Identification of a neutrophil-specific PIK3R1 mutation facilitates targeted treatment in a patient with Sweet syndrome.

BackgroundAcute febrile neutrophilic dermatosis (Sweet syndrome) is a potentially fatal multiorgan inflammatory disease characterized by fever, leukocytosis, and a rash with a neutrophilic infiltrate. The disease pathophysiology remains elusive, and current dogma suggests that Sweet syndrome is a process of reactivity to an unknown antigen. Corticosteroids and steroid-sparing agents remain frontline therapies, but refractory cases pose a clinical challenge.MethodsA 51-year-old woman with multiorgan Sweet syndrome developed serious corticosteroid-related side effects and was refractory to steroid-sparing agents. Blood counts, liver enzymes, and skin histopathology supported the diagnosis. Whole-genome sequencing, transcriptomic profiling, and cellular assays of the patient's skin and neutrophils were performed.ResultsWe identified elevated IL-1 signaling in lesional Sweet syndrome skin caused by a PIK3R1 gain-of-function mutation specifically found in neutrophils. This mutation increased neutrophil migration toward IL-1ß and neutrophil respiratory burst. Targeted treatment of the patient with an IL-1 receptor 1 antagonist resulted in a dramatic therapeutic response and enabled a tapering off of corticosteroids.ConclusionDysregulated PI3K/AKT signaling is the first signaling pathway linked to Sweet syndrome and suggests that this syndrome may be caused by acquired mutations that modulate neutrophil function. Moreover, integration of molecular data across multiple levels identified a distinct subtype within a heterogeneous disease that resulted in a rational and successful clinical intervention. Future patients will benefit from efforts to identify potential mutations. The ability to directly interrogate the diseased skin allows this method to be generalizable to other inflammatory diseases and demonstrates a potential personalized medicine approach for patients with clinically challenging disease.Funding SourcesBerstein Foundation, NIH, Veterans Affairs (VA) Administration, Moseley Foundation, and H.T. Leung Foundation.

Increased expression of cathepsin D is required for L1-mediated colon cancer progression.

Hyperactivation of Wnt/ß-catenin target genes is considered a key step in human colorectal cancer (CRC) development. We previously identified the immunoglobulin-like cell adhesion receptor L1 as a target gene of ß-catenin/TCF transactivation that is localized at the invasive edge of CRC tissue. Using gene arrays, we discovered a number of downstream target genes and signaling pathways conferred by L1 overexpression during colon cancer progression. Here, we have used a proteomic approach to identify proteins in the secretome of L1-overexpressing CRC cells and studied the role of the increase in the aspartate protease cathepsin D (CTSD) in L1-mediated colon cancer development. We found that in addition to the increase in CTSD in the secretome, the RNA and protein levels of CTSD were also induced by L1 in CRC cells. CTSD overexpression resulted in elevated proliferation under stress and increased motility, tumorigenesis and liver metastasis, although to a lesser extent than after L1-transfection. The suppression of endogenous CTSD in L1-expressing cells blocked the increase in the proliferative, motile, tumorigenic and metastatic ability of CRC cells. Enhancing Wnt/ß-catenin signaling by the inhibition of GSK3ß resulted in increased endogenous CTSD levels, suggesting the involvement of the Wnt/ß-catenin pathway in CTSD expression. In human CRC tissue, CTSD was detected in epithelial cells and in the stromal compartment at the more invasive areas of the tumor, but not in the normal mucosa, indicating that CTSD plays an essential role in CRC progression.

ISG15 induction is required during L1-mediated colon cancer progression and metastasis.

