Wilms Tumor Cell Line Research Articles

Modeling high-risk Wilms tumors enables the discovery of therapeutic vulnerability

Wilms tumor (WT) is the most common pediatric kidney cancer treated with standard chemotherapy. However, less-differentiated blastemal type of WT often relapses. To model the high-risk WT for therapeutic intervention, we introduce pluripotency factors into WiT49, a mixed-type WT cell line, to generate partially reprogrammed cells, namely WiT49-PRCs. When implanted into the kidney capsule in mice, WiT49-PRCs form kidney tumors and develop both liver and lung metastases, whereas WiT49 tumors do not metastasize. Histological characterization and gene expression signatures demonstrate that WiT49-PRCs recapitulate blastemal-predominant WTs. Moreover, drug screening in isogeneic WiT49 and WiT49-PRCs leads to the identification of epithelial- or blastemal-predominant WT-sensitive drugs, whose selectivity is validated in patient-derived xenografts (PDXs). Histone deacetylase (HDAC) inhibitors (e.g., panobinostat and romidepsin) are found universally effective across different WT and more potent than doxorubicin in PDXs. Taken together, WiT49-PRCs serve as a blastemal-predominant WT model for therapeutic intervention to treat patients with high-risk WT.

DROSHA Regulates Mesenchymal Gene Expression in Wilms Tumor.

Wilms tumor, the most common pediatric kidney cancer, resembles embryonic renal progenitors. Currently, there are no ways to therapeutically target Wilms tumor driver mutations, such as in the microRNA processing gene DROSHA. In this study, we used a "multiomics" approach to define the effects of DROSHA mutation in Wilms tumor. We categorized Wilms tumor mutations into four mutational subclasses with unique transcriptional effects: microRNA processing, MYCN activation, chromatin remodeling, and kidney developmental factors. In particular, we find that DROSHA mutations are correlated with de-repressing microRNA target genes that regulate differentiation and proliferation and a self-renewing, mesenchymal state. We model these findings by inhibiting DROSHA expression in a Wilms tumor cell line, which led to upregulation of the cell cycle regulator cyclin D2 (CCND2). Furthermore, we observed that DROSHA mutations in Wilms tumor and DROSHA silencing in vitro were associated with a mesenchymal state with aberrations in redox metabolism. Accordingly, we demonstrate that Wilms tumor cells lacking microRNAs are sensitized to ferroptotic cell death through inhibition of glutathione peroxidase 4, the enzyme that detoxifies lipid peroxides. Implications: This study reveals genotype-transcriptome relationships in Wilms tumor and points to ferroptosis as a potentially therapeutic vulnerability in one subset of Wilms tumor.

Targeting TRIP13 in favorable histology Wilms tumor with nuclear export inhibitors synergizes with doxorubicin

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

Abstract 1084: Preclinical testing of a cell adhesion molecule 1 (CADM1) targeting antibody-drug conjugate in osteosarcoma and other pediatric solid tumor models

Abstract Introduction: Survival outcomes of osteosarcoma have remained stagnant for decades, emphasizing the urgent need to identify new cell surface targets and develop novel therapies. Antibody-drug conjugates (ADCs), known for their targeted delivery of cytotoxic agents to specific targets, present an attractive therapeutic class for potential use in osteosarcoma. Methods: We previously identified Cell Adhesion Molecule 1 (CADM1) is highly expressed on the cell surface in osteosarcoma using an integrated proteomic and transcriptomic surfaceome profiling approach. As a proof of concept, a humanized CADM1 antibody (clone PTA021-A1) was conjugated with Tesirine (SG3249), a pyrrolobenzodiazepine (PBD) dimer payload. The antitumor activity of this CADM1 ADC was validated in vivo in 8 osteosarcoma patient-derived xenografts (PDXs). CADM1 is also highly expressed in other pediatric solid malignancies such as neuroblastoma and Wilms tumor. The CADM1 ADC was tested in vitro in neuroblastoma and Wilms tumor models. Negative controls included a CD19 targeting ADC with the same payloads and CADM1 negative Ewing sarcoma models. Results: The expression of the CADM1 was validated by flow cytometry in osteosarcoma, neuroblastoma, and Wilms tumor cell lines, and by immunohistochemistry (IHC) in osteosarcoma patient samples and PDX models. In vivo testing of CADM1 ADC in 8 osteosarcoma PDX models showed that the ADC was well-tolerated and significantly prolonged progressive-free survival in all osteosarcoma models. Complete response (CR) and maintained CR was observed in 3 models. In vitro testing for neuroblastoma and Wilms tumor is ongoing, and preliminary data suggest promising antitumor activities. Conclusions: CADM1 is highly expressed in osteosarcoma and other pediatric solid tumors. CADM1 ADC demonstrated promising antitumor in vitro and in vivo activity in osteosarcoma, and initial in vitro studies in neuroblastoma and Wilms tumor show similarly promising responses. These data support the need for further studies assessing the potential of a CADM1 ADC as a novel agent for the treatment of pediatric solid cancers. Citation Format: Zhongting Zhang, Yifei Wang, Wendong Zhang, Xiangjun Tian, Qi Wang, Rossana N Lazcano Segura, Michael Roth, Jonathan Gill, Douglas Harrison, Zhaohui Xu, Yianhua Yi, Sylvester Jusu, Giuseppe Longo, Xin Zhou, Jing Wang, Richard Gorlick. Preclinical testing of a cell adhesion molecule 1 (CADM1) targeting antibody-drug conjugate in osteosarcoma and other pediatric solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1084.

