YB1 modulates the DNA damage response in medulloblastoma.

Y-box binding protein 1 (YBX1 or YB1) is a therapeutically relevant oncoprotein capable of RNA and DNA binding and mediating protein-protein interactions that drive proliferation, stemness, and resistance to platinum-based therapies. Given our previously published findings, the potential for YB1-driven cisplatin resistance in medulloblastoma (MB), and the limited studies exploring YB1-DNA repair protein interactions, we chose to investigate the role of YB1 in mediating radiation resistance in MB. MB, the most common pediatric malignant brain tumor, is treated with surgical resection, cranio-spinal radiation, and platinum-based chemotherapy, and could potentially benefit from YB1 inhibition. The role of YB1 in the response of MB to ionizing radiation (IR) has not yet been studied but remains relevant for determining potential anti-tumor synergy of YB1 inhibition with standard radiation therapy. We have previously shown that YB1 drives proliferation of cerebellar granular neural precursor cells (CGNPs) and murine Sonic Hedgehog (SHH) group MB cells. While others have demonstrated a link between YB1 and homologous recombination protein binding, functional and therapeutic implications remain unclear, particularly following IR-induced damage. Here we show that depleting YB1 in both SHH and Group 3 MB results not only in reduced proliferation but also synergizes with radiation due to differential response dynamics. YB1 silencing through shRNA followed by IR drives a predominantly NHEJ-dependent repair mechanism, leading to faster γH2AX resolution, premature cell cycle re-entry, checkpoint bypass, reduced proliferation, and increased senescence. These findings show that depleting YB1 in combination with radiation sensitizes SHH and Group 3 MB cells to radiation.

3D-on-2D: A Physiologically Relevant and Gel-Free In Vitro Coculture Method to Assay Antimetastatic Agents.

Metastasis of cancer cells leads to 90% of lethality among cancer patients. A crucial step in the hematogenous spread of metastatic cancer is the detachment of cells from the primary tumor followed by invasion through nearby blood vessels (Wong and Hynes. Cell Cycle 5(8):812-817, 2006). This is common to several solid tumors, including medulloblastoma (Van Ommeren et al. Brain Pathol 30:691-702, 2020). Because invasion is a crucial step in metastasis, the development of assays studying invasion are important for identifying antimetastatic drugs. There is always a need to develop better 3D in vitro models that not only mimic the complexity of in vivo architecture of solid tumors and their microenvironment, but are also simple to execute in medium to high throughput. We developed an in vitro coculture invasion assay that relies on the binary interaction between cancer cells and endothelial cells for research on tumor invasion and antimetastatic drug discovery. The goal of the current protocol is to use the simplicity of a two-dimensional endothelial cell culture to create a gel-free physiological substratum that can facilitate cancer cell invasion from a 3D cancer spheroid. This provides a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. Using this assay, we can compare the effect of invasion inhibitors/activators on cancer spheroids. The results are analyzed by manual scoring of images for the presence or absence of sprouting from cancer spheroids. This enables simple and fast analysis of metastasis, which facilitates multiparameter examination.

In Vitro Screening Assay for Activators of T-Cell Migration to Solid Tumors.

Recently, chimeric antigen receptor (CAR) T-cell therapy has begun to be used for solid tumors such as glioblastoma multiforme. Pediatric malignant brain tumors patients develop extensive long-term morbidity of intensive multimodal curative treatment. CAR T-cells treatments could potentially create favorable outcomes and reduce the toxicity of current treatment. T-cell infiltration of solid tumors has been associated with good prognosis. A largely overlooked area of CAR T-cell therapy targeting solid tumors is enhancing the ability of CAR T-cells to migrate and infiltrate solid tumors. A potential reason could be lack of standard in vitro assays which can screen for genetic modifications that result in enhanced T-cell migration in CAR T-cell therapies. We report a novel coculture assay using 3D tumor spheroids cocultured with T-cells to analyze the effect of activating CAR T-cell interventions on cell migration by a simple imaging based readout. This assay can be applied to several different kinds of cancer cell lines in higher throughput as well as toward measuring the efficiency of currently available CAR T-cell therapies in untested solid tumors.

Preclinical Research Strategy Development for RNAi-Based Therapies in Oncology Using Patient-Centered Information from Public Databases of Human Protein Atlas and R2Genomics Platform.

