TAD hierarchy restricts poised LTR activation and loss of TAD hierarchy promotes LTR co-option in cancer.

Transposable elements (TEs) are abundant in the human genome, and they provide the sources for genetic and functional diversity. The regulation of TEs expression and their functional consequences in physiological conditions and cancer development remain to be fully elucidated. Previous studies suggested TEs are repressed by DNA methylation and chromatin modifications. The effect of 3D chromatin topology on TE regulation remains elusive. Here, by integrating transcriptome and 3D genome architecture studies, we showed that haploinsufficient loss of NIPBL selectively activates alternative promoters at the long terminal repeats (LTRs) of the TE subclasses. This activation occurs through the reorganization of topologically associating domain (TAD) hierarchical structures and recruitment of proximal enhancers. These observations indicate that TAD hierarchy restricts transcriptional activation of LTRs that already possess open chromatin features. In cancer, perturbation of the hierarchical chromatin topology can lead to co-option of LTRs as functional alternative promoters in a context-dependent manner and drive aberrant transcriptional activation of novel oncogenes and other divergent transcripts. These data uncovered a new layer of regulatory mechanism of TE expression beyond DNA and chromatin modification in human genome. They also posit the TAD hierarchy dysregulation as a novel mechanism for alternative promoter-mediated oncogene activation and transcriptional diversity in cancer, which may be exploited therapeutically.

ETV4 mediates dosage-dependent prostate tumor initiation and cooperates with p53 loss to generate prostate cancer.

The mechanisms underlying ETS-driven prostate cancer initiation and progression remain poorly understood due to a lack of model systems that recapitulate this phenotype. We generated a genetically engineered mouse with prostate-specific expression of the ETS factor, ETV4, at lower and higher protein dosage through mutation of its degron. Lower-level expression of ETV4 caused mild luminal cell expansion without histologic abnormalities, and higher-level expression of stabilized ETV4 caused prostatic intraepithelial neoplasia (mPIN) with 100% penetrance within 1 week. Tumor progression was limited by p53-mediated senescence and Trp53 deletion cooperated with stabilized ETV4. The neoplastic cells expressed differentiation markers such as Nkx3.1 recapitulating luminal gene expression features of untreated human prostate cancer. Single-cell and bulk RNA sequencing showed that stabilized ETV4 induced a previously unidentified luminal-derived expression cluster with signatures of cell cycle, senescence, and epithelial-to-mesenchymal transition. These data suggest that ETS overexpression alone, at sufficient dosage, can initiate prostate neoplasia.

PRC2-Inactivating Mutations in Cancer Enhance Cytotoxic Response to DNMT1-Targeted Therapy via Enhanced Viral Mimicry.

Polycomb repressive complex 2 (PRC2) has oncogenic and tumor-suppressive roles in cancer. There is clinical success of targeting this complex in PRC2-dependent cancers, but an unmet therapeutic need exists in PRC2-loss cancer. PRC2-inactivating mutations are a hallmark feature of high-grade malignant peripheral nerve sheath tumor (MPNST), an aggressive sarcoma with poor prognosis and no effective targeted therapy. Through RNAi screening in MPNST, we found that PRC2 inactivation increases sensitivity to genetic or small-molecule inhibition of DNA methyltransferase 1 (DNMT1), which results in enhanced cytotoxicity and antitumor response. Mechanistically, PRC2 inactivation amplifies DNMT inhibitor-mediated expression of retrotransposons, subsequent viral mimicry response, and robust cell death in part through a protein kinase R (PKR)-dependent double-stranded RNA sensor. Collectively, our observations posit DNA methylation as a safeguard against antitumorigenic cell-fate decisions in PRC2-loss cancer to promote cancer pathogenesis, which can be therapeutically exploited by DNMT1-targeted therapy. SIGNIFICANCE: PRC2 inactivation drives oncogenesis in various cancers, but therapeutically targeting PRC2 loss has remained challenging. Here we show that PRC2-inactivating mutations set up a tumor context-specific liability for therapeutic intervention via DNMT1 inhibitors, which leads to innate immune signaling mediated by sensing of derepressed retrotransposons and accompanied by enhanced cytotoxicity. See related commentary by Guil and Esteller, p. 2020. This article is highlighted in the In This Issue feature, p. 2007.

