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Supplementary Table S1: Patients and clinical information. Supplementary Table S2: Genes used to determine the AR signature.

Supplementary Figure S1: RNA sequencing and pathway analysis of CTCs from Bone(+) versus Visceral(+) breast cancer patients. Supplementary Figure S2: Correlation between AR expression and AR signatures. Supplementary Figure S3: ER and PR expression in CTCs from Bone(+) versus Visceral(+) breast cancer patients. Supplementary Figure S4: AR antibody validation. Supplementary Figure S5: AR copy number assessment in breast cancer bone metastasis. Supplementary Figure S6: ER and PR expression in primary and metastatic breast cancer. Supplementary Figure S7: AR expression correlates with bone metastasis independently of ER status. Supplementary Figure S8: Enzalutamide treatment induces downregulation of a subset of AR-dependent genes in breast cancer cells. Supplementary Figure S9: AR shRNA validation. Supplementary Figure S10: AR inhibition does not alter primary tumor growth.

Molecular drivers underlying bone metastases in human cancer are not well understood, in part due to constraints in bone tissue sampling. Here, RNA sequencing was performed of circulating tumor cells (CTC) isolated from blood samples of women with metastatic estrogen receptor (ER)+ breast cancer, comparing cases with progression in bone versus visceral organs. Among the activated cellular pathways in CTCs from bone-predominant breast cancer is androgen receptor (AR) signaling. AR gene expression is evident, as is its constitutively active splice variant AR-v7. AR expression within CTCs is correlated with the duration of treatment with aromatase inhibitors, suggesting that it contributes to acquired resistance to endocrine therapy. In an established breast cancer xenograft model, a bone-tropic derivative displays increased AR expression, whose genetic or pharmacologic suppression reduces metastases to bone but not to lungs. Together, these observations identify AR signaling in CTCs from women with bone-predominant ER+ breast cancer, and provide a rationale for testing androgen inhibitors in this subset of patients. Implications: This study highlights a role for the AR in breast cancer bone metastasis, and suggests that therapeutic targeting of the AR may benefit patients with metastatic breast cancer. Mol Cancer Res; 16(4); 720–7. ©2018 AACR.

Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC-clusters). Existing technologies for CTC enrichment are designed primarily to isolate single CTCs, and while CTC-clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here, we developed a microchip technology (Cluster-Chip) specifically designed to capture CTC-clusters independent of tumor-specific markers from unprocessed blood. CTC-clusters are isolated through specialized bifurcating traps under low shear-stress conditions that preserve their integrity and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identify CTC-clusters in 30–40% of patients with metastatic cancers of the breast, prostate and melanoma. RNA sequencing of CTC-clusters confirms their tumor origin and identifies leukocytes within the clusters as tissue-derived macrophages. Together, the development of a device for efficient capture of CTC-clusters will enable detailed characterization of their biological properties and role in cancer metastasis.

SummaryCirculating tumor cell clusters (CTC clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC clusters have 23- to 50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and though rare, they greatly contribute to the metastatic spread of cancer.

The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases remove methyl groups from tri- and dimethylated lysine 4 of histone H3. Accumulating evidence from primary tumors and model systems supports a role for KDM5A (JARID1A/RBP2) and KDM5B (JARID1B/PLU1) as oncogenic drivers. The KDM5 family is unique among the Jumonji domain-containing histone demethylases in that there is an atypical insertion of a DNA-binding ARID domain and a histone-binding PHD domain into the Jumonji domain, which separates the catalytic domain into two fragments (JmjN and JmjC). Here we demonstrate that internal deletion of the ARID and PHD1 domains has a negligible effect on in vitro enzymatic kinetics of the KDM5 family of enzymes. We present a crystal structure of the linked JmjN-JmjC domain from KDM5A, which reveals that the linked domain fully reconstitutes the cofactor (metal ion and α-ketoglutarate) binding characteristics of other structurally characterized Jumonji domain demethylases. Docking studies with GSK-J1, a selective inhibitor of the KDM6/KDM5 subfamilies, identify critical residues for binding of the inhibitor to the reconstituted KDM5 Jumonji domain. Further, we found that GSK-J1 inhibited the demethylase activity of KDM5C with 8.5-fold increased potency compared with that of KDM5B at 1 mm α-ketoglutarate. In contrast, JIB-04 (a pan-inhibitor of the Jumonji demethylase superfamily) had the opposite effect and was ~8-fold more potent against KDM5B than against KDM5C. Interestingly, the relative selectivity of JIB-04 toward KDM5B over KDM5C in vitro translates to a ~10-50-fold greater growth-inhibitory activity against breast cancer cell lines. These data define the minimal requirements for enzymatic activity of the KDM5 family to be the linked JmjN-JmjC domain coupled with the immediate C-terminal helical zinc-binding domain and provide structural characterization of the linked JmjN-JmjC domain for the KDM5 family, which should prove useful in the design of KDM5 demethylase inhibitors with improved potency and selectivity.

Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targeting miRNAs. This indirect but highly specific mechanism, by which a chromatin regulator that mediates transcriptional activating marks can lead to the downregulation of a critical effector gene, is shared with multiple genes in the p53 pathway. Through such miRNA-dependent effects, SETD1A regulates cell cycle progression in vitro and modulates tumorigenesis in mouse xenograft models. Together, these observations help explain the remarkably specific genetic consequences associated with alterations in generic chromatin modulators in cancer., The p53-inducible antiproliferative gene BTG2 is suppressed in many cancers, in the absence of inactivating gene mutations. Here the authors show that the histone lysine methyltransferase SETD1A suppresses the expression of several p53 target genes including BTG2 by inducing a network of microRNAs.

459 Background: Baseline patient characteristics may impact duration on study and survivorship of patients in immunotherapy trials. In this study, GVAX pancreas (GM-CSF–secreting allogeneic pancreatic tumor cells) was administered with low-dose cyclophosphamide (Cy), with or without subsequent CRS-207 (live-attenuated Listeria monocytogenes–expressing mesothelin) infusion. Baseline clinical and immunological data were examined for two patient subsets based on duration on study. Methods: Previously treated patients with metastatic pancreatic adenocarcinoma were randomly assigned at a ratio of 2:1 to 2 doses of Cy/GVAX followed by 4 doses of CRS-207 or 6 doses of Cy/GVAX every 3 weeks. Stable patients were offered additional courses. Patients were evaluated for post-randomization duration on study of < 60 days (rapid dropouts, RD) and > 18 months (long-term survivors, LTS). Baseline characteristics of these subsets were examined. Results: A total of 93 patients were randomized. Fifteen patients were RD; 13 were LTS. More RDs versus LTS had liver metastasis and lymph node involvement. On average, fewer RD patients had radiotherapy prior to randomization and fewer had prior surgical intervention. RD averaged 1.73 prior metastatic cancer treatment regimens versus 1.31 in LTS. RD also had higher average baseline CA 19-9 levels. Immune analysis showed higher circulating terminal effector CD8+ T cells and lower serum IL-6 in LTS compared to RD. Serum IL-6 concentration remained lower in LTS at four weeks following initiation of treatment. For patients who received CRS-207, CD8+ T cells specific to listeriolysin O (LLO) and mesothelin were higher in LTS than RD, as determined by ELISPOT. Conclusions: Baseline clinical and immunological characteristics may impact patient duration on immunotherapy trials. Immune monitoring and biomarker analysis in this study is ongoing to identify circulating cellular, tumor biomarkers and genomic (miRNA and mRNA) signatures that may correlate with survival. These findings warrant continued exploration of patient characteristics to identify best candidates for immunotherapy trials. Clinical trial information: NCT01417000.

The metastatic spread of breast cancer, typically to bone, lung, liver and brain, accounts for the vast majority of cancer-related deaths. Breast cancer metastases are thought to be derived primarily from individual migratory cancer cells, reaching distant sites through the bloodstream and initiating proliferation within distant organs. In addition to these single circulating tumor cells (CTCs), CTC-clusters have been detected in the blood of patients with cancer. In patients with breast cancer, we find that presence of such CTC-clusters is correlated with decreased progression-free survival. To study their functional role, we used mouse models, demonstrating that breast cancer cells injected intravascularly as clusters are more prone to survive and colonize the lungs than similarly injected single cancer cells. Primary orthotopic mammary tumors comprised of differentially tagged cells give rise to oligoclonal CTC-clusters, with 40-fold increased metastatic potential to the lung, compared with single CTCs. Using in vivo flow cytometry, we show that CTC-clusters are rapidly cleared from peripheral vessels, consistent with their trapping in small capillaries. Together, our observations suggest that primary tumor cells break off into the vasculature as CTC-clusters, and exhibit greatly enhanced metastatic propensity. Citation Format: Nicola Aceto, Aditya Bardia, Joel A. Spencer, Ben S. Wittner, Min Yu, Maria C. Donaldson, Adam Pely, Amanda Engstrom, Huili Zhu, Brian W. Brannigan, Ravi Kapur, Shannon L. Stott, Toshi Shioda, Sridhar Ramaswamy, David T. Ting, Charles P. Lin, Mehmet Toner, Daniel A. Haber, Shyamala Maheswaran. Circulating tumor cell clusters are precursors of breast cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-192. doi:10.1158/1538-7445.AM2014-LB-192