Precision genomic and epigenomic cancer medicine against Glioblastoma-Multiforme (GBM).

Prof Dr John N. Giannios

Hippocrates (460-370 BC), the father of Western Medicine has described cancer in great detail, and he has been a basic proponent of personalized medicine emphasizing the importance of administering different drugs to different patients. Unfortunately today, conventional chemotherapy applies the one size fits all approach or the trial and error strategy prescribing the same drugs to virtually all patients with a particular type of cancer causing more harm than good because most anticancer drugs have a very narrow therapeutic index, which is associated with life threatening toxicities.

Furthermore, conventional chemotherapeutic agents, such as anthracyclines may upregulate chemoresistant genes such as MDR1 that encode drug efflux protein Pgp or cytostatic agents like microtubule-depolymerizers may activate cancer stem cells enhancing metastatic potential (Giannios,2010). Furthermore, cancer monotherapies may induce genetic, and epigenetic alterations creating chemo/radioresistance (Claes et al,2010).

Thus, we need precision cancer medicine with tailored molecular targeting approaches with novel drug or gene delivery systems that will target specifically tumor cells circumventing biological milieu interactions. Now it is well known that cancer is a genetic and epigenetic disease.

GBM like any other cancer is a disease driven by progressive genetic, and epigenetic aberrations that exist as global alterations in chromatin packaging, and specific-promoters which influence the transcription of associated genes. Epigenetics which is a term that is derived from the Greek “epi” which means ‘Upon”, and “genetics”(Yu,2009) involves mitotically, and meiotically heritable alterations in gene-expression that are not coded in the underlying DNA sequence. Thus, in contrast to genetic alterations, the epigenetic modifications are reversible in tumor cells favoring exploitation for drug targeting (Nagarajan and Costello, 2009).

Epigenome is the group of alterations in our genomes that will modify the DNA conformation, which subsequently will change the expression of genes without altering the sequence of the bases in the DNA. Thus, if the genome consists of the alphabet, the epigenome is composed of the typed words in bold that give emphasis to the written text. Epigenetic alterations in cells are implicated in diseases like cancer because the epigenetic mechanisms constitute the basis for development influencing the expression of cancer genes alone or combined with genetic mechanisms. Tumor specific epigenetic mutations or epimutations at global and gene specific level have been exhibited for all the components of the epigenetic machinery including DNA- methylation, RNA covalent histone and noncovalent modifications that interact with each other cooperating with genetic mutations for establishing tumor-cell specific phenotypes, which are characterized by high tumor cell proliferation, resistance to apoptosis after anticancer treatment, inhibition of cell-differentiation, enhanced cellular motility and invasion causing metastasis (Mund and Lyko,2010).

Another epigenetic hallmark includes noncoding-RNA, such as miRNA that may dysregulate gene activity in cancer alone or after interacting synergistically with the rest of epigenetic components by silencing tumor suppressor genes or activating oncogenes both of which have been identified as the major drivers of carcinogenesis, and tumor progression (Claes et al, 2010). It is a suprising fact about noncoding-RNAs such as miRNAs ,that although it was thought that the standard pathway of information-flow in a cell was from DNA to message RNA to ptotein, recent genomic studies involving large-scale complementary-DNA sequencing, and genome tiling-array studies have exhibited that the majority of RNA transcripts are noncoding RNA or not protein coding-RNAs ( Mann,2007; Zhang,2008).

Thus, approximately half of the human genomic-DNA is transcribed into RNA-transcripts of which 98% is noncoding RNAs (ncRNAs), and the remaining 2% is translated into proteins (Mattick and Makunin,2006; Szymanski et al,2003). MicroRNAs(miRNAs) are evolutionarily conserved, endogenous, noncoding small RNAs that act as post-transcriptional gene-regulators leading to mRNA-degradation or repressed-protein production (Wu et al,2008; Osaki et al,2008).

Each miRNA is able to regulate several mRNAs by mechanisms, such as incomplete base-pairing and Post-Transcriptional Gene-Silencing (PTGS) (Wurdinger and Costa,2007). The other post-transcriptional pathway that controls gene-expression consists of the AU-rich element Mediated-Degradation of mRNA (AMD). Expression profiles of human miRNAs demonstrated that many miRNAs are deregulated in incurable solid tumors cancers such as Glioblastoma-Multiforme(GBM), and are differentially expressed in normal-tissues and cancers (Novakova et al,2009).

Glioblastoma-multiforme (GBM) remains one of the most challenging tumors to treat worldwide due to its resistance to conventional chemoradiotherapy (Frosina, 2009). Even with surgery the median-survival after diagnosis is less than fourteen months (Dreyfuss et al, 2009). Thus, a search for more efficient, specific, and personalized-therapy with less systemic-toxicity is urgently needed for this disease of orphan-drug status. Therefore, one of the key goals of this project is to examine if every cellular-process in incurable solid tumors such as GBM is likely to be regulated by an interplay of epigenetic components including microRNAs (Casalini and Iorio, 2009), and how these microRNAs, and the interacting epigenetic mechanisms act as master regulators of protein-coding gene-expression at the post-transcriptional level (Cortez et al, 2010) influencing almost all cellular processes in cancer.

