Collaboration among writers-readers-erasers of m6A regulates the stability of tumor-specific genes.

Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including brain-derived neurotrophic factor (BDNF), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis.

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.

Molecular mechanisms by which fertilization competent acrosome-reacted sperm bind to the oolemma remain uncharacterized. To identify oolemmal binding partner(s) for sperm acrosomal ligands, affinity panning was performed with mouse oocyte lysates using sperm acrosomal protein, SLLP1 as a target. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants, each containing a zinc binding active site and a putative transmembrane domain, with signal peptides in three variants. SAS1B transcripts were ovary specific. SAS1B protein was first detected in early secondary follicles in day 3 ovaries. Immunofluorescence localized SAS1B to the microvillar oolemma of M2 oocytes. After fertilization, SAS1B decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native SLLP1 co-localized with SAS1B to the microvillar domain of ovulated M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western, yeast two-hybrid, recombinant- and native- co-IP analyses. SAS1B bound to SLLP1 with high affinity. SAS1B had protease activity, and SAS1B protein or antibody significantly inhibited fertilization. SAS1B knockout female mice showed a 34% reduction in fertility. The study identified SAS1B-SLLP1 as a pair of novel sperm-egg binding partners involving the oolemma and intra-acrosomal compartment during fertilization.

Despite improvements in overall survival, only a modest percentage of patients survives high-risk medulloblastoma. The devastating side effects of radiation and chemotherapy substantially reduce quality of life for surviving patients. Here, using genomic screens, we identified miR-584-5p as a potent therapeutic adjuvant that potentiates medulloblastoma to radiation and vincristine. MiR-584-5p inhibited medulloblastoma growth and prolonged survival of mice in pre-clinical tumor models. MiR-584-5p overexpression caused cell cycle arrest, DNA damage, and spindle defects in medulloblastoma cells. MiR-584-5p mediated its tumor suppressor and therapy-sensitizing effects by targeting HDAC1 and eIF4E3. MiR-584-5p overexpression or HDAC1/eIF4E3 silencing inhibited medulloblastoma stem cell self-renewal without affecting neural stem cell growth. In medulloblastoma patients, reduced expression of miR-584-5p correlated with increased levels of HDAC1/eIF4E3. These findings identify a previously undefined role for miR-584-5p/HDAC1/eIF4E3 in regulating DNA repair, microtubule dynamics, and stemness in medulloblastoma and set the stage for a new way to treat medulloblastoma using miR-584-5p.

Although decades of research have established that androgen is essential for spermatogenesis, androgen's mechanism of action remains elusive. This is in part because only a few androgen-responsive genes have been definitively identified in the testis. Here, we propose that microRNAs – small, non-coding RNAs – are one class of androgen-regulated trans-acting factors in the testis. Specifically, by using androgen suppression and androgen replacement in mice, we show that androgen regulates the expression of several microRNAs in Sertoli cells. Our results reveal that several of these microRNAs are preferentially expressed in the testis and regulate genes that are highly expressed in Sertoli cells. Because androgen receptor-mediated signaling is essential for the pre- and post-meiotic germ cell development, we propose that androgen controls these events by regulating Sertoli/germ cell-specific gene expression in a microRNA-dependent manner.

Purpose The approaches aimed at inhibiting the ability of cancer cells to repair DNA strand breaks have emerged as promising targets for treating cancers. Here, we assessed the potential of imipramine blue (IB), a novel analogue of anti-depressant imipramine, to suppress breast cancer growth and metastasis by inhibiting the ability of breast cancer cells to repair DNA strand breaks by homologous recombination. Experimental Design The effect of IB on breast cancer growth and metastasis was assessed in vitro as well as in preclinical mouse models. Besides, the therapeutic efficacy and safety of IB was determined in ex-vivo explants from breast cancer patients. The mechanism of action of IB was evaluated by performing gene expression, drug-protein interaction, cell cycle and DNA repair studies. Results We show that the systemic delivery of IB using nanoparticle-based delivery approach suppressed breast cancer growth and metastasis without inducing toxicity in preclinical mouse models. Using ex-vivo explants from breast cancer patients, we demonstrated that IB inhibited breast cancer growth without affecting normal mammary epithelial cells. Furthermore, our mechanistic studies revealed that IB may interact and inhibit the activity of proto-oncogene FoxM1 and associated signaling that play critical roles in homologous recombination-mediated DNA repair. Conclusions These findings highlight the potential of IB to be applied as a safe regimen for treating breast cancer patients. Given that FoxM1 is an established therapeutic target for several cancers, the identification of a compound that inhibits FoxM1 and FoxM1-mediated DNA repair has immense translational potential for treating many aggressive cancers.

Phagocytic clearance of apoptotic germ cells by Sertoli cells is vital for germ cell development and differentiation. Here, using a tissue-specific miRNA transgenic mouse model, we show that interaction between miR-471-5p and autophagy member proteins regulates clearance of apoptotic germ cells via LC3-associated phagocytosis (LAP). Transgenic mice expressing miR-471-5p in Sertoli cells show increased germ cell apoptosis and compromised male fertility. Those effects are due to defective engulfment and impaired LAP-mediated clearance of apoptotic germ cells as miR-471-5p transgenic mice show lower levels of Dock180, LC3, Atg12, Becn1, Rab5 and Rubicon in Sertoli cells. Our results reveal that Dock180 interacts with autophagy member proteins to constitute a functional LC3-dependent phagocytic complex. We find that androgen regulates Sertoli cell phagocytosis by controlling expression of miR-471-5p and its target proteins. These findings suggest that recruitment of autophagy machinery is essential for efficient clearance of apoptotic germ cells by Sertoli cells using LAP.