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ObjectiveSporadic early-onset colorectal cancer (EOCRC) has bad prognosis, yet is poorly represented by cell line models. We examine the key mutational and transcriptomic alterations in an organoid biobank enriched in EOCRCs.DesignWe established paired cancer (n=32) and normal organoids (n=18) from 20 patients enriched in microsatellite-stable EOCRC. Exome and transcriptome analysis was performed.ResultsWe observed a striking diversity of molecular phenotypes, including PTPRK-RSPO3 fusions. Transcriptionally, RSPO fusion organoids resembled normal colon organoids and were distinct from APC mutant organoids, with high BMP2 and low PTK7 expression. Single cell transcriptome analysis confirmed the similarity between RSPO fusion organoids and normal organoids, with a propensity for maturation on Wnt withdrawal, whereas the APC mutant organoids were locked in progenitor stages. CRISPR/Cas9 engineered mutation of APC in normal human colon organoids led to upregulation of PTK7 protein and suppression of BMP2, but less so with an engineered RNF43 mutation. The frequent co-occurrence of RSPO fusions with SMAD4 or BMPR1A mutation was confirmed in TCGA database searches. RNF43 mutation was found in organoid from a leukaemia survivor with a novel mutational signature; and organoids with POLE proofreading mutation displayed ultramutation. The cancer organoid genomes were stable over long culture periods, while normal human colon organoids tended to be subject to clonal dominance over time.ConclusionsThese organoid models enriched in EOCRCs with linked genomic data fill a gap in existing CRC models and reveal distinct genetic profiles and novel pathway cooperativity.

Three articles in Nature show that intestinal stem cells with cancer-promoting mutations could shape the surrounding normal tissue in their favor to promote clonal fixation and field expansion, raising the possibility of developing therapeutic strategies that maintain or enhance the health of normal cells to out-compete the mutant cells.

Precision medicine requires in vitro models which will both faithfully recapitulate the features of an individual's disease and enable drug testing on a wide variety of samples covering the greatest range of phenotypes possible for a particular disease. Organoid technology has immense potential to fulfill this demand, but it will be necessary to develop robust protocols that enable the generation of organoids in a dependable manner from nearly every patient. Here we provide a user's guide, including detailed step-by-step protocols, to the establishment, isolation and verification of gastric cancer organoids. Selection strategies include omission of growth factors, addition of drugs, isolation of distinct phenotypes and generation of monoclonal lines. For confirmation of cancer identity, we use sequencing, drug selection, karyotyping and histology. While we specify these protocols for human gastric cancer organoids here, the methods described are applicable to organoids derived from other tissues as well.

Five years ago, Li et al. (2014) and Gao et al. (2014) reported the power and unique advantages of cancer organoids. We shine a spotlight on the platform's enormous potential for studying cancer biology and as a preclinical human tumor model that can facilitate drug discovery and personalized therapy.

OBJECTIVE: Serrated polyps (hyperplastic polyps, sessile or traditional serrated adenomas), which can arise in a sporadic or polyposis setting, predispose to colorectal cancer (CRC), especially those with microsatellite instability (MSI) due to MLH1 promoter methylation (MLH1(me+)). We investigate genetic alterations in the serrated polyposis pathway. DESIGN: We used a combination of exome sequencing and target gene Sanger sequencing to study serrated polyposis families, sporadic serrated polyps and CRCs, with validation by analysis of The Cancer Genome Atlas (TCGA) cohort, followed by organoid-based functional studies. RESULTS: In one out of four serrated polyposis families, we identified a germline RNF43 mutation that displayed autosomal dominant cosegregation with the serrated polyposis phenotype, along with second-hit inactivation through loss of heterozygosity or somatic mutations in all serrated polyps (16), adenomas (5) and cancer (1) examined, as well as coincidental BRAF mutation in 62.5% of the serrated polyps. Concurrently, somatic RNF43 mutations were identified in 34% of sporadic sessile/traditional serrated adenomas, but 0% of hyperplastic polyps (p=0.013). Lastly, in MSI CRCs, we found significantly more frequent RNF43 mutations in the MLH1(me+) (85%) versus MLH1(me-) (33.3%) group (p

Summary: Arginine methylation is a post-translational modification that plays pivotal roles in signal transduction and gene transcription during cell fate determination. We found protein methyltransferase 6 (PRMT6) to be frequently downregulated in hepatocellular carcinoma (HCC) and its expression to negatively correlate with aggressive cancer features in HCC patients. Silencing of PRMT6 promoted the tumor-initiating, metastasis, and therapy resistance potential of HCC cell lines and patient-derived organoids. Consistently, loss of PRMT6 expression aggravated liver tumorigenesis in a chemical-induced HCC PRMT6 knockout (PRMT6−/−) mouse model. Integrated transcriptome and protein-protein interaction studies revealed an enrichment of genes implicated in RAS signaling and showed that PRMT6 interacted with CRAF on arginine 100, which decreased its RAS binding potential and altered its downstream MEK/ERK signaling. Our work describes a critical repressive function for PRMT6 in maintenance of HCC cells by regulating RAS binding and MEK/ERK signaling via methylation of CRAF on arginine 100. : RAS/RAF/MEK/ERK pathway signaling is known to be frequently activated in cancers, in which it regulates cell growth, malignant transformation, drug resistance, and stemness. Using hepatocellular carcinoma as a model system, Chan et al. describe a mechanism by which this oncogenic signaling pathway is regulated by PRMT6 at the post-translational level via arginine methylation. Keywords: arginine methylation, cancer stemness, tumor-initiating cells, HCC, epigenetics

