Seongho Ryu

Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance.

The role of epithelial-to-mesenchymal transition (EMT) in metastasis is a longstanding source of debate, largely owing to an inability to monitor transient and reversible EMT phenotypes in vivo. Here we establish an EMT lineage-tracing system to monitor this process in mice, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We show that within a predominantly epithelial primary tumour, a small proportion of tumour cells undergo EMT. Notably, lung metastases mainly consist of non-EMT tumour cells that maintain their epithelial phenotype. Inhibiting EMT by overexpressing the microRNA miR-200 does not affect lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment owing to reduced proliferation, apoptotic tolerance and increased 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.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.

Myeloid Progenitor Cells in the Premetastatic Lung Promote Metastases by Inducing Mesenchymal to Epithelial Transition

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(+)Gr1(+) myeloid progenitor cells in the premetastatic lungs. Gene expression profiling revealed that the myeloid cells from metastatic lungs express versican, an extracellular matrix proteoglycan. Notably, versican in metastatic lungs was mainly contributed by the CD11b(+)Ly6C(high) monocytic fraction of the myeloid cells and not the tumor cells or other stromal cells. Versican knockdown in the bone marrow significantly impaired lung metastases in vivo, without impacting their recruitment to the lungs or altering the immune microenvironment. Versican stimulated mesenchymal to epithelial transition of metastatic tumor cells by attenuating phospho-Smad2 levels, which resulted in elevated cell proliferation and accelerated metastases. Analysis of clinical specimens showed elevated versican expression within the metastatic lung of patients with breast cancer. Together, our findings suggest that selectively targeting tumor-elicited myeloid cells or versican represents a potential therapeutic strategy for combating metastatic disease.

Suppression of miRNA-708 by Polycomb Group Promotes Metastases by Calcium-Induced Cell Migration

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.

Bone Marrow-Derived Gr1+ Cells Can Generate a Metastasis-Resistant Microenvironment Via Induced Secretion of Thrombospondin-1

UNLABELLED Metastatic tumors have been shown to establish permissive microenvironments for metastases via recruitment of bone marrow-derived cells. Here, we show that metastasis-incompetent tumors are also capable of generating such microenvironments. However, in these situations, the otherwise prometastatic Gr1(+) myeloid cells create a metastasis-refractory microenvironment via the induction of thrombospondin-1 (Tsp-1) by tumor-secreted prosaposin. Bone marrow-specific genetic deletion of Tsp-1 abolished the inhibition of metastasis, which was restored by bone marrow transplant from Tsp-1(+) donors. We also developed a 5-amino acid peptide from prosaposin as a pharmacologic inducer of Tsp-1 in Gr1(+) bone marrow cells, which dramatically suppressed 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. SIGNIFICANCE The mechanisms of metastasis suppression are poorly understood. Here, we have identified a novel mechanism whereby metastasis-incompetent tumors generate metastasis-suppressive microenvironments in distant organs by inducing Tsp-1 expression in the bone marrow–derived Gr1+myeloid cells. A 5-amino acid peptide with Tsp-1–inducing activity was identified as a therapeutic agent against metastatic cancer.

Discovery of Novel Human Breast Cancer MicroRNAs from Deep Sequencing Data by Analysis of Pri-MicroRNA Secondary Structures

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.

Induction of pulmonary mucosal immune responses with a protein vaccine targeted to the DEC-205/CD205 receptor.

It is of great interest to develop a pneumonic plague vaccine that would induce combined humoral and cellular immunity in the lung. Here we investigate a novel approach based on targeting of dendritic cells using the DEC-205/CD205 receptor (DEC) via the intranasal route as way to improve mucosal cellular immunity to the vaccine. Intranasal administration of Yersinia pestis LcrV (V) protein fused to anti-DEC antibody together with poly IC as an adjuvant induced high frequencies of IFN-γ secreting CD4(+) T cells in the airway and lung as well as pulmonary IgG and IgA antibodies. Anti-DEC:LcrV was more efficient to induce IFN-γ/TNF-α/IL-2 secreting polyfunctional CD4(+) T cells when compared to non-targeted soluble protein vaccine. In addition, the intranasal route of immunization with anti-DEC:LcrV was associated with improved survival upon pulmonary challenge with the virulent CO92 Y. pestis. Taken together, these data indicate that targeting dendritic cells via the mucosal route is a potential new avenue for the development of a mucosal vaccine against pneumonic plague.

