The nucleocytoplasmic transport processes are mediated by soluble transport factors constantly navigating between nuclear and cytoplasmic compartments. Our understanding about nuclear export of ...general ‘nuclear import factors’ that deliver the cargo to the nucleus is still fragmentary. Utilizing green fluorescent protein tagged glucocorticoid receptor (GR) and relA as our working model and with judicious use of LMB, we show in living cells that all the soluble components of the nuclear import machinery exit nucleus via exportin1/CRM1 independent pathway(s).
Abstract
Medulloblastoma (MB) is the most common malignant brain tumor in children that arises from cerebellar neuronal progenitor cells. Despite aggressive treatment involving radiation and ...chemotherapy, the prognosis for high-risk MB remains poor and long-term complications from current therapies are common. Therefore, new effective therapies based on the molecular features of MB are needed to improve therapeutic outcomes. The STAT3 transcription factor is known to be constitutively activated in a variety of human cancers, including MB and functions as an oncoprotein, mediating cancer cell survival, proliferation, migration and drug-resistance. We have delineated the functional role of STAT3 NH2-Terminal Domain (NTD) in MB by using a cell permeable peptide derivative of the STAT3 second helix that specifically binds and perturbs the structure/function of STAT3 and interferes with tetramerization of STAT3 dimers, protein-protein interactions and target genes transcription. Herein, we report that treatment of MB cells with STAT3-NTD inhibitor (S3-NTDi) leads to growth inhibition, cell cycle arrest and apoptosis. The inhibition of STAT3 signaling was also confirmed by downregulation of its downstream targets, including MYC, CCND1, BCl2L1, BCL2, PIM1 and APEX1. Moreover, we observed that S3-NTDi exposure attenuated the migration of MB cells in a wound-healing assay, a prerequisite for tumor invasion and metastasis. We also found that S3-NTDi abrogated IL-6 induced epithelial-mesenchymal transition (EMT) marker protein expression and inhibition of EMT-related transcription factors SNAIL and TWIST. Most importantly, we observed that combination therapy with S3-NTDi and cisplatin significantly decreased the highly aggressive MYC-driven MB cell growth in a dose dependent manner and induced apoptosis by downregulating STAT3 regulated anti-proliferative and anti-apoptotic gene expression. To elucidate the mechanisms of S3-NTDi mediated inhibition, we showed that S3-NTDi upregulated expression of pro-apoptotic gene C/EBP-homologous protein (CHOP) and concomitantly decreased association of the STAT3 transcription factor to endogenous proximal promoter of CCND1 and BCL2 in chromatin immunoprecipitation assay. Furthermore, we determined that S3-NTDi mediated downregulation of miRNA-21 in MB cells, de-repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 which, in turn, attenuated STAT3 signaling pathway. Overall, our results revealed an important role of STAT3 NTD and its downstream effector molecules in regulating MB pathogenesis and disruption of this pathway with S3-NTDi may serves as a promising new candidate for therapeutic interventions in MB therapy, thereby improving the outcomesin high-risk pediatric MB patients.
Citation Format: Sutapa Ray, Don W. Coulter, Shawn D. Gray, Jason A. Sughroue, Nagendra K. Chaturvedi, Shantaram S. Joshi, Kishor K. Bhakat, Timothy R. McGuire, John G. Sharp. STAT3 NH2-terminal domain inhibition sensitizes medulloblastoma cells to chemotherapy abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1064. doi:10.1158/1538-7445.AM2017-1064
Abstract
Neuroblastoma is the most devastating extracranial solid malignancy in children. Despite an intense treatment regimen, the prognosis for high-risk neuroblastoma patients remains poor, with ...less than 40% survival. So far, MYCN status/amplification is considered the most prognostic factor but corresponds to only 25% of neuroblastoma patients. Therefore, it is essential to identify a better prognosis and prediction marker of therapy response in neuroblastoma patients. The identification of master regulators with good prognostic and drug response abilities are not successful due to the complexity and lack of data-driven bioinformatic workflows. We applied robust bioinformatic data mining tools such as Weighted Gene Co-expression Network Analysis, cisTarget, and Single-Cell rEgulatory Network Inference and Clustering on three neuroblastoma patient datasets (n=1252). We found Sin3A Associated Protein 30 (Sap30), a driver transcription factor positively associated with high-risk, progression, stage 4, and poor survival in high-risk neuroblastoma patient cohorts. Tumors of high-risk neuroblastoma patients and relapse-specific patient-derived xenografts showed higher Sap30 levels. The Genomics of Drug Sensitivity in Cancer, The Cancer Cell Line Encyclopedia, and CRISPR-Cas9 screens indicated that Sap30 essentiality is associated with Cisplatin resistance and further showed higher levels in Cisplatin resistant patient-derived xenograft tumor cell lines. The silencing of Sap30 in cells reduced cell growth, induced cell death, mitochondrial membrane potential loss in vitro, and reduced tumorigenicity in vivo. Altogether, these results indicate that Sap30 is a novel prognostic and Cisplatin resistant marker and thus a potential drug target in high-risk neuroblastoma patients.