Identification of Cancer Drivers at CTCF Insulators in 1,962 Whole Genomes.

Recent studies have shown that mutations at non-coding elements, such as promoters and enhancers, can act as cancer drivers. However, an important class of non-coding elements, namely CTCF insulators, has been overlooked in the previous driver analyses. We used insulator annotations from CTCF and cohesin ChIA-PET and analyzed somatic mutations in 1,962 whole genomes from 21 cancer types. Using the heterogeneous patterns of transcription-factor-motif disruption, functional impact, and recurrence of mutations, we developed a computational method that revealed 21 insulators showing signals of positive selection. In particular, mutations in an insulator in multiple cancer types, including 16% of melanoma samples, are associated with TGFB1 up-regulation. Using CRISPR-Cas9, we find that alterations at two of the most frequently mutated regions in this insulator increase cell growth by 40%-50%, supporting the role of this boundary element as a cancer driver. Thus, our study reveals several CTCF insulators as putative cancer drivers.

CHD1 Loss Alters AR Binding at Lineage-Specific Enhancers and Modulates Distinct Transcriptional Programs to Drive Prostate Tumorigenesis.

Deletion of the gene encoding the chromatin remodeler CHD1 is among the most common alterations in prostate cancer (PCa); however, the tumor-suppressive functions of CHD1 and reasons for its tissue-specific loss remain undefined. We demonstrated that CHD1 occupied prostate-specific enhancers enriched for the androgen receptor (AR) and lineage-specific cofactors. Upon CHD1 loss, the AR cistrome was redistributed in patterns consistent with the oncogenic AR cistrome in PCa samples and drove tumor formation in the murine prostate. Notably, this cistrome shift was associated with a unique AR transcriptional signature enriched for pro-oncogenic pathways unique to this tumor subclass. Collectively, these data credential CHD1 as a tumor suppressor in the prostate that constrains AR binding/function to limit tumor progression.

FOXF1 Defines the Core-Regulatory Circuitry in Gastrointestinal Stromal Tumor.

The cellular context that integrates upstream signaling and downstream nuclear response dictates the oncogenic behavior and shapes treatment responses in distinct cancer types. Here, we uncover that in gastrointestinal stromal tumor (GIST), the forkhead family member FOXF1 directly controls the transcription of two master regulators, KIT and ETV1, both required for GIST precursor-interstitial cells of Cajal lineage specification and GIST tumorigenesis. Further, FOXF1 colocalizes with ETV1 at enhancers and functions as a pioneer factor that regulates the ETV1-dependent GIST lineage-specific transcriptome through modulation of the local chromatin context, including chromatin accessibility, enhancer maintenance, and ETV1 binding. Functionally, FOXF1 is required for human GIST cell growth in vitro and murine GIST tumor growth and maintenance in vivo The simultaneous control of the upstream signaling and nuclear response sets up a unique regulatory paradigm and highlights the critical role of FOXF1 in enforcing the GIST cellular context for highly lineage-restricted clinical behavior and treatment response.Significance: We uncover that FOXF1 defines the core-regulatory circuitry in GIST through both direct transcriptional regulation and pioneer factor function. The unique and simultaneous control of signaling and transcriptional circuitry by FOXF1 sets up an enforced transcriptional addiction to FOXF1 in GIST, which can be exploited diagnostically and therapeutically. Cancer Discov; 8(2); 234-51. ©2017 AACR.See related commentary by Lee and Duensing, p. 146This article is highlighted in the In This Issue feature, p. 127.

