Transcription Factor Wilms Research Articles

Not too much, not too little—just the right amount: the story of YAP in the podocyte

Chen etal. identify dysregulation of the transcriptional activator Yes-associated protein in the podocytes of diabetic mouse and human kidneys. Podocyte Yes-associated protein deficiency led to downregulation of the key transcription factor Wilms' tumor 1, and worsened podocyte injury in a mouse model of diabetic kidney injury. Yes-associated protein may therefore play a critical role in diabetic podocyte injury via regulation of Wilms' tumor 1 expression.

Homeobox regulator Wilms Tumour 1 is displaced by androgen receptor at cis-regulatory elements in the endometrium of PCOS patients.

Decidualisation, the process whereby endometrial stromal cells undergo morphological and functional transformation in preparation for trophoblast invasion, is often disrupted in women with polycystic ovary syndrome (PCOS) resulting in complications with pregnancy and/or infertility. The transcription factor Wilms tumour suppressor 1 (WT1) is a key regulator of the decidualization process, which is reduced in patients with PCOS, a complex condition characterized by increased expression of androgen receptor in endometrial cells and high presence of circulating androgens. Using genome-wide chromatin immunoprecipitation approaches on primary human endometrial stromal cells, we identify key genes regulated by WT1 during decidualization, including homeobox transcription factors which are important for regulating cell differentiation. Furthermore, we found that AR in PCOS patients binds to the same DNA regions as WT1 in samples from healthy endometrium, suggesting dysregulation of genes important to decidualisation pathways in PCOS endometrium due to competitive binding between WT1 and AR. Integrating RNA-seq and H3K4me3 and H3K27ac ChIP-seq metadata with our WT1/AR data, we identified a number of key genes involved in immune response and angiogenesis pathways that are dysregulated in PCOS patients. This is likely due to epigenetic alterations at distal enhancer regions allowing AR to recruit cofactors such as MAGEA11, and demonstrates the consequences of AR disruption of WT1 in PCOS endometrium.

Evaluation of glomerular sirtuin-1 and claudin-1 in the pathophysiology of nondiabetic focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is the leading cause of nephrotic syndrome, which is characterized by podocyte injury. Given that the pathophysiology of nondiabetic glomerulosclerosis is poorly understood and targeted therapies to prevent glomerular disease are lacking, we decided to investigate the tight junction protein claudin-1 and the histone deacetylase sirtuin-1 (SIRT1), which are known to be involved in podocyte injury. For this purpose, we first examined SIRT1, claudin-1 and podocin expression in kidney biopsies from patients diagnosed with nondiabetic FSGS and found that upregulation of glomerular claudin-1 accompanies a significant reduction in glomerular SIRT1 and podocin levels. From this, we investigated whether a small molecule activator of SIRT1, SRT1720, could delay the onset of FSGS in an animal model of adriamycin (ADR)-induced nephropathy; 14 days of treatment with SRT1720 attenuated glomerulosclerosis progression and albuminuria, prevented transcription factor Wilms tumor 1 (WT1) downregulation and increased glomerular claudin-1 in the ADR + SRT1720 group. Thus, we evaluated the effect of ADR and/or SRT1720 in cultured mouse podocytes. The results showed that ADR [1 µM] triggered an increase in claudin-1 expression after 30 min, and this effect was attenuated by pretreatment of podocytes with SRT1720 [5 µM]. ADR [1 µM] also led to changes in the localization of SIRT1 and claudin-1 in these cells, which could be associated with podocyte injury. Although the use of specific agonists such as SRT1720 presents some benefits in glomerular function, their underlying mechanisms still need to be further explored for therapeutic use. Taken together, our data indicate that SIRT1 and claudin-1 are relevant for the pathophysiology of nondiabetic FSGS.

A single-nucleus transcriptomic atlas of primate testicular aging reveals exhaustion of the spermatogonial stem cell reservoir and loss of Sertoli cell homeostasis.

The testis is pivotal for male reproduction, and its progressive functional decline in aging is associated with infertility. However, the regulatory mechanism underlying primate testicular aging remains largely elusive. Here, we resolve the aging-related cellular and molecular alterations of primate testicular aging by establishing a single-nucleus transcriptomic atlas. Gene-expression patterns along the spermatogenesis trajectory revealed molecular programs associated with attrition of spermatogonial stem cell reservoir, disturbed meiosis and impaired spermiogenesis along the sequential continuum. Remarkably, Sertoli cell was identified as the cell type most susceptible to aging, given its deeply perturbed age-associated transcriptional profiles. Concomitantly, downregulation of the transcription factor Wilms' Tumor 1 (WT1), essential for Sertoli cell homeostasis, was associated with accelerated cellular senescence, disrupted tight junctions, and a compromised cell identity signature, which altogether may help create a hostile microenvironment for spermatogenesis. Collectively, our study depicts in-depth transcriptomic traits of non-human primate (NHP) testicular aging at single-cell resolution, providing potential diagnostic biomarkers and targets for therapeutic interventions against testicular aging and age-related male reproductive diseases.

Restoration of WT1/miR-769-5p axis by HDAC1 inhibition promotes MMT reversal in mesenchymal-like mesothelial cells

Histone acetylation/deacetylation play an essential role in modifying chromatin structure and in regulating cell plasticity in eukaryotic cells. Therefore, histone deacetylase (HDAC) pharmacological inhibitors are promising tools in the therapy of fibrotic diseases and in cancer. Peritoneal fibrosis is a pathological process characterized by many cellular and molecular alterations, including the acquisition of invasive/pro-fibrotic abilities by mesothelial cells (MCs) through induction of mesothelial to mesenchymal transition (MMT). The aim of this study was to characterize the molecular mechanism of the antifibrotic role of HDAC1 inhibition. Specifically, treatment with MS-275, an HDAC1-3 inhibitor previously known to promote MMT reversal, induced the expression of several TGFBRI mRNA-targeting miRNAs. Among them, miR-769-5p ectopic expression was sufficient to promote MMT reversal and to limit MC migration and invasion, whereas miR-769-5p silencing further enhanced mesenchymal gene expression. These results were confirmed by HDAC1 genetic silencing. Interestingly, miR-769-5p silencing maintained mesenchymal features despite HDAC1 inhibition, thus indicating that it is necessary to drive MMT reversal induced by HDAC1 inhibition. Besides TGFBRI, miR-769-5p was demonstrated to target SMAD2/3 and PAI-1 expression directly. When analyzing molecular mechanisms underlying miR-769-5p expression, we found that the transcription factor Wilms’ tumor 1 (WT1), a master gene controlling MC development, binds to the miR-769-5p promoter favoring its expression. Interestingly, both WT1 expression and binding to miR-769-5p promoter were increased by HDAC1 inhibition and attenuated by TGFβ1 treatment. Finally, we explored the significance of these observations in the cell-to-cell communication: we evaluated the ability of miR-769-5p to be loaded into extracellular vesicles (EVs) and to promote MMT reversal in recipient mesenchymal-like MCs. Treatment of fibrotic MCs with EVs isolated from miR-769-5p over-expressing MCs promoted the down-regulation of specific mesenchymal targets and the reacquisition of an epithelial-like morphology. In conclusion, we highlighted an HDAC1-WT1-miR-769-5p axis potentially relevant for therapies aimed at counteracting organ fibrosis.

WT1 Inhibits Human Renal Carcinoma Cell Proliferation and Induces G2/M Arrest by Upregulating IL-24 Expression.

The transcription factor Wilms' tumor 1 (WT1) is involved in development, tissue homeostasis, and disease. However, the exact roles and the mechanisms of WT1 in renal carcinoma are not well understood. Therefore, in this study, we evaluated the ability of WT1 to block proliferation in renal carcinoma cells in vitro. Experimental analysis showed that WT1 overexpression inhibited the proliferation of renal carcinoma A498 cells and promoted arrest at the G2/M checkpoint. RNA-Seq identified differentially expressed genes, including IL-24, related to both the cell proliferation and the cell cycle. WT1 overexpression upregulated IL-24 expression, and IL-24 overexpression induced G2/M arrest. ChIP-Seq identified JUN as a direct target of WT1 in A498 cells, in which positive regulation was shown by RT-qPCR. It has been shown that the transcription factor JUN can regulate IL-24 expression, and therefore, we hypothesize that WT1 might regulate the IL-24 through JUN. Furthermore, analysis based on TCGA datasets showed that the expression of WT1-regulated genes, including TXNIP and GADD45A, was significantly correlated with the stage and histological grade of tumors, with high levels linked to favorable prognoses. Our results demonstrated that the overexpression of WT1 upregulates IL-24, leading to G2/M checkpoint arrest to reduce proliferation. These results indicate that regulation of IL-24 by WT1 inhibits proliferation and may represent a potential target for treating renal carcinoma.