Citation Format: Philip Prathipati, Anup S. Pathania, Nagendra K. Chaturvedi, Subash C. Gupta, Siddappa N. Byrareddy, Don W. Coulter, Kishore B. Challagundla. SAP30, a novel drug response specific transcription factor in high-risk neuroblastoma 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 2349.
Abstract
Neuroblastoma is the most common pediatric malignancy with poor response to current therapy. The NF-kB/mTOR, hedgehog and PLK1 pathways/molecules are aberrantly expressed and activated in ...high-risk neuroblastoma, thereby targeting these pathways/molecules is an attractive therapeutic strategy. Therefore, we investigated the efficacy and associated molecular mechanism(s) of NF-kB/mTOR dual inhibitor 13-197, hedgehog inhibitor Vismodegib and PLK1 inhibitor BI2536 alone or in combination with Topotecan against high-risk neuroblastoma in vitro and in vivo. Using three neuroblastoma cell lines, the in vitro efficacy of the inhibitors as single agents or in combination with Topotecan on cell growth and apoptosis along with associated molecular mechanism(s) were investigated. In addition, the combined efficacy of Vismodegib and Topotecan was determined in vivo using a xenograft mouse model. Results showed that 13-197, BI2536 and Vismodegib significantly decreased neuroblastoma cell growth and induced apoptosis by targeting associated pathways and molecules in vitro. The 13-197 and BI2536 as single agents showed similar efficacies and were most potent in inhibiting cell growth and survival of neuroblastoma cells. In combination with Topotecan, BI2536 or Vismodegib further significantly decreased neuroblastoma cell growth/survival and neurospheres formation. Corresponding changes in the expression of targeted molecules following therapy were observed. Together, in vitro data demonstrated that hedgehog pathway inhibitor Vismodegib was most efficacious in potentiating Topotecan-induced antineuroblastoma effects. Therefore, as a next logical step, we tested the combined efficacy of Vismodegib and Topotecan against neuroblastoma in vivo using NOD-SCID Gamma deleted (NSG) mice. Our in vivo results showed that Vismodegib combined with Topotecan significantly (p<0.001) reduced tumor growth and increased survival of xenograft mice compared to single agent activity. This combination not only significantly reduced tumor growth and increased survival of the mice but also significantly enhanced antineuroblastoma efficacy by targeting hedgehog pathway components in neuroblastoma in vivo. Thus our studies lay a foundation for taking this therapeutic strategy to clinical setting to determine their efficacies in high-risk neuroblastoma patients.
Citation Format: Nagendra K. Chaturvedi, McGuire R. Timothy, Don W. Coulter, Ashima Shukla, Erin M. McIntyre, J. Graham Sharp, Shantaram S. Joshi. Improved therapy for neuroblastoma using small molecule inhibitors in combination with chemotherapy. abstract. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B05.
Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma (NHL) which is one of the most aggressive lymphomas. Despite recent improvements in therapies, the development of therapy-resistance is ...still a major problem; therefore, in order to understand the molecular basis of therapy-resistance, stable therapy-resistant MCL cell lines have been established by us. Based on the gene expression profiles of these cell lines, Polo-like kinase 1 (PLK1) was chosen as a therapeutic target. In this paper, we demonstrate a significant antilymphoma effect of targeting PLK1 in therapy-resistant MCL cells and primary MCL cells from refractory patients. PLK1 knockdown with the antisense oligonucleotide (ASO)/or small molecule inhibitor BI2536 showed significantly decreased proliferation and increased apoptosis in therapy-resistant MCL cell lines and MCL primary cells. Additionally, the direct protein-protein interaction partners of PLK1 were mapped using ingenuity pathway and confirmed the level of association of these partners with PLK1 based on their expression changes following PLK1 knockdown using real-time PCR. Results suggest that PLK1 is a viable target for the treatment of therapy-resistant MCL.
Chronic Lymphocytic Leukemia (CLL) represents the most common adult leukemia in western countries, affecting approximately 10,000 individuals every year in United States of America. In order to ...develop effective therapeutic strategies there is a need to understand the precise molecular events associated with CLL development and progression. In an attempt to understand the process we performed transcriptome analysis of CLL cells from peripheral blood (PB) of 7 good prognosis and 8 poor prognosis patients. Additionally to validate the results further, we have utilized gene expression profiling data of CLL cells from 77 good or poor prognosis patients. Using transcriptome and gene expression profiling, we identified PR Domain Zinc Finger Protein1 (PRDM1) as one of the candidate tumor suppressor genes in CLL. PRDM1/BLIMP1 is a transcriptional repressor which is crucial for terminal differentiation of mature B cells into plasma cells. PRDM1 has been shown to promote differentiation by repressing genes essential for B cell receptor signaling and cellular proliferation. Our results demonstrated that PRDM1 expression was significantly (p < 0.05) decreased in CLL cells from poor prognosis compared to good prognosis CLL cases (Figure 1). In addition, to determine the clinical significance, the expression levels of PRDM1 were found to correlate with time to treatment in CLL patients (Figure 2). Lower expression of PRDM1 was associated (p=0.001) with shorter time to treatment in CLL (n = 40). Furthermore, using IPA analyses we identified PRDM1 interacting partners. Among those, there was increased expression of BCL2, PAX5, EHMT2, SPIB and decreased expression of BCL6, IRF4, BCR, CASP3, pFOS, TP53 and HDAC2 in CLL cells from poor prognosis patients in comparison with good prognosis. Also we have observed the differential expression of TLE1, a co-repressor molecule associated with PRDM1 and member of the Groucho family of proteins. Display omitted
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Together these results are suggestive of a role for PRDM1 as a negative regulator of CLL aggressiveness and progression. These studies warranted additional investigation to elucidate the mechanism of PRDM1 mediated CLL progression and to identify a therapeutic target.
No relevant conflicts of interest to declare.
Abstract
Butrylcholinesterase (BChE) is a glycoprotein found in the peripheral and central nervous system including in neuroblastoma, a tumor of the peripheral sympathetic nervous system. The use of ...BChE as a tumor biomarker and potential therapeutic target has been proposed. The purpose of this study was to assess the association between BChE and tumorigenicity in two NB cell lines and their respective stem cell populations. Two MYCN amplified neuroblastoma cell lines, BE-2 (highly MYCN amplified) and IMR-32 (intermediately MYCN amplified) were studied along with neuroblastoma stem cells identified by side population (SP) in flow cytometry. MYCN protein, which is a poor prognostic proliferative marker in neuroblastoma, was measured by western blot in each of the two cell lines to characterize and confirm the amplification phenotype. Cell cycle characteristics of each cell line was evaluated using a propidium iodide based analysis by flow cytometry. BChE protein was measured using western blot in the two cell lines as well as in their SP and non-SP (NSP) populations after sorting. An MYCN non-amplified neuroblastoma cell line (SH-SY5Y) was tested for both MYCN and BChE protein as a potential low MYCN and BChE expressing tumor. The metabolic profile (glycolysis and OXPHOS) was determined for both unsorted, SP and