Advanced Bioinformatics Analysis and Genetic Technologies for Targeting Autophagy in Glioblastoma Multiforme

Abstract

As the most malignant primary brain tumor in adults, a diagnosis of glioblastoma multiforme (GBM) continues to carry a poor prognosis. GBM is characterized by cytoprotective homeostatic processes such as the activation of autophagy, capability to confer therapeutic resistance, evasion of apoptosis, and survival strategy even in the hypoxic and nutrient-deprived tumor microenvironment. The current gold standard of therapy, which involves radiotherapy and concomitant and adjuvant chemotherapy with temozolomide (TMZ), has been a game-changer for patients with GBM, relatively improving both overall survival (OS) and progression-free survival (PFS); however, TMZ is now well-known to upregulate undesirable cytoprotective autophagy, limiting its therapeutic efficacy for induction of apoptosis in GBM cells. The identification of targets utilizing bioinformatics-driven approaches, advancement of modern molecular biology technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas9) or CRISPR-Cas9 genome editing, and usage of microRNA (miRNA)-mediated regulation of gene expression led to the selection of many novel targets for new therapeutic development and the creation of promising combination therapies. This review explores the current state of advanced bioinformatics analysis and genetic technologies and their utilization for synergistic combination with TMZ in the context of inhibition of autophagy for controlling the growth of GBM.

Keywords: CRISPR-Cas9 gene editing; autophagy inhibition; bioinformatics analysis; glioblastoma multiforme; mRNAs; novel combination therapy option.

Figure 1

Characteristic stressors in the tumor microenvironment drive selection of tumor phenotypes that are resistant to radiotherapy and chemotherapeutics due to the induction of cytoprotective autophagy and prevention of induction of apoptotic cell death. Identification of autophagy-related genes (ARGs) that are differentially expressed using advanced bioinformatics analysis can enable the selection of novel targets for CRISPR-Cas9 gene editing and miRNA-mediated autophagy inhibition to potentiate TMZ efficacy.

Figure 2

An overview of the CRISPR-Cas9 gene editing technology. The ever-evolving CRISPR-Cas9 gene editing technology has enabled the knockout (KO) and knockdown (KD) of specific genes that are known to promote cytoprotective autophagy in cancers. The protospacer adjacent motif (PAM) sequence and sgRNA are essential for DNA target recognition. The double-strand breaks (DSBs) that result from CRISPR-Cas9 action can lead to either non-homologous end joining (NHEJ) or homology-directed repair (HDR) in the DNA molecule. KO typically involves NHEJ, which produces indels that lead to frameshift mutations, while HDR is another mechanism of DNA repair that involves the insertion of a specific DNA template utilizing homologous regions to rejoin cleaved DNA (homologous regions are shown by X). On the other hand, the use of catalytically deactivated Cas9 (dCas9) enables the reversible KD of ARGs, blocking transcriptional elongation, RNA polymerase binding, and/or transcription factor binding.

Figure 3

A visual representation of pathways with involvement of miRNAs in the induction of cytoprotective autophagy and inhibition of apoptosis in GBM cells. Various miRNAs directly interact with the pathways involved in autophagy progression to either promote or inhibit autophagy. Key pathways include those involving proteins that are members of the Bcl-2 family, such as Bim, Bad, Bax, Bcl-2, Bcl-xL, Beclin 1, and the tumor suppressor p53. Modification of expression of these miRNAs has led to increased TMZ chemosensitivity and synergism in combatting GBM.