Testicular cancer and cryptorchidism

The failure of testicular descent or cryptorchidism is the most common defect in newborn boys. The descent of the testes during development is controlled by insulin-like 3 peptide and steroid hormones produced in testicular Leydig cells, as well as by various genetic and developmental factors. While in some cases the association with genetic abnormalities and environmental causes has been shown, the etiology of cryptorchidism remains uncertain. Cryptorchidism is an established risk factor for infertility and testicular germ cell tumors (TGCT). Experimental animal models suggest a causative role for an abnormal testicular position on the disruption of spermatogenesis however the link between cryptorchidism and TGCT is less clear. The most common type of TGCT in cryptorchid testes is seminoma, believed to be derived from pluripotent prenatal germ cells. Recent studies have shown that seminoma cells and their precursor carcinoma in situ cells express a number of spermatogonial stem cell (SSC) markers suggesting that TGCTs might originate from adult stem cells. We review here the data on changes in the SSC somatic cell niche observed in cryptorchid testes of mouse models and in human patients. We propose that the misregulation of growth factors' expression may alter the balance between SSC self-renewal and differentiation and shift stem cells towards neoplastic transformation

NOTCH1 Gain of Function in Germ Cells Causes Failure of Spermatogenesis in Male Mice

NOTCH1 is a member of the NOTCH receptor family, a group of single-pass trans-membrane receptors. NOTCH signaling is highly conserved in evolution and mediates communication between adjacent cells. NOTCH receptors have been implicated in cell fate determination, as well as maintenance and differentiation of stem cells. In the mammalian testis expression of NOTCH1 in somatic and germ cells has been demonstrated, however its role in spermatogenesis was not clear. To study the significance of NOTCH1 in germ cells, we applied a cre/loxP approach in mice to induce NOTCH1 gain- or loss-of function specifically in male germ cells. Using a Stra8-icretransgene we produced mice with conditional activation of the NOTCH1 intracellular domain (NICD) in germ cells. Spermatogenesis in these mutants was progressively affected with age, resulting in decreased testis weight and sperm count. Analysis of downstream target genes of NOTCH1 signaling showed an increased expression of Hes5, with a reduction of the spermatogonial differentiation marker, Neurog3 expression in the mutant testis. Apoptosis was significantly increased in mouse germ cells with the corresponding elevation of pro-apoptotic Trp53 and Trp63genes\u27 expression. We also showed that the conditional germ cell-specific ablation of Notch1 had no effect on spermatogenesis or male fertility. Our data suggest the importance of NOTCH signaling regulation in male germ cells for their survival and differentiation

Reduced Androgen Receptor Expression Accelerates the Onset of ERBB2 Induced Breast Tumors in Female Mice

Androgen receptor (AR) is commonly expressed in both the epithelium of normal mammary glands and in breast cancers. AR expression in breast cancers is independent of estrogen receptor alpha (ERα) status and is frequently associated with overexpression of the ERBB2 oncogene. AR signaling effects on breast cancer progression may depend on ERα and ERBB2 status. Up to 30% of human breast cancers are driven by overactive ERBB2 signaling and it is not clear whether AR expression affects any steps of tumor progression in this cohort of patients. To test this, we generated mammary specific Ar depleted mice (MARKO) by combining the floxed allele of Ar with the MMTV-cre transgene on an MMTV-NeuNT background and compared them to littermate MMTV-NeuNT, Arfl/+ control females. Heterozygous MARKO females displayed reduced levels of AR in mammary glands with mosaic AR expression in ductal epithelium. The loss of AR dramatically accelerated the onset of MMTV-NeuNT tumors in female MARKO mice. In this report we show that accelerated MMTV-NeuNT-dependent tumorigenesis is due specifically to the loss of AR, as hormonal levels, estrogen and progesterone receptors expression, and MMTV-NeuNT expression were similar between MARKO and control groups. MMTV-NeuNT induced tumors in both cohorts displayed distinct loss of AR in addition to ERα, PR, and the pioneer factor FOXA1. Erbb3 mRNA levels were significantly elevated in tumors in comparison to normal mammary glands. Thus the loss of AR in mouse mammary epithelium accelerates malignant transformation rather than the rate of tumorigenesis

INPP4B protects from metabolic syndrome and associated disorders

A high fat diet and obesity have been linked to the development of metabolic dysfunction and the promotion of multiple cancers. The causative cellular signals are multifactorial and not yet completely understood. In this report, we show that Inositol Polyphosphate-4-Phosphatase Type II B (INPP4B) signaling protects mice from diet-induced metabolic dysfunction. INPP4B suppresses AKT and PKC signaling in the liver thereby improving insulin sensitivity. INPP4B loss results in the proteolytic cleavage and activation of a key regulator in de novo lipogenesis and lipid storage, SREBP1. In mice fed with the high fat diet, SREBP1 increases expression and activity of PPARG and other lipogenic pathways, leading to obesity and non-alcoholic fatty liver disease (NAFLD). Inpp4b−/− male mice have reduced energy expenditure and respiratory exchange ratio leading to increased adiposity and insulin resistance. When treated with high fat diet, Inpp4b−/− males develop type II diabetes and inflammation of adipose tissue and prostate. In turn, inflammation drives the development of high-grade prostatic intraepithelial neoplasia (PIN). Thus, INPP4B plays a crucial role in maintenance of overall metabolic health and protects from prostate neoplasms associated with metabolic dysfunction

