Derek Lee

Long non-coding RNA expression profiles predict clinical phenotypes in glioma.

Glioma is the commonest form of primary brain tumor in adults with varying malignancy grades and histological subtypes. Long non-coding RNAs (lncRNAs) are a novel class of non-protein-coding transcripts that have been shown to play important roles in cancer development. To discover novel tumor-related lncRNAs and determine their associations with glioma subtypes, we first applied a lncRNA classification pipeline to identify 1970 lncRNAs that were represented on Affymetrix HG-U133 Plus 2.0 array. We then analyzed the lncRNA expression patterns in a set of previously published glioma gene expression profiles of 268 clinical specimens, and identified sets of lncRNAs that were unique to different histological subtypes (astrocytic versus oligodendroglial tumors) and malignancy grades. These lncRNAs signatures were then subject to validation in another non-overlapping, independent data set that contained 157 glioma samples. This is the first reported study that correlates lncRNA expression profiles with malignancy grade and histological differentiation in human gliomas. Our findings indicate the potential roles of lncRNAs in the biogenesis, development and differentiation of gliomas, and provide an important platform for future studies.

Inhibition of prolyl 4-hydroxylase, beta polypeptide (P4HB) attenuates temozolomide resistance in malignant glioma via the endoplasmic reticulum stress response (ERSR) pathways

BACKGROUND Glioblastoma multiforme (GBM), the most aggressive malignant primary brain tumor of the central nervous system, is characterized by a relentless disease recurrence despite continued advancement in surgery, radiotherapy, and chemotherapy. Resistance to temozolomide (TMZ), a standard chemotherapeutic agent for GBM, remains a major challenge. Understanding the mechanisms behind TMZ resistance can direct the development of novel strategies for the prevention, monitoring, and treatment of tumor relapse. METHODS AND RESULTS Our research platform, based on the establishment of 2 pairs of TMZ-sensitive/resistant GBM cells (D54-S and D54-R; U87-S and U87-R), has successfully identified prolyl 4-hydroxylase, beta polypeptide (P4HB) over-expression to be associated with an increased IC50 of TMZ. Elevated P4HB expression was verified using in vivo xenografts developed from U87-R cells. Clinically, we found that P4HB was relatively up-regulated in the recurrent GBM specimens that were initially responsive to TMZ but later developed acquired resistance, when compared with treatment-naive tumors. Functionally, P4HB inhibition by RNAi knockdown and bacitracin inhibition could sensitize D54-R and U87-R cells to TMZ in vitro and in vivo, whereas over-expression of P4HB in vitro conferred resistance to TMZ in both D54-S and U87-S cells. Moreover, targeting P4HB blocked its protective function and sensitized glioma cells to TMZ through the PERK arm of the endoplasmic reticulum stress response. CONCLUSIONS Our study identified a novel target together with its functional pathway in the development of TMZ resistance. P4HB inhibition may be used alone or in combination with TMZ for the treatment of TMZ-resistant GBM.

Hypoxia induces myeloid‐derived suppressor cell recruitment to hepatocellular carcinoma through chemokine (C‐C motif) ligand 26

A population of stromal cells, myeloid‐derived suppressor cells (MDSCs), is present in tumors. Though studies have gradually revealed the protumorigenic functions of MDSCs, the molecular mechanisms guiding MDSC recruitment remain largely elusive. Hypoxia, O2 deprivation, is an important factor in the tumor microenvironment of solid cancers, whose growth often exceeds the growth of functional blood vessels. Here, using hepatocellular carcinoma as the cancer model, we show that hypoxia is an important driver of MDSC recruitment. We observed that MDSCs preferentially infiltrate into hypoxic regions in human hepatocellular carcinoma tissues and that hypoxia‐induced MDSC infiltration is dependent on hypoxia‐inducible factors. We further found that hypoxia‐inducible factors activate the transcription of chemokine (C‐C motif) ligand 26 in cancer cells to recruit chemokine (C‐X3‐C motif) receptor 1‐expressing MDSCs to the primary tumor. Knockdown of chemokine (C‐C motif) ligand 26 in cancer cells profoundly reduces MDSC recruitment, angiogenesis, and tumor growth. Therapeutically, blockade of chemokine (C‐C motif) ligand 26 production in cancer cells by the hypoxia‐inducible factor inhibitor digoxin or blockade of chemokine (C‐X3‐C motif) receptor 1 in MDSCs by chemokine (C‐X3‐C motif) receptor 1 neutralizing antibody could substantially suppress MDSC recruitment and tumor growth. Conclusion: This study unprecedentedly reveals a novel molecular mechanism by which cancer cells direct MDSC homing to primary tumor and suggests that targeting MDSC recruitment represents an attractive therapeutic approach against solid cancers. (Hepatology 2016;64:797‐813)

NDUFA4L2 Fine-tunes Oxidative Stress in Hepatocellular Carcinoma.

Purpose: Hepatocellular carcinoma (HCC) lacks effective curative therapy. Hypoxia is commonly found in HCC. Hypoxia elicits a series of protumorigenic responses through hypoxia-inducible factor-1 (HIF1). Better understanding of the metabolic adaptations of HCC cells during hypoxia is essential to the design of new therapeutic regimen. Experimental Design: Expressions of genes involved in the electron transport chain (ETC) in HCC cell lines (20% and 1% O2) and human HCC samples were analyzed by transcriptome sequencing. Expression of NDUFA4L2, a less active subunit in complex I of the ETC, in 100 pairs of HCC and nontumorous liver tissues were analyzed by qRT-PCR. Student t test and Kaplan–Meier analyses were used for clinicopathologic correlation and survival studies. Orthotopic HCC implantation model was used to evaluate the efficiency of HIF inhibitor. Results: NDUFA4L2 was drastically overexpressed in human HCC and induced by hypoxia. NDUFA4L2 overexpression was closely associated with tumor microsatellite formation, absence of tumor encapsulation, and poor overall survival in HCC patients. We confirmed that NDUFA4L2 was HIF1-regulated in HCC cells. Inactivation of HIF1/NDUFA4L2 increased mitochondrial activity and oxygen consumption, resulting in ROS accumulation and apoptosis. Knockdown of NDUFA4L2 markedly suppressed HCC growth and metastasis in vivo. HIF inhibitor, digoxin, significantly suppressed growth of tumors that expressed high level of NDUFA4L2. Conclusions: Our study has provided the first clinical relevance of NDUFA4L2 in human cancer and suggested that HCC patients with NDUFA4L2 overexpression may be suitable candidates for HIF inhibitor treatment. Clin Cancer Res; 22(12); 3105–17. ©2016 AACR.

Effects of progesterone vs. dexamethasone on brain oedema and inflammatory responses following experimental brain resection.

Abstract Background: Dexamethasone (DEXA) is commonly used to reduce brain swelling during neurosurgical procedures. DEXA, however, has many side-effects that can increase the risks of post-operative complications. In contrast, progesterone (PRO) has fewer side-effects and has been found to be neuroprotective on traumatic brain injury (TBI). Whether PRO may be used as an alternative to DEXA during routine procedures has not been fully explored. Object: To compare the effects of DEXA and PRO on surgical brain injury (SBI). Methods: Seventy-five adult male Sprague Dawley rats were randomized into five groups: (1) SBI + drug vehicle (peanut oil, 1 ml kg−1); (2) SBI + DEXA (1 mg kg−1); (3) SBI + low-dose PRO (10 mg kg−1); (4) SBI + high-dose PRO (20 mg kg−1); and (5) sham SBI + drug vehicle. Magnetic resonance imaging study and assessments of brain water content (BWC), blood–brain barrier (BBB) permeability, cellular inflammatory responses and matrix metalloproteinase 9 (MMP-9) expression were conducted. Results: This model consistently resulted in increased BWC and BBB disruption. PRO reduced astrocyte and microglia responses and attenuated brain oedema with preservation of BBB. A significant down-regulation of MMP-9 expression occurred in the PRO 20 group. Conclusions: PRO is as effective as DEXA in reducing brain oedema and inflammation following SBI; 10 mg kg−1 of PRO was demonstrated to be more effective in relieving acute cellular inflammatory responses.

Folate cycle enzyme MTHFD1L confers metabolic advantages in hepatocellular carcinoma

Cancer cells preferentially utilize glucose and glutamine, which provide macromolecules and antioxidants that sustain rapid cell division. Metabolic reprogramming in cancer drives an increased glycolytic rate that supports maximal production of these nutrients. The folate cycle, through transfer of a carbon unit between tetrahydrofolate and its derivatives in the cytoplasmic and mitochondrial compartments, produces other metabolites that are essential for cell growth, including nucleotides, methionine, and the antioxidant NADPH. Here, using hepatocellular carcinoma (HCC) as a cancer model, we have observed a reduction in growth rate upon withdrawal of folate. We found that an enzyme in the folate cycle, methylenetetrahydrofolate dehydrogenase 1–like (MTHFD1L), plays an essential role in support of cancer growth. We determined that MTHFD1L is transcriptionally activated by NRF2, a master regulator of redox homeostasis. Our observations further suggest that MTHFD1L contributes to the production and accumulation of NADPH to levels that are sufficient to combat oxidative stress in cancer cells. The elevation of oxidative stress through MTHFD1L knockdown or the use of methotrexate, an antifolate drug, sensitizes cancer cells to sorafenib, a targeted therapy for HCC. Taken together, our study identifies MTHFD1L in the folate cycle as an important metabolic pathway in cancer cells with the potential for therapeutic targeting.

MicroRNA-210 and Endoplasmic Reticulum Chaperones in the Regulation of Chemoresistance in Glioblastoma.

Glioblastoma multiforme (GBM) is the commonest primary brain tumour in adults characterized by relentless recurrence due to resistance towards the standard chemotherapeutic agent temozolomide (TMZ). Prolyl 4-hydroxylase, beta polypeptide (P4HB), an endoplasmic reticulum (ER) chaperone, is known to be upregulated in TMZ-resistant GBM cells. MicroRNAs (miRNAs) are non-protein-coding transcripts that may play important roles in GBM chemoresistance. We surmised that miRNA dysregulations may contribute to P4HB upregulation, hence chemoresistance. We found that miRNA-210 (miR-210) was P4HB-targeting and was highly downregulated in TMZ-resistant GBM cells. Forced overexpression of miR-210 led to P4HB downregulation and a reduction in TMZ-resistance. A reciprocal relationship between their expressions was also verified in clinical glioma specimens. Our study is the first to demonstrate a potential link between miR-210 and ER chaperone in determining chemosensitivity in GBM. The findings have important translational implications in suggesting new directions of future studies.

Rutin increases the cytotoxicity of temozolomide in glioblastoma via autophagy inhibition

The chemotherapeutic agent temozolomide (TMZ) is widely used in the treatment of glioblastoma multiforme (GBM). Rutin, a citrus flavonoid ecglycoside found in edible plants, has neuroprotective and anticancer activities. This study aimed to investigate the efficacy and the underlying mechanisms of rutin used in combination with TMZ in GBM. In vitro cell viability assay demonstrated that rutin alone had generally low cytotoxic effect, but it enhanced the efficacy of TMZ in a dose-dependent manner. Subcutaneous and orthotopic xenograft studies also showed that tumor volumes were significantly lower in mice receiving combined TMZ/Rutin treatment as compared to TMZ or rutin alone treatment. Moreover, immunoblotting analysis showed that TMZ activated JNK activity to induce protective response autophagy, which was blocked by rutin, resulting in decreased autophagy and increased apoptosis, suggesting that rutin enhances TMZ efficacy both in vitro and in vivo via inhibiting JNK-mediated autophagy in GBM. The combination rutin with TMZ may be a potentially useful therapeutic approach for GBM patient.

Comparison between self-assembling peptide nanofiber scaffold (SAPNS) and fibrin sealant in neurosurgical hemostasis.

RADA16‐I is a synthetic type I self‐assembling peptide nanofiber scaffold (SAPNS) which may serve as a novel biocompatible hemostatic agent. Its application in neurosurgical hemostasis, however, has not been explored. Although RADA16‐I is nontoxic and nonimmunogenic, its intrinsic acidity may potentially provoke inflammation in the surgically injured brain. We conducted an animal study to compare RADA16‐I with fibrin sealant, a commonly used agent, with the hypothesis that the former would be a comparable alternative. Using a standardized surgical brain injury model, 30 Sprague–Dawley rats were randomized into three treatment groups: RADA16‐I, fibrin sealant or gelatin sponge (control). Animals were sacrificed on day 3 and 42. Astrocytic and microglial infiltrations within the cerebral parenchyma adjacent to the operative site were significantly lower in the RADA16‐I and fibrin sealant groups than control. RADA16‐I did not cause more cellular inflammatory response despite its acidity when compared with fibrin sealant. Immunohistochemical studies showed infiltration by astrocytes and microglia into the fibrin sealant and RADA16‐I grafts, suggesting their potential uses as tissue scaffolds. RADA16‐I is a promising candidate for further translational and clinical studies that focus on its applications as a safe and effective hemostat, proregenerative nanofiber scaffold as well as drug and cell carrier.

Chemoresistance in Glioma

Glioma remains one of the most aggressive and lethal form of cancer. Despite the best available treatment options including surgical resection, radiotherapy, and chemotherapy, prognosis is poor with a median overall survival of just over 1 year. Most patients would die of tumour recurrences because of their tumours’ intrinsic or acquired resistance against chemotherapy. Researchers have strived to better understand the molecular mechanisms of chemoresistance in glioma by using different experimental models, and to direct targeted therapeutics in an attempt to overcome treatment resistance. Currently, different pathways that can confer drug resistance have been identified including DNA damage repair, drug efflux, hypoxia, cancer stem cells, and microRNAs (miRNAs). This chapter will discuss how modulation of these signalling pathways may potentially lead to the development of novel approaches for the treatment of this condition.