Human intestinal tract as an alternative route to acquire MERS-CoV infection., Middle East respiratory syndrome coronavirus (MERS-CoV) has caused human respiratory infections with a high case fatality rate since 2012. However, the mode of virus transmission is not well understood. The findings of epidemiological and virological studies prompted us to hypothesize that the human gastrointestinal tract could serve as an alternative route to acquire MERS-CoV infection. We demonstrated that human primary intestinal epithelial cells, small intestine explants, and intestinal organoids were highly susceptible to MERS-CoV and can sustain robust viral replication. We also identified the evidence of enteric MERS-CoV infection in the stool specimen of a clinical patient. MERS-CoV was considerably resistant to fed-state gastrointestinal fluids but less tolerant to highly acidic fasted-state gastric fluid. In polarized Caco-2 cells cultured in Transwell inserts, apical MERS-CoV inoculation was more effective in establishing infection than basolateral inoculation. Notably, direct intragastric inoculation of MERS-CoV caused a lethal infection in human DPP4 transgenic mice. Histological examination revealed MERS-CoV enteric infection in all inoculated mice, as shown by the presence of virus-positive cells, progressive inflammation, and epithelial degeneration in small intestines, which were exaggerated in the mice pretreated with the proton pump inhibitor pantoprazole. With the progression of the enteric infection, inflammation, virus-positive cells, and live viruses emerged in the lung tissues, indicating the development of sequential respiratory infection. Taken together, these data suggest that the human intestinal tract may serve as an alternative infection route for MERS-CoV.

Spermatogenesis is a complex biochemical event, involving the participation of the hypothalamus and the pituitary gland via the secretion of the hypothalamus hormone GnRH, and two pituitary hormones FSH and LH. Thus, the hypothalamus-pituitary-testicular axis is a crucial regulatory axis for testicular function. Recent studies have shown that in the microenvironment of the seminiferous epithelium, wherein each Sertoli cell is supporting ~30-50 germ cells at different stages of their development, locally produced autocrine and paracrine factors are also involved in spermatogenesis, in particular at the level of cell junctions. These cell junctions at the Sertoli-Sertoli and Sertoli-germ cell interface are crucial to coordinate different events of spermatogenesis by sending signals back-and-forth between Sertoli and germ cells, in order to precisely regulate spermatogonial cells renewal by mitosis, cell cycle progression, meiosis, spermiogenesis, germ cell movement across the epithelium, spermiation and germ cell apoptosis. In this minireview, we provide an update on these latest findings for an emerging new concept regarding the presence of a local “apical ectoplasmic specialization – blood-testis barrier – hemidesmosome/basement membrane” functional axis that regulates the events of spermiation and blood-testis barrier (BTB) restructuring via paracrine/autocrine factors and polarity proteins produced locally in the seminiferous epithelium. These findings provide a new window of research for investigators in the field to tackle the functional regulation of spermatogenesis.

During spermatogenesis in the mammalian testis, preleptotene/leptotene spermatocytes differentiate from type B spermatogonia and traverse the blood-testis barrier (BTB) at stage VIII of the seminiferous epithelial cycle for further development. This timely movement of germ cells involves extensive junction restructuring at the BTB. Previous studies have shown that these events are regulated by testosterone (T) and cytokines [e.g., the transforming growth factor (TGF) -βs], which promote and disrupt the BTB assembly, respectively. However, the mechanisms underlying the "opening" of the BTB above a migrating preleptotene/leptotene spermatocyte and the "resealing" of the barrier underneath this cell remain obscure. We now report findings on a novel mechanism utilized by the testes to regulate these events. Using cell surface protein biotinylation coupled with immunoblotting and immunofluorescent microscopy, we assessed the kinetics of endocytosis and recycling of BTB-associated integral membrane proteins: occludin, JAM-A, and N-cadherin. It was shown that these proteins were continuously endocytosed and recycled back to the Sertoli cell surface via the clathrin-mediated but not the caveolin-mediated pathway. When T or TGF-β2 was added to Sertoli cell cultures with established functional BTB, both factors accelerated the kinetics of internalization of BTB proteins from the cell surface, perhaps above the migrating preleptotene spermatocyte, thereby opening the BTB. Likewise, T also enhanced the kinetics of recycling of internalized biotinylated proteins back to the cell surface, plausibly relocating these proteins beneath the migrating spermatocyte to reassemble the BTB. In contrast, TGF-β2 targeted internalized biotinylated proteins to late endosomes for degradation, destabilizing the BTB. In summary, the transient opening of the BTB that facilitates germ cell movement is mediated via the differential effects of T and cytokines on the kinetics of endocytosis and recycling of integral membrane proteins at the BTB. The net result of these interactions, in turn, determines the steady-state protein levels at the Sertoli-Sertoli cell interface at the BTB. © FASEB., link_to_OA_fulltext

