GILZ-dependent modulation of mTORC1 regulates spermatogonial maintenance.

Male fertility is dependent on spermatogonial stem cells (SSCs) that self-renew and produce differentiating germ cells. Growth factors produced within the testis are essential for SSC maintenance but intrinsic factors that dictate the SSC response to these stimuli are poorly characterised. Here, we have studied the role of GILZ, a TSC22D family protein and spermatogenesis regulator, in spermatogonial function and signalling. Although broadly expressed in the germline, GILZ was prominent in undifferentiated spermatogonia and Gilz deletion in adults resulted in exhaustion of the GFRα1+ SSC-containing population and germline degeneration. GILZ loss was associated with mTORC1 activation, suggesting enhanced growth factor signalling. Expression of deubiquitylase USP9X, an mTORC1 modulator required for spermatogenesis, was disrupted in Gilz mutants. Treatment with an mTOR inhibitor rescued GFRα1+ spermatogonial failure, indicating that GILZ-dependent mTORC1 inhibition is crucial for SSC maintenance. Analysis of cultured undifferentiated spermatogonia lacking GILZ confirmed aberrant activation of ERK MAPK upstream mTORC1 plus USP9X downregulation and interaction of GILZ with TSC22D proteins. Our data indicate an essential role for GILZ-TSC22D complexes in ensuring the appropriate response of undifferentiated spermatogonia to growth factors via distinct inputs to mTORC1.

Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling.

Spermatogenesis is a dynamic process involving self-renewal and differentiation of spermatogonial stem cells, meiosis, and ultimately, the differentiation of haploid spermatids into sperm. Centrosomal protein 55 kDa (CEP55) is necessary for somatic cell abscission during cytokinesis. It facilitates equal segregation of cytoplasmic contents between daughter cells by recruiting endosomal sorting complex required for transport machinery (ESCRT) at the midbody. In germ cells, CEP55, in partnership with testes expressed-14 (TEX14) protein, has also been shown to be an integral component of intercellular bridge before meiosis. Various in vitro studies have demonstrated a role for CEP55 in multiple cancers and other diseases. However, its oncogenic potential in vivo remains elusive. To investigate, we generated ubiquitously overexpressing Cep55 transgenic ( Cep55Tg/Tg) mice aiming to characterize its oncogenic role in cancer. Unexpectedly, we found that Cep55Tg/Tg male mice were sterile and had severe and progressive defects in spermatogenesis related to spermatogenic arrest and lack of spermatids in the testes. In this study, we characterized this male-specific phenotype and showed that excessively high levels of Cep55 results in hyperactivation of PI3K/protein kinase B (Akt) signaling in testis. In line with this finding, we observed increased phosphorylation of forkhead box protein O1 (FoxO1), and suppression of its nuclear retention, along with the relative enrichment of promyelocytic leukemia zinc finger (PLZF) -positive cells. Independently, we observed that Cep55 amplification favored upregulation of ret ( Ret) proto-oncogene and glial-derived neurotrophic factor family receptor α-1 ( Gfra1). Consistent with these data, we observed selective down-regulation of genes associated with germ cell differentiation in Cep55-overexpressing testes at postnatal day 10, including early growth response-4 ( Egr4) and spermatogenesis and oogenesis specific basic helix-loop-helix-1 ( Sohlh1). Thus, Cep55 amplification leads to a shift toward the initial maintenance of undifferentiated spermatogonia and ultimately results in progressive germ cell loss. Collectively, our findings demonstrate that Cep55 overexpression causes change in germ cell proportions and manifests as a Sertoli cell only tubule phenotype, similar to that seen in many azoospermic men.-Sinha, D., Kalimutho, M., Bowles, J., Chan, A.-L., Merriner, D. J., Bain, A. L., Simmons, J. L., Freire, R., Lopez, J. A., Hobbs, R. M., O'Bryan, M. K., Khanna, K. K. Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling.

c-Abl phosphorylates E6AP and regulates its E3 ubiquitin ligase activity.

In human papillomavirus (HPV)-infected cells, the p53 tumor suppressor is tightly regulated by the HPV-E6-E6AP complex, which promotes it for proteasomal degradation. We previously demonstrated that c-Abl tyrosine kinase protects p53 from HPV-E6-E6AP complex-mediated ubiquitination and degradation under stress conditions. However, the underlying mechanism was not defined. In this study, we explored the possibility that c-Abl targets E6AP and thereby protects p53. We demonstrated that c-Abl interacts with and phosphorylates E6AP. We determined that the E3 ligase activity of E6AP is impaired in response to phosphorylation by c-Abl. We mapped the phosphorylation site to tyrosine 636 within the HECT catalytic domain of E6AP, and using substitution mutants, we showed that this residue dictates the E3 ligase activity of E6AP, in a substrate-specific manner. On the basis of the crystal structure of the HECT domain of E6AP, we propose a model in which tyrosine 636 plays a regulatory role in the oligomerization of E6AP, which is a process implicated in its E3 ubiquitin ligase activity. Our results suggest that c-Abl protects p53 from HPV-E6-E6AP complex-mediated degradation by phosphorylating E6AP and impairing its E3 ligase activity, thus providing a molecular explanation for the stress-induced protection of p53 in HPV-infected cells.

Distinctive molecular features of regenerative stem cells in the damaged male germline.

Maintenance of male fertility requires spermatogonial stem cells (SSCs) that self-renew and generate differentiating germ cells for production of spermatozoa. Germline cells are sensitive to genotoxic drugs and patients receiving chemotherapy can become infertile. SSCs surviving treatment mediate germline recovery but pathways driving SSC regenerative responses remain poorly understood. Using models of chemotherapy-induced germline damage and recovery, here we identify unique molecular features of regenerative SSCs and characterise changes in composition of the undifferentiated spermatogonial pool during germline recovery by single-cell analysis. Increased mitotic activity of SSCs mediating regeneration is accompanied by alterations in growth factor signalling including PI3K/AKT and mTORC1 pathways. While sustained mTORC1 signalling is detrimental for SSC maintenance, transient mTORC1 activation is critical for the regenerative response. Concerted inhibition of growth factor signalling disrupts core features of the regenerative state and limits germline recovery. We also demonstrate that the FOXM1 transcription factor is a target of growth factor signalling in undifferentiated spermatogonia and provide evidence for a role in regeneration. Our data confirm dynamic changes in SSC functional properties following damage and support an essential role for microenvironmental growth factors in promoting a regenerative state.

DDX5 plays essential transcriptional and post-transcriptional roles in the maintenance and function of spermatogonia.

Mammalian spermatogenesis is sustained by mitotic germ cells with self-renewal potential known as undifferentiated spermatogonia. Maintenance of undifferentiated spermatogonia and spermatogenesis is dependent on tightly co-ordinated transcriptional and post-transcriptional mechanisms. The RNA helicase DDX5 is expressed by spermatogonia but roles in spermatogenesis are unexplored. Using an inducible knockout mouse model, we characterise an essential role for DDX5 in spermatogonial maintenance and show that Ddx5 is indispensable for male fertility. We demonstrate that DDX5 regulates appropriate splicing of key genes necessary for spermatogenesis. Moreover, DDX5 regulates expression of cell cycle genes in undifferentiated spermatogonia post-transcriptionally and is required for cell proliferation and survival. DDX5 can also act as a transcriptional co-activator and we demonstrate that DDX5 interacts with PLZF, a transcription factor required for germline maintenance, to co-regulate select target genes. Combined, our data reveal a critical multifunctional role for DDX5 in regulating gene expression programmes and activity of undifferentiated spermatogonia.

Identification of dynamic undifferentiated cell states within the male germline.

The role of stem cells in tissue maintenance is appreciated and hierarchical models of stem cell self-renewal and differentiation often proposed. Stem cell activity in the male germline is restricted to undifferentiated A-type spermatogonia (Aundiff); however, only a fraction of this population act as stem cells in undisturbed testis and Aundiff hierarchy remains contentious. Through newly developed compound reporter mice, here we define molecular signatures of self-renewing and differentiation-primed adult Aundiff fractions and dissect Aundiff heterogeneity by single-cell analysis. We uncover an unappreciated population within the self-renewing Aundiff fraction marked by expression of embryonic patterning genes and homeodomain transcription factor PDX1. Importantly, we find that PDX1 marks a population with potent stem cell capacity unique to mature, homeostatic testis and demonstrate dynamic interconversion between PDX1+ and PDX1- Aundiff states upon transplant and culture. We conclude that Aundiff exist in a series of dynamic cell states with distinct function and provide evidence that stability of such states is dictated by niche-derived cues.

