A MYC-ZNF148-ID1/3 regulatory axis modulating cancer stem cell traits in aggressive breast cancer

The MYC proto-oncogene (MYC) is one of the most frequently overexpressed genes in breast cancer that drives cancer stem cell-like traits, resulting in aggressive disease progression and poor prognosis. In this study, we identified zinc finger transcription factor 148 (ZNF148, also called Zfp148 and ZBP-89) as a direct target of MYC. ZNF148 suppressed cell proliferation and migration and was transcriptionally repressed by MYC in breast cancer. Depletion of ZNF148 by short hairpin RNA (shRNA) and CRISPR/Cas9 increased triple-negative breast cancer (TNBC) cell proliferation and migration. Global transcriptome and chromatin occupancy analyses of ZNF148 revealed a central role in inhibiting cancer cell de-differentiation and migration. Mechanistically, we identified the Inhibitor of DNA binding 1 and 3 (ID1, ID3), drivers of cancer stemness and plasticity, as previously uncharacterized targets of transcriptional repression by ZNF148. Silencing of ZNF148 increased the stemness and tumorigenicity in TNBC cells. These findings uncover a previously unknown tumor suppressor role for ZNF148, and a transcriptional regulatory circuitry encompassing MYC, ZNF148, and ID1/3 in driving cancer stem cell traits in aggressive breast cancer

Loss of the nuclear receptor corepressor SLIRP compromises male fertility

Nuclear receptors (NRs) and their coregulators play fundamental roles in initiating and directing gene expression influencing mammalian reproduction, development and metabolism. SRA stem Loop Interacting RNA-binding Protein (SLIRP) is a Steroid receptor RNA Activator (SRA) RNA-binding protein that is a potent repressor of NR activity. SLIRP is present in complexes associated with NR target genes in the nucleus; however, it is also abundant in mitochondria where it affects mitochondrial mRNA transcription and energy turnover. In further characterisation studies, we observed SLIRP protein in the testis where its localization pattern changes from mitochondrial in diploid cells to peri-acrosomal and the tail in mature sperm. To investigate the in vivo effects of SLIRP, we generated a SLIRP knockout (KO) mouse. This animal is viable, but sub-fertile. Specifically, when homozygous KO males are crossed with wild type (WT) females the resultant average litter size is reduced by approximately one third compared with those produced by WT males and females. Further, SLIRP KO mice produced significantly fewer progressively motile sperm than WT animals. Electron microscopy identified disruption of the mid-piece/annulus junction in homozygous KO sperm and altered mitochondrial morphology. In sum, our data implicates SLIRP in regulating male fertility, wherein its loss results in asthenozoospermia associated with compromised sperm structure and mitochondrial morphology

microRNA-7-5p inhibits melanoma cell proliferation and metastasis by suppressing RelA/NF-κB

microRNA-7-5p (miR-7-5p) is a tumor suppressor in multiple cancer types and inhibits growth and invasion by suppressing expression and activity of the epidermal growth factor receptor (EGFR) signaling pathway. While melanoma is not typically EGFR-driven, expression of miR-7-5p is reduced in metastatic tumors compared to primary melanoma. Here, we investigated the biological and clinical significance of miR-7-5p in melanoma. We found that augmenting miR-7-5p expression in vitro markedly reduced tumor cell viability, colony formation and induced cell cycle arrest. Furthermore, ectopic expression of miR-7-5p reduced migration and invasion of melanoma cells in vitro and reduced metastasis in vivo. We used cDNA microarray analysis to identify a subset of putative miR-7-5p target genes associated with melanoma and metastasis. Of these, we confirmed nuclear factor kappa B (NF-κB) subunit RelA, as a novel direct target of miR-7-5p in melanoma cells, such that miR-7- 5p suppresses NF-κB activity to decrease expression of canonical NF-κB target genes, including IL-1β, IL-6 and IL-8. Importantly, the effects of miR-7-5p on melanoma cell growth, cell cycle, migration and invasion were recapitulated by RelA knockdown. Finally, analysis of gene array datasets from multiple melanoma patient cohorts revealed an association between elevated RelA expression and poor survival, further emphasizing the clinical significance of RelA and its downstream signaling effectors. Taken together, our data show that miR-7-5p is a potent inhibitor of melanoma growth and metastasis, in part through its inactivation of RelA/NF-κB signaling. Furthermore, miR-7-5p replacement therapy could have a role in the treatment of this disease

SLIRP KO males produce smaller litters and have fewer progressively motile sperm.