Apical ectoplasmic specialization (ES) is a testis-specific hybrid cell/cell actin-based adherens junction and cell/matrix focal contact anchoring junction type restricted to the interface between Sertoli cells and developing spermatids. Recent studies have shown that laminin γ3, restricted to elongating spermatids, is a putative binding partner of α 6β 1-integrin localized in Sertoli cells at the apical ES. However, the identity of the α and β chains, which constitute a functional laminin ligand with the γ3 chain at the apical ES, is not known. Using reverse transcription-PCR and immunoblotting to survey all laminin chains in cells of the seminiferous epithelium, it was noted that α 2, α 3, β1, β2, β3, and γ3 chains were found in germ cells, whereas α 1, α 2, α 4, α 5, β1, β2, γ1, γ2, and γ3 chains were found in Sertoli cells, implying that α 3 and β3 are the plausible laminin chains restricted to germ cells that may be the bona fide partners of γ3. To verify this postulate, recombinant proteins based on domain G of α 3 and domain I of β3 and γ3 chains were produced and used to obtain the corresponding specific polyclonal antibodies. Additional studies have demonstrated that the laminin α 3, β3, and γ3 chains indeed are restricted to germ cells at the apical ES, co-localizing with each other and with β1-integrin. Furthermore, co-immunoprecipitation studies have confirmed the interactions among laminin α 3, β3, and γ3, as well as β1-integrin. When the functional laminin ligand at the apical ES was disrupted via blocking antibodies, such as using anti-laminin α 3 or γ3 IgG, this treatment perturbed adhesion between Sertoli and germ cells (mostly spermatids), leading to germ cell loss from the epithelium. More important, a transient disruption of the blood-testis barrier was also detected.

In the mammalian testis, the blood–testis barrier (BTB), unlike the blood–brain and blood–retina barriers, is composed of coexisting tight junctions (TJs) and adherens junctions (AJs). Yet these junctions must open (or disassemble) to accommodate the migration of preleptotene and leptotene spermatocytes across the BTB during spermatogenesis while maintaining its integrity. In this report, we show that the BTB utilizes a unique “engagement” and “disengagement” mechanism to permit the disruption of AJ that facilitates germ cell movement without compromising the BTB integrity. For instance, both TJ (e.g., occludin and JAM-1) and AJ (e.g., N-cadherin) integral membrane proteins were colocalized to the same site at the BTB. Although these TJ- and AJ-integral membrane proteins did not physically interact with each other, they were structurally linked by means of peripheral adaptors (e.g., ZO-1 and α- and γ-catenins). As such, these proteins are structurally “engaged” under physiological conditions to reinforce the BTB. When rats were exposed to Adjudin to induce AJ restructuring that eventually led to germ cell loss from the epithelium, this structural interaction between occludin and N-cadherin by means of their adaptors became “disengaged” while their protein levels were significantly induced. In short, when the epithelium is under assault, such as by Adjudin or plausibly at the time of germ cell migration across the BTB during spermatogenesis, the TJ- and AJ-integral membrane proteins can be disengaged. Thus, this mechanism is used by the testis to facilitate AJ restructuring to accommodate germ cell migration while maintaining the BTB integrity.

Hepatocellular carcinoma (HCC), the major type of liver cancer, remains one of the most prevalent and deadliest cancer types in the world. Contemporary challenge in treating HCC has been the common therapy resistance and recurrence after therapy, all of which have been reported to be associated with stem-like behavior of cancer cells. Our group has previously identified a functional liver cancer stem cell (CSC) subset marked by the CD133 cell surface phenotype. Utilizing a PCR array encompassing diverse human chromatin modifiers, protein arginine methyltransferase 6 (PRMT6) was found to be differentially down-regulated in CD133+ liver CSCs of human HCC cells as well as CD133 enriched chemoresistant hepatospheres as compared to their counterparts. Clinically, reduced PRMT6 expression was detected in HCC specimens and correlated with a higher risk of metastasis. PRMT6 negatively regulated diverse in vitro cancer stem cell properties of HCC cells including self-renewal, therapy resistance, metastasis and expression of CSC and pluripotency markers. In addition, PRMT6 also suppressed in vivo tumor initiation and serial transplantation. Surprising, contrary to its usual localization in the nucleus as a chromatin modification enzyme mediating histone H3R2 methylation, we found PRMT6 to be predominantly expressed in the cytoplasm in normal liver and HCC cells. Through tandem affinity purification and subsequent mass spectrometry profiling, we identified CRAF, a serine/threonine-protein kinase, as a novel cytoplasmic protein partner of PRMT6. Binding of PRMT6 to CRAF inhibited its kinase activity through site-specific arginine methylation, resulting in inhibition of ERK-mediated CSC plasticity in HCC, demonstrated through in vivo / in vitro methylation assays, kinase assay and functional rescue experiments with the ERK inhibitor U0126. The link between PRMT6, ERK and cancer stemness was further substantiated in primary human normal liver and HCC organoids with or without PRMT6 modulated. Taken together, we found PRMT6 to be down-regulated in the liver CSC subset and to be functionally involved in regulating liver CSC plasticity via an unprecedented role in the cytoplasm through suppression of CRAF/ERK cascade. Citation Format: Lok Hei Chan, Lei Zhou, Kai Yu Ng, Tin Lok Wong, Stella Chai, Terence K Lee, Xin Yuan Guan, Yick Pang Ching, Chung Mau Lo, Kwan Man, Benedetta Artegiani, Hans Clevers, Helen H Yan, Suet Yi Leung, Stèphane Richard, Michael SY Huen, Stephanie Ma. PRMT6-dependent CRAF/ERK signaling regulates cancer stem cell plasticity in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-139. doi:10.1158/1538-7445.AM2017-LB-139