Hyperactivation of Wnt/ß-catenin target gene expression is a hallmark of colorectal cancer (CRC) development. We identified L1-CAM (L1) and Nr-CAM, members of the immunoglobulin family of nerve cell adhesion receptors, as target genes of the Wnt/ß-catenin pathway in CRC cells. L1 overexpression in CRC cells enhances their motile and tumorigenic capacity and promotes liver metastasis. L1 is often localized at the invasive edge of CRC tissue. Using gene arrays and proteomic analyses we identified downstream signaling pathways and targets of L1-mediated signaling. Here, we found that the expression of interferon-stimulated gene 15 (ISG15) that operates much like ubiquitin (is conjugated to proteins by ISGylation), is elevated in the conditioned medium and in CRC cells overexpressing L1. Suppression of endogenous ISG15 levels in L1-expressing cells blocked the increased proliferative, motile, tumorigenic and liver metastatic capacities of CRC cells. ISG15 overexpression, on its own, could enhance these properties in CRC cells, but only to a much lower extent compared to L1. We show that NF-κB signaling is involved in the L1-mediated increase in ISG15, since blocking the NF-κB pathway abolished the induction of ISG15 by L1. Point mutations in the L1 ectodomain that interfere with its binding to L1 ligands, also inhibited the increase in ISG15. We detected high levels of ISG15 in human CRC tissue cells and in the adjacent stroma, but not in the normal mucosa. The results suggest that ISG15 is involved in L1-mediated CRC development and is a potential target for CRC therapy.

Testosterone augments FSH signaling by upregulating the expression and activity of FSH-Receptor in Pubertal Primate Sertoli cells.

The synergistic actions of Testosterone (T) and FSH via testicular Sertoli cells (Sc) regulate male fertility. We have previously reported that the actions of these hormones (T and FSH) in infant monkey testes are restricted only to the expansion of Sc and spermatogonial cells. The robust differentiation of male Germ cells (Gc) occurs after pubertal maturation of testis. The present study was aimed to investigate the molecular basis of the synergy between T and FSH action in pubertal primate (Macaca mulatta) Sc. Using primary Sc culture, we here have demonstrated that T (but not FSH) downregulated AMH and Inhibin-ß-B (INHBB) mRNAs in pubertal Sc. We also found that, prolonged stimulation of T in pubertal Sc significantly elevated the expression of genes involved in FSH signaling pathway like FSH-Receptor (FSHR), GNAS and RIC8B, and this was associated with a rise in cAMP production. T also augmented FSH induced expression of genes like SCF, GDNF, ABP and Transferrin (TF) in pubertal Sc. We therefore conclude that T acts in synergy with FSH signaling in pubertal Sc. Such a coordinated network of hormonal signaling in Sc may facilitate the timely onset of the first spermatogenic wave in pubertal primates and is responsible for quantitatively and qualitatively normal spermatogenesis.

Cell-cell adhesion: linking Wnt/ß-catenin signaling with partial EMT and stemness traits in tumorigenesis.

Changes in cell adhesion and motility are considered key elements in determining the development of invasive and metastatic tumors. Co-opting the epithelial-to-mesenchymal transition (EMT) process, which is known to occur during embryonic development, and the associated changes in cell adhesion properties in cancer cells are considered major routes for tumor progression. More recent in vivo studies in tumor tissues and circulating tumor cell clusters suggest a stepwise EMT process rather than an "all-or-none" transition during tumor progression. In this commentary, we addressed the molecular mechanisms underlying the changes in cell adhesion and motility and adhesion-mediated signaling and their relationships to the partial EMT states and the acquisition of stemness traits by cancer cells.

The intestinal stem cell regulating gene ASCL2 is required for L1-mediated colon cancer progression.

Aberrant Wnt/ß-catenin signaling is a common event during human colorectal cancer (CRC) development. Previously, we characterized members of the L1 family of cell adhesion receptors as targets of ß-catenin-LEF1/TCF transactivation that are expressed at the invasive CRC tissue edge. Overexpression of L1 in CRC cells confers enhanced motility, tumorigenesis and liver metastasis. We identified several downstream targets of L1-mediated signaling that are considered key intestinal stem cell signature genes. Here, we investigated the involvement of ASCL2, a Wnt target gene and key determinant of intestinal stem cell state, in L1-mediated CRC progression. In L1 overexpressing CRC cells we found an increase in ASCL2, a decrease in E-cadherin and accumulation of nuclear ß-catenin, ß-catenin-LEF1/TCF transactivation and target gene expression. The increase in ASCL2 by L1 overexpression enhanced ASCL2 target gene expression, conferred increased motility, tumorigenesis and metastasis, similar to L1 overexpression. Suppression of ASCL2 in cells expressing L1 blocked these tumorigenic properties. In human CRC tissue, ASCL2 was detected in the nuclei of cells at invasive areas of the tumor that also expressed L1. The results suggest that increased ASCL2 expression is a critical step in L1-mediated CRC progression.