NSP cells using Seahorse® metabolic analysis. BE-2 and IMR-32 tumorigenicity was characterized by growing cells subcutaneously in the flanks of NSG immunodeficient mice. BE-2c tumors developed earlier and grew more rapidly than IMR-32 tumors after injection of a similar number of cells. MYCN protein was similarly expressed in BE-2c and IMR-32 despite higher in-vivo tumorigenicity of BE-2c. In contrast, BChE was higher in BE-2 compared to IMR-32 corresponding to the order of tumorigenicity in-vivo. BE-2 and IMR-32 SP stem cells were relatively quiescent as measured by lower BChE protein compared to NSP cells and lower metabolic rates as measured by extracellular acidification (glycolysis) and oxygen consumption (OXPHOS metabolism). Cell cycle analysis and cell growth curves support BE-2 being a more proliferative cell line than IMR-32. MYCN has been the traditional marker to identify aggressive poor prognostic NB but data presented suggests BChE may also identify high risk disease generally correlating with growth of tumor in NSG mice and cellular growth characteristics. The use of small molecule inhibitors of BChE are currently under pre-clinical evaluation and given the relationship between BChE expression and tumorigenicity, BChE may be a promising new target. Ongoing work in our laboratories is investigating mechanisms of association between BChE and high-risk features of NB.
Citation Format: Katie Greenwood, Timothy R. McGuire, Donald Coulter, Erin McIntyre, Nagendra K. Chaturvedi, Paul Kortylewicz, Shantaram S. Joshi, Xiaoyu Chen, John G. Sharp, Janina Baranowska-Kortylewicz. Butyrylcholinesterase (BChE) expression as a marker of cellular proliferation across neuroblastoma cells lines: new prognostic marker and therapeutic target. abstract. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A127.
Abstract
Mantle cell lymphoma (MCL) is a rare but aggressive form of B cell non-Hodgkin lymphoma in which therapy resistance is common. New therapeutic options have extended survival in refractory ...MCL but have not provided durable remission. Tools are needed to assess the molecular and genetic changes associated with therapy resistance. Therefore, therapy-resistant MCL cell lines were established from the liver, kidney and lungs of human Granta 519-bearing NOD-SCID (non-obese diabetic-severe combined immunodeficiency) mice following treatment with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy in combination with bortezomib. The cytomorphologies, immunophenotypes, growth patterns in semi-solid agar, cytogenetic profiles and gene expression differences between these cell lines were characterized to identify major changes associated with therapy resistance. Therapy-resistant cell lines exhibit more aggressive growth patterns and markedly different gene expression profiles compared to parental Granta 519 cells. Thus, these stable therapy-resistant cell lines are useful models to further study the molecular basis of drug resistance and to identify clinically relevant molecular targets in MCL.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Abstract 63
Mantle cell lymphoma (MCL) is one of the most aggressive B-cell non-Hodgkin lymphomas (NHL) with a median survival of less than five years. Currently, there is no curative therapy ...available for refractory MCL because of relapse from therapy-resistant tumor cells. It has been well documented that the NF-κB and mTOR pathways are constitutively active in MCL leading to increased survival, proliferation and decreased apoptosis. Therefore, in an effort to improve therapy for refractory MCL, we investigated the antilymphoma activity in vitro and in vivo and associated molecular mechanism of action of 13–197, a quinoxaline analog that specifically perturbs IκB kinase (IKK) β, an upstream kinase of the NF-κB and mTOR pathways.
Established therapy-resistant from Granta 519 (Ahrens and Chaturvedi et al, Leukemia and Lymphoma doi:10.3109/10428194.2012.691481), other MCL cell lines Mino and Rec-1 and primary MCL cells from patients were used in this study. These MCL cells were treated in vitro with varying concentrations of 13–197 for the different time points. Cellular proliferation/viability, cytomorphology, frequency of cells undergoing apoptosis in treated and control cells were evaluated using 3H-thymidine uptake, MTT assay, cytomorphology and Annexin-V staining methods respectively. The status of key molecules in the NF-κB and mTOR pathways were examined in therapy-resistant and parental MCL cells following treatment with 13–197 using western blot analyses. The results of these analyses were compared to untreated control cells as appropriate and statistical significance of the results were determined using student‘t' test. In addition, in vivo therapeutic efficacy of 13–197 was investigated using NOD-SCID mouse bearing therapy-resistant MCL.