Serrated polyps can arise in a sporadic or familial polyposis setting and predispose to colorectal cancer (CRC). Recently, we have identified RNF43 germline mutation in a family with Serrated Polyposis Syndrome (Yan et al, GUT 2016). The presence of second somatic hit in all serrated polyps examined from members of this family further confirmed the pathogenicity of RNF43 germline mutation. In an attempt to further delineate the global genomic alterations in these serrated polyps, so as to understand the RNF43 driven serrated neoplasia pathway for the development of colorectal cancer, we performed whole exome sequencing (WES) on two sessile serrated adenomas (SSAs) resected from an RNF43 germline mutation carrier, together with the paired blood DNA. Consistent with the results from our previous Sanger sequencing study, WES detected complete inactivation of RNF43 through a 2nd hit somatic mutation, c.461 C>T P154L, or loss of heterozygosity (LOH) in the two SSAs, respectively. We also identified 51 and 58 somatic mutations in the two SSAs, respectively. BRAFV600E mutation was the only shared mutation between the two polyps. Interestingly, each of the SSAs carried a truncating mutation in a histone-methyltransferase gene, either PRDM9 or SETD1B, respectively. Other truncating or deleterious mutations included chromatin modifiers (CHD2, CHD4, TSPYL2) or other genes with methyltransferase activity (TRMT2B, METTL12, METTL14, ECE2). In addition, we found few instances of chromosomal aberration or LOH, apart from a region of LOH at chromosome 17 encompassing RNF43 in one of the SSAs. Overall, we have revealed the genomic landscape of two sessile serrated adenomas resected from a germline RNF43 mutation carrier. The results confirmed somatic RNF43 2nd hit and BRAFV600E mutation as the key events, along with putative roles for histone methyltransferase, as well as other chromatin modifiers. These findings highlight the potentially important role of an altered chromatin in the oncogenic pathway of serrated neoplasia. Citation Format: Helen Hoi Ning Yan, Jeffrey C W Lai, Siu Lun Ho, Anthony K W Chan, Wai Yin Tsui, Annie S Y Chan, Siu Tsan Yuen, Suet Yi Leung. Unraveling the oncogenic pathway of serrated polyposis syndrome driven by RNF43 germline mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1448. doi:10.1158/1538-7445.AM2017-1448

With recent advances in 3D organoid culture techniques, normal epithelial and tumor cells can be directly cultured from clinical specimens in vitro and expanded long-term with a very high success rate. Thus, this constitutes a good platform for various mechanistic functional studies and clinical applications. A previous karyotyping and gene expression profiling study has indicated that organoid culture of normal epithelial cells can be propagated for long periods of time without genomic alterations. However, data on the genomic stability of tumor organoids in long-term culture are not yet available. We have successfully established organoid cultures derived from either primary tumor or lymph node metastasis from 4 gastric cancer patients. For each sample, we performed long-term culture for at least 6 months. DNA was extracted from blood, as well as the early and late passages of these organoids, and submitted for whole-exome sequencing (WES), achieving a mean coverage approaching 50X. Somatic mutations detected in early versus late passage gastric cancer organoids were compared. Furthermore, we examined the organoids for copy number variations based on the coverage and loss of heterozygosity (LOH) information derived from exome sequencing. We detected between 80 to 228 somatic mutations in the 4 early passage organoids, in which over 84-99% of them were retained during long-term culture. We detected 16, 20, 31 and 98 new mutations in the 4 long-term cultures, respectively. The one case with a substantially large number of new mutations (n=98) appeared to have emerged from a subclone of TP53 wild-type cells in a TP53 mutant tumor. Notably, the C>T mutation was the most dominant mutation spectrum in this case, constituting 48%. Despite the presence of frequent long segment LOH and chromosomal aberrations in early passage organoids, indicating the presence of chromosomal instability, these patterns of aberrations were stably maintained in long-term passage. Overall, with the current organoid culture protocol, tumor genomes are mostly stably maintained with the accumulation of only a small number of new mutations. Our results indicate that this is a reliable and stable in vitro cell culture model for various cancer-related and clinical studies. Citation Format: Sarah Siu Kuen Yue, Helen Hoi Ning Yan, Hoi Cheong Siu, Siu Lun Ho, Wai Yin Tsui, Dessy Chan, Annie Shuk Yee Chan, Bernard Chi Hang Lee, Anthony Kin Wang Chan, Suet Yi Leung. Gastric cancer organoid culture shows preserved genomic stability in long-term passage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4378. doi:10.1158/1538-7445.AM2017-4378

Gastric cancer is a heterogeneous disease with diverse molecular and histological subtypes. We performed whole-genome sequencing in 100 tumor-normal pairs, along with DNA copy number, gene expression and methylation profiling, for integrative genomic analysis. We found subtype-specific genetic and epigenetic perturbations and unique mutational signatures. We identified previously known (TP53, ARID1A and CDH1) and new (MUC6, CTNNA2, GLI3, RNF43 and others) significantly mutated driver genes. Specifically, we found RHOA mutations in 14.3% of diffuse-type tumors but not in intestinal-type tumors (P < 0.001). The mutations clustered in recurrent hotspots affecting functional domains and caused defective RHOA signaling, promoting escape from anoikis in organoid cultures. The top perturbed pathways in gastric cancer included adherens junction and focal adhesion, in which RHOA and other mutated genes we identified participate as key players. These findings illustrate a multidimensional and comprehensive genomic landscape that highlights the molecular complexity of gastric cancer and provides a road map to facilitate genome-guided personalized therapy.