Defective Wnt3 expression by testicular Sertoli cells compromise male fertility.

Testicular Sertoli cells make a niche for the division and differentiation of germ cells. Sertoli cells respond to increased follicle-stimulating hormone (FSH) and testosterone (T) levels at the onset of puberty by producing paracrine factors which affect germ cells and trigger robust onset of spermatogenesis. Such paracrine support to germ cells is absent during infancy, despite Sertoli cells being exposed to high FSH and T within the infant testis. This situation is similar to certain cases of male idiopathic infertility where post-pubertal Sertoli cells fail to support germ cell division and differentiation in spite of endogenous or exogenous hormonal support. Defective Sertoli cells in such individuals may fail to express the full complement of their paracrine repertoire. Identification and supplementation with such factors may overcome Sertoli cells deficiencies and help trigger quantitatively and qualitatively normal differentiation of germ cells. To this end, we compared the transcriptome of FSH- and T-treated infant and pubertal monkey Sertoli cells by DNA microarray. Expression of Wnt3, a morphogen of the Wnt/ß-catenin pathway, was higher in pubertal Sertoli cells relative to infant Sertoli cells. Transgenic mice were generated by us in which Wnt3 expression was curtailed specifically in post-pubertal Sertoli cells by shRNA. Subfertility and oligozoospermia were noticed in such animals with low Wnt3 expression in post-pubertal Sertoli cells along with diminished expression of Connexin43, a gap-junctional molecule essential for germ cell development. We report that the FSH- and T-targetedf Wnt3 governs Sertoli cell-mediated regulation of spermatogenesis and hence is crucial for fertility.

Wnt signaling in cancer stem cells and colon cancer metastasis.

Overactivation of Wnt signaling is a hallmark of colorectal cancer (CRC). The Wnt pathway is a key regulator of both the early and the later, more invasive, stages of CRC development. In the normal intestine and colon, Wnt signaling controls the homeostasis of intestinal stem cells (ISCs) that fuel, via proliferation, upward movement of progeny cells from the crypt bottom toward the villus and differentiation into all cell types that constitute the intestine. Studies in recent years suggested that cancer stem cells (CSCs), similar to ISCs of the crypts, consist of a small subpopulation of the tumor and are responsible for the initiation and progression of the disease. Although various ISC signature genes were also identified as CRC markers and some of these genes were even demonstrated to have a direct functional role in CRC development, the origin of CSCs and their contribution to cancer progression is still debated. Here, we describe studies supporting a relationship between Wnt-regulated CSCs and the progression of CRC.

Low levels of Gαs and Ric8b in testicular sertoli cells may underlie restricted FSH action during infancy in primates.

FSH acts via testicular Sertoli cells (Sc) bearing FSH receptor (FSH-R) for regulating male fertility. Despite an adult-like FSH milieu in infant boys and monkeys, spermatogenesis is not initiated until the onset of puberty. We used infant and pubertal monkey Sc to reveal the molecular basis underlying developmental differences of FSH-R signaling in them. Unlike pubertal Sc, increasing doses of FSH failed to augment cAMP production by infant Sc. The expression of Gαs subunit and Ric8b, which collectively activate adenylyl cyclase (AC) for augmenting cAMP production and gene transcription, were significantly low in infant Sc. However, forskolin, which acts directly on AC bypassing FSH-R, augmented cAMP production and gene transcription uniformly in both infant and pubertal Sc. FSH-induced Gαs mRNA expression was higher in pubertal Sc. However, Gαi-2 expression was down-regulated by FSH in pubertal Sc, unlike infant Sc. FSH failed, but forskolin or 8-Bromoadenosine 3',5'-cyclic monophosphate treatment to infant Sc significantly augmented the expression of transferrin, androgen binding protein, inhibin-ß-B, stem cell factor, and glial-derived neurotropic factor, which are usually up-regulated by FSH in pubertal Sc during spermatogenic onset. This suggested that lack of FSH mediated down-regulation of Gαi-2 expression and limited expression of Gαs subunit as well as Ric8b may underlie limited FSH responsiveness of Sc during infancy. This study also divulged that intracellular signaling events downstream of FSH-R are in place and can be activated exogenously in infant Sc. Additionally, this information may help in the proper diagnosis and treatment of infertile individuals having abnormal G protein-coupled FSH-R.