Oncogenic Cells of Renal Embryonic Lineage Sensitive to the Small-Molecule Inhibitor QC6352 Display Depletion of KDM4 Levels and Disruption of Ribosome Biogenesis.

The histone lysine demethylases KDM4A-C are involved in physiologic processes including stem cell identity and self-renewal during development, DNA damage repair, and cell-cycle progression. KDM4A-C are overexpressed and associated with malignant cell behavior in multiple human cancers and are therefore potential therapeutic targets. Given the role of KDM4A-C in development and cancer, we aimed to test the potent, selective KDM4A-C inhibitor QC6352 on oncogenic cells of renal embryonic lineage. The anaplastic Wilms tumor cell line WiT49 and the tumor-forming human embryonic kidney cell line HEK293 demonstrated low nanomolar QC6352 sensitivity. The cytostatic response to QC6352 in WiT49 and HEK293 cells was marked by induction of DNA damage, a DNA repair-associated protein checkpoint response, S-phase cell-cycle arrest, profound reduction of ribosomal protein gene and rRNA transcription, and blockade of newly synthesized proteins. QC6352 caused reduction of KDM4A-C levels by a proteasome-associated mechanism. The cellular phenotype caused by QC6352 treatment of reduced migration, proliferation, tumor spheroid growth, DNA damage, and S-phase cell-cycle arrest was most closely mirrored by knockdown of KDM4A as determined by siRNA knockdown of KDM4A-C. QC6352 sensitivity correlated with high basal levels of ribosomal gene transcription in more than 900 human cancer cell lines. Targeting KDM4A may be of future therapeutic interest in oncogenic cells of embryonic renal lineage or cells with high basal expression of ribosomal protein genes.

Abstract 3521: CCND2 promotes proliferation in Drosha and DICER1 driven tumors

Abstract Mutations in microRNA processing genes such as DROSHA and DICER1 consist of 1.5-2% of pediatric cancers including a brain tumor called pineoblastoma. We developed a genetically engineered mouse model of Drosha- and Dicer1- driven pineal tumors. These tumors develop without microRNAs and express early pineal developmental markers. Gene set enrichment analysis revealed E2F target genes to be highly expressed in these tumors. One of the most upregulated genes in these tumors is cyclin D2 (Ccnd2). Additionally, these tumors stain positively for Rb phosphorylation. In a Wilms tumor cell line (WiT49) with Drosha knocked down, RNA sequencing revealed that CCND2 was the most upregulated gene. Similarly, in patient-derived xenografts (PDXs) of DICER1-related tumors such as pleuropulmonary blastoma (PPB), we observed high expression of CCND2 and positive staining for Rb phosphorylation. To test the dependence of Rb phosphorylation in Drosha and Dicer tumors, we next treated a Drosha pineal tumor allograft with a CDK4/6 inhibitor, palbociclib, and observed tumor growth suppression. Additionally, treatment of DICER1 mutant PPB PDXs with palbociclib had similar results. These experiments suggest that CDK4/6 is important for tumor proliferation in Drosha and DICER1 driven cancers. Citation Format: Claudette R. Fraire, Uma Obalapuram, Patricia D. Tiburcio, Kavita Desai, Kenneth S. Chen. CCND2 promotes proliferation in Drosha and DICER1 driven tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3521.