<p>(A) Litter sizes resulting from breeding wild type (WT) females with WT or SLIRP knockout (KO) males (*, significant difference by Maximum Likelihood analysis of repeated measures, p = 0.025). (B) Comparison of daily sperm production (B), motility (C) and progressive motility (D) between WT and SLIRP KO animals. **, ANOVA, p&lt;0.001.</p

SLIRP is expressed in the mouse testis.

<p>(A &amp; B) Detection of SLIRP by immunohistochemistry in Leydig cells, spermatogonia (green arrow), early spermatocytes (yellow arrows), round spermatids (red arrows), elongate spermatids and sperm lining the lumen (white arrows). (C, F, I) Immunofluorescent staining for SLIRP (green), (D) Hsp60 (red), PCNA (G, red) and SP56 (J, red) overlayed in E, H &amp; K respectively. Yellow indicates colocalization in overlayed panels. Nuclear DAPI staining in blue E, H &amp; K. White bars: A, 80 µm, B, H &amp; K, 10 µm, E, 20 µm.</p

SLIRP localization in epididymal sperm.

<p>(A) Immunofluorescent staining for SLIRP (green) and Sp56 (B, red) in sperm heads (C, overlay). (D) SLIRP (green) and Septin 4 (E, red) staining of sperm tails (F, overlay). White bars: C, 10 µm; F, 1 µm.</p

Generation of the SLIRP KO mouse.

<p>(A) Wild type (WT), floxed SLIRP and SLIRP knockout (KO) mouse SLIRP locus configurations. Floxed mice contain a cDNA for SLIRP exons 2 to 4 (Ex 2–4) with polyadenylation signal (poly A) preceded by loxp and splice acceptor (SA) sites and followed by loxp, SA, Flag epitope, stop codon and c-<i>fos</i> sequences inserted within the first SLIRP intron (intron length in kb, nt, nucleotides). (B) Southern analysis to detect a 31.3 kb EcoRV fragment in WT (+/+) and heterozygous (+/−) mice and a 13.3 kb recombinant band in heterozygous and SLIRP homozygous KO (−/−) animals. (C) WT (334 nt) and recombinant (247 nt) RT-PCR products generated from WT and recombinant SLIRP mouse liver cDNAs. (D) Western analysis of WT and recombinant SLIRP mouse testis lysates for SLIRP and β–actin.</p

Annulus and mitochondrial disruption in SLIRP KO sperm.

<p>Electron micrographs of the distal mid-piece and annulus region of (A) WT and (B) SLIRP KO sperm. White arrows, annulus; white bar, abnormal mid-piece/annulus junction in KO sperm; black arrows, electron light and dense areas in WT and KO mitochondria respectively. Black bars, 0.5 µm.</p

Variation in reproductive success across captive populations: methodological differences, potential biases and opportunities

Our understanding of fundamental organismal biology has been disproportionately influenced by studies of a relatively small number of ‘model’ species extensively studied in captivity. Laboratory populations of model species are commonly subject to a number of forms of past and current selection that may affect experimental outcomes. Here, we examine these processes and their outcomes in one of the most widely used vertebrate species in the laboratory – the zebra finch (Taeniopygia guttata). This important model species is used for research across a broad range of fields, partly due to the ease with which it can be bred in captivity. However despite this perceived amenability, we demonstrate extensive variation in the success with which different laboratories and studies bred their subjects, and overall only 64% of all females that were given the opportunity, bred successfully in the laboratory. We identify and review several environmental, husbandry, life-history and behavioural factors that potentially contribute to this variation. The variation in reproductive success across individuals could lead to biases in experimental outcomes and drive some of the heterogeneity in research outcomes across studies. The zebra finch remains an excellent captive animal system and our aim is to sharpen the insight that future studies of this species can provide, both to our understanding of this species and also with respect to the reproduction of captive animals more widely. We hope to improve systematic reporting methods and that further investigation of the issues we raise will lead both to advances in our fundamental understanding of avian reproduction as well as to improvements in future welfare and experimental efficiency