The mechanisms that alter the expression of noncoding-RNAs, such as miRNAs including gross-genomic aberrations, epigenetic cross-talk, and mutations or functional polymorphisms in the mRNAs targeted by miRNAs (miSNPs) are very important for pharmaco-epigenomics, and pharmacogenomics (Claes et al,2010;Brid et al, 2010). Noncoding-RNAs such as miRNAs are categorized according to their function in oncomirs, metastamirs, apoptomirs, hypoxamirs,and angiomirs. These categories of miRNAs, and the other epigenetic components must be used as prognostic biomarkers for survival, and predictive-biomarkers for treatment leading to personalized cancer medicine approaches (Tili et al,2007).

Another very important issue is the significance of epigenetic-profiling including miRNA-profiling ,and functional-analysis of incurable solid tumors such as GBM for discovering multiple-epigenetic targets that affect all the diverse biological-processes of these deadly tumors (Godlewski et al, 2009). Very crucial in cancer is the phenomenon of oncomiR-addiction, synthetic sickness/lethality (SSL) relationship, and synergistic outcome determination (SOD) for identifying new therapeutic-approaches against incurable solid tumors, such as GBM (Rehman et al, 2010; Xiong et al, 2010).

There is an interactive interplay between abnormalities of the epigenome that consists of DNA methylation, and covalent/noncovalent histone modifications, and the miRNome which consists of epi-miRNAs (Meenakshisundaram et al,2009; Valeri N et al,2009) for creating new therapeutic avenues of Translational Medicine towards a personalized therapeutic approach mediated by tailored molecular targeting strategies against lethal malignancies, such as GBM (Fabbri et al,2008).

Precision medicine approaches of GBM may be mediated by liquid biopsy by isolating exosomes.  

 Since , the epigenome differs over an individual’s lifetime due to influences by environmental chemicals, drugs, diet and aging, we need technologically advanced microarrays for identifying epigenetic changes among different individuals, tissues, developmental stages, and cancer types. This way under a personalized cancer medicine approach, epigenetic drugs targeting histone methyltransferases, deacetylases, demethylases, sirtuins, and methylating genes (DNMT1, DNMT3a, DNMT3b, etc) combined with miRNA mimetics or antagomirs will enable efficient individualized cancer treatment using molecular targeting that can act synergistically with conventional therapy circumventing resistant mechanisms for the eradication of incurable tumour cells, such as GBM.

Emphasis must be given on the identification of biologically relevant targets of microRNAs, and other epigenetic hallmarks, and how the use of miRNA-mimics or anti-microRNAs combined with inhibitors of DNMT, HDAC, histone-methyltransferases, histone-demethylases, and histone-acetyltransferases may interfere efficiently with key molecular-pathways involved in incurable solid tumors, such as GBM (Garofalo and Croce, 2011).

Thus, efficient identification of the most crucial epigenetic lesions for every cancer patient may translate them into effective precision epigenomic medicine approaches (Claes et al,2010). Another priority is to examine viral, and non-viral gene delivery systems such as nanosomes, and how we can circumvent the blood-brain-barrier (BBB),and all the extracellular or intracellular-barriers for achieving an efficient-delivery to incurable solid tumors, such as GBM of novel epigenetic drugs including epigenetic-enzyme inhibitors, miRNA mimics, and miRNA-antagomirs.

More analytically, similarly to cancer-genes that encode proteins, deregulation of miRNA-encoding-genes is associated with genetic or epigenetic-alterations, including deletions, amplifications, point-mutations , aberrant-DNA methylation, histone covalent, and noncovalent modifications (Negrini et al, 2007). Recent evidence has shown that epigenetic alterations including deregulation of miRNAs may correlate with certain features of malignancy such as tumorigenesis, differentiation status, and outcome of tumor patients, and indicates that miRNAs can act as oncogenes or tumor suppressors (Yu et al,2007).

It is very important to elucidate the role of epigenetic interplay on the regulation of multiple-genes that are associated with diverse biological-processes, such as cell-proliferation and development, cell-cycle progression, differentiation, survival, apoptosis, invasion, metastasis, angiogenesis, epithelial-to-mesenchymal transition, DNA-damage repair, immune-escape, chemoradiotherapy-resistance, stem-cell behavior and self-renewal of incurable solid tumors, such as glioblastoma-multiforme that is the most common and aggressive primary brain-tumor with very poor-patient median-survival despite treatment with surgery, radiation, and chemotherapy (Srinivasan et al,2011).

 Thus, by identifying the emerging-roles of epigenetic alterations in the hallmarks of incurable solid tumors may lead to a clearer understanding of the mechanisms involved in the interplay between the epigenetic constituents, which may be targeted with epigenetic drugs combined with other therapies that may lead to synthetic lethality by circumvention of oncogene dominant-effects and/or re-expression of apoptotic tumor suppressor genes.