Mechanisms of INSL3 signaling in male reproductive organs

Global ablation of INSL3 hormone or its receptor RXFP2 in male mice results in cryptorchidism and infertility. Using novel LacZ knock-in Rxfp2 allele we demonstrated a strong expression of this gene in postmeiotic germ cells. RXFP2 was expressed in embryonic and neonatal gubernaculum. No RXFP2 expression was detected in cremaster muscles in adult mice. We produced a floxed allele of Rxfp2 and then deleted this gene in male germ cells in testes located in normal scrotal position. No differences in fertility or spermatogenesis of such males were found, suggesting non-essential role of INSL3 signaling in germ cell differentiation in adult males. We have also produced shRNA transgenic mice with reduced RXFP2 expression Such males manifested various degree of uni- and bilateral cryptorchidism. Total gene expression analysis of the mutant cremasteric sacs indicated misexpression of a significant number of genes in Wnt/β-catenin and NOTCH pathways. Conditional deletion of β-catenin or Notch1 genes in male gubernacular ligament resulted in its abnormal development. Our data suggest that β-catenin and NOTCH1 pathways are potential targets of INSL3 signaling during gubernacular development

Activation of relaxin family receptor 1 from different mammalian species by relaxin peptide and small-molecule agonist ML290

Relaxin peptide (RLN), which signals through the relaxin family peptide 1 (RXFP1) GPCR receptor, has shown therapeutic effects in an acute heart failure clinical trial. We have identified a small-molecule agonist of human RXFP1, ML290; however, it does not activate the mouse receptor. To find a suitable animal model for ML290 testing and to gain mechanistic insights into the interaction of various ligands with RXFP1, we have cloned rhesus macaque, pig, rabbit, and guinea pig RXFP1s and analyzed their activation by RLN and ML290. HEK293T cells expressing macaque or pig RXFP1 responded to relaxin and ML290 treatment as measured by an increase of cAMP production. Guinea pig RXFP1 responded to relaxin but had very low response to ML290 treatment only at highest concentrations used. The rabbit RXFP1 amino acid sequence was the most divergent, with a number of unique substitutions within the ectodomain and the seven-transmembrane domain (7TM). Two splice variants of rabbit RXFP1 derived through alternative splicing of the fourth exon were identified. In contrast to the other species, rabbit RXFP1s were activated by ML290, but not with human, pig, mouse, or rabbit RLNs. Using FLAG-tagged constructs, we have shown that both rabbit RXFP1 variants are expressed on the cell surface. No binding of human Eu-labeled RLN to rabbit RXFP1 was detected, suggesting that in this species, RXFP1 might be non-functional. We used chimeric rabbit–human and guinea pig–human constructs to identify regions important for RLN or ML290 receptor activation. Chimeras with the human ectodomain and rabbit 7TM domain were activated by RLN, whereas substitution of part of the guinea pig 7TM domain with the human sequence only partially restored ML290 activation, confirming the allosteric mode of action for the two ligands. Our data demonstrate that macaque and pig models can be used for ML290 testing

Relaxin family peptide receptors in GtoPdb v.2023.1

Relaxin family peptide receptors (RXFP, nomenclature as agreed by the NC-IUPHAR Subcommittee on Relaxin family peptide receptors [23, 119]) may be divided into two pairs, RXFP1/2 and RXFP3/4. Endogenous agonists at these receptors are heterodimeric peptide hormones structurally related to insulin: relaxin-1, relaxin, relaxin-3 (also known as INSL7), insulin-like peptide 3 (INSL3) and INSL5. Species homologues of relaxin have distinct pharmacology and relaxin interacts with RXFP1, RXFP2 and RXFP3, whereas mouse and rat relaxin selectively bind to and activate RXFP1 [260]. relaxin-3 is the ligand for RXFP3 but it also binds to RXFP1 and RXFP4 and has differential affinity for RXFP2 between species [259]. INSL5 is the ligand for RXFP4 but is a weak antagonist of RXFP3. relaxin and INSL3 have multiple complex binding interactions with RXFP1 [267] and RXFP2 [132] which direct the N-terminal LDLa modules of the receptors together with a linker domain to act as a tethered ligand to direct receptor signaling [262]. INSL5 and relaxin-3 interact with their receptors using distinct residues in their B-chains for binding, and activation, respectively [321, 152]

Relaxin-like peptides in male reproduction: a human perspective

The relaxin family of peptide hormones and their cognate GPCRs are becoming physiologically well-characterized in the cardiovascular system and particularly in female reproductive processes. Much less is known about the physiology and pharmacology of these peptides in male reproduction, particularly as regards humans. H2-relaxin is involved in prostate function and growth, while insulin-like peptide 3 (INSL3) is a major product of the testicular Leydig cells and, in the adult, appears to modulate steroidogenesis and germ cell survival. In the fetus, INSL3 is a key hormone expressed shortly after sex determination and is responsible for the first transabdominal phase of testicular descent. Importantly, INSL3 is becoming a very useful constitutive biomarker reflecting both fetal and post-natal development. Nothing is known about roles for INSL4 in male reproduction and only very little about relaxin-3, which is mostly considered as a brain peptide, or INSL5. The former is expressed at very low levels in the testes, but has no known physiology there, whereas the INSL5 knockout mouse does exhibit a testicular phenotype with mild effects on spermatogenesis,probably due to a disruption of glucose homeostasis. INSL6 is a major product of male germ cells, although it is relatively unexplored with regard to its physiology or pharmacology, except that in mice disruption of the INSL6 gene leads to a disruption of spermatogenesis. Clinically, relaxin analogues may be useful in the control of prostate cancer, and both relaxin and INSL3 have been considered as sperm adjuvants for in vitro fertilization

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery