MicroRNAs miR-29a, miR-29b, and miR-29c as Novel Regulators of NK-cell Immune Response in Neuroblastoma

Background: Neuroblastoma is a challenging cancer to treat in pediatric patients. Despite intense treatment regimens, the prognosis for high-risk pediatric neuroblastoma patients remains poor, with less than 40% survival. Significance of the Problem: One of the major obstacles to effective immunotherapy in neuroblastoma is the defective immune cells. Neuroblastoma tumors generally have impaired T-cell anti-tumor activity due to restricted MHC class I expression on tumor cells. This makes natural killer (NK) cells an attractive alternative for neuroblastoma immunotherapy, as they are not restricted by MHC class I expression on tumor cells. However, the overexpression of immune checkpoint molecules like B7-H3 (gene: CD276) helps tumor cells to escape NK immune surveillance, hindering the effectiveness of NK cell-mediated immunotherapy in neuroblastoma. Objective: Micro RNAs (miRNAs or miR) play critical roles in nervous system development and post-transcriptional regulation of genes involved in neuroblastoma development. Therefore, exploring the role of upstream miRNAs that can target B7-H3 and regulate NK-mediated anti-tumor immune response in neuroblastoma could lead to the identification of novel therapeutic targets for neuroblastoma treatment. Methods: Using the TARGET, neuroblastoma patient dataset, we applied the robust bioinformatic workflows incorporating differential expression, co-expression, survival, heatmaps, and box plots. Results: We present here the role of miRNAs belonging to the miR-29 family, including miR-29a, miR-29b, and miR-29c, in regulating B7-H3 and antitumor immunity in neuroblastoma. Using different neuroblastoma patients\u27 microarray data sets, we show that miR-29a, miR-29b, and miR-29c levels in the tumors were associated with good clinical outcomes and inversely correlated with B7-H3. Higher B7-H3 mRNA was associated with disease progression and poor survival in patients with neuroblastoma. MiR-29a, miR-29b, and miR-29c inhibited B7-H3 expression in neuroblastoma cells. B7-H3 downregulation induced NK cell activation and enhanced its cytotoxic functions against neuroblastoma cells, boosting NK-mediated antitumor immunity. Furthermore, miR-29a, miR-29b, and miR-29c treated neuroblastoma tumors had a large influx of infiltering activated NK and T cells, which is associated with tumor shrinkage, reduced tumor microvessel density, low macrophage infiltration, and enhanced tumor cell apoptosis. In cell culture, overexpression of miR-29a, miR-29b, and miR-29c inhibited proliferation, colony formation, migration, and neurospheres forming ability of neuroblastoma cell lines. Conclusions: Overall, our findings highlight the therapeutic potential of miR-29a, miR-29b, and miR-29c to boost NK and T cell-mediated immune surveillance of neuroblastoma tumors, strengthening natural anti-tumor immunity and response to anticancer therapies.https://digitalcommons.unmc.edu/chri_forum/1071/thumbnail.jp

SAP30, a Novel Oncogenic Transcription Factor in High-Risk Neuroblastoma: Clinical Significance and Role in Tumor-Progression, Survival, and Drug Resistance

