Targeting genetic tool for long non-coding RNA of cancer stem cells with aptamer-guided nanocarriers

1. Introduction

Colloquially termed ‘immortal tumor-initiating cells,’ cancer stem cells represent tumor subpopulations that possess unlimited self-renewal and pluripotency attributes [1]. The classic cancer phenotypes, their aggressive nature, and chemotherapy resistance are largely attributable to cancer stem cells. Recent reviews by Shibata and Hoque (2019) and Zhou et al. (2017) highlighted that cancer stem cells’ rapid self-renewal and differentiation into diverse cancer lineages are the cradle to cancer initiation, progression, and heterogeneous expression [2,3]. The cancer stem cells, with altered metabolism and immune response evasion, give rise to chemo-resistant tumor populations [4]. They can enter into a state of quiescence that shields them from death which in turn facilitates tumor relapse [2,3]. A study by Nair and colleagues (2017) reported that cancer-associated fibroblasts, a key stromal component of tumor microenvironments that support cancer growth and survival, originate from cancer stem cells [5]. The cancer stem cells can rapidly recapitulate the migrating parent tumor through fibroblastic activities to succeed metastasis [6]. On this note, eradicating cancer stem cells is imperative in cancer treatment.

Small non-coding RNAs (sncRNAs; < 200 nucleotides long) are the common vehicle in gene expression and translation [4]. Long non-coding RNAs (lncRNAs), particularly circular RNAs (circRNAs), have recently garnered interest. The once mistaken for ‘junk RNA,’ lncRNAs, are now emerging as important epigenetic regulators of numerous biochemical events in cancer development, including those of cancer stem cells [1]. The lncRNAs such as CUDR, SOX2OT, HOTAIR, and H19 play key roles in cancer stem cells’ self-renewal and stemness. The lncRNAs like MALAT-1, HOTAIR, H19, UCA1, and ROR are involved in the generation of cancer stem cells from non-stem cancer cells via TGF-β, Wnt, PI3K/Akt, and p53 signaling that promotes epithelial-mesenchymal transition [4]. The lncRNAs are also implicated in chemoresistance. HOTAIR and UCA1 are dramatically upregulated in cisplatin- and doxorubicin-resistant lung, ovarian, bladder, and gastric cancers and are found to inhibit the expression of miRNAs miR-126, miR-326, and miR-27b. H19 contributes to doxorubicin resistance in hepatocellular carcinoma by regulating DNA methylation at the promoter region of the P-glycoprotein gene [4]. The newly discovered lncRNA NRAD1 has been shown to regulate the production of cancer stem cells-associated aldehyde dehydrogenases that contribute to multi-drug resistance [7].

The lncRNAs in cancer stem cells, as epigenetic targets, warrant research investigation in order to efficiently eradicate the cancer stem cells and the associated cancer. Unlike conventional chemotherapeutics that are known to be the substrates of P-glycoprotein and function by damaging the nuclear and mitochondrial DNA, targeting lncRNAs with genetic tools, such as sncRNAs (siRNA and shRNA), silence the gene expression and offer to bypass many inherent chemoresistance pathways [8]. Lipid nanocarriers such as stable nucleic acid-lipid particles, constituting of a cationic and fusogenic lipid bilayer coated with diffusible polyethylene glycol, are promising for siRNA delivery [9]. Polymeric nanocarriers, such as polyethylene glycol/transferrin-functionalized, adamantane-stabilized cyclodextrin nanoparticles, have been advocated as the vehicle of sncRNAs where they improve the serum stability and genetic transfection efficiency [9]. The intracellular action of sncRNAs is often short-live due to RNase degradation. The transient silencing effect, nonetheless, could have sufficiently sensitized the cancer stem cells to the co-administering chemotherapy [8]. Nanocarriers, such as cationic liposomes, polymeric (e.g. polyethylene glycol), and peptide (e.g. arginine) nanoparticles, have been adopted to deliver CRISPR/Cas9 systems to cancer cells with the aim to knockout lncRNA genes [10,11]. Similar therapeutic and delivery systems may be applied in the case of cancer stem cells.

The cellular interaction and uptake profiles of nanocarriers are dependent on the physicochemical attributes of the latter such as size, shape, surface morphology, charges and chemistry [12]. Organ, tissue and cell targeting can be collectively effected in order to drive the therapeutics to the closest site of action at the cellular level [12–14]. With reference to cancer stem cells, scarce targeting cells, insufficient particulate drug penetration of tumor core and inefficient targeting are the main drug delivery hurdles [3,15,16].

In recent years, aptamers (short single-stranded oligonucleotides) have emerged as promising non-protein ligands for cancer stem cell targeting [17]. The aptamers fold into unique tertiary structures and bind to their targets with a high affinity and specificity. They exhibit many advantages over antibody ligands (~150 kD) including cheaper synthesis cost, low toxicity, high serum stability and non-immunogenicity. They are relatively small in size (< 25 kD) and their use as targeting ligand in nanocarrier is expected not hindering the processes of particle diffusion and penetration into tumor cores to reach the cancer stem cell populations [3]. The aptamers are stable over a wide pH range (4–9) [18]. It allows pH-responsive nanocarriers with acid-cleavable drug conjugate to be developed for site-specific release in response to cancer microenvironment. Aptamer (EpCAM- and CD133-targeting)-doxorubicin conjugates have been shown to exhibit cancer stem cell targeting with reduced chemoresistance and systemic adverse effects [18]. Aptamer-functionalized pH-responsive cationic liposomes, consisting of cholesterol-based twin cationic lipid, zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and amphiphilic palmitoyl homocysteine, have been designed with improved payload delivery in EpCAM-expressing MCF-7 (breast cancer), COLO 205 (colorectal adenocarcinoma), Caco-2 (colorectal adenocarcinoma), and MDA-MB-231 (adenocarcinoma) cancer stem cells compared to non-functionalized counterparts, and with minimal cytotoxicity in normal HEK293T cells compared to free doxorubicin [15]. Overall, the aptamer-guided nanocarriers for cancer stem cell targeting are a relatively new concept [10,18]. Further explorations are pending to succeed the relevant science.

2. Expert opinion

The cancer stem cell markers have a different expression from non-stem cancer cells and normal stem cells. They are indeed promising as cancer stem cell-specific targets. Examples of the targetable cancer stem cell-specific markers are EpCAM, BORIS sf6, CD133, and DNAJB8 [6]. While some cancer stem cell markers, particularly EpCAM and CD133, have shown promising aptamer-based targetability [15,17,18], others are still subjected to investigation for potential application in aptamer targeting. Cell targeting by means of ligand-decorated nanocarriers is not a new drug delivery strategy and research platform. Targeting cancer stem cells however is met with challenges in the availability of functional markers that can act as the target site for therapeutical nanocarriers via their affinity for specific ligands. Aptamer-guided nanocarrier as the vehicle of genetic tools for lncRNAs of cancer stem cells could well be a powerful tool to finally cure cancer. Single targeting ligand approach nonetheless may not be ideal, under the constraint of possibly limited number population of specific markers in cancer stem cells. Exploring targetable cancer stem cell-specific markers for aptamer as well as new ligands appears imperative, where multi-ligand targeting approach is envisaged to promote the interaction propensity between the cancer stem cells and nanocarrier.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties

Additional information

Funding

The paper was funded by the Ministry of Science, Technology & Innovation Malaysia MOSTI (IF0402Q1226).