developed metastases to lungs, liver, lymph nodes, or the gastro-intestinal tract. In conclusion, we show that inducible and conditional Lgr6-driven inactivation of E-cadherin and p53 in mice causes squamous cell carcinomas of the skin in approximately 40% of the mice and an occasional ductal-type mammary carcinoma after long latency periods.

NCAM-induced focal adhesion assembly: a functional switch upon loss of E-cadherin.

Loss of expression of the cell-cell adhesion molecule E-cadherin is a hallmark of epithelial-mesenchymal transition (EMT) in development and in the progression from epithelial tumours to invasive and metastatic cancers. Here, we demonstrate that the loss of E-cadherin function upregulates expression of the neuronal cell adhesion molecule (NCAM). Subsequently, a subset of NCAM translocates from fibroblast growth factor receptor (FGFR) complexes outside lipid rafts into lipid rafts where it stimulates the non-receptor tyrosine kinase p59(Fyn) leading to the phosphorylation and activation of focal adhesion kinase and the assembly of integrin-mediated focal adhesions. Ablation of NCAM expression during EMT inhibits focal adhesion assembly, cell spreading and EMT. Conversely, forced expression of NCAM induces epithelial cell delamination and migration, and high NCAM expression correlates with tumour invasion. These results establish a mechanistic link between the loss of E-cadherin expression, NCAM function, focal adhesion assembly and cell migration and invasion.

Spatial collagen stiffening promotes collective breast cancer cell invasion by reinforcing extracellular matrix alignment.

The tumor micro-environment often contains stiff and irregular-bundled collagen fibers that are used by tumor cells to disseminate. It is still unclear how and to what extent, extracellular matrix (ECM) stiffness versus ECM bundle size and alignment dictate cancer cell invasion. Here, we have uncoupled Collagen-I bundling from stiffness by introducing inter-collagen crosslinks, combined with temperature induced aggregation of collagen bundling. Using organotypic models from mouse invasive ductal and invasive lobular breast cancers, we show that increased collagen bundling in 3D induces a generic increase in breast cancer invasion that is independent of migration mode. However, systemic collagen stiffening using advanced glycation end product (AGE) crosslinking prevents collective invasion, while leaving single cell invasion unaffected. Collective invasion into collagen matrices by ductal breast cancer cells depends on Lysyl oxidase-like 3 (Loxl3), a factor produced by tumor cells that reinforces local collagen stiffness. Finally, we present clinical evidence that collectively invading cancer cells at the invasive front of ductal breast carcinoma upregulate LOXL3. By uncoupling the mechanical, chemical, and structural cues that control invasion of breast cancer in three dimensions, our data reveal that spatial control over stiffness and bundling underlie collective dissemination of ductal-type breast cancers.

Small molecules to regulate the GH/IGF1 axis by inhibiting the growth hormone receptor synthesis.

Growth hormone (GH) and insulin-like growth factor-1 (IGF1) play an important role in mammalian development, cell proliferation and lifespan. Especially in cases of tumor growth there is an urgent need to control the GH/IGF1 axis. In this study we screened a 38,480-compound library, and in two consecutive rounds of analogues selection, we identified active lead compounds based on the following criteria: inhibition the GH receptor (GHR) activity and its downstream effectors Jak2 and STAT5, and inhibition of growth of breast and colon cancer cells. The most active small molecule (BM001) inhibited both the GH/IGF1 axis and cell proliferation with an IC50 of 10-30 nM of human cancer cells. BM001 depleted GHR in human lymphoblasts. In preclinical xenografted experiments, BM001 showed a strong decrease in tumor volume in mice transplanted with MDA-MB-231 breast cancer cells. Mechanistically, the drug acts on the synthesis of the GHR. Our findings open the possibility to inhibit the GH/IGF1 axis with a small molecule.

Loss of E-cadherin leads to Id2-dependent inhibition of cell cycle progression in metastatic lobular breast cancer.

Invasive lobular breast carcinoma (ILC) is characterized by proliferative indolence and long-term latency relapses. This study aimed to identify how disseminating ILC cells control the balance between quiescence and cell cycle re-entry. In the absence of anchorage, ILC cells undergo a sustained cell cycle arrest in G0/G1 while maintaining viability. From the genes that are upregulated in anchorage independent ILC cells, we selected Inhibitor of DNA binding 2 (Id2), a mediator of cell cycle progression. Using loss-of-function experiments, we demonstrate that Id2 is essential for anchorage independent survival (anoikis resistance) in vitro and lung colonization in mice. Importantly, we find that under anchorage independent conditions, E-cadherin loss promotes expression of Id2 in multiple mouse and (organotypic) human models of ILC, an event that is caused by a direct p120-catenin/Kaiso-dependent transcriptional de-repression of the canonical Kaiso binding sequence TCCTGCNA. Conversely, stable inducible restoration of E-cadherin expression in the ILC cell line SUM44PE inhibits Id2 expression and anoikis resistance. We show evidence that Id2 accumulates in the cytosol, where it induces a sustained and CDK4/6-dependent G0/G1 cell cycle arrest through interaction with hypo-phosphorylated Rb. Finally, we find that Id2 is indeed enriched in ILC when compared to other breast cancers, and confirm cytosolic Id2 protein expression in primary ILC samples. In sum, we have linked mutational inactivation of E-cadherin to direct inhibition of cell cycle progression. Our work indicates that loss of E-cadherin and subsequent expression of Id2 drive indolence and dissemination of ILC. As such, E-cadherin and Id2 are promising candidates to stratify low and intermediate grade invasive breast cancers for the use of clinical cell cycle intervention drugs.