miRNA-211 maintains metabolic homeostasis in medulloblastoma through its target gene long-chain acyl-CoA synthetase 4.

The prognosis of childhood medulloblastoma (MB) is often poor, and it usually requires aggressive therapy that adversely affects quality of life. microRNA-211 (miR-211) was previously identified as an important regulator of cells that descend from neural cells. Since medulloblastomas primarily affect cells with similar ontogeny, we investigated the role and mechanism of miR-211 in MB. Here we showed that miR-211 expression was highly downregulated in cell lines, PDXs, and clinical samples of different MB subgroups (SHH, Group 3, and Group 4) compared to normal cerebellum. miR-211 gene was ectopically expressed in transgenic cells from MB subgroups, and they were subjected to molecular and phenotypic investigations. Monoclonal cells stably expressing miR-211 were injected into the mouse cerebellum. miR-211 forced expression acts as a tumor suppressor in MB both in vitro and in vivo, attenuating growth, promoting apoptosis, and inhibiting invasion. In support of emerging regulatory roles of metabolism in various forms of cancer, we identified the acyl-CoA synthetase long-chain family member (ACSL4) as a direct miR-211 target. Furthermore, lipid nanoparticle-coated, dendrimer-coated, and cerium oxide-coated miR-211 nanoparticles were applied to deliver synthetic miR-211 into MB cell lines and cellular responses were assayed. Synthesizing nanoparticle-miR-211 conjugates can suppress MB cell viability and invasion in vitro. Our findings reveal miR-211 as a tumor suppressor and a potential therapeutic agent in MB. This proof-of-concept paves the way for further pre-clinical and clinical development.

The LIN28B-let-7-PBK pathway is essential for group 3 medulloblastoma tumor growth and survival.

Children with Group 3 medulloblastoma (G3 MB) have a very poor prognosis, and many do not survive beyond 5 years after diagnosis. A factor that may contribute to this is the lack of available targeted therapy. Expression of protein lin-28 homolog B (LIN28B), a regulator of developmental timing, is upregulated in several cancers, including G3 MB, and is associated with worse survival in this disease. Here, we investigate the role of the LIN28B pathway in G3 MB and demonstrate that the LIN28B-lethal-7 (let-7; a microRNA that is a tumor suppressor)-lymphokine-activated killer T-cell-originated protein kinase (PBK; also known as PDZ-binding kinase) axis promotes G3 MB proliferation. LIN28B knockdown in G3-MB-patient-derived cell lines leads to a significant reduction in cell viability and proliferation in vitro and in prolonged survival of mice with orthotopic tumors. The LIN28 inhibitor N-methyl-N-[3-(3-methyl-1,2,4-triazolo[4,3-b]pyridazin-6-yl)phenyl]acetamide (1632) significantly reduces G3 MB cell growth and demonstrates efficacy in reducing tumor growth in mouse xenograft models. Inhibiting PBK using HI-TOPK-032 also results in a significant reduction in G3 MB cell viability and proliferation. Together, these results highlight a critical role for the LIN28B-let-7-PBK pathway in G3 MB and provide preliminary preclinical results for drugs targeting this pathway.

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.

IGFBP2 promotes proliferation and cell migration through STAT3 signaling in Sonic hedgehog medulloblastoma.

Medulloblastoma (MB) is the most common pediatric brain malignancy and is divided into four molecularly distinct subgroups: WNT, Sonic Hedgehog (SHHp53mut and SHHp53wt), Group 3, and Group 4. Previous reports suggest that SHH MB features a unique tumor microenvironment compared with other MB groups. To better understand how SHH MB tumor cells interact with and potentially modify their microenvironment, we performed cytokine array analysis of culture media from freshly isolated MB patient tumor cells, spontaneous SHH MB mouse tumor cells and mouse and human MB cell lines. We found that the SHH MB cells produced elevated levels of IGFBP2 compared to non-SHH MBs. We confirmed these results using ELISA, western blotting, and immunofluorescence staining. IGFBP2 is a pleiotropic member of the IGFBP super-family with secreted and intracellular functions that can modulate tumor cell proliferation, metastasis, and drug resistance, but has been understudied in medulloblastoma. We found that IGFBP2 is required for SHH MB cell proliferation, colony formation, and cell migration, through promoting STAT3 activation and upregulation of epithelial to mesenchymal transition markers; indeed, ectopic STAT3 expression fully compensated for IGFBP2 knockdown in wound healing assays. Taken together, our findings reveal novel roles for IGFBP2 in SHH medulloblastoma growth and metastasis, which is associated with very poor prognosis, and they indicate an IGFBP2-STAT3 axis that could represent a novel therapeutic target in medulloblastoma.

The long noncoding RNA lnc-HLX-2-7 is oncogenic in Group 3 medulloblastomas.

