Novel patient-derived models of DSRCT enable validation of ERBB signaling as a potential therapeutic vulnerability

AbstractDesmoplastic small round cell tumor (DSRCT) is characterized by the t(11;22)(p13;q12) chromosomal translocation, which fuses the transcriptional regulatory domain of EWSR1 with the zinc finger DNA-binding domain of WT1, resulting in the oncogenic transcription factor EWS-WT1. DSRCT primarily affects young males and has a 5-year overall survival of about 15%. Typical treatment approaches for patients with DSRCT involve a multi-modal combination of surgery, chemotherapy and radiation. The paucity of DSRCT disease models has hampered functional and pre-clinical therapeutic studies in this aggressive cancer. Here, we developed robust preclinical disease models and mined DSRCT expression profiling data to identify genetic vulnerabilities that could be leveraged for the identification of rational therapies. Specifically, we developed four new DSRCT cell lines and one patient-derived xenograft (PDX) model. Transcriptomic and proteomic profiling showed evidence of activation of the ERBB pathway. Ectopic expression of EWSR1-WT1 resulted in upregulation of ERRB family ligands and downstream signaling. Treatment of DSRCT cell lines with ERBB ligands resulted in activation of EGFR, ERBB2, ERK1/2 and AKT, and stimulation of cell growth. Conversely, targeting of EGFR using shRNA, small molecule inhibitors (afatinib, neratinib) or an anti-EGFR antibody (cetuximab) inhibited growth and induced apoptosis in DSRCT cells. Finally, treatment of mice bearing DSRCT xenografts with a combination of cetuximab and afatinib significantly reduced tumor growth. These data provide a rationale for the clinical evaluation of EGFR antagonists in patients with DSRCT.

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Different regulation of chromatin modification guide cellular reprogramming into a rate-limited step after initiation phase

10.7287/peerj.preprints.1991 ◽

2016 ◽

Author(s):

Rong Hu ◽

Xianhua Dai ◽

Zhiming Dai ◽

Qian Xiang

Keyword(s):

Cell Cycle ◽

Cell Adhesion ◽

Cell Lines ◽

Expression Profiles ◽

Chromatin Modification ◽

Ectopic Expression ◽

Gene Expression Profiles ◽

Cellular Reprogramming ◽

Ips Cell ◽

Initiation Phase

Background. In the early and late stages of cell reprogramming to induced pluripotent stem cells (iPSCs) ectopic expression of Oct4, Sox2, Klf4 and Myc (OSKM) aroused two peaks of transcriptional and epigenetic change respectively. However, it was relatively quiet in the intermediate stage. In this paper our aim is to gain insight into the molecular events that occur after the initiation phase of pluripotency induction. Methods. GSE42379 containing 28 gene expression profiles and GSE42477 containing 10 genome binding/occupancy profiles of mouse embryonic fibroblasts (MEF) were downloaded from GEO. These datasets included untreated MEFs, OSKM-induced MEFs progressing and refractory to reprogram at 3, 6, 9, 12 day post-transduction, and iPS cell lines. Differentially expressed genes (DEGs) were identified between different cell lines. The Chip-seq peaks and putative target gene were obtained from GeneProf website. Gene ontology analysis was performed on the Ensemble website. Result. Compared with the progressing cells, the refractory cells obtained more than two times DEGs at 6 day post-transduction, in particular, down-regulated DEGs related to cell cycle, cell adhesion and development were over 4 times of that in progressing cells. The expression of the DEGs which could only be detected in refractory cells at 6 day were traced back to day 3, and we found the expression of the up-regulated DEGs at 3 day were higher in refractory cells, whereas the expression of the down-regulated DEGs at 3 day were lower in refractory cells. The analysis of histone modification in genome-wide and in DEGs showed that during the reprogramming process the increase of bivalent sites in genome were mainly attributed to gaining of H3K27me3 and losing of H3K4me3. Different regulation of H3K27me3 in DEGs was the key to regulate the expression differently in progressing and refractory cells. The expression of chromatin modifiers in the two cell populations were checked and found to be differential regulated at different time point during reprogramming process. Conclusion. Genes related to immune response, cell adhesion, DNA replication and cell cycle in the refractory cells responded to the induction factor earlier than in the progressing cells, which led to excessive conversion rate. We supposed that after initiation phase cellular reprogramming required to undergo a rate-limited step guided by different regulation of chromatin modification.