Dysregulated overexpression of Sox9 induces fibroblast activation in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease associated with unremitting fibroblast activation including fibroblast-to-myofibroblast transformation (FMT), migration, resistance to apoptotic clearance, and excessive deposition of extracellular matrix (ECM) proteins in the distal lung parenchyma. Aberrant activation of lung-developmental pathways is associated with severe fibrotic lung disease; however, the mechanisms through which these pathways activate fibroblasts in IPF remain unclear. Sry-box transcription factor 9 (Sox9) is a member of the high-mobility group box family of DNA-binding transcription factors that are selectively expressed by epithelial cell progenitors to modulate branching morphogenesis during lung development. We demonstrate that Sox9 is upregulated via MAPK/PI3K-dependent signaling and by the transcription factor Wilms’ tumor 1 in distal lung-resident fibroblasts in IPF. Mechanistically, using fibroblast activation assays, we demonstrate that Sox9 functions as a positive regulator of FMT, migration, survival, and ECM production. Importantly, our in vivo studies demonstrate that fibroblast-specific deletion of Sox9 is sufficient to attenuate collagen deposition and improve lung function during TGF-α–induced pulmonary fibrosis. Using a mouse model of bleomycin-induced pulmonary fibrosis, we show that myofibroblast-specific Sox9 overexpression augments fibroblast activation and pulmonary fibrosis. Thus, Sox9 functions as a profibrotic transcription factor in activating fibroblasts, illustrating the potential utility of targeting Sox9 in IPF treatment.

Overexpression of Wilms tumor 1 promotes IL-1β expression by upregulating histone acetylation in keratinocytes

Psoriasis is a common inflammatory skin disease. Infiltration of inflammatory cells and excessive proliferation of keratinocytes are the histopathological markers of psoriasis. The transcription factor Wilms Tumor 1 (WT1) is overexpressed in several tumor types, and plays an important part in the proliferation and apoptosis of cells. Studies have found that, compared with normal skin, WT1expression in the skin lesions of patients with psoriasis are increased significantly. Knockdown of WT1 inhibited the proliferation of a human epidermal keratinocyte cell line (HaCaT cells) and promoted their apoptosis, whereas WT1 overexpression exhibited the opposite effect. WT1 was overexpressed or inhibited in HaCaT cells by transfection with the WT1 plasmid or WT1 small interferring RNA (siRNA) using Lipofectamine 2000. Transcriptome sequencing and bioinformatics analysis revealed significant differences in IL-1β expression between the experimental group and control group. Real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assays showed that expression of IL-1β and WT1 were consistent. Subsequently, IL-1β was demonstrated to be a target of WT1 by chromatin immunoprecipitation (ChIP)-sequencing and luciferase reporter assay. ChIP-qPCR showed that WT1 regulated IL-1β expression by altering acetylation. Expression of WT1 mRNA was positively correlated with expression of IL-1β mRNA in psoriatic skin lesions. Our study suggested that WT1 likely promotes psoriasis development by regulating its target gene IL-1β, which shows high expression in psoriatic lesions and is involved in psoriasis development. These findings provide a new target for psoriasis treatment.

Generation of Monogenic Candidate Genes for Human Nephrotic Syndrome Using 3 Independent Approaches