BACKGROUND: Medulloblastoma (MB) is an aggressive brain tumor that predominantly affects children. Recent high-throughput sequencing studies suggest that the noncoding RNA genome, in particular long noncoding RNAs (lncRNAs), contributes to MB subgrouping. Here we report the identification of a novel lncRNA, lnc-HLX-2-7, as a potential molecular marker and therapeutic target in Group 3 MBs. METHODS: Publicly available RNA sequencing (RNA-seq) data from 175 MB patients were interrogated to identify lncRNAs that differentiate between MB subgroups. After characterizing a subset of differentially expressed lncRNAs in vitro and in vivo, lnc-HLX-2-7 was deleted by CRISPR/Cas9 in the MB cell line. Intracranial injected tumors were further characterized by bulk and single-cell RNA-seq. RESULTS: Lnc-HLX-2-7 is highly upregulated in Group 3 MB cell lines, patient-derived xenografts, and primary MBs compared with other MB subgroups as assessed by quantitative real-time, RNA-seq, and RNA fluorescence in situ hybridization. Depletion of lnc-HLX-2-7 significantly reduced cell proliferation and 3D colony formation and induced apoptosis. Lnc-HLX-2-7-deleted cells injected into mouse cerebellums produced smaller tumors than those derived from parental cells. Pathway analysis revealed that lnc-HLX-2-7 modulated oxidative phosphorylation, mitochondrial dysfunction, and sirtuin signaling pathways. The MYC oncogene regulated lnc-HLX-2-7, and the small-molecule bromodomain and extraterminal domain family‒bromodomain 4 inhibitor Jun Qi 1 (JQ1) reduced lnc-HLX-2-7 expression. CONCLUSIONS: Lnc-HLX-2-7 is oncogenic in MB and represents a promising novel molecular marker and a potential therapeutic target in Group 3 MBs.

Regulation of poly ADP-ribosylation of VEGF by an interplay between PARP-16 and TNKS-2.

The biological activity of vascular endothelial growth factor (VEGF), the major cytokine regulating the process of angiogenesis is tightly controlled at multiple levels including processes involving post-translational modification such as ADP-ribosylation and glycosylation. ADP-ribosylation is a reversible NAD+-dependent modification, catalyzed by poly ADP-ribose polymerase (PARP) or ADP-ribosyl transferase (ADPRTs) and has been reported by us and others as a modification that reduces the biological activity of VEGF. The factors responsible for any such modification should occur in the secretory pathway, i.e., in the endoplasmic reticulum and Golgi. Our investigation carried out in this direction revealed that ADP-ribosylation of VEGF requires the interplay between members of poly ADP-ribose polymerase (PARP) family in the secretory pathway, viz., ER associated PARP-16 and Golgi associated Tankyrase-2 (TNKS-2). The data presented in this manuscript suggest that PARP-16 catalysis the priming mono ADP-ribosylation of VEGF which is a prerequisite for poly ADP-ribosylation of VEGF by TNKS-2.

The Paradoxical Behavior of microRNA-211 in Melanomas and Other Human Cancers.

Cancer initiation, progression, and metastasis leverage many regulatory agents, such as signaling molecules, transcription factors, and regulatory RNA molecules. Among these, regulatory non-coding RNAs have emerged as molecules that control multiple cancer types and their pathologic properties. The human microRNA-211 (MIR211) is one such molecule, which affects several cancer types, including melanoma, glioblastoma, lung adenocarcinomas, breast, ovarian, prostate, and colorectal carcinoma. Previous studies suggested that in certain tumors MIR211 acts as a tumor suppressor while in others it behaves as an oncogenic regulator. Here we summarize the known molecular genetic mechanisms that regulate MIR211 gene expression and molecular pathways that are in turn controlled by MIR211 itself. We discuss how cellular and epigenetic contexts modulate the biological effects of MIR211, which exhibit pleiotropic effects. For example, up-regulation of MIR211 expression down-regulates Warburg effect in melanoma tumor cells associated with an inhibition of the growth of human melanoma cells in vitro, and yet these conditions robustly increase tumor growth in xenografted mice. Signaling through the DUSP6-ERK5 pathway is modulated by MIR211 in BRAFV600E driven melanoma tumors, and this function is involved in the resistance of tumor cells to the BRAF inhibitor, Vemurafenib. We discuss several alternate but testable models, involving stochastic cell-to-cell expression heterogeneity due to multiple equilibria involving feedback circuits, intracellular communication, and genetic variation at miRNA target sties, to reconcile the paradoxical effects of MIR211 on tumorigenesis. Understanding the precise role of this miRNA is crucial to understanding the genetic basis of melanoma as well as the other cancer types where this regulatory molecule has important influences. We hope this review will inspire novel directions in this field.

ER stress mediated regulation of miR23a confer Hela cells better adaptability to utilize glycolytic pathway.