Aberrant Activation of a Gastrointestinal Transcriptional Circuit in Prostate Cancer Mediates Castration Resistance.

Prostate cancer exhibits a lineage-specific dependence on androgen signaling. Castration resistance involves reactivation of androgen signaling or activation of alternative lineage programs to bypass androgen requirement. We describe an aberrant gastrointestinal-lineage transcriptome expressed in ∼5% of primary prostate cancer that is characterized by abbreviated response to androgen-deprivation therapy and in ∼30% of castration-resistant prostate cancer. This program is governed by a transcriptional circuit consisting of HNF4G and HNF1A. Cistrome and chromatin analyses revealed that HNF4G is a pioneer factor that generates and maintains enhancer landscape at gastrointestinal-lineage genes, independent of androgen-receptor signaling. In HNF4G/HNF1A-double-negative prostate cancer, exogenous expression of HNF4G at physiologic levels recapitulates the gastrointestinal transcriptome, chromatin landscape, and leads to relative castration resistance.

Egress of sperm autoantigen from seminiferous tubules maintains systemic tolerance.

Autoimmune responses to meiotic germ cell antigens (MGCA) that are expressed on sperm and testis occur in human infertility and after vasectomy. Many MGCA are also expressed as cancer/testis antigens (CTA) in human cancers, but the tolerance status of MGCA has not been investigated. MGCA are considered to be uniformly immunogenic and nontolerogenic, and the prevailing view posits that MGCA are sequestered behind the Sertoli cell barrier in seminiferous tubules. Here, we have shown that only some murine MGCA are sequestered. Nonsequestered MCGA (NS-MGCA) egressed from normal tubules, as evidenced by their ability to interact with systemically injected antibodies and form localized immune complexes outside the Sertoli cell barrier. NS-MGCA derived from cell fragments that were discarded by spermatids during spermiation. They egressed as cargo in residual bodies and maintained Treg-dependent physiological tolerance. In contrast, sequestered MGCA (S-MGCA) were undetectable in residual bodies and were nontolerogenic. Unlike postvasectomy autoantibodies, which have been shown to mainly target S-MGCA, autoantibodies produced by normal mice with transient Treg depletion that developed autoimmune orchitis exclusively targeted NS-MGCA. We conclude that spermiation, a physiological checkpoint in spermatogenesis, determines the egress and tolerogenicity of MGCA. Our findings will affect target antigen selection in testis and sperm autoimmunity and the immune responses to CTA in male cancer patients.

BO-1055, a novel DNA cross-linking agent with remarkable low myelotoxicity shows potent activity in sarcoma models.

DNA damaging agents cause rapid shrinkage of tumors and form the basis of chemotherapy for sarcomas despite significant toxicities. Drugs having superior efficacy and wider therapeutic windows are needed to improve patient outcomes. We used cell proliferation and apoptosis assays in sarcoma cell lines and benign cells; γ-H2AX expression, comet assay, immunoblot analyses and drug combination studies in vitro and in patient derived xenograft (PDX) models. BO-1055 caused apoptosis and cell death in a concentration and time dependent manner in sarcoma cell lines. BO-1055 had potent activity (submicromolar IC50) against Ewing sarcoma and rhabdomyosarcoma, intermediate activity in DSRCT (IC50 = 2-3µM) and very weak activity in osteosarcoma (IC50 >10µM) cell lines. BO-1055 exhibited a wide therapeutic window compared to other DNA damaging drugs. BO-1055 induced more DNA double strand breaks and γH2AX expression in cancer cells compared to benign cells. BO-1055 showed inhibition of tumor growth in A673 xenografts and caused tumor regression in cyclophosphamide resistant patient-derived Ewing sarcoma xenografts and A204 xenografts. Combination of BO-1055 and irinotecan demonstrated synergism in Ewing sarcoma PDX models. Potent activity on sarcoma cells and its relative lack of toxicity presents a strong rationale for further development of BO-1055 as a therapeutic agent.

