MicroRNAs (miRNA) aberrantly expressed in tumors may offer novel therapeutic approaches to treatment. miR-145 is downregulated in various cancers including colon carcinoma in which in vitro studies have established proapoptotic and antiproliferative roles. miR-33a was connected recently to cancer through its capacity to downregulate the oncogenic kinase Pim-1. To date, miRNA replacement therapy has been hampered by the lack of robust nonviral delivery methods for in vivo administration. Here we report a method of miRNA delivery by using polyethylenimine (PEI)-mediated delivery of unmodified miRNAs, using miR-145 and miR-33a to preclinically validate the method in a mouse model of colon carcinoma. After systemic or local application of low molecular weight PEI/miRNA complexes, intact miRNA molecules were delivered into mouse xenograft tumors, where they caused profound antitumor effects. miR-145 delivery reduced tumor proliferation and increased apoptosis, with concomitant repression of c-Myc and ERK5 as novel regulatory target of miR-145. Similarly, systemic injection of PEI-complexed miR-33a was validated as a novel therapeutic targeting method for Pim-1, with antitumor effects comparable with PEI/siRNA-mediated direct in vivo knockdown of Pim-1 in the model. Our findings show that chemically unmodified miRNAs complexed with PEI can be used in an efficient and biocompatible strategy of miRNA replacement therapy, as illustrated by efficacious delivery of PEI/miR-145 and PEI/miR-33a complexes in colon carcinoma. Cancer Res; 71(15); 5214-24. Ó2011 AACR.

The discovery of oestrogen receptor b (ERb/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ERa (ESR1) raised hopes for improved endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ERb antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically targets ERb in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ERb protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. We do not find evidence of expression in normal or cancerous human breast. This expression pattern aligns well with RNA-seq data, but contradicts a multitude of studies. Our study highlights how inadequately validated antibodies can lead an exciting field astray.

Chronic inflammation of the colon (colitis) is a risk factor for colorectal cancer (CRC). Hormone‐replacement therapy reduces CRC incidences, and the estrogen receptor beta (ERβ/ESR2) has been implicated in this protection. Gut microbiota is altered in both colitis and CRC and may influence the severity of both. Here we test the hypothesis that intestinal ERβ impacts the gut microbiota. Mice with and without intestine‐specific deletion of ERβ (ERβKO Vil ) were generated using the Cre‐LoxP system. Colitis and CRC were induced with a single intraperitoneal injection of azoxymethane (AOM) followed by administration of three cycles of dextran sulfate sodium (DSS) in drinking water. The microbiota population were characterized by high‐throughput 16S rRNA gene sequencing of DNA extracted from fecal samples ( N = 39). Differences in the microbiota due to AOM/DSS and absence of ERβ were identified through bioinformatic analyses of the 16S‐Seq data, and the distribution of bacterial species was corroborated using qPCR. We demonstrate that colitis‐induced CRC reduced the gut microbiota diversity and that loss of ERβ enhanced this process. Further, the Bacteroidetes genus Prevotellaceae_ UCG_001 was overrepresented in AOM/DSS mice compared to untreated controls (3.5‐fold, p = 0.004), and this was enhanced in females and in ERβKO Vil mice. Overall, AOM/DSS enriched for microbiota impacting immune system diseases and metabolic functions, and lack of ERβ in combination with AOM/DSS enriched for microbiota impacting carbohydrate metabolism and cell motility, while reducing those impacting the endocrine system. Our data support that intestinal ERβ contributes to a more favorable microbiome that could attenuate CRC development.