Cancer cells exhibit increased dependency on aerobic glycolysis, a phenomenon referred as the "Warburg effect" and therefore, blocking glycolysis by using non-metabolizable analogues of glucose, like 2-Deoxy glucose (2-DG), has been proposed to be of huge therapeutic importance. One of the major drawbacks of using 2-DG as a chemotherapeutic agent is that it can induce ER stress. ER stress is a hall mark in many solid tumors and the unfolded protein response (UPR) associated with it initiates many survival mechanisms in cancer cells. In the present study, we report a novel survival mechanism associated with ER stress, by which the cancer cells become more adapted to aerobic glycolysis. When ER stress was induced in Hela cells by treating them with 2-DG or Thapsigargin (TG) the expression and activity of LDH was significantly up regulated, conferring the cells a greater glycolytic potential. A simultaneous decrease was observed in the expression of miR-23a, which was predicted in silico to have target site on the 3'UTR of LDH A and B mRNAs. miRNA over expression studies and mRNA degradation assays suggest that miR-23a could target LDH A and LDH B mRNAs. Further on the basis of our results and previous scientific reports, we propose that "c-Myc," which is over expressed during ER stress, repress the expression of miR-23a, which in turn regulates the expression of its target genes viz., LDH A and LDH B, thereby making the cells more competent to survive in tumor microenvironment, which requires efficient use of aerobic glycolysis.

Horizontal transfer of miR-23a from hypoxic tumor cell colonies can induce angiogenesis.

Neo vessel formation by angiogenesis is an important event during many pathological conditions including cancer, where it is indispensable for tumor growth and survival. Although, various pro-angiogenic cytokines and soluble factors, secreted by tumor cells, have been reported to promote angiogenesis, recent studies have shown regulatory role of exosomes, secreted by tumor cells in the process of angiogenesis. These exosomes are capable of carrying nucleic acids, proteins, etc., as their cargo. Under the light of these facts and considering the presence of miRNAs, the non-coding RNAs capable of regulating target gene expression, as one of the major cargos in the exosomes, we investigated, whether exosomes derived from normoxic and hypoxic tumor cell colonies exhibit difference in levels of miR-23∼27∼24 cluster members and if so, to check the significance of their horizontal transfer on the process of angiogenesis. Results of our study showed that exosomes secreted by hypoxic tumor cell colonies possess significantly higher levels of miR23a and can induce angiogenesis. Further, we have shown that exosomes secreted by cells that ectopically over express miR23a is capable of inducing angiogenesis in different angiogenic model systems such as CAM, in ovo Xenograft and HUVEC models systems. Further, mechanistic analysis revealed that miR23a driven regulation of angiogenesis is brought about by down regulation of SIRT1 in the recipient cells. Collectively, the results presented here suggest that exosomal transfer of miR23a from tumor cell colonies can induce the process of angiogenesis by targeting SIRT1 in the recipient endothelial cells.

MicroRNA106a regulates matrix metalloprotease 9 in a sirtuin-1 dependent mechanism.

Cellular migration is important during many physiological as well as pathological conditions and is regulated very tightly by an intricate network of signaling and effector molecules. One of the important players during cellular migration are matrix metalloproteases and their levels have been reported to be important in determining the cellular migratory properties during metastasis. MMPs and regulators of MMPs therefore, present themselves as potent candidates for manipulation, to control conditions where they get dysregulated. Micro RNAs are a group of micro regulators that can modulate expression of a gene through transcriptional and post transcriptional regulations. Owing to the fact that many microRNAs have already been reported to regulate MMPs and that miR106a, a member of oncomir17 family has been implicated in metastatic conditions, the present study intended to analyze if miR106a can regulate levels of MMP9, an important inducible matrix metalloproteinase. The results of the in vitro experiments demonstrated that under conditions of migration cells showed elevated levels of miR106a, which could regulate the expression of major MMP9 regulator, SIRT-1. Decreased levels of SIRT1thus resulted in an increase in the expression and activity of MMP9. Over expression and mRNA stability studies carried out also suggested regulatory role of miR106a. The overall results thus suggested that the levels of miR106a gets modulated during cellular migration, causing a change in the levels of SIRT-1 mRNA by affecting its stability and the levels of SIRT-1 in turn can regulate the levels of MMP9.

2-Deoxy Glucose Modulates Expression and Biological Activity of VEGF in a SIRT-1 Dependent Mechanism.

Reprogramming of energy metabolism particularly switching over of cells to aerobic glycolysis leading to accumulation of lactate is a hallmark of cancer. Lactate can induce angiogenesis, an important process underlying tumor growth and metastasis. VEGF is one of the most important cytokines which regulate this process and the present study was designed to examine if blocking glycolytic pathway in tumor cells can affect its angiogenic potency with respect to VEGF. For this, the expression and biological activity of VEGF synthesized and secreted by tumor derived cell lines in the presence or absence of 2-deoxy glucose (2-DG), an inhibitor of glycolysis was determined. The results suggested that inhibition of glycolysis using sub-lethal doses of 2-DG down-regulated the expression of VEGF and also significantly reduced its biological activity. Further mechanistic studies revealed that the down regulation of VEGF gene expression by 2-DG was due to an increase in SIRT-1 activity and the reduced biological activity was found to be due to an increase in the PAR modification of VEGF. Activity of SIRT-1 and PAR modification of VEGF in turn, was found to be correlated to the cellular NAD+ levels. The results presented here therefore suggest that treatment of cancer cells with 2-DG can significantly reduce its overall angiogenic potency through transcriptional and post-translational mechanisms. J. Cell. Biochem. 118: 252-262, 2017. © 2016 Wiley Periodicals, Inc.