Adjudin--A Male Contraceptive with Other Biological Activities.

BACKGROUND: Adjudin has been explored as a male contraceptive for the last 15 years since its initial synthesis in the late 1990s. More than 50 papers have been published and listed in PubMed in which its mechanism that induces exfoliation of germ cells from the seminiferous epithelium, such as its effects on actin microfilaments at the apical ES (ectoplasmic specialization, a testis-specific actin-rich anchoring junction) has been delineated. OBJECTIVE: Recent studies have demonstrated that, besides its activity to induce germ cell exfoliation from the seminiferous epithelium to cause reversible infertility in male rodents, adjudin possesses other biological activities, which include anti-cancer, anti-inflammation in the brain, and anti-ototoxicity induced by gentamicin in rodents. Results of these findings likely spark the interest of investigators to explore other medical use of this and other indazole-based compounds, possibly mediated by the signaling pathway(s) in the mitochondria of mammalian cells following treatment with adjudin. In this review, we carefully evaluate these recent findings. METHODS: Papers published and listed at www.pubmed.org and patents pertinent to adjudin and its related compounds were searched. Findings were reviewed and critically evaluated, and summarized herein. RESULTS: Adjudin is a novel compound that possesses anti-spermatogenetic activity. Furthermore, it possesses anti-cancer, anti-inflammation, anti-neurodegeneration, and anti-ototoxicity activities based on studies using different in vitro and in vivo models. CONCLUSION: Studies on adjudin should be expanded to better understand its biological activities so that it can become a useful drug for treatment of other ailments besides serving as a male contraceptive.

Alternative transcription initiation leads to expression of a novel ALK isoform in cancer.

Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ∼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALK(ATI). In ALK(ATI)-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALK(ATI) is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALK(ATI) transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALK(ATI) stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALK(ATI), suggesting that patients with ALK(ATI)-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

Differential effects of c-Src and c-Yes on the endocytic vesicle-mediated trafficking events at the Sertoli cell blood-testis barrier: an in vitro study.

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. However, it undergoes cyclic restructuring during the epithelial cycle of spermatogenesis in which the "old" BTB located above the preleptotene spermatocytes being transported across the immunological barrier is "disassembled," whereas the "new" BTB found behind these germ cells is rapidly "reassembled," i.e., mediated by endocytic vesicle-mediated protein trafficking events. Thus, the immunological barrier is maintained when preleptotene spermatocytes connected in clones via intercellular bridges are transported across the BTB. Yet the underlying mechanism(s) in particular the involving regulatory molecules that coordinate these events remains unknown. We hypothesized that c-Src and c-Yes might work in contrasting roles in endocytic vesicle-mediated trafficking, serving as molecular switches, to effectively disassemble and reassemble the old and the new BTB, respectively, to facilitate preleptotene spermatocyte transport across the BTB. Following siRNA-mediated specific knockdown of c-Src or c-Yes in Sertoli cells, we utilized biochemical assays to assess the changes in protein endocytosis, recycling, degradation and phagocytosis. c-Yes was found to promote endocytosed integral membrane BTB proteins to the pathway of transcytosis and recycling so that internalized proteins could be effectively used to assemble new BTB from the disassembling old BTB, whereas c-Src promotes endocytosed Sertoli cell BTB proteins to endosome-mediated protein degradation for the degeneration of the old BTB. By using fluorescence beads mimicking apoptotic germ cells, Sertoli cells were found to engulf beads via c-Src-mediated phagocytosis. A hypothetical model that serves as the framework for future investigation is thus proposed.

Cytokines, polarity proteins, and endosomal protein trafficking and signaling-the sertoli cell blood-testis barrier system in vitro as a study model.