IntroductionSteroid-resistant nephrotic syndrome (SRNS) is the second most common cause of chronic kidney disease during childhood. Identification of 63 monogenic human genes has delineated 12 distinct pathogenic pathways.MethodsHere, we generated 2 independent sets of nephrotic syndrome (NS) candidate genes to augment the discovery of additional monogenic causes based on whole-exome sequencing (WES) data from 1382 families with NS.ResultsWe first identified 63 known monogenic causes of NS in mice from public databases and scientific publications, and 12 of these genes overlapped with the 63 known human monogenic SRNS genes. Second, we used a set of 64 genes that are regulated by the transcription factor Wilms tumor 1 (WT1), which causes SRNS if mutated. Thirteen of these WT1-regulated genes overlapped with human or murine NS genes. Finally, we overlapped these lists of murine and WT1 candidate genes with our list of 120 candidate genes generated from WES in 1382 NS families, to identify novel candidate genes for monogenic human SRNS. Using this approach, we identified 7 overlapping genes, of which 3 genes were shared by all datasets, including SYNPO. We show that loss-of-function of SYNPO leads to decreased CDC42 activity and reduced podocyte migration rate, both of which are rescued by overexpression of wild-type complementary DNA (cDNA), but not by cDNA representing the patient mutation.ConclusionThus, we identified 3 novel candidate genes for human SRNS using 3 independent, nonoverlapping hypotheses, and generated functional evidence for SYNPO as a novel potential monogenic cause of NS.

Expansion of the renal capsular stroma, ureteric bud branching defects and cryptorchidism in mice with Wilms tumor 1 gene deletion in the stromal compartment of the developing kidney.

Development of the mammalian kidney is orchestrated by reciprocal interactions of stromal and nephrogenic mesenchymal cells with the ureteric bud epithelium. Previous work showed that the transcription factor Wilms tumor 1 (WT1) acts in the nephrogenic lineage to maintain precursor cells, to drive the epithelial transition of aggregating precursors into a renal vesicle and to specify and maintain the podocyte fate. However, WT1 is expressed not only in the nephrogenic lineage but also transiently in stromal progenitors in the renal cortex. Here we report that specific deletion of Wt1 in the stromal lineage using the Foxd1cre driver line results at birth in cryptorchidism and hypoplastic kidneys that harbour fewer and enlarged ureteric bud tips and display an expansion of capsular stroma into the cortical region. In vivo and ex vivo analysis at earlier stages revealed that stromal loss of Wt1 reduces stromal proliferation, and delays and alters branching morphogenesis, resulting in a variant architecture of the collecting duct tree with an increase of single at the expense of bifurcated ureteric bud tips. Molecular analysis identified a transient reduction of Aldh1a2 expression and of retinoic acid signalling activity in stromal progenitors, and of Ret in ureteric bud tips. Administration of retinoic acid partly rescued the branching defects of mutant kidneys in culture. We propose that WT1 maintains retinoic acid signalling in the cortical stroma, which, in turn, assures proper levels and dynamics of Ret expression in the ureteric bud tips, and thus normal ramification of the ureteric tree. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Analysis of wt1a reporter line expression levels during proepicardium formation in the zebrafish.

The epicardium is the outer mesothelial layer of the heart. It covers the myocardium and plays important roles in both heart development and regeneration. It is derived from the proepicardium (PE), groups of cells that emerges at early developmental stages from the dorsal pericardial layer (DP) close to the atrio-ventricular canal and the venous pole of the heart-tube. In zebrafish, PE cells extrude apically into the pericardial cavity as a consequence of DP tissue constriction, a process that is dependent on Bmp pathway signaling. Expression of the transcription factor Wilms tumor-1, Wt1, which is a leader of important morphogenetic events such as apoptosis regulation or epithelial-mesenchymal cell transition, is also necessary during PE formation. In this study, we used the zebrafish model to compare intensity level of the wt1a reporter line epi:GFP in PE and its original tissue, the DP. We found that GFP is present at higher intensity level in the PE tissue, and differentially wt1 expression at pericardial tissues could be involved in the PE formation process. Our results reveal that bmp2b overexpression leads to enhanced GFP level both in DP and in PE tissues.

