Metastasis to distant tissues is the chief driver of breast cancer-related mortality, but little is known about the systemic physiologic dynamics that regulate this process. To investigate the role of neuroendocrine activation in cancer progression, we used in vivo bioluminescence imaging to track the development of metastasis in an orthotopic mouse model of breast cancer. Stress-induced neuroendocrine activation had a negligible effect on growth of the primary tumor but induced a 30-fold increase in metastasis to distant tissues including the lymph nodes and lung. These effects were mediated by β-adrenergic signaling, which increased the infiltration of CD11b + F4/80 + macrophages into primary tumor parenchyma and thereby induced a prometastatic gene expression signature accompanied by indications of M2 macrophage differentiation. Pharmacologic activation of β-adrenergic signaling induced similar effects, and treatment of stressed animals with the β-antagonist propranolol reversed the stress-induced macrophage infiltration and inhibited tumor spread to distant tissues. The effects of stress on distant metastasis were also inhibited by in vivo macrophage suppression using the CSF-1 receptor kinase inhibitor GW2580. These findings identify activation of the sympathetic nervous system as a novel neural regulator of breast cancer metastasis and suggest new strategies for antimetastatic therapies that target the β-adrenergic induction of prometastatic gene expression in primary breast cancers.

Polycomb group protein enhancer of zeste 2 (EZH2) is a master regulatory protein that plays a critical role in development as part of the polycomb repressive complex 2. Polycomb repressive complex 2 controls numerous cell cycle and regulatory genes through trimethylation of histone 3, which results in chromatin condensation and transcriptional silencing. EZH2 overexpression has been correlated with high incidence of more aggressive, metastatic prostate cancers. Although this correlation means EZH2 could prove valuable as a biomarker in clinical settings, the question remains whether EZH2 is actually responsible for the initiation of these more aggressive tumor types. In this study, EZH2-mediated neoplastic transformation of the normal prostate epithelial cell line benign prostate hyperplasia 1 (BPH1) was confirmed by in vivo tumor growth and in vitro colony formation. Furthermore, EZH2 transformation resulted in increased invasive behavior of BPH1 cells, indicating that EZH2 may be responsible for aggressive behavior in prostate cancers. BPH1 was also transformed with the classic oncogenes myristoylated Akt and activated Ras(V12) to allow phenotype comparisons with the EZH2-transformed cells. This study marks the first demonstration of neoplastic transformation in prostate cells mediated by EZH2 and establishes that EZH2 possesses stronger transforming activity than Akt but weaker activity than activated Ras.

Background Although most prostate cancers respond well to initial treatments, a fraction of prostate cancers are more aggressive and will recur and metastasize. At that point, there are few treatment options available. Significant efforts have been made to identify biomarkers that will identify these more aggressive cancers to tailor a more vigorous treatment in order to improve outcome. Polycomb Group protein Enhancer of Zeste 2 (EZH2) was found to be overexpressed in metastatic prostate tumors, and is considered an excellent candidate for such a biomarker. Scattered studies have found that EZH2 overexpression causes neoplastic transformation, invasion, and growth of prostate cells. However, these studies utilized different systems and cell lines, and so are difficult to correlate with one another. Methods In this study, a comprehensive evaluation of the phenotypic effects of EZH2 in a panel of five prostate cancer cell lines was performed. By using multiple cell lines, and examining overexpression and knockdown of EZH2 concurrently, a broad view of EZH2's role in prostate cancer was achieved. Results Overexpression of EZH2 led to more aggressive behaviors in all prostate cell lines tested. In contrast, downregulation of EZH2 reduced invasion and tumorigenicity of androgen-independent cell lines CWR22Rv1, PC3, and DU145, but not of androgen-dependent cell lines LAPC4 and LNCaP. Conclusions Findings from this study suggest androgen-independent prostate tumors are more dependent on EZH2 expression than androgen-dependent tumors. Our observations provide an explanation for the strong correlation between EZH2 overexpression and advanced stage, aggressive prostate cancers.

Better intraprostatic cancer imaging techniques are needed to guide clinicians in prostate cancer treatment decisions. Because many genes are specifically overexpressed in cancer cells, one strategy to improve prostate cancer detection is to image intraprostatic cancer-specific transcriptional activity. Because of the obstacles of weak cancer-or tissue-specific promoter activity and bladder clearance of many PET tracers, intraprostatic PET of gene transcriptional activity has not been previously reported. Methods: The two-step transcriptional amplification (TSTA) system that amplifies the prostate-specific antigen promoter activity was used for PET imaging of the reporter gene herpes simplex virus type-1 sr39 thymidine kinase (HSV1-sr39tk). The TSTA-sr39tk system was injected directly into prostates or prostatic tumors as a replication-incompetent adenovirus (AdTSTA-sr39tk) and imaged using PET. Results: AdTSTA-sr39tk was able to image prostate-specific antigen promoter transcriptional activity by 9-(4-18 F-fluoro-3-[hydroxymethyl]butyl)guanine PET, in both mouse and canine prostates in vivo. Ex vivo small-animal PET images, scintigraphic counts, and sr39tk expression analysis confirmed the specificity of the observed signal. Conclusion: Here, by combining the TSTA-amplified signal with a protocol for tracer administration, we show that in vivo PET detection of transcriptional activity is possible in both mouse and immunocompetent canine prostates. These results suggest that imaging applications using transcription-based tumor-specific promoters should be pursued to better visualize cancer foci that escape detection by conventional biopsies.