Neuroblastoma is the most common devastating extracranial solid malignancy in children, accounting for 15% of childhood cancer-related mortality. Despite an intense treatment regimen, approximately 50% of children treated for high-risk neuroblastoma have more aggressive tumor relapse with less than 20% five-year overall survival. Amplification of the oncogene MYCN is associated with a high risk of relapse. However, only 25% of high-risk neuroblastomas are MYCN-amplified, indicating that the rest are driven by factors other than MYCN. Therefore, it is essential to identify novel driver transcription factors but not passenger genes that improve prediction efficacy of therapy response and association with high-risk, progression, stage 4, and survival in neuroblastoma patients. We used three neuroblastoma patient datasets (n=1252 patients) and applied robust bioinformatic data mining tools such as Weighted Gene Co-expression Network Analysis (WGCNA), cisTarget, and Single-Cell Regulatory Network Inference and Clustering (SCENIC) to identify driver transcription factors (regulon) that associate with high-risk, progression, stage, and survival in neuroblastoma patients. Based on the regulon specificity score, we derived a 10-transcription factor signature and prioritized Sin3A Associated Protein 30 (SAP30), given its highest regulon specificity score, especially in high-risk and aggressive stage cohorts. Higher SAP30 expression was found in high-risk neuroblastoma patients and progression-specific patient-derived xenograft tumors than their respective controls. The advanced pharmacogenomic analysis and CRISPR-Cas9 screens indicated that SAP30 essentiality correlated with Cisplatin resistance and further validated in Cisplatin resistant patient-derived xenograft tumor-derived cell lines. SAP30 silencing inhibited cell proliferation, slowed growth and induced cell death in vitro, and reduced tumor burden and size in vivo. Overall, our results indicate that SAP30 is a better prognostic and Cisplatin resistant marker associated with high-risk, stage 4 progression, and poor survival in neuroblastoma patients.https://digitalcommons.unmc.edu/chri_forum/1057/thumbnail.jp

A novel quinazolinone derivative induces cytochrome c interdependent apoptosis and autophagy in human leukemia MOLT-4 cells

Crosstalk between apoptosis and autophagy is budding as one of the novel strategies in the cancer therapeutics. The present study tinted toward the interdependence of autophagy and apoptosis induce by a novel quinazolinone derivative 2,3-dihydro-2-(quinoline-5-yl) quinazolin-4(1H)-one structure [DQQ] in human leukemia MOLT-4 cells. DQQ induces cytochrome c arbitrated apoptosis and autophagy in MOLT-4 cells. Apoptosis induces by DQQ was confirmed through a battery of assay e.g. cellular and nuclear microscopy, annexin-V assay, cell cycle analysis, loss of mitochondrial membrane potential and immune-expression of cytochrome c, caspases and PARP. Furthermore, acridine orange staining, LC3 immunofluorescence and western blotting of key autophagy proteins revealed the autophagic potential of DQQ. A universal caspase inhibitor, Z-VAD-FMK and cytochrome c silencing, strongly inhibited the DQQ induce autophagy and apoptosis. Beclin1 silencing through siRNA partially reversed the cell death, which was not as significant as by cytochrome c silencing. Although, it partially reversed the PARP cleavage induced by DQQ, indicating the role of autophagy in the regulation of apoptosis. The present study first time portrays the negative feedback potential of cytochrome c regulated autophagy and the importance of quinazolinone derivative in discovery of novel anticancer therapeutics

The synthetic tryptanthrin analogue suppresses STAT3 signaling and induces caspase dependent apoptosis via ERK up regulation in human leukemia HL-60 cells.

Tryptanthrin is a natural product which has been reported to have several medicinal properties. In this study, we tried to investigate the detailed molecular mechanism of its bromo analogue (TBr), a potent cytotoxic agent in the induction of cancer cell death. It was found that TBr primarily targets STAT3 and ERK signaling during the induction of apoptosis in several human leukemia cell lines. In HL-60 cells, TBr treatment caused early down regulation of p-STAT3 with concomitant up regulation of p-ERK which led to the activation of intrinsic and extrinsic pathways of apoptosis. The mechanism of TBr mediated inhibition of p-STAT3 was found to be due to the activation of ubiquitin dependent degradation of tyrosine 705 and serine 727 p-STAT3. As IL-6 is the main driver of the STAT3 pathway, the effect of TBr on cell death was subdued when treated in the combination with IL-6 in HL60 cells. Interestingly, PD98059 significantly reduced the apoptotic effects of TBr, thus showing the direct involvement of p-ERK in TBr mediated cell death. It was further shown that apoptotic protein Bax silencing in HL-60 cells resists TBr mediated ERK dependent apoptosis. In summary, for the first time we report the mechanism of TBr mediated cell death in human leukemia cell lines by targeting STAT3 and ERK pathways

Semisynthesis of Mallotus B from Rottlerin: Evaluation of Cytotoxicity and Apoptosis-Inducing Activity