Abstract 2689: Targeting the proteasome pathway in dactinomycin resistant Wilms tumors

Abstract Nephroblastomas or Wilms tumors are the most common pediatric renal cancer. Across all cases, the 5-year overall survival is over 90%. This is largely in part due to brute-force, chemotherapy regimens such as dactinomycin (actD) that have been routinely used to treat Wilms tumors. However, those that do respond to treatment still face a 15% chance of relapse. Patients with anaplastic tumor histology require more toxic therapies, and yet they still face a worse prognosis of only 66% 4-year survival and relapse rate of 43%. Furthermore, recurrence cuts survival rate down to 50%. With the widespread use of actD in Wilms tumors and other childhood malignancies, understanding the mechanism to recalcitrance against this chemotherapy can potentially reveal important vulnerabilities that can be exploited in the clinic. While transcriptomic and genomic analyses have been done in Wilms tumors, no targetable pathways have been identified in actD chemotherapy-resistant and anaplastic tumor cases. To that end, we performed ribosome sequencing of actD-treated anaplastic Wilms tumor cell line WiT49 and uncovered that the proteasome pathway is preferentially translated. Moreover, loss of proteasome subunits in a CRISPR library dropout screen sensitized the cells to actD. Consistent with these findings we found upregulation of proteasome subunit levels and proteasome enzymatic activity in Wilms tumor lines following actD treatment. To interrogate the effect of cotreating bortezomib with dactinomycin in vivo, we subcutaneously implanted anaplastic Wilms tumor patient-derived xenograft (PDX) lines in NOD/SCID Gamma (NSG) mice. Combinatorial treatment was found to be retard tumor growth compared to vehicle and single drug conditions. With further investigation, our findings can provide a foundation for exploring proteasome inhibitors with dactinomycin in clinical trials. Our study has important implications to improving the outcomes for not just for recurrent and anaplastic Wilms tumors, but to other pediatric cancers that use actD as well. Citation Format: Patricia Denise Ballesteros Tiburcio, Kenneth S. Chen. Targeting the proteasome pathway in dactinomycin resistant Wilms tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2689.

Gene expression studies of WT1 mutant Wilms tumor cell lines in the frame work of published kidney development data reveals their early kidney stem cell origin.

In order to get a better insight into the timing of WT1 mutant Wilms tumor development, we compared the gene expression profiles of nine established WT1 mutant Wilms tumor cell lines with published data from different kidney cell types during development. Publications describing genes expressed in nephrogenic precursor cells, ureteric bud cells, more mature nephrogenic epithelial cells and interstitial cell types were used. These studies uncovered that the WT1 mutant Wilms tumor cells lines express genes from the earliest nephrogenic progenitor cells, as well as from more differentiated nephron cells with the highest expression from the stromal/interstitial compartment. The expression of genes from all cell compartments points to an early developmental origin of the tumor in a common stem cell. Although variability of the expression of specific genes was evident between the cell lines the overall expression pattern was very similar. This is likely dependent on their different genetic backgrounds with distinct WT1 mutations and the absence/presence of mutant CTNNB1.

IL-2 Combined with IL-15 Enhanced the Expression of NKG2D Receptor on Patient Autologous NK Cells to Inhibit Wilms' Tumor via MAPK Signaling Pathway.