Reticular Fibroblasts Expressing the Transcription Factor WT1 Define a Stromal Niche that Maintains and Replenishes Splenic Red Pulp Macrophages

Located within red pulp cords, splenic red pulp macrophages (RPMs) are constantly exposed to the blood flow, clearing senescent red blood cells (RBCs) and recycling iron from hemoglobin. Here, we studied the mechanisms underlying RPM homeostasis, focusing on the involvement of stromal cells as these cells perform anchoring and nurturing macrophage niche functions in lymph nodes and liver. Microscopy revealed that RPMs are embedded in a reticular meshwork of red pulp fibroblasts characterized by the expression of the transcription factor Wilms' Tumor 1 (WT1) and colony stimulating factor 1 (CSF1). Conditional deletion of Csf1 in WT1+ red pulp fibroblasts, but not white pulp fibroblasts, drastically altered the RPM network without altering circulating CSF1 levels. Upon RPM depletion, red pulp fibroblasts transiently produced the monocyte chemoattractants CCL2 and CCL7, thereby contributing to the replenishment of the RPM network. Thus, red pulp fibroblasts anchor and nurture RPM, a function likely conserved in humans.

Wilms tumor 1 regulates lipid accumulation in human endometrial stromal cells during decidualization

We previously reported that the transcription factor Wilms tumor 1 (WT1) regulates the expression of insulin-like growth factor-binding protein-1 (IGFBP-1) and prolactin (PRL) during decidualization of human endometrial stromal cells (ESCs). However, other roles of WT1 in decidualization remain to be fully clarified. Here, we investigated how WT1 regulates the physiological functions of human ESCs during decidualization. We incubated ESCs isolated from proliferative-phase endometrium with cAMP to induce decidualization, knocked down WT1 with siRNA, and generated three types of treatments (nontreated cells, cAMP-treated cells, and cAMP-treated + WT1-knockdown cells). To identify WT1-regulated genes, we used gene microarrays and compared the transcriptome data obtained among these three treatments. We observed that WT1 up-regulates 121 genes during decidualization, including several genes involved in lipid transport. The WT1 knockdown inhibited lipid accumulation (LA) in the cAMP-induced ESCs. To examine the mechanisms by which WT1 regulates LA, we focused on very low-density lipoprotein receptor (VLDLR), which is involved in lipoprotein uptake. We found that cAMP up-regulates VLDLR and that the WT1 knockdown inhibits it. Results of ChIP assays revealed that cAMP increases the recruitment of WT1 to the promoter region of the VLDLR gene, indicating that WT1 regulates VLDLR expression. Moreover, VLDLR knockdown inhibited cAMP-induced LA, and VLDLR overexpression reverted the suppression of LA caused by the WT1 knockdown. Taken together, our results indicate that WT1 enhances lipid storage by up-regulating VLDLR expression in human ESCs during decidualization.

A Stromal Niche Defined by Expression of the Transcription Factor WT1 Mediates Programming and Homeostasis of Cavity-Resident Macrophages

Tissue-resident macrophages require specific milieus for the maintenance of defining gene-expression programs. Expression of the transcription factor GATA6 is required for the homeostasis, function and localization of peritoneal cavity-resident macrophages. Gata6 expression is maintained in a non-cell autonomous manner and is elicited by the vitamin A metabolite, retinoic acid. Here, we found that the GATA6 transcriptional program is a common feature of macrophages residing in all visceral body cavities. Retinoic acid-dependent and -independent hallmark genes of GATA6+ macrophages were induced by mesothelial and fibroblastic stromal cells that express the transcription factor Wilms' Tumor 1 (WT1), which drives the expression oftwo rate-limiting enzymes in retinol metabolism. Depletion of Wt1+ stromal cellsreduced the frequency of GATA6+ macrophages in the peritoneal, pleural and pericardial cavities. Thus, Wt1+mesothelial and fibroblastic stromal cells constitute essential niche components supporting the tissue-specifying transcriptional landscape and homeostasis of cavity-resident macrophages.

Overexpression mRNA of HMGA2 and WT1 Genes in Whole Blood Samples As Potential Diagnostic Markers in MPN