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ABT-806 derived antibody drug conjugates (ADCs) inhibit growth of malignant mesothelioma in-vivo.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e14677 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e14677-e14677

Author(s):

Puey Ling Chia ◽

Diana Cao ◽

Hui Kong Gan ◽

Edward B. Reilly ◽

Andrew Phillips ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Malignant Mesothelioma ◽

Xenograft Model ◽

Drug Conjugates ◽

Pdx Model ◽

Anti Tumor Activity ◽

One Way Anova ◽

Egfr Antibody

e14677 Background: Malignant mesothelioma (MM) is an aggressive malignancy of the pleura with limited therapeutic options, and is associated with a poor prognosis. EGFR is known to be highly over-expressed in mesothelioma with reported EGFR overexpression between 44 to 97%. We have investigated an anti-EGFR antibody (ABT-806), which is tumor specific and robustly inhibits EGFR-expressing tumors. We have previously shown that ABT-806 ADCs demonstrate potent anti-tumor activity in 806 immunohistochemistry (IHC) positive MSTO-211H MM cell line xenograft model. We present data in MM using ABT-806 novel ADCs [ABT-414 (ABT-806- monomethyl auristatin F), ABBV-221 (ABT-806- monomethyl auristatin E) and ABBV-321 (ABT-806- pyrrolobenzodiazepine)] in MM patient derived xenografts (PDX). Methods: We evaluated expression of EGFR and mAb 806 IHC in MM cell lines and PDXs. PDXs were implanted into 5 to 10 NOD-Scid mice per group and treated with control ADC, saline, cisplatin or ABT-806 ADCs and followed longitudinally with caliper measurements. Comparative statistics were performed in Graphpad prism. Results: Three PDX models were selected according to their 806 IHC statuses (2 epithelioid 806 IHC positive, 1 biphasic histology 806 IHC negative). In one epithelioid PDX model, ABBV-321 resulted in significantly reduced tumor growth on day 27 post therapy with median tumor volumes of 180 mm3 (ADC control) compared with 78mm3 (ABBV-321; p = 0.0159 two-sided). Moreover, the median survival was also significantly longer in ABBV-321 treated models (p = 0.018). In the other epithelioid PDX model, ABBV-321 also resulted in significant responses (median 428mm3 (ADC control) vs 167mm3 (ABBV-321, p = 0.0201). In the 806 IHC negative PDX model, the differences in tumor volumes between all groups were found to be non-statistically significant (ADC control vs ABT-414, ADC control vs ABBV-221, ADC-control vs ABBV-321 groups) with p = 0.0597 for one-way ANOVA. MSTO211H cell line xenograft model also demonstrated significant anti-tumor response to both ABT-414 and ABBV-221 (p < 0.01). Conclusions: In a disease with limited therapies, ABT-806 targeting ADCs in MM demonstrated significant responses in 806+ PDX and cell lines. These data support clinical expansion of these compounds in 806+ MM patients.

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Potential Eradication of Myeloma Stem Cells by Combining ATRA with WNT and HH Signaling Pathways Inhibitors.

Blood ◽

10.1182/blood.v114.22.2839.2839 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2839-2839 ◽

Cited By ~ 1

Author(s):

Guido J. Tricot ◽

Siqing Wang ◽

Lei Shi ◽

Hongwei Xu ◽

Chunjiao Xu ◽

...

Keyword(s):

Stem Cells ◽

Signaling Pathways ◽

Cell Lines ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Stem Cell Population ◽