Our results showed that 13–197 significantly decreased the proliferation and induced a ∼four-fold (P<0.005) increase in apoptosis in parental and therapy-resistant MCL cells compared to control cells. At the molecular level, we observed down-regulation of IκBα phosphorylation and inhibition of NF-κB nuclear translocation by the 13–197 in MCL cells. In addition, NF-κB regulated genes such as cyclin D1, Bcl-XL and Mcl-1 were down-regulated in 13–197-treated cells. 13–197 also inhibited the phosphorylation of S6K and 4E-BP1, the downstream molecules of mTOR pathway that are also activated in refractory MCL. Further, to investigate the therapeutic efficacy of 13–197 against therapy-resistant MCL in vivo, we treated NOD-SCID mice bearing therapy-resistant MCL with 13–197; there was significantly reduced tumor burden in the kidney (p>0.05), liver (p>0.01), and lungs (p>0.03) of 13–197 treated mice compared to vehicle treated mice. Indeed, 13–197 treatment significantly increased the survival (p>0.001) of MCL transplanted mice. Taken together, our results suggest that 13–197 targets IKKβ which leads to both the transcriptional (NF-κB) and translational (mTOR) downregulation of gene products (cyclin D1, Bcl-XL and Mcl-1) misregulated in therapy-resistant MCL.
Overall, results suggest that 13–197 perturbs the NF-κB and mTOR pathways leading significant antilymphoma effects in vitro and in vivo thus demonstrates its potentials to be a therapeutic agent for refractory MCL.
(This work was supported by the Lymphoma Research Foundation New York, NY)
No relevant conflicts of interest to declare.
Abstract 1781
Chronic Lymphocytic Leukemia (CLL), the most prevalent adult B-cell malignancy in western countries, is a highly heterogeneous with a very variable clinical outcome. Emerging evidence ...indicates that the stromal tumor microenvironment (STME) play important roles in the pathogenesis of CLL. However, the precise mechanism and molecules of STME involved in this process remain unknown. In an attempt to explore the role of STME in this process, we examined the expression levels of stromal associated genes using gene expression profiling (GEP) of CLL cells from 53 patients’ lymph node (LN) (n=15), bone marrow (BM) (n =18), and peripheral blood (PB) (n=20). Using significant analyses of microarray (SAM), gene set enrichment analyses (GSEA), and ingenuity pathway analyses (IPA), among the major pathways associated with the differentially expressed genes, a cytoskeleton genes associated with stromal signatures are the focus of this report. Of these molecules, a significant number of molecules including: LUM, MMP9, MYLK, ITGA9, CAV1, CAV2, FBN1, PARVA, CALD1, ITGB5 and EHD2 were overexpressed and ITGB2, DLC1 and ITGA6 are under expressed in LN-CLL compared to BM-CLL and PB-CLL indicating a role of LN-mediated TME in CLL cell survival/progression. Among these genes, expression of myosin light chain kinase (MYLK), caveolin 1 (CAV1) and caveolin 2 (CAV2) correlated with clinical outcome (see adjacent Figure) as determined by time to treatment. We recently reported the role of a CAV1 in LN microenvironment-induced immune tolerance in CLL and possibility of their involvement in CLL cytoskeleton (Gilling et al, 2012). In the present study we report aberrant expression of other cytoskeleton genes such as MYLK and CAV2 are involved in the regulation of CLL cell survival in the stromal microenvironment affecting other members of the cytoskeletal signature via actin cytoskeleton signaling, integrin signaling and Pak signaling. In addition, MYLK and CAV2 are also involved in regulation of CLL proliferation. Together our studies show that members of the stromal signature particularly in the CLL cells from lymph nodes regulate the CLL cell survival and proliferation and thus leukemic progression.
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No relevant conflicts of interest to declare.
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