Objective The dysfunction of immune surveillance, a hot spot in cancer research, could lead to the occurrence and development in multicancers. However, the potential mechanisms of immunity in Wilms' tumor (WT) remain unclear on Wilms' tumor (WT). In this study, we aim to investigate the immune cell in WT and explore the underlying treatment strategy. Method We quantified stromal and immune scores by using ESTIMATE algorithm based on gene expression matrix of WT patients in TCGA and GEO databases. Different expression genes (DEGs) and functional enrichments were analyzed by R studio and DAVID tools. Flow cytometry, immunofluorescence staining, ELISA assay, and qRT-PCR were used for detecting the NK cells, cytotoxic cytokines (INF-γ, PRF, and GZMB), and NK cell receptor expression, respectively. WT patient autologous NK cells were stimulated by IL-2 and IL-15, and the cytotoxicity of NK cells against WT cell lines was detected by LDH assay. Western blot experiment was used for measuring the MAPK signaling pathway protein maker in NK cells. Results ESTIMATE indicated that WT tissue had a lower immune score than adjacent kidney tissue. Meanwhile, the low immune score group was associated with poorly outcomes. DEG functional enrichment analysis showed that NK cell-mediated cytotoxicity was significantly different in low and high immune score groups. Although few of proportion of NK cells in WT patients were increased, most of that were significantly lower than normal children. Moreover, the proportion of NK cells and the expression level of INF-γ, PRF, and GZMB in WT tissue were lower than adjacent kidney tissue. Importantly, the NKG2D expression level of NK cells was significantly lower in WT tissue. Furthermore, in vitro, compared with uncultured NK cells, IL-2 and IL-15 could effectively enhance the cytotoxicity of NK cells on killing the WT cell lines. The FACS and WB results showed that the NKG2D and p-PI3K ratio PI3K, MEK1/2, and p-ERK1/2 ratio ERK1/2 were significantly increased in IL-2 and IL15 group compared with uncultured groups. Conclusion The abnormal NK cell-mediated cytotoxicity may cause the occurrence of WT. Costimulation of WT patients autologous NK cells could effectively enhance the antitumor reaction which involved in activation of NKG2D-mediated MAPK signaling pathway.

KHSRP modulated cell proliferation and cell cycle via regulating PPP2CA and p27 expression in Wilms tumor

Wilms tumor (WT) is the most common renal malignancy in children, and the survival rate of high-risk WT patients was still low despite multimodality therapy. KHSRP, an RNA-binding protein, has been proved to be relative to tumor progression in different kinds of malignancies, but the function of KHSRP in WT remained unclear. Here, our study aimed to explore and clarify the function of KHSRP in WT cells and its molecular mechanism. Thus, our results showed that KHSRP was highly expressed in WT tumor tissues compared to normal kidney tissues and correlated with poor prognosis in WT patients. Downregulation of KHSRP using siRNAs in WT cell line SK-NEP-1 and Wit49 resulted in inhibition of cell proliferation and cell cycle arrest via stabilizing and upregulating p27 protein. Furthermore, mechanistic analyses revealed that KHSRP bound to 3’UTR of PPP2CA mRNA and modulating its mRNA stability, resulting in regulation of the phosphorylation level and protein stability of p27 in WT cell lines. In conclusion, our results demonstrated that KHSRP played an important role in WT and modulated cell proliferation and cell cycle via regulating the expression of PPP2CA and p27.

Abstract 1810: Targeting of the nuclear export protein XPO1 represents a non-genetically encoded vulnerability in malignant rhabdoid and Wilms tumors

Abstract Introduction: Malignant rhabdoid (MRT) and Wilms tumor (WT) comprise more than 5% of all pediatric cancers. Despite intensive multimodality therapy, outcomes remain dismal for a subset of patients with aggressive or high-risk molecular features. Characteristic of most pediatric cancers, MRT and WT demonstrate relatively low frequencies of somatic mutations compared to adult tumors and generally lack therapeutically targetable genetic alterations. Hence, we applied a systems biology approach to identify and evaluate non-genetically encoded vulnerabilities in MRT and WT. Methods: MetaVIPER analysis was performed to computationally infer protein activity from MRT and WT whole transcriptomic data available in the TARGET database. Expanded metaVIPER analysis of TARGET and TCGA cohorts demonstrated XPO1 as having consistently high inferred activity in MRT and WT. Functional in vitro studies using a selective inhibitor of XPO1, selinexor, were performed on a panel of MRT and WT cell lines to evaluate the effects of XPO1 inhibition on proliferation, cell cycle transition and apoptosis induction. In vivo validation of anti-tumor activity following XPO1 inhibition were performed in cell line-derived (CDX) and patient-derived xenograft (PDX) models of MRT and WT. Results: MetaVIPER analysis identified consistent high inferred activity of XPO1 in MRT and WT compared to other tumor types. MRT and WT cell lines demonstrated in vitro sensitivity to selinexor treatment resulting in cell cycle arrest and apoptosis induction. Furthermore, protein expression analysis showed increased nuclear sequestration of tumor suppressors proteins following treatment with selinexor. In vivo treatment of panel of MRT and WT CDX and PDX models with selinexor and a next-generation XPO1 inhibitor, eltanexor, resulted in significant abrogation of tumor growth with associated decreases in inferred XPO1 activity. Pharmacodynamic analysis of treated PDX tumors show decreased levels of XPO1, RB1-pSer780, and increased p53, p27 and p21 protein levels. Based on promising preclinical data, we describe a case report of a child with relapsed and progressive Wilms tumor who experienced a sustained complete remission on maintenance selinexor therapy. Conclusion: XPO1 represents a non-genetically encoded vulnerability in MRT and WT. Promising preclinical activity in MRT and WT models has provided the preclinical rationale for evaluation of XPO1 inhibition in an investigator-initiated clinical trial of Selinexor in pediatric MRT and WT. Citation Format: Diego F. Coutinho, Chelsey Burke, Prabhjot Mundi, Michael V. Ortiz, Kelly L. Vallance, Matthew Long, Nestor Rosales, Glorymar Ibanez, Lianna J. Marks, Daniel Diolaiti, Andoyo Ndengu, Daoqi You, Armaan Siddiquee, Ervin S. Gaviria, Allison R. Rainey, Andrea Califano, Andrew L. Kung, Filemon S. Dela Cruz. Targeting of the nuclear export protein XPO1 represents a non-genetically encoded vulnerability in malignant rhabdoid and Wilms tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1810.