Molecular Characteristics ◽

Myeloma Cells ◽

Hh Signaling ◽

Stimulation Of

Abstract Abstract 2839 Poster Board II-815 Multiple myeloma (MM) remains largely incurable. The existence of the putative myeloma stem cell population may account for drug-resistance and relapse. Previous investigation showed that the CD138-negative (CD138-) MM cell fraction contains the putative MM stem cells. However, little is known about the molecular characteristics of MM stem cells, which makes it difficult to specifically target such cells. In this study, by comparing gene expression profiles (GEP) of CD138+ and CD138- cells isolated from 10 MM cell lines, we discovered that RARα is the top one over-expressed gene in CD138- MM stem cells. RARα has two major isoforms, RARα1 and RARalpha2. Real-time PCR detected significantly higher expression of RARalpha2 but not of RARalpha1 in CD138- MM cells compared with CD138+ cells. We recently reported that increased RARalpha2 expression at diagnosis resulted in a significantly shorter overall-survival (P = 0.003); importantly, ATRA selectively killed RARalpha2-positive myeloma cells, but not RARalpha2-negative cells. These results suggested that ATRA could be used to eradicate specifically RARalpha2-over-expressing MM stem cells. Indeed, we found that ATRA selectively killed CD138- MM stem cells (P < 0.01) from ARP-1 and OCI-MY5 human myeloma cell lines and 5T33 murine myeloma cells but spared the CD138+ tumor cells from these cell lines. WNT and Hedgehog (HH) signaling pathways were activated in CD138- stem cells. To our surprise, ATRA treatment (10-6M) further increased WNT and HH signaling pathways in CD138- myeloma cells, based on increased protein levels of β-catenin and cleaved Shh/Ihh in ATRA-treated cells. Stimulation of these signaling pathways by LiCl (5 mM) and/or SHH(20 μg/mL) partially abrogated ATRA-induced cytotoxities on CD138- MM cells, demonstrating that stimulation of WNT and HH signaling induced partial ATRA-resistance in CD138- cells. A combinational treatment of ATRA (1 μM), a COX-2 inhibitor celecoxib (shown to inhibit WNT signaling, 50 μM) and a HH signaling inhibitor cyclopamine (10 μM) induced synergistic effects on cell death and growth inhibition of CD138- and RARalpha2-over-expressing MM cells in-vitro. In the 5T33 murine myeloma model, the combination of ATRA (20mg/Kg), celecoxib (10mg/Kg) and cyclopamine (20mg/Kg) induced synergistic inhibition of tumor growth in-vivo. Thus, our study provides novel approaches to specifically target MM stem cells. Disclosures: No relevant conflicts of interest to declare.

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Different regulation of chromatin modification guide cellular reprogramming into a rate-limited step after initiation phase

10.7287/peerj.preprints.1991v1 ◽

2016 ◽

Author(s):

Rong Hu ◽

Xianhua Dai ◽

Zhiming Dai ◽

Qian Xiang

Keyword(s):

Cell Cycle ◽

Cell Adhesion ◽

Cell Lines ◽

Expression Profiles ◽

Chromatin Modification ◽

Ectopic Expression ◽

Gene Expression Profiles ◽

Cellular Reprogramming ◽

Ips Cell ◽

Initiation Phase

Background. In the early and late stages of cell reprogramming to induced pluripotent stem cells (iPSCs) ectopic expression of Oct4, Sox2, Klf4 and Myc (OSKM) aroused two peaks of transcriptional and epigenetic change respectively. However, it was relatively quiet in the intermediate stage. In this paper our aim is to gain insight into the molecular events that occur after the initiation phase of pluripotency induction. Methods. GSE42379 containing 28 gene expression profiles and GSE42477 containing 10 genome binding/occupancy profiles of mouse embryonic fibroblasts (MEF) were downloaded from GEO. These datasets included untreated MEFs, OSKM-induced MEFs progressing and refractory to reprogram at 3, 6, 9, 12 day post-transduction, and iPS cell lines. Differentially expressed genes (DEGs) were identified between different cell lines. The Chip-seq peaks and putative target gene were obtained from GeneProf website. Gene ontology analysis was performed on the Ensemble website. Result. Compared with the progressing cells, the refractory cells obtained more than two times DEGs at 6 day post-transduction, in particular, down-regulated DEGs related to cell cycle, cell adhesion and development were over 4 times of that in progressing cells. The expression of the DEGs which could only be detected in refractory cells at 6 day were traced back to day 3, and we found the expression of the up-regulated DEGs at 3 day were higher in refractory cells, whereas the expression of the down-regulated DEGs at 3 day were lower in refractory cells. The analysis of histone modification in genome-wide and in DEGs showed that during the reprogramming process the increase of bivalent sites in genome were mainly attributed to gaining of H3K27me3 and losing of H3K4me3. Different regulation of H3K27me3 in DEGs was the key to regulate the expression differently in progressing and refractory cells. The expression of chromatin modifiers in the two cell populations were checked and found to be differential regulated at different time point during reprogramming process. Conclusion. Genes related to immune response, cell adhesion, DNA replication and cell cycle in the refractory cells responded to the induction factor earlier than in the progressing cells, which led to excessive conversion rate. We supposed that after initiation phase cellular reprogramming required to undergo a rate-limited step guided by different regulation of chromatin modification.