Recent studies have demonstrated the presence of a functional axis that coordinates the events of spermiation and blood-testis barrier (BTB) restructuring which take place simultaneously at the opposite ends of the seminiferous epithelium at stage VIII of the epithelial cycle of spermatogenesis in the rat testis. In short, the disruption of the apical ectoplasmic specialization (apical ES) at the Sertoli cell-elongated spermatid interface, which facilitates the release of sperm at spermiation near the tubule lumen, is coordinated with restructuring at the BTB to accommodate the transit of preleptotene spermatocytes across the immunological barrier near the basement membrane. These two events are likely coordinated by a functional axis involving hemidesmosome at the Sertoli cell-basement membrane interface, and it was designated the apical ES-BTB-hemidesmosome axis. It was demonstrated that fragments of laminin chains (e.g., laminin β3 or γ3 chains) derived from the α6β1-integrin-laminin333 protein complex at the apical ES, which were likely generated via the action of MMP-2 (matrix metalloprotease-2, MMP2) prior to spermiation, acted as biologically active peptides to perturb the BTB permeability function by accelerating protein endocytosis (e.g., occludin) at the site, thereby destabilizing the BTB integrity to facilitate the transit of preleptotene spermatocytes. These laminin fragments also perturbed hemidesmosome function via their action on β1-integrin, a component of hemidesmosome in the testis, which in turn, sent a signal to further destabilize the BTB function. As such, the events of spermiation and BTB restructuring are coordinated via this functional axis. Recent studies using animal models treated with toxicants, such as mono-(2-ethylhexyl) phthalate (MEHP), or adjudin, a male contraceptive under investigation, have also supported the presence of this functional axis in the mouse. In this short review, we critically evaluate the role of this local functional axis in the seminiferous epithelium in spermatogenesis. We also provide molecular modeling information on the interactions between biologically active laminin fragments and β1-integrin, which will be important to assist in the design of more potent laminin-based peptides to disrupt this axis, thereby perturbing spermatogenesis for male contraception and to understand the underlying biology that coordinates spermiation and BTB restructuring during spermatogenesis.

The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other blood-tissue barriers, such as the blood-brain barrier and the blood-ocular (or blood-retina) barrier which formed by tight junctions (TJ) between endothelial cells of the microvessels, the BTB is constituted by coexisting TJ, basal ectoplasmic specialization (basal ES), desmosomes and gap junctions between adjacent Sertoli cells near the basement membrane of the seminiferous tubule. The BTB also divides the seminiferous epithelium into the apical (or adluminal) and basal compartments so that meiosis I and II and post-meiotic germ cell development can all take place in a specialized microenvironment in the apical compartment behind the BTB. While the unusual anatomical features of the BTB have been known for decades, the physiological function of the coexisting junctions, in particular the desmosome and gap junction, that constitute the BTB was unknown until recently. Based on recently published findings, we critically evaluate the role of the desmosome and gap junction that serve as a signaling platform to coordinate the 'opening' and 'closing' of the TJ-permeability barrier conferred by TJ and basal ES during the seminiferous epithelial cycle of spermatogenesis. This is made possible by polarity proteins working in concert with nonreceptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and c-Src, at the site to regulate endosome-mediated protein trafficking events (e.g., endocytosis, transcytosis, recycling or protein degradation). These events not only serve to destabilize the existing 'old' BTB above preleptotene spermatocytes in transit in 'clones' at the BTB, but also contribute to the assembly of 'new' BTB below the transiting spermatocytes. Furthermore, hemidesmosomes at the Sertoli cell-basement membrane interface also contribute to the BTB restructuring events at stage VIII of the epithelial cycle. Additionally, the findings that a gap junction at the BTB provides a possible route for the passage of toxicants [e.g., bisphenol A (BPA)] and potential male contraceptives (e.g., adjudin) across the BTB also illustrate that these coexisting junctions, while helpful to maintain the immunological barrier integrity during the transit of spermatocytes, can be the 'gateway' to making the BTB so vulnerable to toxicants and/or chemicals, causing male reproductive dysfunction., link_to_OA_fulltext

Environmental toxicants, such as cadmium and bisphenol A (BPA) are endocrine disruptors. In utero, perinatal or neonatal exposure of BPA to rats affect the male reproductive function, such as the blood-testis barrier (BTB) integrity. This effect of BPA on BTB integrity in immature rats is likely mediated via a loss of gap junction function at the BTB, failing to coordinate tight junction and anchoring junction function at the site to maintain the immunological barrier integrity. This in turn activates the extracellular signal-regulated kinases 1/2 (Erk1/2) downstream and an increase in protein endocytosis, destabilizing the BTB. The cadmium-induced disruption of testicular dysfunction is mediated initially via its effects on the occludin/ZO-1/focal adhesion kinase (FAK) complex at the BTB, causing redistribution of proteins at the Sertoli-Sertoli cell interface, leading to the BTB disruption. The damaging effects of these toxicants to testicular function are mediated by mitogen-activated protein kinases (MAPK) downstream, which in turn perturbs the actin bundling and accelerates the actin-branching activity, causing disruption of the Sertoli cell tight junction (TJ)-barrier function at the BTB and perturbing spermatid adhesion at the apical ectoplasmic specialization (apical ES, a testis-specific anchoring junction type) that leads to premature release of germ cells from the testis. However, the use of specific inhibitors against MAPK was shown to block or delay the cadmium-induced testicular injury, such as BTB disruption and germ cell loss. These findings suggest that there may be a common downstream p38 and/or Erk1/2 MAPK-based signaling pathway involving polarity proteins and actin regulators that is shared between different toxicants that induce male reproductive dysfunction. As such, the use of inhibitors and/or antagonists against specific MAPKs can possibly be used to "manage" the illnesses caused by these toxicants and/or "protect" industrial workers being exposed to high levels of these toxicants in their work environment.