 Therefore, the epigenetic signature of incurable solid tumors must be used for precision medicine translational approaches including development of potential epigenetic diagnostic or prognostic-biomarkers, and therapeutic-approaches, such as inhibitors of epigenetic enzymes, and miRNA-mimics or antagomirs that might be molecularly-targeted with delivery-systems against cancer epigenetic lesions under a personalized-basis aiming to eradicate incurable solid tumors, such as GBM which is a fatal tumor known as the “terminator” due to its extremely high and unavoidable fatality.

The most crucial research question is of how epigenetic biomarkers will lead to the development of “theranostics” which consist of diagnostic-testing for tailoring the correct epigenetic treatment, and dose on an individualized/personalized basis against incurable solid tumors. Effective precision cancer medicine requires the discovery and development mediated by Translational Cancer Medicine of the most effective treatments against lethal cancers, such as GBM that have failed all other therapeutic options.

These should be novel epigenetically based therapeutic agents combined with molecular genetic-based therapeutic approaches, and standard chemotherapy for circumvention of chemoresistance by molecular targeting of signaling pathways on an individualized basis that will be facilitated by biomarker signatures derived from pharmaco-epigenomics, and pharmacogenomics.

Epigenetic cancer therapy including miRNA-based approached may even eradicate lethal tumors by interfering with mutant oncogenic drivers that affect one or more of the 12 core cancer signaling pathways (Cho,2010) with RNA modification therapy which may be facilitated by categories of Nucleic Acid Drugs (Jarald et al, 2004), such as First Generation Antisense Oligonucleotides including  Phosphorothioate (PS) oligodeoxynucleotides (ASON), Second Generation Antisense Oligonucleotides such as 2’-O-Alkyl-RNA, Third Generation Antisense Oligonucleotides including Peptide Nucleic Acids(PNAs) (Giannios,2008;Giannios et al,2008), Phosphoroamidates(NPs) such as  2-Deoxy-2’-fluoro-b-d-arabino nucleic acid (FANA), and Locked Nucleic Acid (LNA) (Giannios,2010), RNA interference (siRNA) (Giannios,2010), Antigene Nucleic Acid Compounds, Segmental Trans-Splicing (STS)(Nakayama,2005), Trans-Splicing (Agabian,1990), Ribozyme Nucleic acids including Hairpin Ribozymes (Giannios and Ginopoulos,2000) and Hammer Head Ribozymes (Giannios,2000), Triplex Forming Oligonucleotide (TFO)(Brunet et al,2005; Svinarchuk et al,1997; Giannios,2005; Giannios et al, 2005), Morpholinos Antisense Oligos (PMO) (Summerton et al,1977), and Oligonucleotide Aptamers like Intracellular Antibodies (Intrabodies) that can specifically bind to mutant oncoproteins inactivating them (Giannios and Ginopoulos,1999; Giannios et al,2005; Giannios et al,2007).

Other Genetically- based Therapeutic Approaches include Gene Augmentation Therapy (GAT) or Gene Transfer by Insertion of a wild type copy of an inactivated tumour suppressor gene for induction of apoptosis. The Gene Therapy can be in-vivo, ex-vivo or in-vitro (Giannios and Ioannidou,1998). The intracellular delivery of all of the above therapeutic agents can be mediated by Drug Delivery Systems, such as Viral Vectors (Oncoretroviral vectors, Adeno-associated virus vectors, Adenovirus vectors, Lentivirus), and Non-Viral Vector Systems for Gene Therapy such as niosomes, cationic lipid complexes, nanosomes, and liposomes (Giannios and Ginopoulos,2000) which with the proper manipulation of their lipid composition may become more stable especially with use of high Tc phospholipids, and more hydrophilic with PEGylation.

Specific molecular targeting can be achieved by linking antibodies on the surface of these colloidal carriers, such as Polyclonal,Monoclonal, Chimeric, Single Stranded, and Fab Fragments (Giannios et al,2000; Giannios et al,2004; Giannios and Ginopoulos,1999). Thus,emphasis should be given to the development of efficient drug delivery systems which may improve the molecular targeting process, and the therapeutic drug index by circumventing biological milieu interactions minimizing systemic toxicity.

All these therapeutic molecules, and delivery systems may be used for targeting epigenetic targets which will modulate genetic pathways for circumventing resistant to treatment mechanisms leading to eradication of incurable tumour cells such as GBM by inducing apoptosis or type I programmed cell death (PCD) under a precision cancer medicine approach. Thus, we must define the interaction of these potential epigenomic targets with the genomic ones exploring their diagnostic and therapeutic potentialities against incurable solid tumors by elucidating their unkown molecular pathology aspects characterized by potent resistance to conventional anticancer therapy.

Thus, pending issues which will require immediate answers involve  definition of the Precision Translational Cancer Medicine (PTCM) opportunities, and challenges that need to be resolved for the efficient translation of microRNAs and their interactions with the rest of the noncoding RNA epigenomic machinery, and the genomic-pathways involving coding RNA genes which are translated into proteins affecting a plethora of vital downstream signaling pathways for their validated facilitation from bench to clinic as molecular biomarkers for screening, prevention, diagnosis, management, monitoring and prognosis of incurable and lethal tumors, such as GBM under a precision medicine approach mediated by evidence of pharmaco-genomics and mainly pharmaco-epigenomics.

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