Homeobox genes encode transcription factors that are essential for normal development and are often dysregulated in cancers. The molecular mechanisms that cause their misregulation in cancers are largely unknown. In this study, we investigate the mechanism by which the Six1 homeobox protein, which has a crucial role during development, is frequently deregulated in several poor outcome, aggressive, metastatic adult human cancers, including breast cancer, ovarian cancer, hepatocellular carcinoma and pediatric malignancies such as rhabdomyosarcoma and Wilms' tumor. Our results reveal that miRNA-185 translationally represses Six1 by binding to its 3 0 -untranslated region. Analyses of ovarian cancers, pediatric renal tumors and multiple breast cancer cell lines showed decreased miR-185 expression, paralleling an increase in Six1 levels. Further investigation revealed that miR-185 impedes anchorage-independent growth and cell migration, in addition to suppressing tumor growth in vivo, implicating it to be a potent tumor suppressor. Our results indicate that miR-185 mediates its tumor suppressor function by regulating cell-cycle proteins and Six1 transcriptional targets c-myc and cyclin A1. Furthermore, we show that miR-185 sensitizes Six1-overexpressing resistant cancer cells to apoptosis in general and tumor necrosis factor-related apoptosisinducing ligand (TRAIL)-mediated apoptosis in particular. Together, our findings suggest that the altered expression of the novel tumor suppressor miR-185 may be one of the central events that leads to dysregulation of oncogenic protein Six1 in human cancers.

A thorough understanding of the events during mammalian spermatogenesis requires studying specific molecular signatures of individual testicular cell populations as well as their interaction in co-cultures. However, most purification techniques to isolate specific testicular cell populations are time-consuming, require large numbers of animals, and/or are only able to isolate a few cell types. Here we describe a cost-effective and timesaving approach that uses a single protocol to enrich multiple testicular cell populations (Sertoli, Leydig, and several spermatogenic cell populations) from as few as one mouse. Our protocol combines rigorous enzymatic digestion of seminiferous tubules with counter-current centrifugal elutriation, yielding specific testicular cell populations with >80%–95% purity.

Though roles of β-catenin signaling during testis development have been well established, relatively little is known about its role in postnatal testicular physiology. Even less is known about its role in post-meiotic germ cell development and differentiation. Here, we report that β-catenin is highly expressed in post-meiotic germ cells and plays an important role during spermiogenesis in mice. Spermatid-specific deletion of β-catenin resulted in significantly reduced sperm count, increased germ cell apoptosis and impaired fertility. In addition, ultrastructural studies show that the loss of β-catenin in post-meiotic germ cells led to acrosomal defects, anomalous release of immature spermatids and disruption of adherens junctions between Sertoli cells and elongating spermatids (apical ectoplasmic specialization; ES). These defects are likely due to altered expression of several genes reportedly involved in Sertoli cell-germ cell adhesion and germ cell differentiation, as revealed by gene expression analysis. Taken together, our results suggest that β-catenin is an important molecular link that integrates Sertoli cell-germ cell adhesion with the signaling events essential for post-meiotic germ cell development and maturation. Since β-catenin is also highly expressed in the Sertoli cells, we propose that binding of germ cell β-catenin complex to β-catenin complex on Sertoli cell at the apical ES surface triggers a signaling cascade that regulates post-meiotic germ cell differentiation.

Decoupling of transcription and translation during postmeiotic germ cell differentiation is critical for successful spermatogenesis. Here we establish that the interaction between microRNAs and actin-associated protein Arpc5 sets the stage for an elaborate translational control mechanism by facilitating the sequestration of germ cell mRNAs into translationally inert ribonucleoprotein particles until they are later translated. Our studies reveal that loss of microRNA-dependent regulation of Arpc5, which controls the distribution of germ cell mRNAs between translationally active and inactive pools, results in abnormal round spermatid differentiation and impaired fertility. Interestingly, Arpc5 functions as a broadly acting translational suppressor, as it inhibits translation initiation by blocking 80S formation and facilitates the transport of mRNAs to chromatoid/P bodies. These findings identify a unique role for actin-associated proteins in translational regulation, and suggest that mRNA-specific and general translational control mechanisms work in tandem to regulate critical germ cell differentiation events and diverse somatic cell functions.spermiogenesis | messenger ribonucleoprotein | Arp2/3 | processing body

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MicroRNAs (miRNAs) are naturally occurring, short non-coding RNAs believed to be involved in many biological processes including normal development and adult cancer. It seems logical that miRNAs would be particularly important in pediatric cancers as these malignancies generally involve perturbations of normal developmental and differentiation processes, yet relatively little is known about the role of miRNAs in pediatric cancers. Here, we show that oncogenesis in Wilms’ tumor - the most common pediatric renal cancer - is mediated by miRNAs. MiRNA microarray analysis identified a cohort of miRNAs that show altered expression in Wilms’ tumors when compared with normal matched control kidney. Our results reveal that several of these miRNAs show similar expression patterns both in Wilms’ tumors and fetal kidney, suggesting that they may play an important regulatory role in kidney development and Wilms’ tumorigenesis. Supporting this notion are our findings that show a number of differentially expressed miRNAs to target genes known to play a role in kidney development and Wilms’ tumorigenesis, including the Six1 homeobox gene, a potent oncogene reported to be overexpressed in several solid tumors. Analyses of primary Wilms’ tumors and ovarian cancer tissues as well as multiple tumor cell lines from breast cancer, ovarian cancer and rhabdomyosarcoma showed decreased expression of miRNAs targeting the Six1 3′UTR, paralleling an increase in Six1 levels. Further investigation revealed a potent tumor suppressor role for Six1 targeting miRNAs, as ectopic expression suppressed the ability of cancer cell lines to form colonies and tumor growth in nude mice. Interestingly, the reduced expression of these miRNAs in Wilms’ tumors was due in part to genomic loss of loci that contain these miRNAs, as revealed by high resolution comparative genomic hybridization analysis. In summary, our findings suggest that miRNAs are integral to the molecular circuitry that plays an important role in Wilms’ tumorigenesis by regulating events during normal kidney development. In addition, our discovery of novel tumor suppressor miRNAs targeting the Six1 oncogene promises to unravel a unique regulatory mechanism for Six1 deregulation in pediatric as well adult tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2107.