Recent declines in male fertility, as evidenced by decreased sperm counts, in industrialized nations have been attributed partly to the exposure of men to environmental toxicants, such as cadmium, bisphenol A, and others. These environmental toxicants are found in drinking water, food, dairy products, and many utensils (e.g., plastics and glass bottles). It is of interest to note that while animal models clearly show effects of environmental toxicants on male reproductive function, linking specific environmental exposures in humans to adverse effects on the male reproductive system is very tenuous for the vast majority of environmental chemicals since human exposure is often orders of magnitude lower than dose levels used in animal studies. Nonetheless, environment toxicants have become an integrated part of our day-to-day routine and food/water intakes. This thus sparks interest in the field to assess if acute and chronic exposure of these toxicants to laboratory animals would cause reproductive damage, and whether such damage can be reversed and/or rescued. In this review, we summarize recent findings in the field regarding damage that are caused by these toxicants to the testis via their actions at the cell–cell interface, thereby inducing premature loss of germ cells from the seminiferous epithelium which leads to reduced sperm counts in semen. Some of these studies have identified specific signaling pathways that are used by these toxicants to induce disruption at the Sertoli–Sertoli and/or Sertoli–germ cell interface, perturbing the blood–testis barrier (BTB) function and germ cell adhesion. This information should be helpful in future studies to design compounds that can ‘reverse’ and/or ‘reduce’ toxicant toxicity to the testis.

Spermatogenesis takes place in the seminiferous tubules in adult testes such as rats, in which developing germ cells must traverse the seminiferous epithelium while spermatogonia (2n, diploid) undergo mitotic and meiotic divisions, and differentiate into elongated spermatids (1n, haploid). It is conceivable that this event involves extensive junction restructuring particularly at the blood-testis barrier (BTB, a structure that segregates the seminiferous epithelium into the basal and the adluminal compartments) that occurs at stages VII-VIII of the seminiferous epithelial cycle. As such, cross-talk between tight (TJ) and anchoring junctions [e.g., basal ectoplasmic specialization (basal ES), adherens junction (AJ), desmosome-like junction (DJ)] at the BTB must occur to coordinate the transient opening of the BTB to facilitate preleptotene spermatocyte migration. Interestingly, while there are extensively restructuring at the BTB during the epithelial cycle, the immunological barrier function of the BTB must be maintained without disruption even transiently. Recent studies using the androgen suppression and Adjudin models have shown that anchoring junction restructuring that leads to germ cell loss from the seminiferous epithelium also promotes the production of AJ (e.g., basal ES) proteins (such as N-cadherins, catenins) at the BTB site. We postulate the testis is using a similar mechanism during spermatogenesis at stage VIII of the epithelial cycle that these induced basal ES proteins, likely form a "patch" surrounding the BTB, transiently maintain the BTB integrity while TJ is "opened", such as induced by TGF-b3 or TNFa, to facilitate preleptotene spermatocyte migration. However, in other stages of the epithelial cycle other than VII and VIII when the BTB remains "closed" (for approximately 10 days), anchoring junctions (e.g., AJ, DJ, and apical ES) restructuring continues to facilitate germ cell movement. Interestingly, the mechanism(s) that governs this communication between TJ and anchoring junction (e.g., basal ES and AJ) in the testis has remained obscure until recently. Herein, we provide a critical review based on the recently available data regarding the cross-talk between TJ and anchoring junction to allow simultaneous maintenance of the BTB and germ cell movement across the seminiferous epithelium.

The mechanism(s) that regulate and coordinate the events of spermiation and blood-testis barrier (BTB) restructuring in the seminiferous epithelium that occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis are unknown. In this report, fragments derived from the laminin complex composed of laminin α3, β3, and γ3 chains (laminin-333) at the apical ectoplasmic specialization (apical ES) were shown to modulate BTB dynamics directly and/or indirectly via hemidesmosome. Experiments were performed using cultured Sertoli cells with functional tight junction (TJ) barrier and the ultrastructural features of the BTB but not apical ES. Recombinant protein fragments of laminin β3 and γ3 chains were shown to reduce the protein levels of occludin and β1-integrin dose dependently at the Sertoli-Sertoli and Sertoli-basement membrane interface, respectively, thereby destabilizing the BTB permeability function. These results were corroborated by transient overexpression of laminin fragments in Sertoli cells. To further assess the role of β1-integrin in hemidesmosome, knockdown of β1-integrin in Sertoli cells by RNAi was found to associate with occludin redistribution at the Sertoli-Sertoli cell interface, wherein occludin moved away from the cell surface and became associated with endosomes, thereby destabilizing the BTB. In short, an apical ES-BTB-hemidesmosome autocrine regulatory axis was identified in testes, coordinating the events of spermiation and BTB restructuring that occur at the opposite ends of the seminiferous epithelium during spermatogenesis. © 2008 by The National Academy of Sciences of the USA., link_to_OA_fulltext