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Characterization of Two Ethephon-Induced IDA-Like Genes from Mango, and Elucidation of Their Involvement in Regulating Organ Abscission

Genes ◽

10.3390/genes12030439 ◽

2021 ◽

Vol 12 (3) ◽

pp. 439

Author(s):

Avinash Chandra Rai ◽

Eyal Halon ◽

Hanita Zemach ◽

Tali Zviran ◽

Isaac Sisai ◽

...

Keyword(s):

Expression Profiles ◽

Mangifera Indica ◽

Ectopic Expression ◽

Fruit Trees ◽

Floral Organ ◽

Cytosolic Ph ◽

Early Increase ◽

Encoding Genes ◽

Fruitlet Abscission

In mango (Mangifera indica L.), fruitlet abscission limits productivity. The INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) peptide acts as a key component controlling abscission events in Arabidopsis. IDA-like peptides may assume similar roles in fruit trees. In this study, we isolated two mango IDA-like encoding-genes, MiIDA1 and MiIDA2. We used mango fruitlet-bearing explants and fruitlet-bearing trees, in which fruitlets abscission was induced using ethephon. We monitored the expression profiles of the two MiIDA-like genes in control and treated fruitlet abscission zones (AZs). In both systems, qRT-PCR showed that, within 24 h, both MiIDA-like genes were induced by ethephon, and that changes in their expression profiles were associated with upregulation of different ethylene signaling-related and cell-wall modifying genes. Furthermore, ectopic expression of both genes in Arabidopsis promoted floral-organ abscission, and was accompanied by an early increase in the cytosolic pH of floral AZ cells—a phenomenon known to be linked with abscission, and by activation of cell separation in vestigial AZs. Finally, overexpression of both genes in an Atida mutant restored its abscission ability. Our results suggest roles for MiIDA1 and MiIDA2 in affecting mango fruitlet abscission. Based on our results, we propose new possible modes of action for IDA-like proteins in regulating organ abscission.

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Identification and Characterization of Alternatively Spliced Transcript Isoforms of IRX4 in Prostate Cancer

Genes ◽

10.3390/genes12050615 ◽

2021 ◽

Vol 12 (5) ◽

pp. 615

Author(s):

Achala Fernando ◽

Chamikara Liyanage ◽

Afshin Moradi ◽

Panchadsaram Janaththani ◽

Jyotsna Batra

Keyword(s):

Prostate Cancer ◽

Cell Lines ◽

Expression Profiles ◽

Association Studies ◽

Protein Isoforms ◽

The Cancer Genome Atlas ◽

Mass Spectrometry Analysis ◽

Functional Domains ◽

Homeodomain Protein ◽

Genome Wide Association Studies

Alternative splicing (AS) is tightly regulated to maintain genomic stability in humans. However, tumor growth, metastasis and therapy resistance benefit from aberrant RNA splicing. Iroquois-class homeodomain protein 4 (IRX4) is a TALE homeobox transcription factor which has been implicated in prostate cancer (PCa) as a tumor suppressor through genome-wide association studies (GWAS) and functional follow-up studies. In the current study, we characterized 12 IRX4 transcripts in PCa cell lines, including seven novel transcripts by RT-PCR and sequencing. They demonstrate unique expression profiles between androgen-responsive and nonresponsive cell lines. These transcripts were significantly overexpressed in PCa cell lines and the cancer genome atlas program (TCGA) PCa clinical specimens, suggesting their probable involvement in PCa progression. Moreover, a PCa risk-associated SNP rs12653946 genotype GG was corelated with lower IRX4 transcript levels. Using mass spectrometry analysis, we identified two IRX4 protein isoforms (54.4 kDa, 57 kDa) comprising all the functional domains and two novel isoforms (40 kDa, 8.7 kDa) lacking functional domains. These IRX4 isoforms might induce distinct functional programming that could contribute to PCa hallmarks, thus providing novel insights into diagnostic, prognostic and therapeutic significance in PCa management.

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Genome-Wide Role of HSF1 in Transcriptional Regulation of Desiccation Tolerance in the Anhydrobiotic Cell Line, Pv11

International Journal of Molecular Sciences ◽

10.3390/ijms22115798 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5798

Author(s):

Shoko Tokumoto ◽

Yugo Miyata ◽

Ruslan Deviatiiarov ◽

Takahiro G. Yamada ◽

Yusuke Hiki ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Desiccation Tolerance ◽

Expression Profiles ◽

Insect Cell Line ◽

Gene Expression Profiles ◽

Late Embryogenesis Abundant ◽

Heat Shock Factor 1 ◽

Genome Wide ◽

A Genome

The Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.

