Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14 + cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14+ epithelial tumor cell clusters disseminate collectively to colonize distant organs.uring metastasis, cancer cells escape the primary tumor, travel through the circulation, and colonize distant organs. Conventional models of cancer progression propose that each metastasis arises from the clonal outgrowth of a single tumor cell and this conceptual framework is a foundation for models, such as epithelial-mesenchymal transition (EMT) and migratory cancer stem cells (1).Challenging the generality of the single-cell/single-metastasis model are long-standing clinical observations that tumor cell clusters (also termed "tumor clumps") are also observed across the stages of metastasis. Tumor cell clusters are detected in the bloodstream of cancer patients (2), clusters can efficiently seed metastases (3), and though rare, circulating tumor cell (CTC) clusters have prognostic significance (4, 5). Furthermore, metastases are composed of multiple genetically distinct tumor cell clones, in mouse models of breast, pancreas, and small cell carcinoma (5-7), and in human metastatic prostate cancer patients (8). Taken together, these observations provide accumulating evidence that tumor cell clusters contribute to metastasis. However, they leave unresolved two important questions: how do tumor cell clusters emerge from the primary tumor, and which molecular features identify cell clusters that metastasize?An important clinical observation is that cancer cells invade the surrounding stroma as cohesive clusters in the majority of ...

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.

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.

Invasive lobular carcinoma (ILC) accounts for up to 15% of all breast cancer (BC) cases and responds well to endocrine treatment when estrogen receptor α-positive (ER+) yet differs in many biological aspects from other ER+ BC subtypes. Up to 30% of patients with ILC will develop late-onset metastatic disease up to ten years after initial tumor diagnosis and may experience failure of systemic therapy. Unfortunately, preclinical models to study ILC progression and predict the efficacy of novel therapeutics are scarce. Here, we review the current advances in ILC modeling, including cell lines and organotypic models, genetically engineered mouse models, and patient-derived xenografts. We also underscore four critical challenges that can be addressed using ILC models: drug resistance, lobular tumor microenvironment, tumor dormancy, and metastasis. Finally, we highlight the advantages of shared experimental ILC resources and provide essential considerations from the perspective of the European Lobular Breast Cancer Consortium (ELBCC), which is devoted to better understanding and translating the molecular cues that underpin ILC to clinical diagnosis and intervention. This review will guide investigators who are considering the implementation of ILC models in their research programs.

In human cancers, FGFR signaling is frequently hyperactivated by deregulation of FGF ligands or by activating mutations in the FGFR receptors such as gene amplifications, point mutations, and gene fusions. As such, FGFR inhibitors are considered an attractive therapeutic strategy for patients with mutations in FGFR family members. We previously identified as a key driver of invasive lobular carcinoma (ILC) in an insertional mutagenesis screen using the transposon system. Here we explore whether these FGFR-driven ILCs are sensitive to the FGFR inhibitor AZD4547 and use transposon mutagenesis in these tumors to identify potential mechanisms of resistance to therapy. Combined with RNA sequencing-based analyses of AZD4547-resistant tumors, our approach identified several known and novel potential resistance mechanisms to FGFR inhibition, most of which converged on reactivation of the canonical MAPK-ERK signaling cascade. Observed resistance mechanisms included mutations in the tyrosine kinase domain of FGFR2, overexpression of MET, inactivation of RASA1, and activation of the drug-efflux transporter ABCG2. ABCG2 and RASA1 were identified only from transposon insertions acquired during AZD4547 treatment, demonstrating that insertional mutagenesis in mice is an effective tool for identifying potential mechanisms of resistance to targeted cancer therapies. These findings demonstrate that a combined approach of transcriptomics and insertional mutagenesis is an effective method for identifying potential targets to overcome resistance to therapy in the clinic. .

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 (PP1) 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, YAP1 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. Statement of significanceTruncated 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 YAP1 activation enhances tumor progression.

Although the 5-year survival rates for sarcoma patients have improved, the proportion of patients relapsing after first line treatment remains high and survival of patients with metastatic disease is dismal. Moreover, the extensive molecular heterogeneity of the multiple different sarcoma subtypes poses a substantial challenge to developing more personalized treatment strategies. From immunohistochemical staining of a large set of 625 human soft tissue sarcomas we demonstrate strong tumor cell staining of the Endo180 (MRC2) receptor in a high proportion of samples, findings echoed in gene expression datasets showing a significantly increased expression in both soft tissue and bone sarcomas compared to normal tissue. Endo180 is a constitutively recycling transmembrane receptor and therefore an ideal target for an antibody-drug conjugate (ADC). An anti-Endo180 monoclonal antibody conjugated to the anti-mitotic agent, MMAE via a cleavable linker, is rapidly internalized into target cells and trafficked to the lysosome for degradation, causing cell death specifically in Endo180 expressing sarcoma cell lines. In a sarcoma tumor xenograft model, the Endo180-vc-MMAE ADC, but not an Isotype-vc-MMAE control or the unconjugated Endo180 antibody, drives on target cytotoxicity resulting in tumor regression and a significant impairment of metastatic colonization of the lungs, liver and lymph nodes. These data, together with the lack of a phenotype in mice with an Mrc2 genetic deletion, provide pre-clinical proof-of-principle evidence for the future development of an Endo180-ADC as a therapeutic strategy in a broad range of sarcoma subtypes and, importantly, with potential impact both on the primary tumor and in metastatic disease.

<p>Endo180 protein and gene expression in healthy human tissue compared to sarcoma subtypes.</p>