Drug Repositioning Inferred from E2F1-Coregulator Interactions Studies for the Prevention and Treatment of Metastatic Cancers.

Metastasis management remains a long-standing challenge. High abundance of E2F1 triggers tumor progression by developing protein-protein interactions (PPI) with coregulators that enhance its potential to activate a network of prometastatic transcriptional targets. Methods: To identify E2F1-coregulators, we integrated high-throughput Co-immunoprecipitation (IP)/mass spectometry, GST-pull-down assays, and structure modeling. Potential inhibitors of PPI discovered were found by bioinformatics-based pharmacophore modeling, and transcriptome profiling was conducted to screen for coregulated downstream targets. Expression and target gene regulation was validated using qRT-PCR, immunoblotting, chromatin IP, and luciferase assays. Finally, the impact of the E2F1-coregulator complex and its inhibiting drug on metastasis was investigated in vitro in different cancer entities and two mouse metastasis models. Results: We unveiled that E2F1 forms coactivator complexes with metastasis-associated protein 1 (MTA1) which, in turn, is directly upregulated by E2F1. The E2F1:MTA1 complex potentiates hyaluronan synthase 2 (HAS2) expression, increases hyaluronan production and promotes cell motility. Disruption of this prometastatic E2F1:MTA1 interaction reduces hyaluronan synthesis and infiltration of tumor-associated macrophages in the tumor microenvironment, thereby suppressing metastasis. We further demonstrate that E2F1:MTA1 assembly is abrogated by small-molecule, FDA-approved drugs. Treatment of E2F1/MTA1-positive, highly aggressive, circulating melanoma cells and orthotopic pancreatic tumors with argatroban prevents metastasis and cancer relapses in vivo through perturbation of the E2F1:MTA1/HAS2 axis. Conclusion: Our results propose argatroban as an innovative, E2F-coregulator-based, antimetastatic drug. Cancer patients with the infaust E2F1/MTA1/HAS2 signature will likely benefit from drug repositioning.

BAT Exosomes: Metabolic Crosstalk with Other Organs and Biomarkers for BAT Activity.

In the last decade, exosomes have gained interest as a new type of intercellular communication between cells and tissues. Exosomes are circulating, cell-derived lipid vesicles smaller than 200 nm that contain proteins and nucleic acids, including microRNAs (miRNAs), and are able to modify cellular targets. Exosomal miRNAs function as signalling molecules that regulate the transcription of their target genes and can cause phenotypic transformation of recipient cells. Recent studies have shown that brown fat secretes exosomes as a form of communication with other metabolic organs such as the liver. Moreover, it has been shown that levels of miRNAs in BAT-derived exosomes change after BAT activation in vitro and in vivo. Thus, BAT-derived exosomes can be used as potential biomarkers of BAT activity. Here, we review the present knowledge about BAT-derived exosomes and their role in metabolism.

MicroRNAs in brown and beige fat.

Brown adipose tissue (BAT) dissipates energy as heat and its activity correlates with leanness in human adults. Understanding the mechanisms behind the activation of BAT and the process of "browning", i.e. the appearance of inducible brown adipocytes called beige or brite (brown-in-white) cells in white adipose tissue (WAT), is of great interest for developing novel therapies to combat obesity. MicroRNAs (miRNAs) are small transcriptional regulators that control gene expression in a variety of tissues, including WAT and BAT. Recently, miRNAs were reported to regulate browning. Nevertheless, further studies are needed to fully understand the miRNA networks that are involved in the control of brown and beige/brite adipocytes. Particularly, most miRNAs have so far been studied in mice, underlining the importance of additional human studies. In this review, we focus on the regulation of brown fat by miRNAs including their role in promoting or inhibiting the browning process. In recent years, RNA-based therapeutical approaches have entered clinical trials for treatment of other diseases, thus miRNAs could potentially be used to enhance brown and beige fat mass and activity as novel therapies against overweight and its complications.

E2F1 Signalling is Predictive of Chemoresistance and Lymphogenic Metastasis in Penile Cancer: A Pilot Functional Study Reveals New Prognostic Biomarkers.

BACKGROUND: For penile cancer (PC) there are no known molecular predictors of lymphatic spread and/or chemoresistance. OBJECTIVE: To identify functional biomarkers that can predict malignant progression and treatment responsiveness. DESIGN, SETTING, AND PARTICIPANTS: We used four patient-derived PC cell lines and measured invasion and capillary tube formation, chemoresponsiveness, and mRNA and protein expression. Data were further validated in E2F1 transcription factor knockdown and overexpression experiments. We quantified E2F1 transcript levels in a set of nonmetastatic tumours (NM), metastasised primary tumours (PT), and lymph node metastases (M) from 24 patients. E2F1 immunohistochemistry was performed in another set of 13 PC biopsies. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Relationships between different parameters were analysed using Student t tests. Transcript levels in patient samples were compared using Mann-Whitney U tests. Significance was set at p<0.05. RESULTS AND LIMITATIONS: In cell lines established from lymph node metastases, E2F1 was more abundantly expressed, pRB was inactivated, and CDK2, CDK4, and cyclins D and E were elevated in comparison to cells from primary PC. Overexpression of E2F1 enhanced migratory capacity and lymphatic endothelial tubule formation, while depletion reduced invasiveness and increased chemosensitivity. VEGFR-3 and VEGF-C and mesenchymal markers were upregulated by high E2F1. E2F1 was clearly upregulated in infiltrative and metastatic primary tumours and metastases (NM vs PT, p<0.05; NM vs M, p<0.0005). E2F1 Quick scores increased from grade I to grade III tumours. A limitation of the study is the small number of patients. CONCLUSIONS: E2F1 is a driver of invasion and lymphatic dissemination and promotes chemoresistance. E2F1-related biomarkers might assist in stratifying PC patients for different treatment regimens. PATIENT SUMMARY: The availability of penile cancer cell lines allows molecular research on the mechanisms underlying metastasis and chemotherapy. A critical pathway involved in both features has been identified and may lead to better patient stratification for treatment selection.