BackgroundHigh-mobility group AT-hook protein 2 (HMGA2) gene codes for a non-histone protein which expressed during embryogenesis and early hematopoiesis but suppressed in differentiated cells. Overexpression of HMGA2 as well as transcription factor Wilms' tumor 1 (WT1) gene has been found in a variety of human cancers, but only a few reports has included myeloproliferative neoplasms (MPNs) (Guglielmelli P. et al., 2007; Chen C-C. et al., 2017; Tasdemir S. et al, 2015). Furthermore, there were no any data about co-expression of mRNA both genes in blood in patients with MPNs.Aims: evaluate of the diagnostic potential of the WT1 and HMGA2 mRNA level in MPN patients.MethodsIn our retrospective study was analysed 115 archived frozen peripheral blood samples with RNAse inhibitor from 62 MPN patients: 29 with polycythemia vera (PV), 16 with essential thrombocythemia (ET), and 17 with myelofibrosis (MF) were included. Samples of 28 healthy volunteers were tested as control group. Total RNA was isolated using “Ribo-zol-D” (Aplisens) and then were transcribed using “Reverta-L” (Aplisens). Synthesized cDNA was analysed by real-time PCR to detect of WT1 and HMGA2 expression levels on the CFX96 (Bio-Rad) housekeeping kits using. The results were calculated utilizing the delta Ct method in the software package of “R”. The threshold cycles (Ct) WT1 and HMGA2 genes and reference gene ABL determined using Cy0 method. Results are presented as median and range (25% and 75%). Comparisons between the patient and control group were made using the Mann-Whitney U test. Statistical analysis was performed using Statistica 12.0 software.ResultsThe level of HMGA2 mRNA in five samples of venous blood in PV patients and 12 samples in MF patients was higher than in healthy subjects (Figure 1). Expression of WT1 mRNA (≥ 0.01%) was observed only in samples among patients with MF (in 19 of 28). In 12 blood samples of patients with MF, transcripts of both genes were detected together (Table 1). The increased level of HMGA2 mRNA expression was associated with a higher level of erythrocytosis in patients with PV (p<0.05). The co-expression of HMGA2 mRNA and WT1 mRNA in patients with MF was associated with a low level of white and red blood cell (p<0.05). Interestingly that in MF patients the most pronounced thrombocytopenia was observed if only WT1 mRNA was detected. In all cases of repeated testing of patients, the growth of HMGA2 or WT1 mRNA level was accompanied by decline of the cellular blood composition. The level of HMGA2 mRNA and WT1 mRNA in our study did not depend on the driver mutations presence or their allele burden. It should be noted that 4 out of 12 patients with MF died within 5 months after the first detection of WT1 mRNA and one patient died more after 15 months. At that time only one MF patient without WT1 or HGMA2 transcripts died during of 20 months' observation.Summary and ConclusionsExpression of the WT1 and HMGA2 genes is involved in the pathogenesis and progression of the disease and can be used as diagnostic markers. The question of the independent prognostic significance of these transcripts in blood samples for various phenotypes of MPN is promising for further study. DisclosuresNo relevant conflicts of interest to declare.

Overexpression of Wilms' tumor 1 in skin lesions of psoriasis is associated with abnormal proliferation and apoptosis of keratinocytes.

Psoriasis vulgaris (PV) is a chronic inflammatory skin disease, which is characterized by the abnormal proliferation and apoptosis of keratinocytes. Previous studies have demonstrated that transcription factor Wilms' tumor 1 (WT1) is involved in a number of pathophysiological processes, including organ development, tumorigenesis and cell proliferation. However, the role of WT1 in PV remains unclear. In the present study, WT1 expression was analyzed by reverse transcription‑quantitative polymerase chain reaction and western blot analyses. WT1 was stably overexpressed or inhibited in HaCaT cells using Lipofectamine® 2000, and cell proliferation and apoptosis were determined using a Cell Counting Kit‑8 or Fluorescein Isothiocyanate Annexin V Apoptosis Detection kit II, respectively. We demonstrated that compared with normal controls, the mRNA and protein expression levels of WT1 were significantly increased in non‑lesional skins (human, P<0.0001 and P=0.0291, respectively; mouse, P=0.0020 and P=0.0150, respectively) and lesional skins (human, P<0.0001 and P=0.0060, respectively; mouse, P=0.0010 and P=0.0172, respectively) of patients with PV, in addition to the imiquimod (IMQ)‑induced psoriasis‑like mouse model. WT1 mRNA and protein expression levels in lesional skins were slightly increased compared with those in non‑lesional skins from patients with psoriasis (P=0.2510 and P=0.1690, respectively) and IMQ‑treated mice (P=0.9590 and P=0.2552, respectively), although there were no statistical differences. Knockdown of WT1 inhibited the proliferation of HaCaT cells [day (D)4, P=0.0454; D5, P=0.0021] and promoted their apoptosis (P=0.0007), while overexpressing WT1 exhibited the opposite effects (proliferation D3, P=0.0216; D4, P=0.0356; D5, P=0.0188; apoptosis, P=0.0003). Furthermore, it was identified that the inflammatory cytokines interleukin‑17A (IL‑17A), interferon‑γ and IL‑22 induced the overexpression of WT1 in HaCaT cells. The results of the present study suggested that inflammatory cytokine‑induced WT1 overexpression may promote the formation of psoriatic skin lesions via regulation of the proliferation and apoptosis of keratinocytes.