Background & Aims Repulsive guidance molecule member A (RGMA) is a glycosylphosphatidylinositol-anchored glycoprotein and axon guidance molecule that signals through its receptor, neogenin (NEO1), a homologue of the deleted-in-colorectal cancer ( DCC ) gene. RGMA also functions as a bone morphogenetic protein (BMP) coreceptor. We studied the potential roles of RGMA and NEO1 in colorectal cancer (CRC) pathogenesis. Methods We analyzed expression of RGMA and NEO1 , as well as their epigenetic and genetic changes, in a large series of CRC samples, normal colon tissues, adenomas, and cell lines. These studies were accompanied by in vitro functional assay. Results RGMA and NEO1 expression were significantly down-regulated in most CRCs, adenomas, and cell lines. RGMA was frequently silenced by promoter methylation in CRCs (86.7%), adenomas (90.9%), and CRC cell lines (92.3%) but not in normal colon tissues; allelic imbalance of RGMA and NEO1 was observed in 40% and 49% of CRCs, respectively. In CRC samples, reduced RGMA levels were significantly associated with mismatch repair deficiency or mutations in KRAS or BRAF . Exposure to 5-aza-2′-deoxycytidine restored RGMA expression in CRC cell lines. Transfection of RGMA into CRC cells suppressed cell proliferation, migration, and invasion and also increased apoptosis in response to DNA-damaging agent. Conclusions The frequent genetic and epigenetic inactivation of RGMA in CRCs and adenomas along with its in vitro function collectively support its role as a tumor suppressor in colon cells. These findings add to the expanding list of axon guidance molecules with disrupted function during colon carcinogenesis and create new opportunities for early detection and drug development.

Spermatogenesis that occurs in the seminiferous epithelium of adult mammalian testes is associated with extensive junction restructuring at the Sertoli-Sertoli cell, Sertoli-germ cell, and Sertoli-basement membrane interface. While this morphological phenomenon is known and has been described in great details for decades, the biochemical and molecular changes as well as the mechanisms/signaling pathways that define changes at the cell-cell and cell-matrix interface remain largely unknown until recently. In this chapter, we summarize and discuss findings in the field regarding the coordinated efforts of the anchoring [e.g., adherens junction (AJ), such as basal ectoplasmic specialization (basal ES)] and tight junctions (TJs) that are present in the same microenvironment, such as at the blood-testis barrier (BTB), or at distinctly opposite ends of the Sertoli cell epithelium, such as between apical ectoplasmic specialization (apical ES) in the apical compartment, and the BTB adjacent to the basal compartment of the epithelium. These efforts, in turn, regulate and coordinate different cellular events that occur during the seminiferous epithelial cycle. For instance, the events of spermiation and of preleptotene spermatocyte migration across the BTB both take place concurrently at stage VIII of the epithelial cycle of spermatogenesis. Recent findings suggest that these events are coordinated by protein complexes found at the apical and basal ES and TJ, which are located at different ends of the Sertoli cell epithelium. Besides, we highlight important areas of research that can now be undertaken, and functional studies that can be designed to tackle different issues pertinent to junction restructuring during spermatogenesis.

The ectoplasmic specialization (ES) is a testis-specific, actin-based hybrid anchoring and tight junction. It is confined to the interface between Sertoli cells at the blood-testis barrier, known as the basal ES, as well as between Sertoli cells and developing spermatids designated the apical ES. The ES shares features of adherens junctions, tight junctions and focal contacts. By adopting the best features of each junction type, this hybrid nature of ES facilitates the extensive junction-restructuring events in the seminiferous epithelium during spermatogenesis. For instance, the α6β1-integrin- laminin 333 complex, which is usually limited to the cell-matrix interface in other epithelia to facilitate cell movement, is a putative apical ES constituent. Furthermore, JAM-C and CAR, two tight junction integral membrane proteins, are also components of apical ES involving in spermatid orientation. We discuss herein the mechanisms that maintain the cross-talk between ES and blood-testis barrier to facilitate cell movement and orientation in the seminiferous epithelium. © 2006 Wiley Periodicals, Inc., postprint

MicroRNAs (miRNAs) are small non-coding RNAs which exert their effects by post-transcriptionally silencing target mRNAs. Deregulation of miRNA expression is a frequent event in tumorigenesis. MiR-125b is a highly conserved miRNA among various species and is composed of three homologs: hsa-miR-125a, hsa-miR-125b-1 and hsa-miR-125-2. The tumorigenic roles of miR-125b have been studied in various cancers including prostate, colon, glioma etc, and studies have demonstrated that it can act as a tumor suppressor or an oncogene depending on the cellular context. It was characterized as an oncogene in prostate cancer and glioma through down-regulation of pro-apoptotic regulators BAK1 and Bcl-2 modifying factor (BMF), respectively. In colon cancer, a recent clinico-pathological study showed that high expression of miR-125b was associated with tumor invasion and poor prognosis. However, the localization of miR125b in normal colon and tumors is currently unknown. Therefore, we aimed to address the precise expression and the functional role of miR-125b in normal colon, which may provide insight on its potential oncogenic effects during carcinogenesis. We performed gene expression analysis of miR-125b in normal colon tissues from 16 pairs of colon top versus basal crypts and 4 pairs of crypts versus stroma by real-time RT-PCR. Colon top and basal crypts were microdissected from frozen sections, whereas pure normal crypts and stromal fractions were isolated from freshly resected human colon specimens. We found that miR-125b was significantly enriched in the basal crypts (p expression may contribute to carcinogenesis through disruption of this pathway. Note: This abstract was not presented at the meeting. Citation Format: Helen H N Yan, Jackie K Y Lau, Annie S Y Chan, Wai Yin Tsui, Tsun Leung Chan, Suet Yi Leung. Regulation of stromal miR-125b on normal colonic epithelial cell renewal and its putative role in tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 179. doi:10.1158/1538-7445.AM2015-179