Experimental anticancer agents have a history of failing in the late stages of clinical development, which has led to significantly increased losses to stakeholders during the drug development process. A bioinformatics-based approach to predict and derisk a drug development program can save time, effort, and expenses resulting from failure of experimental anticancer agents in preclinical/early clinical stages. We present a two-step in silico ensemble method, involving the comparison of localized gene expression from surrounding tissue with tumor tissue, and subsequent correlation with patient survival data, which can help predict safety and efficacy for siRNA-based drug delivery to internal cancer tissues. This is achieved by reducing the possible off-target effects due to reduced or minimal expression of the drug target in surrounding tissue, and increasing survival probability for patients whose cancers can be controlled/eliminated by siRNA-mediated inhibition of drug target. This kind of approach can be useful for more efficient drug development efforts in oncology through reduction of investment in expensive experimentation during the discovery and preclinical phases; and ultimately support the intended clinical trial design.

STAT3 is required for Smo-dependent signaling and mediates Smo-targeted treatment resistance and tumorigenesis in Shh medulloblastoma.

Sonic hedgehog (Shh)-driven medulloblastoma (Shh MB) cells are dependent on constitutive Shh signaling, but targeted treatment of Shh MB has been ineffective due to drug resistance. The purpose of this study was to address the critical role of signal transducer and activator of transcription 3 (STAT3) in Shh signaling and drug resistance in Shh MB cells. Herein, we show that STAT3 is required for Smoothened (Smo)-dependent Shh signaling and, in turn, is reciprocally regulated by Shh signaling, and demonstrate that STAT3 activity is critical for expression of HCK proto-oncogene, Src family tyrosine kinase (Hck) in Shh MB. We also demonstrate that maintained STAT3 activity suppresses p21 expression and promotes colony formation of Shh MB cells, whereas dual treatment with inhibitors of both Smo and STAT3 results in marked synergistic killing and overcomes drug resistance in vitro of Smo antagonist-resistant Shh MB cells. Finally, STAT3 inhibitor treatment significantly prevents in vivo tumor formation in genetically engineered Shh MB mice. Collectively, we show that STAT3 is necessary to maintain Shh signaling and thus is a potential therapeutic target to treat Shh MB and overcome anti-Smo drug resistance.

A Novel Therapeutic Approach to Corneal Alkaline Burn Model by Targeting Fidgetin-Like 2, a Microtubule Regulator.

Purpose: The purpose of this study was to determine the efficacy of nanoparticle-encapsulated Fidgetin-like 2 (FL2) siRNA (FL2-NPsi), a novel therapeutic agent targeting the FL2 gene, for the treatment of corneal alkaline chemical injury. Methods: Eighty 12-week-old, male Sprague-Dawley rats were divided evenly into 8 treatment groups: prednisolone, empty nanoparticles, control-NPsi (1 µM, 10 µM, and 20 µM) and FL2-NPsi (1 µM, 10 µM, and 20 µM). An alkaline burn was induced onto the cornea of each rat, which was then treated for 14 days according to group assignment. Clinical, histopathologic, and immunohistochemical analyses were conducted to assess for wound healing. FL2-NPsi-mediated knockdown of FL2 was confirmed by in vitro quantitative polymerase chain reaction (qPCR). Toxicity assays were performed to assess for apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling [TUNEL] assay) and nerve damage (whole mount immunochemical staining). Statistical analyses were performed using Student's t-test and ANOVA. Results: Compared with controls, FL2-NPsi-treated groups demonstrated enhanced corneal wound healing, with the 10 and 20 µM FL2-NPsi-treated groups demonstrating maximum rates of corneal re-epithelialization as assessed by ImageJ software, enhanced corneal transparency, and improved stromal organization on histology. Immunohistochemical analysis of vascular endothelial cells, macrophages, and neutrophils did not show significant differences between treatment groups. FL2-NPsi was not found to be toxic to nerves or induce apoptosis (p = 0.917). Conclusions: Dose-response studies found both 10 and 20 µM FL2-NPsi to be efficacious in this rat model. FL2-NPsi may offer a novel treatment for corneal alkaline chemical injuries. Translational Relevance: Basic cell biology findings about the microtubule cytoskeleton were used to design a therapeutic to enhance corneal cell migration, highlighting the promise of targeting microtubules to regulate corneal wound healing.

Engineered biomimetic nanoparticle for dual targeting of the cancer stem-like cell population in sonic hedgehog medulloblastoma.