Amelioration of Diabetic Nephropathy Using a Retinoic Acid Receptorβ2 Agonist

Vitamin A (VA) and its derivatives, known as retinoids, play critical roles in renal development through retinoic acid receptor β2 (RARβ2). Disruptions in VA signaling pathways are associated with the onset of diabetic nephropathy (DN). Despite the known role of RARβ2 in renal development, the effects of selective agonists for RARβ2 in a high-fat diet (HFD) model of DN are unknown. Here we examined whether AC261066 (AC261), a highly selective agonist for RARβ2, exhibited therapeutic effects in a HFD model of DN in C57BL/6 mice. Twelve weeks of AC261 administration to HFD-fed mice was well tolerated with no observable side effects. Compared with HFD-fed mice, HFD + AC261-treated mice had improved glycemic control and reductions in proteinuria and urine albumin-to-creatinine ratio. Several cellular hallmarks of DN were mitigated in HFD + AC261-treated mice, including reductions in tubule lipid droplets, podocyte (POD) effacement, endothelial cell collapse, mesangial expansion, and glomerular basement membrane thickening. Mesangial and tubule interstitial expression of the myofibroblast markers α-smooth muscle actin (α-SMA) and type IV collagen (Col-IV) was lower in HFD + AC261-treated mice compared with HFD alone. Ultrastructural and immunohistochemistry analyses showed that, compared with HFD-fed mice, HFD + AC261-treated mice showed preservation of POD foot process and slit-diaphragm morphology, an increase in the levels of slit-diagram protein podocin, and the transcription factor Wilms tumor-suppressor gene 1 in PODs. Given the need for novel DN therapies, our results warrant further studies of the therapeutic properties of AC261 in DN.

Abstract 3100: Replacing fetal calf serum by human platelet lysate in cancer research and toxicology

Abstract Experiments with cultured mammalian cells represent a common in vitro alternative to animal experiments. Fetal calf serum (FCS) is the most commonly used medium supplement. FCS contains a mixture of largely undefined growth factors and cytokines. Since FCS is received from unborn calves older than three months, it represents a massive burden for the pregnant cows and their fetuses. A needle is inserted into the heart of the fetus to collect blood. Since the animal is not under anesthesia, it may suffer pain and discomfort. Furthermore, the undefined nature of FCS is a source of experimental variation, undesired immune responses, and possible contaminations. Thus, alternative, defined, valid, and reliable medium supplements should be identified. We follow the principles of the 3Rs (Replacement, Reduction, Refinement). We established a method to receive human platelet lysate (hPL) from human blood platelets. We investigated if hPL represents potential true alternative for FCS in cell culture experiments and thereby the cruel slaughtering of unborn fetuses. hPL is received by freeze and thawing cycles and centrifugation steps. We successfully adapted three human leukemia cell lines (K562, NB4 and MV4-11) and HCT116 colon cancer cells to culture medium containing 1-5% hPL. We analyzed cell morphology by microscopy. Cell growth kinetics were examined by counting and cell cycle distributions were investigated by flow cytometry. Protein expression patterns were determined globally by global scale proteomics. We additionally determined possible FCS- or hPL-dependent reactions of cells to the well-established anti-cancer drugs Irinotecan, Hydroxyurea and Imatinib, and to epigenetic modulators of the HDAC inhibitor family (FK228 and the new, highly selective HDAC6 inhibitor Marbostat-100) by flow cytometry and Western blot. In these tests, we particularly focused on the expression of the pan-leukemic marker and transcription factor Wilms tumor 1 (WT1) and on the tumor suppressor p53. All tested cell lines grow with 5% FCS and 5% hPL. Cell morphology, cell cycle distribution, and protein expression patterns were not affected by such different culture conditions in resting and treated cells. We further show that the class I HDACs HDAC1-3, but not HDAC6, regulate WT1 and p53. Our findings demonstrate that hPL is a promising replacement for animal serum. Citation Format: Oliver H. Krämer. Replacing fetal calf serum by human platelet lysate in cancer research and toxicology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3100.