Endosomal signaling is emerging as one of the most important cellular events that regulate signaling function in mammalian cells or an epithelium in response to changes in environment such as the presence of stimuli mediated by cytokines, toxicants, heat, ions during growth and development, and other cellular processes such as cytokinesis and spermatogenesis. Recent studies have shown that protein endocytosis-the initial step of endosomal signaling-involves the participation of polarity proteins, such as partitioning defective protein 6 (Par6), Cdc42 and 14-3-3 (also known as Par5), which in turn is regulated by cytokines (e.g., TGF-ß2, TGF-ß3) and testosterone at the Sertoli cell blood-testis barrier (BTB) in the mammalian testis. In this short method paper, we provide a detailed protocol of assessing protein endocytosis, the initial and also the most critical step of endosomal signaling at the Sertoli cell BTB. This biochemical endocytosis assay summarizes our experience for the last decade, which should likely be performed in conjunction with the dual-labeled immunofluorescence analysis to assess protein endocytosis. While we are using a Sertoli cell in vitro system that mimics the BTB in vivo, this approach should be applicable to virtually all mammalian cells.

Breast cancer resistance protein regulates apical ectoplasmic specialization dynamics stage specifically in the rat testis.

Drug transporters determine the bioavailability of drugs in the testis behind the blood-testis barrier (BTB). Thus, they are crucial for male contraceptive development if these drugs (e.g., adjudin) exert their effects behind the BTB. Herein breast cancer resistance protein (Bcrp), an efflux drug transporter, was found to be expressed by both Sertoli and germ cells. Interestingly, Bcrp was not a component of the Sertoli cell BTB. Instead, it was highly expressed by peritubular myoid cells at the tunica propria and also endothelial cells of the microvessels in the interstitium at all stages of the epithelial cycle. Unexpectedly, Bcrp was found to be expressed at the Sertoli-step 18-19 spermatid interface but limited to stage VI-early VIII tubules, and an integrated component of the apical ectoplasmic specialization (apical ES). Apparently, Bcrp is being used by late-stage spermatids to safeguard their completion of spermiogenesis by preventing harmful drugs to enter these cells while they transform to spermatozoa. Also, the association of Bcrp with actin, Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein), and Arp3 (actin-related protein 3, a component of the Arp2/3 complex known to induce branched actin polymerization) at the apical ES suggest that Bcrp may be involved in regulating the organization of actin filament bundles at the site. Indeed, a knockdown of Bcrp by RNAi in the testis perturbed the apical ES function, disrupting spermatid polarity and adhesion. In summary, Bcrp is a regulator of the F-actin-rich apical ES in the testis.

Focal adhesion kinase and actin regulatory/binding proteins that modulate F-actin organization at the tissue barrier: Lesson from the testis.

Focal adhesion kinase (FAK), as its name implied, is an important mediator of integrin-based signaling function in mammalian cells at the focal adhesion complex (FAC, also known as focal contact) at the cell-extracellular matrix interface. FAK is intimately related to cell movement, such as in macrophages, fibroblasts and also tumor cells. In the testis, however, FAK and two of its phosphorylated forms, p-FAK-Tyr(407) and -Tyr(397), are not found at the FAC since there is no ultrastructure analogous or similar to FAC in the mammalian testis vs. other epithelia. Instead, FAK and its two phosphorylated forms are detected along the seminiferous epithelium in the rat testis at the cell-cell interface in a testis-specific adherens junction (AJ) known as the ectoplasmic specialization (ES). ES is an F-actin-rich ultrastructure in which bundles of actin filaments are sandwiched in-between plasma membrane and cisternae of endoplasmic reticulum not found in other mammalian epithelial/endothelial cells. The ES is restricted to the interface of Sertoli cells and spermatids (step 8-19) known as the apical ES, and to the Sertoli cell-cell interface known as the basal ES. Interestingly, the basal ES is also an integrated component of the blood-testis barrier (BTB), coexisting with tight junction (TJ) and gap junction (GJ), and it is conceivable that actin filament bundles at the ES undergo extensive organization, converting from their "bundled" to "de-bundled/branching" configuration to facilitate transport of germ cells across the epithelium and at the BTB during the epithelial cycle. A recent report (Lie et al. PNAS 109:12562-12567, 2012) has demonstrated that the stage-specific and spatiotemporal expression of p-FAK-Tyr(407) and -Tyr(397) are crucial to the regulation of these events via their stage-specific and spatiotemporal expression during the epithelial cycle mediated by their effects on the organization of the actin filament bundles at the ES, involving actin binding/regulatory proteins. In this Commentary, we will critically evaluate these findings in light of other recent reports in the field. While these ideas are based on studies in the BTB in the rat testis, this information should be applicable and helpful to investigators studying other tissue barriers.