Germline Stem Cell Activity Is Sustained by SALL4-Dependent Silencing of Distinct Tumor Suppressor Genes.

Sustained spermatogenesis in adult males and fertility recovery following germ cell depletion are dependent on undifferentiated spermatogonia. We previously demonstrated a key role for the transcription factor SALL4 in spermatogonial differentiation. However, whether SALL4 has broader roles within spermatogonia remains unclear despite its ability to co-regulate genes with PLZF, a transcription factor required for undifferentiated cell maintenance. Through development of inducible knockout models, we show that short-term integrity of differentiating but not undifferentiated populations requires SALL4. However, SALL4 loss was associated with long-term functional decline of undifferentiated spermatogonia and disrupted stem cell-driven regeneration. Mechanistically, SALL4 associated with the NuRD co-repressor and repressed expression of the tumor suppressor genes Foxl1 and Dusp4. Aberrant Foxl1 activation inhibited undifferentiated cell growth and survival, while DUSP4 suppressed self-renewal pathways. We therefore uncover an essential role for SALL4 in maintenance of undifferentiated spermatogonial activity and identify regulatory pathways critical for germline stem cell function.

E6AP promotes prostate cancer by reducing p27 expression.

Prostate cancer (PC) is the most common cancer in men. Elevated levels of E3 ligase, E6-Associated Protein (E6AP) were previously linked to PC, consistent with increased protein expression in a subset of PC patients. In cancers, irregular E3 ligase activity drives proteasomal degradation of tumor suppressor proteins. Accordingly, E3 ligase inhibitors define a rational therapy to restore tumor suppression. The relevant tumor suppressors targeted by E6AP in PC are yet to be fully identified. In this study we show that p27, a key cell cycle regulator, is a target of E6AP in PC. Down regulation of E6AP increases p27 expression and enhances its nuclear accumulation in PC. We demonstrate that E6AP regulates p27 expression by inhibiting its transcription in an E2F1-dependent manner. Concomitant knockdown of E6AP and p27 partially restores PC cell growth, supporting the contribution of p27 to the overall effect of E6AP on prostate tumorigenesis. Overall, we unravelled the E6AP-p27 axis as a new promoter of PC, exposing an attractive target for therapy through the restoration of tumor suppression.

The E3-ligase E6AP Represses Breast Cancer Metastasis via Regulation of ECT2-Rho Signaling.

Metastatic disease is the major cause of breast cancer-related death and despite many advances, current therapies are rarely curative. Tumor cell migration and invasion require actin cytoskeletal reorganization to endow cells with capacity to disseminate and initiate the formation of secondary tumors. However, it is still unclear how these migratory cells colonize distant tissues to form macrometastases. The E6-associated protein, E6AP, acts both as an E3 ubiquitin-protein ligase and as a coactivator of steroid hormone receptors. We report that E6AP suppresses breast cancer invasiveness, colonization, and metastasis in mice, and in breast cancer patients, loss of E6AP associates with poor prognosis, particularly for basal breast cancer. E6AP regulates actin cytoskeletal remodeling via regulation of Rho GTPases, acting as a negative regulator of ECT2, a GEF required for activation of Rho GTPases. E6AP promotes ubiquitination and proteasomal degradation of ECT2 for which high expression predicts poor prognosis in breast cancer patients. We conclude that E6AP suppresses breast cancer metastasis by regulating actin cytoskeleton remodeling through the control of ECT2 and Rho GTPase activity. These findings establish E6AP as a novel suppressor of metastasis and provide a compelling rationale for inhibition of ECT2 as a therapeutic approach for patients with metastatic breast cancer. Cancer Res; 76(14); 4236-48. ©2016 AACR.