Integrated Loss of miR-1/miR-101/miR-204 Discriminates Metastatic from Nonmetastatic Penile Carcinomas and Can Predict Patient Outcome.

PURPOSE: Penile squamous cell carcinoma is a rare but aggressive cancer. Little is known about pivotal events in tumor pathogenesis and metastasis. Lymph node metastasis is the prevailing prognostic factor while clinical detection in patients remains difficult. Our aim was to identify distinct miRNAs that are differentially expressed in metastatic vs nonmetastatic penile carcinoma, which may serve as diagnostic biomarkers for disease progression. MATERIALS AND METHODS: TaqMan® arrays and quantitative polymerase chain reaction were applied to analyze miRNA profiles in penile squamous cell carcinoma specimens and glans tissue from 24 patients. The prognostic value of deregulated miRNAs was analyzed using the Kaplan-Meier method. The Spearman test was applied to determine a potential linkage between distinctive miRNAs in individual patients. RESULTS: Loss of miR-1 (p = 0.0048), miR-101 (p = 0.0001) and miR-204 (p = 0.0004) in metastasizing tumors and associated metastases (p = 0.0151, 0.0019 and 0.0003, respectively) distinguished patients with metastatic and nonmetastatic penile squamous cell carcinoma. These 3 miRNAs showed a coherent expression pattern. Consistently, patients with low levels of all 3 miRNAs had worse survival (p = 0.03). We identified a coordinately regulated miRNA target hub that is over expressed in penile squamous cell carcinoma and associated with lymphovascular invasion. CONCLUSIONS: Our results provide evidence of a novel multiple miRNA based signature associated with lymph node metastasis and unfavorable prognosis of penile squamous cell carcinoma. The integrated loss of miR-1, miR-101 and miR-204 may predict the formation of metastases in penile cancer at an early stage.

Effect of 10 different polymorphisms on preoperative volumetric characteristics of glioblastoma multiforme.

There is a distinct diversity between the appearance of every glioblastoma multiforme (GBM) on pretreatment magnetic resonance imaging (MRI) with a potential impact on clinical outcome and survival of the patients. The object of this study was to determine the impact of 10 different single nucleotide polymorphisms (SNPs) on various volumetric parameters in patients harboring a GBM. We prospectively analyzed 20 steroid-naïve adult patients who had been treated for newly diagnosed GBM. The volumetry was performed using MRI with the help of a semiautomated quantitative software measuring contrast enhancing tumor volume including necrosis, central necrosis alone and peritumoral edema (PTE). We calculated ratios between the tumor volume and edema (ETR), respectively necrosis (NTR). SNP analysis was done using genomic DNA extracted from peripheral blood genotyped via PCR and sequencing. There was a strong correlation between tumor volume and PTE (p < 0.001), necrosis (p < 0.001) and NTR (p = 0.003). Age and sex had no influence on volumetric data. The Aquaporin 4-31G > A SNP had a significant influence on the ETR (p = 0.042) by decreasing the measured edema compared with the tumor volume. The Interleukin 8-251A > T SNP was significantly correlated with an increased tumor (p = 0.048), PTE (p = 0.033) and necrosis volume (p = 0.028). We found two SNPs with a distinct impact on pretreatment tumor characteristics, presenting a potential explanation for the individual diversity of GBM appearance on MRI and influence on survival.

Epigenetic factor EPC1 is a master regulator of DNA damage response by interacting with E2F1 to silence death and activate metastasis-related gene signatures.

Transcription factor E2F1 is a key regulator of cell proliferation and apoptosis. Recently, it has been shown that aberrant E2F1 expression often detectable in advanced cancers contributes essentially to cancer cell propagation and characterizes the aggressive potential of a tumor. Conceptually, this requires a subset of malignant cells capable of evading apoptotic death through anticancer drugs. The molecular mechanism by which the pro-apoptotic activity of E2F1 is antagonized is widely unclear. Here we report a novel function for EPC1 (enhancer of polycomb homolog 1) in DNA damage protection. Depletion of EPC1 potentiates E2F1-mediated apoptosis in response to genotoxic treatment and abolishes tumor cell motility. We found that E2F1 directly binds to the EPC1 promoter and EPC1 vice versa physically interacts with bifunctional E2F1 to modulate its transcriptional activity in a target gene-specific manner. Remarkably, nuclear-colocalized EPC1 activates E2F1 to upregulate the expression of anti-apoptotic survival genes such as BCL-2 or Survivin/BIRC5 and inhibits death-inducing targets. The uncovered cooperativity between EPC1 and E2F1 triggers a metastasis-related gene signature in advanced cancers that predicts poor patient survival. These findings unveil a novel oncogenic function of EPC1 for inducing the switch into tumor progression-relevant gene expression that may help to set novel therapies.