Ya-Fang Chang

To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44(+) cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy.

Abstract Circulating tumor cells (CTC) seed cancer metastases; however, the underlying cellular and molecular mechanisms remain unclear. CTC clusters were less frequently detected but more metastatic than single CTCs of patients with triple-negative breast cancer and representative patient-derived xenograft models. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding. Aggregated tumor cells exhibited enriched expression of the breast cancer stem cell marker CD44 and promoted tumorigenesis and polyclonal metastasis. Depletion of CD44 effectively prevented tumor cell aggregation and decreased PAK2 levels. The intercellular CD44–CD44 homophilic interactions directed multicellular aggregation, requiring its N-terminal domain, and initiated CD44–PAK2 interactions for further activation of FAK signaling. Our studies highlight that CD44+ CTC clusters, whose presence is correlated with a poor prognosis of patients with breast cancer, can serve as novel therapeutic targets of polyclonal metastasis. Significance: CTCs not only serve as important biomarkers for liquid biopsies, but also mediate devastating metastases. CD44 homophilic interactions and subsequent CD44–PAK2 interactions mediate tumor cluster aggregation. This will lead to innovative biomarker applications to predict prognosis, facilitate development of new targeting strategies to block polyclonal metastasis, and improve clinical outcomes. See related commentary by Rodrigues and Vanharanta, p. 22. This article is highlighted in the In This Issue feature, p. 1

The functional role of progesterone receptor (PR) and its impact on estrogen signaling in breast cancer remain controversial. In primary ER(+) (estrogen receptor-positive)/PR(+) human tumors, we report that PR reprograms estrogen signaling as a genomic agonist and a phenotypic antagonist. In isolation, estrogen and progestin act as genomic agonists by regulating the expression of common target genes in similar directions, but at different levels. Similarly, in isolation, progestin is also a weak phenotypic agonist of estrogen action. However, in the presence of both hormones, progestin behaves as a phenotypic estrogen antagonist. PR remodels nucleosomes to noncompetitively redirect ER genomic binding to distal enhancers enriched for BRCA1 binding motifs and sites that link PR and ER/PR complexes. When both hormones are present, progestin modulates estrogen action, such that responsive transcriptomes, cellular processes, and ER/PR recruitment to genomic sites correlate with those observed with PR alone, but not ER alone. Despite this overall correlation, the transcriptome patterns modulated by dual treatment are sufficiently different from individual treatments, such that antagonism of oncogenic processes is both predicted and observed. Combination therapies using the selective PR modulator/antagonist (SPRM) CDB4124 in combination with tamoxifen elicited 70% cytotoxic tumor regression of T47D tumor xenografts, whereas individual therapies inhibited tumor growth without net regression. Our findings demonstrate that PR redirects ER chromatin binding to antagonize estrogen signaling and that SPRMs can potentiate responses to antiestrogens, suggesting that cotargeting of ER and PR in ER(+)/PR(+) breast cancers should be explored.

Abstract Purpose: Effective targeting of cancer stem cells is necessary and important for eradicating cancer and reducing metastasis-related mortality. Understanding of cancer stemness-related signaling pathways at the molecular level will help control cancer and stop metastasis in the clinic. Experimental Design: By analyzing miRNA profiles and functions in cancer development, we aimed to identify regulators of breast tumor stemness and metastasis in human xenograft models in vivo and examined their effects on self-renewal and invasion of breast cancer cells in vitro. To discover the direct targets and essential signaling pathways responsible for miRNA functions in breast cancer progression, we performed microarray analysis and target gene prediction in combination with functional studies on candidate genes (overexpression rescues and pheno-copying knockdowns). Results: In this study, we report that hsa-miR-206 suppresses breast tumor stemness and metastasis by inhibiting both self-renewal and invasion. We identified that among the candidate targets, twinfilin (TWF1) rescues the miR-206 phenotype in invasion by enhancing the actin cytoskeleton dynamics and the activity of the mesenchymal lineage transcription factors, megakaryoblastic leukemia (translocation) 1 (MKL1), and serum response factor (SRF). MKL1 and SRF were further demonstrated to promote the expression of IL11, which is essential for miR-206's function in inhibiting both invasion and stemness of breast cancer. Conclusions: The identification of the miR-206/TWF1/MKL1-SRF/IL11 signaling pathway sheds lights on the understanding of breast cancer initiation and progression, unveils new therapeutic targets, and facilitates innovative drug development to control cancer and block metastasis. Clin Cancer Res; 23(4); 1091–103. ©2016 AACR.