The role of epithelial to mesenchymal transition (EMT) in metastasis is a longstanding source of controversy, largely due to an inability to monitor transient and reversible EMT phenotypes in vivo. We established an EMT lineage tracing system to monitor this process, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We confirmed that within a predominantly epithelial primary tumor, a small portion of tumor cells undergo EMT. Strikingly, lung metastases mainly consisted of non-EMT tumor cells maintaining their epithelial phenotype. Inhibiting EMT by overexpressing miR-200 did not impact lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment due to reduced proliferation, apoptotic tolerance, and elevated expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment.

Tumors systemically initiate metastatic niches in distant target metastatic organs. These niches, composed of bone marrow-derived hematopoietic cells, provide permissive conditions for future metastases. However, the mechanisms by which these cells mediate outgrowth of metastatic tumor cells are not completely known. Using mouse models of spontaneous breast cancer, we show enhanced recruitment of bone marrow-derived CD11b

The progression of cancer to metastatic disease is a major cause of death. We identified miR-708 being transcriptionally repressed by polycomb repressor complex 2-induced H3K27 trimethylation in metastatic breast cancer. miR-708 targets the endoplasmic reticulum protein neuronatin to decrease intracellular calcium level, resulting in reduction of activation of ERK and FAK, decreased cell migration, and impaired metastases. Ectopic expression of neuronatin refractory to suppression by miR-708 rescued cell migration and metastasis defects. In patients with breast cancer, miR-708 expression was decreased in lymph node and distal metastases, suggesting a metastasis-suppressive role. Our findings uncover a mechanistic role for miR-708 in metastasis and provide a rationale for developing miR-708 as a therapeutic agent against metastatic breast cancer.

Metastatic tumors have been shown to establish permissive microenvironments for metastases via recruitment of bone marrow (BM)- derived cells. Here, we show that metastasis-incompetent tumors are also capable of generating such microenvironments. However, in these situations the otherwise pro-metastatic Gr1+ myeloid cells create a metastasis-refractory microenvironment via the induction of thrombospondin-1 (Tsp-1) by tumor-secreted prosaposin. (BM)-specific genetic deletion of Tsp-1 abolished the inhibition of metastasis, which was restored by BM transplant from Tsp-1+ donors. We also developed a 5-amino acid peptide from prosaposin as a pharmacological inducer of Tsp-1 in Gr1+ BM cells, which dramatically suppresses metastasis. These results provide mechanistic insights into why certain tumors are deficient in metastatic potential and implicate recruited Gr1+ myeloid cells as the main source of Tsp-1. The results underscore the plasticity of Gr1+ cells, which, depending on the context, promote or inhibit metastasis, and suggest that the peptide could be a potential therapeutic agent against metastatic cancer.

Emerging studies have begun to demonstrate that reprogrammed stromal cells play pivotal roles in tumor growth, metastasis, and resistance to therapy. However, the contribution of stromal cells to non-small-cell lung cancer (NSCLC) has remained underexplored. We used an orthotopic model of Kras-driven NSCLC to systematically dissect the contribution of specific hematopoietic stromal cells in lung cancer. RNA deep-sequencing analysis of individually sorted myeloid lineage and tumor epithelial cells revealed cell-type-specific differentially regulated genes, indicative of activated stroma. We developed a computational model for crosstalk signaling discovery based on ligand-receptor interactions and downstream signaling networks and identified known and novel tumor-stroma paracrine and tumor autocrine crosstalk-signaling pathways in NSCLC. We provide cellular and molecular insights into components of the lung cancer microenvironment that contribute to carcinogenesis. This study has the potential for development of therapeutic strategies that target tumor-stroma interactions and may complement conventional anti-cancer treatments.

Highlights d Blockade of EZH2 catalytic activity reduces dissemination and metastasis d EZH2 high tumor subpopulation exhibits enhanced metastatic potential d EZH2-mediated GATA3 suppression maintains EZH2 high metastatic cells d TNBC subtypes exhibit differential sensitivity to EZH2 therapy

Purpose: Treatment with PD-(L)1 blockade can produce remarkably durable responses in nonsmall cell lung cancer (NSCLC) patients. However, a significant fraction of long-term responders ultimately progress and predictors of late progression are unknown. We hypothesized that circulating tumor DNA (ctDNA) analysis of long-term responders to PD-(L)1 blockade may differentiate those who will achieve ongoing benefit from those at risk of eventual progression. Experimental Design: In patients with advanced NSCLC achieving long-term benefit from PD-(L)1 blockade (PFS≥12 months), plasma was collected at a surveillance timepoint late during/after treatment to interrogate ctDNA by Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq). Tumor tissue was available for 24 patients and was profiled by wholeexome sequencing (n=18) or by targeted sequencing (n=6).Results: 31 NSCLC patients with long-term benefit to PD-(L)1 blockade were identified and ctDNA was analyzed in surveillance blood samples collected at a median of 26.7 months after initiation of therapy. Nine patients also had baseline plasma samples available, and all had detectable ctDNA prior to therapy initiation. At the surveillance timepoint, 27 patients had undetectable ctDNA and 25 (93%) have remained progression-free; by contrast, all four patients with detectable ctDNA eventually progressed (Fisher's p<0.0001; PPV 1 [95% CI 0.51-1]; NPV 0.93 [95% CI 0.80-0.99]).Conclusions: ctDNA analysis can noninvasively identify minimal residual disease in patients with long-term responses to PD-(L)1 and predict the risk of eventual progression. If validated, ctDNA surveillance may facilitate personalization of the duration of immune checkpoint blockade and enable early intervention in patients at high risk for progression.

MicroRNAs (miRNAs) are key regulators of gene expression and contribute to a variety of biological processes. Abnormal miRNA expression has been reported in various diseases including pathophysiology of breast cancer, where they regulate protumorigenic processes including vascular invasiveness, estrogen receptor status, chemotherapy resistance, invasion and metastasis. The miRBase sequence database, a public repository for newly discovered miRNAs, has grown rapidly with approximately >10,000 entries to date. Despite this rapid growth, many miRNAs have not yet been validated, and several others are yet to be identified. A lack of a full complement of miRNAs has imposed limitations on recognizing their important roles in cancer, including breast cancer. Using deep sequencing technology, we have identified 189 candidate novel microRNAs in human breast cancer cell lines with diverse tumorigenic potential. We further show that analysis of 500-nucleotide pri-microRNA secondary structure constitutes a reliable method to predict bona fide miRNAs as judged by experimental validation. Candidate novel breast cancer miRNAs with stem lengths of greater than 30 bp resulted in the generation of precursor and mature sequences in vivo. On the other hand, candidates with stem length less than 30 bp were less efficient in producing mature miRNA. This approach may be used to predict which candidate novel miRNA would qualify as bona fide miRNAs from deep sequencing data with approximately 90% accuracy.