The sonic hedgehog subtype of medulloblastoma (SHH MB) is associated with treatment failure and poor outcome. Current strategies utilizing whole brain radiation therapy result in deleterious off-target effects on the normal developing childhood brain. Most conventional chemotherapies remain limited by ineffective blood-brain barrier (BBB) penetrance. These challenges signify an unmet need for drug carriers that can cross the BBB and deliver drugs to targeted sites with high drug-loading efficiency and long-term stability. We herein leverage the enhanced stability and targeting ability of engineered high-density lipoprotein-mimetic nanoparticles (eHNPs) to cross the BBB and deliver a SHH inhibitor effectively to the cancer stem-like cell population in SHH MB. Our microfluidic technology enabled highly reproducible production of multicomponent eHNPs incorporated with apolipoprotein A1, anti-CD15, and a SHH inhibitor (LDE225). We demonstrate the dual-targeted delivery and enhanced therapeutic effect of eHNP-A1-CD15-LDE225 via scavenger receptor class B type 1 (SR-B1) and CD15 on brain SHH MB cells in vitro, ex vivo, and in vivo. Moreover, we show that eHNP-A1 not only serves as a stable drug carrier, but also has a therapeutic effect itself through SR-B1-mediated intracellular cholesterol depletion in SHH MB cells. Through the facilitated and targeted cellular uptake of drugs and direct therapeutic role of this engineered biomimetic nanocarrier in SHH MB, our multifunctional nanoparticle provides intriguing therapeutic promise as an effective and potent nanomedicine for the treatment of SHH MB.

Sonic Hedgehog Signaling Drives Mitochondrial Fragmentation by Suppressing Mitofusins in Cerebellar Granule Neuron Precursors and Medulloblastoma.

UNLABELLED: Sonic hedgehog (Shh) signaling is closely coupled with bioenergetics of medulloblastoma, the most common malignant pediatric brain tumor. Shh-associated medulloblastoma arises from cerebellar granule neuron precursors (CGNP), a neural progenitor whose developmental expansion requires signaling by Shh, a ligand secreted by the neighboring Purkinje neurons. Previous observations show that Shh signaling inhibits fatty acid oxidation although driving increased fatty acid synthesis. Proliferating CGNPs and mouse Shh medulloblastomas feature high levels of glycolytic enzymes in vivo and in vitro. Because both of these metabolic processes are closely linked to mitochondrial bioenergetics, the role of Shh signaling in mitochondrial biogenesis was investigated. This report uncovers a surprising decrease in mitochondrial membrane potential (MMP) and overall ATP production in CGNPs exposed to Shh, consistent with increased glycolysis resulting in high intracellular acidity, leading to mitochondrial fragmentation. Ultrastructural examination of mitochondria revealed a spherical shape in Shh-treated cells, in contrast to the elongated appearance in vehicle-treated postmitotic cells. Expression of mitofusin 1 and 2 was reduced in these cells, although their ectopic expression restored the MMP to the nonproliferating state and the morphology to a fused, interconnected state. Mouse Shh medulloblastoma cells featured drastically impaired mitochondrial morphology, restoration of which by ectopic mitofusin expression was also associated with a decrease in the expression of Cyclin D2 protein, a marker for proliferation. IMPLICATIONS: This report exposes a novel role for Shh in regulating mitochondrial dynamics and rescue of the metabolic profile of tumor cells to that of nontransformed, nonproliferating cells and represents a potential avenue for development of medulloblastoma therapeutics.

Co-evolution of RNA polymerase with RbpA in the phylum Actinobacteria.

The role of RbpA in the backdrop of M. smegmatis showed that it rescues mycobacterial RNA polymerase from rifampicin-mediated inhibition (Dey et al., 2010; Dey et al., 2011). Paget and co-workers (Paget et al., 2001; Newell et al., 2006) have revealed that RbpA homologs occur exclusively in actinobacteria. Newell et al. (2006) showed that MtbRbpA, when complemented in a ∆rbpA mutant of S. coelicolor, showed a low recovery of MIC (from 0.75 to 2 µg/ml) as compared to complementation by native RbpA of S. coelicolor (MIC increases from 0.75 to 11 µg/ml). Our studies on MsRbpA show that it is a differential marker for M. smegmatis RNA polymerase as compared to E. coli RNA polymerase at IC50 levels of rifampicin. A recent sequence-based analysis by Lane and Darst (2010) has shown that RNA polymerases from Proteobacteria and Actinobacteria have had a divergent evolution. E. coli is a representative of Proteobacteria and M. smegmatis is an Actinobacterium. RbpA has an exclusive occurrence in Actinobacteria. Since protein-protein interactions might not be conserved across different species, therefore, the probable reason for the indifference of MsRbpA toward E. coli RNA polymerase could be the lineage-specific differences between actinobacterial and proteobacterial RNA polymerases. These observations led us to ask the question as to whether the evolution of RbpA in Actinobacteria followed the same route as that of RNA polymerase subunits from actinobacterial species. We show that the exclusivity of RbpA in Actinobacteria and the unique evolution of RNA polymerase in this phylum share a co-evolutionary link. We have addressed this issue by a blending of experimental and bioinformatics based approaches. They comprise of induction of bacterial cultures coupled to rifampicin-tolerance, transcription assays and statistical comparison of phylogenetic trees for different pairs of proteins in actinobacteria.