In adult rat testes, blood–testis barrier (BTB) restructuring facilitates the migration of preleptotene spermatocytes from the basal to the adluminal compartment that occurs at stage VIII of the epithelial cycle. Structural proteins at the BTB must utilize an efficient mechanism (e.g. endocytosis) to facilitate its transient ‘opening’. Dynamin II, a large GTPase known to be involved in endocytosis, was shown to be a product of Sertoli and germ cells in the testis. It was also localized to the BTB, as well as the apical ectoplasmic specialization (apical ES), during virtually all stages of the epithelial cycle. By co-immunoprecipitation, dynamin II was shown to associate with occludin, N-cadherin, zonula occludens-1 (ZO-1), β-catenin, junctional adhesion molecule-A, and p130Cas, but not nectin-3. An in vivo model in rats previously characterized for studying adherens junction (AJ) dynamics in the testes by adjudin (formerly called AF-2364, 1-(2,4-dichlorobenzyl)-1H-indazole-3-car-hohydrizide) treatment was used in our studies. At the time of germ cell loss from the seminiferous epithelium as a result of adjudin-induced AJ restructuring without disrupting the BTB integrity, a significant decline in the steady-state dynamin II protein level was detected. This change was associated with a concomitant increase in the levels of two protein complexes at the BTB, namely occludin/ZO-1 and N-cadherin/β-catenin. Interestingly, these changes were also accompanied by a significant increase in the structural interaction of dynamin II with β-catenin and ZO-1. β-Catenin and ZO-1 are adaptors that structurally link the cadherin- and occludin-based protein complexes together at the BTB in an ‘engaged’state to reinforce the barrier function in normal testes. However, β-catenin and ZO-1 were ‘disengaged’ from each other but bound to dynamin II during adjudin-induced AJ restructuring in the testis. The data reported herein suggest that dynamin II may assist the ‘disengagement’ of β-catenin from ZO-1 during BTB restructuring. Thus, this may permit the occludin/ZO-1 complexes to maintain the BTB integrity when the cadherin/catenin complexes are dissociated to facilitate germ cell movement.

Colorectal cancer (CRC) is one of the major causes of cancer death worldwide. Whilst adjuvant chemotherapy can improve survival in Dukes’ C patients, its application on Dukes’ B patients is controversial. Identification of factors that can predict recurrence in the latter group may facilitate classification of high risk patients for receiving adjuvant therapy. Retinoic acid receptor responder 3 (Rarres3) was firstly identified as the retinoid induced class II tumor suppressor in human keratinocytes and psoriatic lesions. In breast, head and neck, lung and gastric cancer cell lines, induced expression of Rarres3 by retinoid can inhibit cell proliferation. Less is known of its effect on colon cancer. Using real-time quantitative RT-PCR, coupled with in vitro functional assays, we have characterized the role of Rarres3 in colorectal cancer development. By comparing the mRNA expression level of Rarres3 in 405 CRCs and 19 colon cancer cell lines with 63 paired normal colons, Rarres3 is down-regulated in most of the CRCs and cell lines. Loss of Rarres3 expression is strongly associated with Dukes’ D patients (p=0.009) and well or moderately differentiated tumor type (p=0.016). Kaplan-Meier analysis demonstrated an improved overall survival (p=0.001) for patients with high expression of Rarres3 by log-rank test. Specifically, there is a prolonged overall survival (p=0.007) and disease free (p=0.012) for Dukes’ B patients (n=158). In multivariate Cox Regression analysis, Rarres3 is an independent factor to predict overall survival (HR=0.473, p=0.001) and disease free (HR=0.557, p=0.047) apart from lymphovascular invasion and Dukes’ stages. Rarres3 protein expression can be induced in colon cancer cell lines by treatment with either interferon-γ (IFNγ) or all-trans retinoic acid (ATRA). Stable transfection of Rarres3 into SW480 colon cancer cells led to inactivation of Akt signaling pathway, suppression of cell growth and enhancement of cell apoptosis via capase-1 activated apoptotic pathway in response to 5-fluorouracil treatment. In addition, we found that Rarres3 is a Wnt target in which its expression is suppressed during Wnt activation and vice versa in colon cancer cell lines. In summary, Rarres3 expression can be induced by IFNγ and ATRA, causing suppression of cell growth and sensitization of cancer cells to apoptosis. Loss of Rarres3 expression in CRCs is predictive of tumor recurrence and poor prognosis especially in Dukes’ B patients, thus aids in molecular stratification of high risk patients. The results also raise the possibility for the use of IFNγ and ATRA in CRC treatment especially in the high risk Dukes’ B subgroup. 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 5016.