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Dengue Virus Induces the Expression and Release of Endocan from Endothelial Cells by an NS1–TLR4-Dependent Mechanism

Microorganisms ◽

10.3390/microorganisms9061305 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1305

Author(s):

Carlos Alonso Domínguez-Alemán ◽

Luis Alberto Sánchez-Vargas ◽

Karina Guadalupe Hernández-Flores ◽

Andrea Isabel Torres-Zugaide ◽

Arturo Reyes-Sandoval ◽

...

Keyword(s):

Endothelial Cell ◽

Mrna Expression ◽

Cell Lines ◽

Cell Activation ◽

Dengue Infection ◽

Elevated Serum ◽

Fold Increase ◽

Endothelial Cell Activation ◽

Stimulation Of

A common hallmark of dengue infections is the dysfunction of the vascular endothelium induced by different biological mechanisms. In this paper, we studied the role of recombinant NS1 proteins representing the four dengue serotypes, and their role in promoting the expression and release of endocan, which is a highly specific biomarker of endothelial cell activation. We evaluated mRNA expression and the levels of endocan protein in vitro following the stimulation of HUVEC and HMEC-1 cell lines with recombinant NS1 proteins. NS1 proteins increase endocan mRNA expression 48 h post-activation in both endothelial cell lines. Endocan mRNA expression levels were higher in HUVEC and HMEC-1 cells stimulated with NS1 proteins than in non-stimulated cells (p < 0.05). A two-fold to three-fold increase in endocan protein release was observed after the stimulation of HUVECs or HMEC-1 cells with NS1 proteins compared with that in non-stimulated cells (p < 0.05). The blockade of Toll-like receptor 4 (TLR-4) signaling on HMEC-1 cells with an antagonistic antibody prevented NS1-dependent endocan production. Dengue-infected patients showed elevated serum endocan levels (≥30 ng/mL) during early dengue infection. High endocan serum levels were associated with laboratory abnormalities, such as lymphopenia and thrombocytopenia, and are associated with the presence of NS1 in the serum.

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Polyamine-modulated c-Myc expression in normal intestinal epithelial cells regulates p21Cip1 transcription through a proximal promoter region

Biochemical Journal ◽

10.1042/bj20060217 ◽

2006 ◽

Vol 398 (2) ◽

pp. 257-267 ◽

Cited By ~ 37

Author(s):

Lan Liu ◽

Xin Guo ◽

Jaladanki N. Rao ◽

Tongtong Zou ◽

Bernard S. Marasa ◽

...

Keyword(s):

Cell Proliferation ◽

Transcriptional Repression ◽

Critical Role ◽

Ectopic Expression ◽

Proximal Region ◽

Proximal Promoter ◽

Transcriptional Level ◽

Intestinal Epithelial ◽

Major Player ◽

Stimulation Of

Maintenance of intestinal mucosal epithelial integrity requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest and apoptosis. Our previous studies have shown that polyamines are essential for expression of the c-myc gene and that polyamine-induced c-Myc plays a critical role in stimulation of normal IEC (intestinal epithelial cell) proliferation, but the exact downstream targets of induced c-Myc are still unclear. The p21Cip1 protein is a major player in cell cycle control, which is primarily regulated at the transcriptional level. The current study was designed to determine whether induced c-Myc stimulates normal IEC proliferation by repressing p21Cip1 transcription following up-regulation of polyamines. Overexpression of the ODC (ornithine decarboxylase) gene increased levels of cellular polyamines, induced c-Myc expression and inhibited p21Cip1 transcription, as indicated by repression of p21Cip1 promoter activity and a decrease in p21Cip1 protein levels. In contrast, depletion of cellular polyamines by inhibiting ODC enzyme activity with α-difluoromethylornithine decreased c-Myc, but increased p21Cip1 transcription. Ectopic expression of wild-type c-myc not only inhibited basal levels of p21Cip1 transcription in control cells, but also prevented increased p21Cip1 in polyamine-deficient cells. Experiments using different p21Cip1 promoter mutants showed that transcriptional repression of p21Cip1 by c-Myc was mediated through Miz-1- and Sp1-binding sites within the proximal region of the p21Cip1 promoter in normal IECs. These findings confirm that p21Cip1 is one of the direct mediators of induced c-Myc following increased polyamines and that p21Cip1 repression by c-Myc is implicated in stimulation of normal IEC proliferation.

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