Downregulation of APOBEC3G by xenotropic murine leukemia-virus related virus (XMRV) in prostate cancer cells.

BACKGROUND: Xenotropic murine leukemia virus (MLV)-related virus (XMRV) is a gammaretrovirus that was discovered in prostate cancer tissues. Recently, it has been proposed that XMRV is a laboratory contaminant and may have originated via a rare recombination event. Host restriction factor APOBEC3G (A3G) has been reported to severely restrict XMRV replication in human peripheral blood mononuclear cells. Interestingly, XMRV infects and replicates efficiently in prostate cancer cells of epithelial origin. It has been proposed that due to lack off or very low levels of A3G protein XMRV is able to productively replicate in these cells. FINDINGS: This report builds on and challenges the published data on the absence of A3G protein in prostate epithelial cells lines. We demonstrate the presence of A3G in prostate epithelial cell lines (LNCaP and DU145) by western blot and mass spectrometry. We believe the discrepancy in A3G detection is may be due to selection and sensitivity of A3G antibodies employed in the prior studies. Our results also indicate that XMRV produced from A3G expressing LNCaP cells can infect and replicate in target cells. Most importantly our data reveal downregulation of A3G in XMRV infected LNCaP and DU145 cells. CONCLUSIONS: We propose that XMRV replicates efficiently in prostate epithelial cells by downregulating A3G expression. Given that XMRV lacks accessory proteins such as HIV-1 Vif that are known to counteract A3G function in human cells, our data suggest a novel mechanism by which retroviruses can counteract the antiviral effects of A3G proteins.

Molecular insights into the mechanism of phenotypic tolerance to rifampicin conferred on mycobacterial RNA polymerase by MsRbpA.

The protein MsRbpA from Mycobacterium smegmatis rescues RNA polymerase (RNAP) from the inhibitory effect of rifampicin (Rif). We have reported previously that MsRbpA interacts with the ß-subunit of RNAP and that the effect of MsRbpA on Rif-resistant (Rif(R)) RNAP is minimal. Here we attempted to gain molecular insights into the mechanism of action of this protein with respect to its role in rescuing RNAP from Rif-mediated transcription inhibition. Our experimental approach comprised multiple-round transcription assays, fluorescence spectroscopy, MS and surface plasmon resonance in order to meet the above objective. Based on our molecular studies we propose here that Rif is released from its binding site in the RNAP-Rif complex in the presence of MsRbpA. Biophysical studies reveal that the location of MsRbpA on RNAP is at the junction of the ß- and ß'-subunits, close to the Rif-binding site and the (i+1) site on RNAP.

Role of an RNA polymerase interacting protein, MsRbpA, from Mycobacterium smegmatis in phenotypic tolerance to rifampicin.

Rifampicin and its derivatives are at the forefront of the current standard chemotherapeutic regimen for active tuberculosis; they act by inhibiting the transcription activity of prokaryotic RNA polymerase. Rifampicin is believed to interact with the beta subunit of RNA polymerase. However, it has been observed that protein-protein interactions with RNA polymerase core enzyme lead to its reduced susceptibility to rifampicin. This mechanism became more diversified with the discovery of RbpA, a novel RNA polymerase-binding protein, in Streptomyces coelicolor that could mitigate the effect of rifampicin on RNA polymerase activity. MsRbpA is a homologue of RbpA in Mycobacterium smegmatis. On deciphering the role of MsRbpA in M. smegmatis we found that it interacts with RNA polymerase and increases the rifampicin tolerance levels, both in vitro and in vivo. It interacts with the beta subunit of RNA polymerase. However, it was found to be incapable of rescuing rifampicin-resistant RNA polymerases in the presence of rifampicin at the respective IC(50).