TERT Expression in Wilms Tumor Is Regulated by Promoter Mutation or Hypermethylation, WT1, and N-MYC.

Simple SummaryThe telomerase enzyme adds repetitive genetic sequences to the ends of chromosomes called telomeres to prevent cellular senescence. Gain of telomerase function is one of the hallmarks of human cancer. The telomerase protein is coded by the gene TERT and increased TERT RNA levels have been associated with disease relapse in Wilms tumor, the most common kidney cancer of childhood. This study aimed to determine the mechanisms of increased TERT expression in Wilms tumor. This study found mutations in the TERT promoter, increased methylation of the TERT promoter, and genomic copy number amplifications of TERT as potential mechanisms of TERT activation. Conversely, this study found that inactivating WT1 mutation was associated with low TERT RNA levels and telomerase activity. N-MYC overexpression in Wilms tumor cells resulted in increased TERT promoter activity and TERT transcription. TERT transcription is associated with molecular and histologic subgroups in Wilms tumor and telomere-targeted therapies warrant future investigation.Increased TERT mRNA is associated with disease relapse in favorable histology Wilms tumor (WT). This study sought to understand the mechanism of increased TERT expression by determining the association between TERT and WT1 and N-MYC, two proteins important in Wilms tumor pathogenesis that have been shown to regulate TERT expression. Three out of 45 (6.7%) WTs and the corresponding patient-derived xenografts harbored canonical gain-of-function mutations in the TERT promoter. This study identified near ubiquitous hypermethylation of the TERT promoter region in WT compared to normal kidney. WTs with biallelic inactivating mutations in WT1 (7/45, 15.6%) were found to have lower TERT expression by RNA-seq and qRT-PCR and lower telomerase activity determined by the telomerase repeat amplification protocol. Anaplastic histology and increased percentage of blastema were positively correlated with higher TERT expression and telomerase activity. In vitro shRNA knockdown of WT1 resulted in decreased expression of TERT, reduced colony formation, and decreased proliferation of WiT49, an anaplastic WT cell line with wild-type WT1. CRISPR-Cas9-mediated knockout of WT1 resulted in decreased expression of telomere-related gene pathways. However, an inducible Wt1-knockout mouse model showed no relationship between Wt1 knockout and Tert expression in normal murine nephrogenesis, suggesting that WT1 and TERT are coupled in transformed cells but not in normal kidney tissues. N-MYC overexpression resulted in increased TERT promoter activity and TERT transcription. Thus, multiple mechanisms of TERT activation are involved in WT and are associated with anaplastic histology and increased blastema. This study is novel because it identifies potential mechanisms of TERT activation in Wilms tumor that could be of therapeutic interests.

Bromodomain 4 inhibition leads to MYCN downregulation in Wilms tumor.