Secreted Frizzled-related protein 1 (sFRP1) regulates spermatid adhesion in the testis via dephosphorylation of focal adhesion kinase and the nectin-3 adhesion protein complex.

Development of spermatozoa in adult mammalian testis during spermatogenesis involves extensive cell migration and differentiation. Spermatogonia that reside at the basal compartment of the seminiferous epithelium differentiate into more advanced germ cell types that migrate toward the apical compartment until elongated spermatids are released into the tubule lumen during spermiation. Apical ectoplasmic specialization (ES; a testis-specific anchoring junction) is the only cell junction that anchors and maintains the polarity of elongating/elongated spermatids (step 8-19 spermatids) in the epithelium. Little is known regarding the signaling pathways that trigger the disassembly of the apical ES at spermiation. Here, we show that secreted Frizzled-related protein 1 (sFRP1), a putative tumor suppressor gene that is frequently down-regulated in multiple carcinomas, is a crucial regulatory protein for spermiation. The expression of sFRP1 is tightly regulated in adult rat testis to control spermatid adhesion and sperm release at spermiation. Down-regulation of sFRP1 during testicular development was found to coincide with the onset of the first wave of spermiation at approximately age 45 d postpartum, implying that sFRP1 might be correlated with elongated spermatid adhesion conferred by the apical ES before spermiation. Indeed, administration of sFRP1 recombinant protein to the testis in vivo delayed spermiation, which was accompanied by down-regulation of phosphorylated (p)-focal adhesion kinase (FAK)-Tyr(397) and retention of nectin-3 adhesion protein at the apical ES. To further investigate the functional relationship between p-FAK-Tyr(397) and localization of nectin-3, we overexpressed sFRP1 using lentiviral vectors in the Sertoli-germ cell coculture system. Consistent with the in vivo findings, overexpression of sFRP1 induced down-regulation of p-FAK-Tyr(397), leading to a decline in phosphorylation of nectin-3. In summary, this report highlights the critical role of sFRP1 in regulating spermiation via its effects on the FAK signaling and retention of nectin-3 adhesion complex at the apical ES.

The Scribble/Lgl/Dlg polarity protein complex is a regulator of blood-testis barrier dynamics and spermatid polarity during spermatogenesis.

During spermatogenesis, spermiogenesis that releases sperm into the tubule lumen and restructuring of the blood-testis barrier (BTB) that accommodates the transit of preleptotene spermatocytes take place simultaneously, but at the opposite ends of the seminiferous epithelium. These events are tightly regulated and coordinated; however, neither the underlying mechanism(s) nor the involving molecules are known. Herein, the Scribble/Lgl (Lethal giant larvae)/Dlg (Discs large) polarity complex was shown to regulate spermatid polarity during spermiogenesis and tight junction (TJ)-permeability barrier via changes in protein distribution at the apical ectoplasmic specialization and the BTB during the epithelial cycle, respectively. Scribble, Lgl2, and Dlg1 were found to be expressed by Sertoli and germ cells. Scribble also displayed stage-specific expression at the BTB, being highest at stages VII-VIII, colocalizing with TJ proteins occludin and ZO-1. Unlike components of other polarity complex modules, such as partitioning-defective 6, the knockdown of which by RNA interference was found to impede Sertoli cell TJ barrier, a knockdown of the Scribble complex (i.e. simultaneous knockdown of Scribble, Lgl and Dlg or Lgl alone; but not Scribble or Dlg alone) both in vitro and in vivo promoted the TJ integrity. This was mediated by reorganizing actin filament network at the Sertoli cell-cell interface, which, in turn, affected changes in the localization and/or distribution of occludin and/or ß-catenin at the BTB. These knockdowns also perturbed F-actin organization at the Sertoli cell-spermatid interface, thereby modulating spermatid adhesion and polarity at the apical ectoplasmic specialization. In summary, the Scribble/Lgl/Dlg complex participates in the regulation of BTB dynamics and spermatid adhesion/polarity in the testis.