Development of single-nucleotide polymorphism-based phylum-specific PCR amplification technique: Application to the community analysis using ciliates as a reference organism

Despite recent advance in mass sequencing technologies such as pyrosequencing, assessment of culture-independent microbial eukaryote community structures using universal primers remains very difficult due to the tremendous richness and complexity of organisms in these communities. Use of a specific PCR marker targeting a particular group would provide enhanced sensitivity and more in-depth evaluation of microbial eukaryote communities compared to what can be achieved with universal primers. We discovered that many phylum- or groupspecific single-nucleotide polymorphisms (SNPs) exist in small subunit ribosomal RNA (SSU rRNA) genes from diverse eukaryote groups. By applying this discovery to a known simple allele-discriminating (SAP) PCR method, we developed a technique that enables the identification of organisms belonging to a specific higher taxonomic group (or phylum) among diverse types of eukaryotes. We performed an assay using two complementary methods, pyrosequencing and clone library screening. In doing this, specificities for the group (ciliates) targeted in this study in bulked environmental samples were 94.6% for the clone library and 99.2% for pyrosequencing, respectively. In particular, our novel technique showed high selectivity for rare species, a feature that may be more important than the ability to identify quantitatively predominant species in community structure analyses. Additionally, our data revealed that a target-specific library (or ciliate-specific one for the present study) can better explain the ecological features of a sampling locality than a universal library.

Single-cell PCR on protargol-impregnated euplotid ciliates: a combined approach of morphological and molecular taxonomy

Ciliates are considered one of the most diverse protozoa and play significant roles in ecology. For successful taxonomic study of these microscopic eukaryotes, a staining procedure is necessary, due mainly to intrinsic difficulties in recognizing characteristics from living cells. Although molecular taxonomy has been used to resolve the ambiguities associated with traditional morphology-based taxonomy, extraction of genomic DNA from stained ciliate cells is not available yet. In the present study, we describe a method to extract genomic DNA from a single protargol-impregnated euplotid cell. By using HgCl2 as a fixative and modulating the exposure time of bleach solution in the protargol impregnation, high-quality genomic DNA can successfully be extracted from a stained single cell with minimal loss of morphological integrity. This technique will contribute to the effectiveness of combined approaches of molecular and morphological taxonomy from single ciliate cells.

Identification of Reprogrammed Myeloid Cell Transcriptomes in NSCLC

Lung cancer is the leading cause of cancer related mortality worldwide, with non-small cell lung cancer (NSCLC) as the most prevalent form. Despite advances in treatment options including minimally invasive surgery, CT-guided radiation, novel chemotherapeutic regimens, and targeted therapeutics, prognosis remains dismal. Therefore, further molecular analysis of NSCLC is necessary to identify novel molecular targets that impact prognosis and the design of new-targeted therapies. In recent years, tumor “activated/reprogrammed” stromal cells that promote carcinogenesis have emerged as potential therapeutic targets. However, the contribution of stromal cells to NSCLC is poorly understood. Here, we show increased numbers of bone marrow (BM)-derived hematopoietic cells in the tumor parenchyma of NSCLC patients compared with matched adjacent non-neoplastic lung tissue. By sorting specific cellular fractions from lung cancer patients, we compared the transcriptomes of intratumoral myeloid compartments within the tumor bed with their counterparts within adjacent non-neoplastic tissue from NSCLC patients. The RNA sequencing of specific myeloid compartments (immature monocytic myeloid cells and polymorphonuclear neutrophils) identified differentially regulated genes and mRNA isoforms, which were inconspicuous in whole tumor analysis. Genes encoding secreted factors, including osteopontin (OPN), chemokine (C-C motif) ligand 7 (CCL7) and thrombospondin 1 (TSP1) were identified, which enhanced tumorigenic properties of lung cancer cells indicative of their potential as targets for therapy. This study demonstrates that analysis of homogeneous stromal populations isolated directly from fresh clinical specimens can detect important stromal genes of therapeutic value.

Abstract 5078: EZH2 methyltransferase regulates disseminating tumor cells in breast cancer metastasis

Triple negative breast cancer (TNBC, ER-, PR-, HER2-) exhibits the worst outcome due to higher rates of metastasis compared to non TNBC subtypes. Despite this clinical significance, there is a conspicuous lack of FDA approved molecularly targeted anti-metastatic therapies for TNBC. The enhancer of zeste homolog 2 (EZH2), a catalytic core subunit of the Polycomb repressive complex 2 (PRC2) with histone methyltransferase (HMT) activity is associated with the worst clinical outcome in breast cancer patients. Using a combination of genetic and pharmacological approaches, we show that EZH2 HMT blockade did not impact primary tumor growth, but significantly reduced distal metastases. Metastasis suppression was associated with a marked reduction of tumor-initiating cells (TICs) in primary tumor, circulating tumor cells (CTCs) in the blood and impaired lung colonization. Using a SOX2/OCT4 promoter reporter system, we identified EZH2-sensitive metastatic cells with GATA3 low luminal progenitor phenotypes in the primary tumor, and EZH2 HMT blockade restored GATA3 expression, promoted differentiation of luminal progenitors and impaired metastasis. These key preliminary findings have led to the hypothesis that EZH2 promotes metastasis, and that inhibition of EZH2 HMT may constitute a viable anti-metastatic approach. We will also discuss the potential of EZH2 inhibition in combination with chemotherapy as an effective strategy against TNBC metastasis. Citation Format: Shira Yomtoubian, Seongho Ryu, Sharrell Lee, Geoff Markowitz, Dingcheng Gao, Vivek Mittal. EZH2 methyltransferase regulates disseminating tumor cells in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5078. doi:10.1158/1538-7445.AM2017-5078