Wilms tumor is the most common childhood kidney cancer. Two distinct histological subtypes of Wilms tumor have been described: tumors lacking anaplasia (the favorable subtype) and tumors displaying anaplastic features (the unfavorable subtype). Children with favorable disease generally have a very good prognosis, whereas those with anaplasia are oftentimes refractory to standard treatments and suffer poor outcomes, leading to an unmet clinical need. MYCN dysregulation has been associated with a number of pediatric cancers including Wilms tumor. In this context, we undertook a functional genomics approach to uncover novel therapeutic strategies for those patients with anaplastic Wilms tumor. Genomic analysis and in vitro experimentation demonstrate that cell growth can be reduced by modulating MYCN overexpression via bromodomain 4 (BRD4) inhibition in both anaplastic and nonanaplastic Wilms tumor models. We observed a time-dependent reduction of MYCN and MYCC protein levels upon BRD4 inhibition in Wilms tumor cell lines, which led to cell death and proliferation suppression. BRD4 inhibition significantly reduced tumor volumes in Wilms tumor patient-derived xenograft (PDX) mouse models. We suggest that AZD5153, a novel dual-BRD4 inhibitor, can reduce MYCN levels in both anaplastic and nonanaplastic Wilms tumor cell lines, reduces tumor volume in Wilms tumor PDXs, and should be further explored for its therapeutic potential.

MiR-378c suppresses Wilms tumor development via negatively regulating CAMKK2.

Wilms tumor is a rare kidney malignancy primarily developed in children. Treatment for Wilms tumor includes surgery, radiotherapy, and chemotherapy. Recent studies have demonstrated that microRNAs (miRNAs) play important roles in regulating Wilms tumor development. In this study, we aimed to elucidate the expression and function of miR-378c in Wilms tumor. Quantitative real-time PCR (qRT-PCR) results showed that miR-378c was downregulated in Wilms tumor tissues and cell lines. Functionally, further CCK-8, would healing, and transwell assays revealed that overexpression of miR-378c impaired Wilms tumor cell growth and metastasis in vitro. In addition, xenograft assay showed that miR-378c overexpression inhibited Wilms tumor development in vivo. Mechanistically, luciferase reporter assay confirmed that miR-378c directly targets CAMKK2 in Wilms tumor. qRT-PCR and western blot assays demonstrated that CAMKK2 was highly expressed in Wilms tumor tissues and cell lines. Rescue experiments were performed to further evaluate the functional relationship between miR-378c and CAMKK2. Overexpression of miR-378c suppressed Wilms tumor cell metastasis via negatively regulating CAMKK2 expression. Consistently, inhibition of miR-378c enhanced Wilms tumor cell malignancy behavior via augmenting CAMKK2 expression, which could be abrogated by CAMKK2 knockdown. In summary, our findings suggest that miR-378c inhibits the development and metastasis of Wilms tumor via negatively regulating CAMKK2 expression, which could be utilized to develop new therapy strategy.

Effect of piRNA NU13 in regulating biological behaviors of human Wilms tumor cells in vitro

To investigate the role of the differential piRNA NU13 derived from piwil2-induced cancer stem-like cells (piwil2-iCSCs) in regulating biological behaviors of Wilms tumor cells (G401). The expressions of piRNA NU13 and NOP56 were detected in Wilms tumor cell line G401 using RT-qPCR. G401 cells were transfected with piRNA NU13 mimics and inhibitor for its over-expression and inhibition, and the transfection efficiency was verified with RT-qPCR. The changes in proliferation of G401 cells after transfection were detected using CCK8 assay, and cell apoptosis was analyzed using flow cytometry. Wound healing assay and Transwell assay were performed to examine the changes in migration and invasion abilities of the transfected cells. The binding of NOP56 and piRNA NU13 was detected using dual luciferase experiment. The protein expressions of MMP2, MMP9, BAX, Bcl2, and NOP56 in the cells were detected with Western blotting. RTqPCR showed that the expression of piRNA NU13 decreased significantly in human Wilms tumor G401 cells as compared with that in renal tubular epithelial cell line HK2 (P < 0.05), and NOP56 was highly expressed in G401 cells and Wilms tumor tissues (P < 0.05). Over-expression of piRNA NU13 significantly suppressed the proliferation, migration and invasion of G401 cells, promoted cell apoptosis (P < 0.05), inhibited the expression of MMP2, MMP9 and Bcl2, and enhanced the expression of BAX (P < 0.05). The results of dual luciferase experiment showed that piRNA NU13 did not bind to NOP56 directly but regulated the expression of NOP56 in an indirect manner. piRNA NU13 is down-regulated and NOP56 is highly expressed in Wilms tumor. piNU13 may regulate the expression of NOP56 indirectly to inhibit the proliferation, migration and invasion and promote apoptosis of Wilms tumor cells in vitro.

Comprehensive Biology and Genetics Compendium of Wilms Tumor Cell Lines with Different WT1 Mutations.

Simple SummaryWilms tumor is a childhood kidney tumor arising from embryonal cells. Wilms tumors are heterogeneous with several distinct subgroups that differ in their response to treatment. The genetic basis for these diverse forms of Wilms tumor is not fully understood. One subgroup of Wilms tumors is associated with mutations in the WT1 gene, encoding a transcription factor with a role in early kidney differentiation. Patients with WT1 mutant Wilms tumor may harbor germline mutations in this gene. Cell lines from Wilms tumors are notoriously difficult to establish and only few exist. We developed a method to cultivate cells from the WT1 mutant subtype of Wilms tumors and have established 11 cell lines with different mutations in WT1 to date. These cells will be instrumental to study the biology and genetics ultimately to develop precision treatmentsPurpose: WT1 mutant Wilms tumors represent a distinct subgroup, frequently associated with CTNNB1 mutations. The genetic basis for the development of this subtype is currently not fully understood. Methods: Live WT1 mutant Wilms tumors were collected during surgery of patients and cell cultures established in mesenchymal stem cell medium. They were studied for mutations in WT1 and CTNNB1, their differentiation capacity and protein activation status. Four cell lines were immortalized with a triple mutant ts SV40 largeT antigen and Telomerase. Results: 11 cell lines were established from Wilms tumors of nine patients, including a left and right tumor from the same patient and a primary and second tumor from another patient. Six patients had germ line and three were tumor specific mutations. All cell lines harbored only mutant or deleted WT1 genes. CTNNB1 was wild type in three, all others carried mutations affecting amino acid S45. They had variable and limited capacities for mesenchymal differentiation, a high migratory capacity and a low invasive potential. All cells showed an activation of multiple receptor tyrosine kinases and downstream signaling pathways. Conclusions: These cell lines represent an important new tool to study WT1 mutant Wilms tumors, potentially leading to new treatment approaches.

Inhibition of Wilms' Tumor Proliferation and Invasion by Blocking TGF-β Receptor I in the TGF-β/Smad Signaling Pathway.

Wilms' tumor (WT) is a common embryonal tumor, and nephrogenic rests play a critical role in WT development. The transforming growth factor β (TGF-β) signaling pathway is fundamental to embryo development and cell growth and proliferation. Moreover, TGF-β contributes to WT development, but the mechanisms of disease pathogenicity are unknown. This study investigated whether the TGF-β signaling pathway was involved in WT and whether blocking TβRI receptor inhibited WT growth, proliferation, and invasion. A total of 60 WT patients with clinical data and surgical specimens were evaluated. Immunohistochemistry (IHC) was used to detect the expression of TGF-β1 and P-smad2/3. In vitro, the proliferation, migration, apoptosis, and epithelial-mesenchymal transition (EMT) protein expression were analyzed using the CCK8 assay, wound healing assay, transwell assay, flow cytometry, and western blot, respectively. In vivo, tumor morphology, tumor size, toxicity, and EMT protein expression were analyzed in tumor-bearing mice treated with a TβRI kinase inhibitor or PBS. High protein levels of TGF-β1 and P-samd2/3 were associated with clinical stage and metastasis or invasion. TβRI inhibition effectively suppressed WT proliferation and migration and promoted apoptosis in the human WT cell line G401, consequently decreasing EMT protein expression. In addition, the TβRI kinase inhibitor significantly impaired the subcutaneous growth of WT. It is worth noting that treatment with the TβRI kinase inhibitor did not cause liver and kidney injury. Our results indicate that the TGF-β/Smad signaling pathway plays a crucial role in WT progression. Blocking the TβRI receptor may be a novel strategy to treat and prevent WT.

LncRNA XIST promotes migration of Wilms' tumor cells through modulation of microRNA-193a-5p.