Polarity protein complex Scribble/Lgl/Dlg and epithelial cell barriers.

The Scribble polarity complex or module is one of the three polarity modules that regulate cell polarity in multiple epithelia including blood-tissue barriers. This protein complex is composed of Scribble, Lethal giant larvae (Lgl) and Discs large (Dlg), which are well conserved across species from fruitflies and worms to mammals. Originally identified in Drosophila and C. elegans where the Scribble complex was found to work with the Par-based and Crumbs-based polarity modules to regulate apicobasal polarity and asymmetry in cells and tissues during embryogenesis, their mammalian homologs have all been identified in recent years. Components of the Scribble complex are known to regulate multiple cellular functions besides cell polarity, which include cell proliferation, assembly and maintenance of adherens junction (AJ) and tight junction (TJ), and they are also tumor suppressors. Herein, we provide an update on the Scribble polarity complex and how this protein complex modulates cell adhesion with some emphasis on its role in Sertoli cell blood-testis barrier (BTB) function. It should be noted that this is a rapidly developing field, in particular the role of this protein module in blood-tissue barriers, and this short chapter attempts to provide the information necessary for investigators studying reproductive biology and blood-tissue barriers to design future studies. We also include results of recent studies from flies and worms since this information will be helpful in planning experiments for future functional studies in the testis to understand how Scribble-based proteins regulate BTB dynamics and spermatogenesis.

Regulation of blood-testis barrier dynamics by TGF-beta3 is a Cdc42-dependent protein trafficking event.

In the testis, the blood-testis barrier (BTB) is constituted by specialized junctions between adjacent Sertoli cells in the seminiferous epithelium near the basement membrane. Although the BTB is one of the tightest blood-tissue barriers in the mammalian body, it undergoes extensive restructuring at stage VIII of the seminiferous epithelial cycle to facilitate the transit of preleptotene spermatocytes. Thus, meiosis and postmeiotic germ cell development take place in the seminiferous epithelium behind the BTB. Cytokines (e.g., TGF-beta3) are known to regulate BTB dynamics by enhancing the endocytosis of integral membrane proteins and their intracellular degradation. This thus reduces the levels of proteins above the spermatocytes in transit at the BTB, causing its disruption after testosterone-induced new tight junction (TJ) fibrils are formed behind these cells. By using Sertoli cells cultured in vitro with an established TJ permeability barrier that mimicked the BTB in vivo, Cdc42 was shown to be a crucial regulator that mediated the TGF-beta3-induced BTB disruption. TGF-beta3 was shown to activate Cdc42 to its active GTP-bound form. However, an inactivation of Cdc42 by overexpressing its dominant-negative mutant T17N in Sertoli cell epithelium was shown to block the TGF-beta3-induced acceleration in protein endocytosis. Consequently, this prevented the disruption of Sertoli cell TJ permeability barrier and redistribution of TJ proteins (e.g., CAR and ZO-1) from the cell-cell interface to cell cytosol caused by TGF-beta3. In summary, Cdc42 is a crucial regulatory component in the TGF-beta3-mediated cascade of events that leads to the disruption of the TJ fibrils above the preleptotene spermatocytes to facilitate their transit.