A non-surgical approach for male germ cell mediated gene transmission through transgenesis.

Microinjection of foreign DNA in male pronucleus by in-vitro embryo manipulation is difficult but remains the method of choice for generating transgenic animals. Other procedures, including retroviral and embryonic stem cell mediated transgenesis are equally complicated and have limitations. Although our previously reported technique of testicular transgenesis circumvented several limitations, it involved many steps, including surgery and hemicastration, which carried risk of infection and impotency. We improved this technique further, into a two step non-surgical electroporation procedure, for making transgenic mice. In this approach, transgene was delivered inside both testes by injection and modified parameters of electroporation were used for in-vivo gene integration in germ cells. Using variety of constructs, germ cell integration of the gene and its transmission in progeny was confirmed by PCR, slot blot and immunohistochemical analysis. This improved technique is efficient, requires substantially less time and can be easily adopted by various biomedical researchers.

A switch in Sertoli cell responsiveness to FSH may be responsible for robust onset of germ cell differentiation during prepubartal testicular maturation in rats.

FSH and Testosterone (T) regulate spermatogenesis via testicular Sertoli cells (Sc), which bear receptors for these hormones. Despite sufficient circulating levels of FSH and T postnatally, predominant appearance of spermatogonia B and spermatocytes is not discernible until 11 and 18 days of postnatal age, respectively, in rat testes. In an attempt to explore the underlying causes, we cultured Sc from neonatal (5- and 9-day-old) and prepubertal (12- and 19-day-old) rat testes and compared the status of FSH receptor (FSH-R) and androgen receptor (AR) signaling. Protein and mRNA levels of FSH-R and AR remained uniform in cultured Sc from all age groups. Androgen binding ability of AR was similar, and T-induced nuclear localization of AR was discernible in Sc from all age groups. Binding of FSH to FSH-R, subsequent production of cAMP, and mRNA of stem cell factor (SCF) and glial cell line-derived neurotrophic factor (GDNF), known to be essential for the robust differentiation of repopulating spermatogonia, were significantly augmented in prepubertal Sc compared with those in neonatal Sc. However, treatment of neonatal Sc with cholera toxin or forskolin, which stimulate cAMP production bypassing FSH-R, demonstrated a concomitant rise in SCF and GDNF mRNA expression, which was similar to the FSH-mediated rise observed in prepubertal Sc. These observations suggested that, during prepubertal Sc maturation, the ability of FSH-R to respond to FSH is significantly augmented and is associated with the robust differentiation of repopulating spermatogonia, and such a switch in Sc from FSH-resistant to FSH-responsive mode during prepubertal development may underlie the initiation of robust spermatogenesis.

A method for rapid generation of transgenic animals to evaluate testis genes during sexual maturation.

In certain forms of idiopathic infertility, there is failure of follicle stimulating hormone (FSH) and testosterone (T) to initiate spermatogenesis despite the presence of Sertoli cells and germ cells in the testis. In postnatal rats (up to 11 days of age) and infant monkeys (3-4 months old), robust division and differentiation of spermatogonial stem cells is not discerned, even though serum levels of FSH and T are similar to those found during adulthood. Lack of spermatogenesis together with normal hormone levels is a situation similar to that found in certain categories of male infertility. To investigate this intriguing situation, Sertoli cells were cultured from infant and pubertal rats and monkeys and differential gene expression by testicular Sertoli cells was evaluated by DNA microarray using the Agilent microarray system. To determine the role of candidate genes in regulation of spermatogenesis, transgenic animals over-expressing these genes must be generated. However, present techniques for generation of transgenic animals have limited utility for production of several transgenic animals within a short period of time. Therefore, we have developed a technique for making transgenic animals by the testicular route which is less labor intensive and less time consuming. This technique is also ethically superior since fewer mice are required than in existing alternative methods of transgenesis.