Mallotus B (<b>2d</b>) is a prenylated dimeric phloroglucinol compound isolated from <i>Mallotus philippensis.</i> There have been no reports on the synthesis or biological activity of this compound. In the present paper, a semisynthetic preparation of mallotus B is reported via base-mediated intramolecular rearrangement of rottlerin (<b>1</b>), which is one of the major constituents of <i>M. philippensis</i>. The homodimer “rottlerone” was also formed as one of the products of this base-mediated intramolecular reaction. Rottlerin (<b>1</b>), along with rottlerone (<b>2c</b>) and mallotus B (<b>2d</b>), was evaluated for cytotoxicity against a panel of cancer cell lines including HEPG2, Colo205, MIAPaCa-2, PC-3, and HL-60 cells. Mallotus B (<b>2d</b>) displayed cytotoxicity for MIAPaCa-2 and HL-60 cells with IC₅₀ values of 9 and 16 μM, respectively. Microscopic studies in HL-60 cells indicated that mallotus B (<b>2d</b>) induces cell cycle arrest at the G1 phase and causes defective cell division. It also induces apoptosis, as evidenced by distinct changes in cell morphology

TBr down regulated STAT3 target genes and promoted ubiquitin dependent degradation of activated STAT3.

<p>(A) HL-60, MOLT-4 and K-562 cells were treated with TBr at indicated concentrations for 6 h. Lysates were prepared and immunoblotting of indicated proteins were performed. β actin was used as the loading control. (B) HL-60 cells were treated with TBr at 2 and 3 µM concentrations along with the proteasomal inhibitor MG132 (0.07 µM) for 6 h. Protein lysates were prepared and p-STAT3 (Y705) was immunoprecipitated with its respective antibody. Immunoblotting was used to detect antiubiquitin (P4D1, Santacruz) and p-STAT3 (Y705) antibodies. A predominant ubiquitination of the tyrosine705 phosphorylated STAT3 is seen in the TBr treated cells under proteasomal inhibition. (C) MG132 pre-treatment rescued decreased p-STAT3 expression in TBr treated HL-60 cells. Data are mean ± SD of three similar experiments, *p<0.05, **p<0.01 for TBr vs TBr with MG132.</p

Bax silencing rescued the TBr-mediated cytotoxic effect in HL-60 cells.

<p>(A) Cells containing control and Bax siRNA were treated with TBr at indicated concentrations for 24 h and MTT assay was performed. (B) Cell cycle analysis of TBr treated Bax positive and Bax silenced cells. (C) PARP-1 and Caspase-3 expression in Bax positive and Bax silenced TBr treated cells. β actin was used as a positive control. Data are mean ± SD of three similar experiments, *p<0.05, **p<0.01 for control vs Bax siRNA.</p

TBr induced p-ERK dependent apoptosis in HL-60 cells.

<p>(A) IL-6 treatment increased cell viability in TBr treated cells. Cells were pre-treated with IL-6 (50 ng/ml) for 40 min followed by TBr (3 µM) addition for another 24 h. MTT dye was added 3 h before the experiment termination. Data are mean ± SD from three experiments. (B) TBr (3 µM) induced p-ERK expression in HL-60 cells (C) MTT assay of TBr treated cells along with ERK inhibitor PD98059 (30 µM) at indicated time periods. PD98059 was added 1 h before TBr addition. (D) Effect of PD98059 on subG1 population in TBr treated cells. After treatment, cells were stained with PI and DNA fluorescence was determined by flow cytometery. (E) Effect of PD98059 on Bax and PARP-1 cleavage in TBr treated HL-60 cells. Cells were treated with TBr (3 µM) and PD98059 (30 µM) at indicated time intervals. Whole, mitochondrial and cytosolic protein lystaes were prepared and equal amount of proteins were loaded and resolved on SDS PAGE followed by immunoblotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110411#s2" target="_blank">material and methods</a> section. (F) Effect of PD98059 (10 µM) and U0126 (10 µM) on STAT3 expression in TBr treated cells. β actin was used as a positive control. Data are mean ± SD of three similar experiments and statistical comparisons of TBr vs. TBr with PD98059/IL-6, was made by using the Bonferroni method, *p<0.05, **p<0.01.</p