Loss of Wilms tumor 1 protein is a marker for apoptosis in response to replicative stress in leukemic cells

A remaining expression of the transcription factor Wilms tumor 1 (WT1) after cytotoxic chemotherapy indicates remaining leukemic clones in patients. We determined the regulation and relevance of WT1 in leukemic cells exposed to replicative stress and DNA damage. To induce these conditions, we used the clinically relevant chemotherapeutics hydroxyurea and doxorubicin. We additionally treated cells with the pro-apoptotic kinase inhibitor staurosporine. Our data show that these agents promote apoptosis to a variable extent in a panel of 12 leukemic cell lines and that caspases cleave WT1 during apoptosis. A chemical inhibition of caspases as well as an overexpression of mitochondrial, anti-apoptotic BCL2 family proteins significantly reduces the processing of WT1 and cell death in hydroxyurea-sensitive acute promyelocytic leukemia cells. Although the reduction of WT1 correlates with the pharmacological efficiency of chemotherapeutics in various leukemic cells, the elimination of WT1 by different strategies of RNA interference (RNAi) does not lead to changes in the cell cycle of chronic myeloid leukemia K562 cells. RNAi against WT1 does also not increase the extent of apoptosis and the accumulation of γH2AX in K562 cells exposed to hydroxyurea. Likewise, a targeted genetic depletion of WT1 in primary oviduct cells does not increase the levels of γH2AX. Our findings position WT1 as a downstream target of the apoptotic process that occurs in response to cytotoxic forms of replicative stress and DNA damage.

Serine hydroxymethyl transferase 1 stimulates pro-oncogenic cytokine expression through sialic acid to promote ovarian cancer tumor growth and progression

High-grade serous (HGS) ovarian cancer accounts for 90% of all ovarian cancer-related deaths. However, factors that drive HGS ovarian cancer tumor growth have not been fully elucidated. In particular, comprehensive analysis of the metabolic requirements of ovarian cancer tumor growth has not been performed. By analyzing The Cancer Genome Atlas mRNA expression data for HGS ovarian cancer patient samples, we observed that six enzymes of the folic acid metabolic pathway were overexpressed in HGS ovarian cancer samples compared with normal ovary samples. Systematic knockdown of all six genes using short hairpin RNAs (shRNAs) and follow-up functional studies demonstrated that serine hydroxymethyl transferase 1 (SHMT1) was necessary for ovarian cancer tumor growth and cell migration in culture and tumor formation in mice. SHMT1 promoter analysis identified transcription factor Wilms tumor 1 (WT1) binding sites, and WT1 knockdown resulted in reduced SHMT1 transcription in ovarian cancer cells. Unbiased large-scale metabolomic analysis and transcriptome-wide mRNA expression profiling identified reduced levels of several metabolites of the amino sugar and nucleotide sugar metabolic pathways, including sialic acid N-acetylneuraminic acid (Neu5Ac), and downregulation of pro-oncogenic cytokines interleukin-6 and 8 (IL-6 and IL-8) as unexpected outcomes of SHMT1 loss. Overexpression of either IL-6 or IL-8 partially rescued SHMT1 loss-induced tumor growth inhibition and migration. Supplementation of culture medium with Neu5Ac stimulated expression of IL-6 and IL-8 and rescued the tumor growth and migratory phenotypes of ovarian cancer cells expressing SHMT1 shRNAs. In agreement with the ovarian tumor-promoting role of Neu5Ac, treatment with Neu5Ac-targeting glycomimetic P-3Fax-Neu5Ac blocked ovarian cancer growth and migration. Collectively, these results demonstrate that SHMT1 controls the expression of pro-oncogenic inflammatory cytokines by regulating sialic acid Neu5Ac to promote ovarian cancer tumor growth and migration. Thus, targeting of SHMT1 and Neu5Ac represents a precision therapy opportunity for effective HGS ovarian cancer treatment.