The aim of this study was to investigate long non-coding RNA (lncRNA) XIST expression in Wilms' tumor (WT) and to further explore its relationship with clinical features and prognosis of WT patients. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was carried out to examine the expression level of XIST in tumor tissue samples and paracancerous ones collected from 43 patients with renal cell carcinoma, and the interplay between XIST expression and clinical indicators, as well as prognosis of patients was analyzed. Meanwhile, XIST level in the nephroblast cancer cell line was further confirmed by qRT-PCR. In addition, XIST knockdown model was constructed using lentivirus in the WT cell lines, including HFWT and 17-94, and the influence of XIST on WT cell functions was analyzed through transwell assay. Finally, we investigated whether lncRNA XIST plays a role in the progression of WT by modulating microRNA-193a-5p. In this research, qRT-PCR results revealed a significantly higher expression of lncRNA XIST in tumor tissue specimens of patients with renal cell carcinoma than that in adjacent ones. Compared with patients with low expression of lncRNA XIST, those with high XIST expression had a higher incidence of distant metastasis and a lower overall survival rate. Compared with the negative control group, the metastatic ability of WT cells in the lncRNA XIST knockdown group was markedly weakened. In addition, the results of qPCR showed that mRNA expression of lncRNA XIST and microRNA-193a-5p were negatively correlated in renal cell carcinoma tissue specimens. At the same time, silencing microRNA-193a-5p reversed the reduced metastasis ability of WT cells induced by knockdown of XIST. LncRNA XIST expression is dramatically enhanced in WT tissues and cell lines, which is closely associated with the incidence of distant metastasis and patients' poor prognosis. In addition, we demonstrated that lncRNA XIST may accelerate the malignant progression of WT via inhibiting microRNA-193a-5p.

MiR-30d Induced Apoptosis by Targeting Sox4 to Inhibit the Proliferation, Invasion and Migration of Nephroblastoma.

BackgroundWilms tumor (WT) is an embryonic malignant tumor, and its related mechanism is still unclear. microRNA (miR), as a short-chain non-coding RNA, has low expression in various tumors. In this study, WT differential miR was screened by multi-chip in GEO database and its mechanism was explored to provide potential therapeutic targets and ideas for clinic.MethodsWe logged into GEO database and downloaded GSE57370 and GSE48137 chip matrix files to analyze potential differences in miR. TargetScan, miRDB, miRTarBase and starBase were applied to predict the target genes of miR with significant differences. qRT-PCR was applied to determine the expression of miR-30d and Sox4 in WT tissue and cell line (G401). The interaction of miR-30d with Sox4 was confirmed by qRT-PCR, Western blot and luciferase assay, respectively. CCK-8, Transwell and flow cytometry were applied to determine the proliferation, invasion, migration and apoptosis of cells.ResultsWe found that miR-30d was low expressed in two chips. qRT-PCR showed that miR-30d was down-regulated and SOX4 was up-regulated in WT tissues and cells. The online target gene prediction software showed there was a targeted binding site between Sox4 and miR-30d. Sox4 was negatively controlled by miR-30d. Subsequent studies found that over-expression of miR-30d inhibited the proliferation, invasion, migration and induced apoptosis of C64 and WiT49 cells. In addition, Sox4 could reverse the proliferation, invasion and migration of C64 and WiT49 induced by miR-30d and induce apoptosis.ConclusionmiR-30d is poorly expressed in WT and can induce apoptosis and inhibit proliferation, invasion and migration by mediating Sox4.

MiR-22-3p regulates the proliferation and invasion of Wilms' tumor cells by targeting AKT3.

In this study, the regulatory mechanism of miR-22-3p/AKT3 in the development of Wilms' tumor (WT) was investigated. Twenty-seven pairs of surgical tumor specimens and adjacent normal tissues were obtained from Jining No. 1 People's Hospital. The expression level of miR-22-3p in WT tissues and cell lines was measured by quantitative RT-PCR. MTT and transwell assays were performed to analyze cell proliferation and invasion in WT. The relationship between miR-22-3p and AKT3 was verified by a Dual-Luciferase assay. The protein expression of AKT3 was evaluated by Western blotting analysis. MiR-22-3p was downregulated and AKT3 was upregulated in WT. Functionally, overexpression of miR-22-3p inhibited cell proliferation and invasion in WT. Moreover, miR-22-3p directly targets AKT3. The knockdown of AKT3 suppressed cell proliferation and invasion in WT. In addition, upregulation of AKT3 restored the tumor suppressive effect of miR-22-3p in WT. MiR-22-3p inhibits the proliferation and invasion of WT cells by downregulating AKT3, indicating that miR-22-3p may be developed as a new biomarker for the diagnosis of WT.