Cancer Letters

Cancer Letters

Volume 330, Issue 1, 1 March 2013, Pages 84-95
Cancer Letters

RNA aptamers targeting cancer stem cell marker CD133

https://doi.org/10.1016/j.canlet.2012.11.032Get rights and content

Abstract

The monoclonal antibody against the AC133 epitope of CD133 has been widely used as a cell surface marker of cancer stem cells in several different cancer types. Here, we describe the isolation and characterisation of two RNA aptamers, including the smallest described 15 nucleotide RNA aptamer, which specifically recognise the AC133 epitope and the CD133 protein with high sensitivity. As well, both these aptamers show superior tumour penetration and retention when compared to the AC133 antibody in a 3-D tumour sphere model. These novel CD133 aptamers will aid future development of cancer stem cell targeted therapeutics and molecular imaging.

Highlights

Cancer stem cells (CSCs) are thought to be responsible for post-therapy relapse of cancer. ► AC133, a CSC marker, represents a unique target for the treatment of various tumour types. ► Aptamers generated against the CD133 protein bind to two separate epitopes. ► These aptamers represent the ideal molecule for use as targeted theranostics.

Introduction

CD133, also known as Prominin-1, is a pentaspan, highly glycosylated, membrane glycoprotein that is associated with cholesterol in the plasma membrane [1], [2]. Though this protein is known to define a broad population of cells, including somatic stem and progenitor cells, and is expressed in various developing epithelial and differentiated cells, its exact function is still being elucidated. It has, however, been linked to the Notch-signalling pathway which is critical for binary cell fate, differentiation of intestinal epithelium, and lymphopoiesis [3]. CD133 has gained its prominence in the cancer research field due to its reported role as a marker of cancer stem cells (CSCs) in glioblastomas [4]. Indeed, growing evidence has shown that CD133 is an important cell surface marker for CSCs in a variety of solid cancers, including those of the brain, prostate, pancreas, melanoma, colon, liver, lung and ovarian cancers [5] by virtue of the enhanced tumourigenic potential of CD133+ cells versus their negative counterparts in immunodeficient mice [6].

Most of the work in isolating CD133+ putative cancer stem cell subpopulation from the bulk cancer cells utilises one monoclonal antibody, AC133 [7]. However, there has been some controversy regarding the notion that CD133 can be used as a marker for CSCs, as investigators from independent laboratories showed that the CD133 cells are also tumourigenic in immunocompromised mice [8], [9], [10]. This contention is further complicated by the near ubiquitous expression of CD133 on non-CSCs as well as CSCs, especially in tumours of the colon [11]. A recent study has shed some light on this, with a conformational change postulated to hide an epitope on the second extracellular membrane loop of CD133 during the differentiation process [5]. Kemper and co-workers suggested that the CD133 protein becomes differentially folded as a result of glycosylation, thus masking the AC133 epitope. These results were further supported by later studies [5], [12] which suggested that the AC133 epitope, rather than the complete CD133 protein, is the marker for CSCs. As well, a recent report has shown that the AC133 epitope is lost upon cell differentiation, suggesting that this epitope is a marker of primitive cells [5]. While the AC133 epitope has been shown to be a marker for CSCs, not all cells positive for the AC133 epitope are CSCs. In fact, the first description of this epitope was by Yin et al., in 1997, who described this as a marker of haematopoietic stem and progenitor cells [13]. However, the expression of AC133 on these cells is approximately 1000-fold lower than that observed in CSCs [14]. Despite the on-going debate of the utility of using AC133 to identify cancer stem cells, a retrospective study on colorectal patients showed that a high level of AC133 expression was associated with a poorer prognosis [15], though the sole use of the AC133 antibody is not recommended as it is thought to underestimate the level of CD133 expression [1], [16].

AC133-positive cells have been shown to have an increased resistance to radiation therapy due to activation of the DNA damage checkpoint proteins, and an increased chemoresistance due to an increased Akt/PKB and Bcl-2 cell survival response [17]. These data suggest that a more targeted response is required to eradicate this population of cells, especially given the increasing evidence regarding the roles that CSCs play in the relapse of cancer after initial treatments. Immunotherapy has had a great impact on the treatment of cancer in recent years [18], [19]. However, the use of antibodies, even humanised antibodies, can lead to adverse side effects with fatal consequences [20]. This has led to the search for ‘bigger and better’ options. There have been several attempts to use nucleic acids as therapeutics though these have met with disappointing results, not least because of the failure of these nucleic acids to enter the cell [21]. The reports in 1990 by two separate groups describing the generation of nucleic acids that can bind target molecules in the same manner as antibodies seemed to be the answer [22], [23]. These chemical antibodies, termed aptamers, have been increasingly utilised for clinical applications recently. Indeed, one RNA aptamer has been approved by the FDA and several more are in clinical trials [21], [24]. The increased interest in these aptamers is due to the fact that they exhibit no immunogenicity, little batch-to-batch variation due to being chemically synthesised, and are more stable than conventional antibodies. Due to their small size, aptamers also show superior tumour penetration. One important feature of these chemical antibodies is their versatility as they can be attached to nanoparticles, drugs, imaging agents or other nucleic acid therapeutics without loss-of-function [25], [26]. This functionalisation is leading to new and more targeted therapies, with fewer side effects than current treatment modalities [25]. When compared to conventional treatment which is largely a passive process, targeted delivery systems are much more effective. For an aptamer to be an effective drug delivery agent, the aptamer must be efficiently internalised upon binding to its target on the cell surface [27].

In this study, we performed iterative rounds of an in vitro selection process, known as the systematic evolution of ligands by exponential enrichment (SELEX), to identify RNA aptamers that specifically bind to CD133. Further studies identified one aptamer, CD133-A15, specifically bound to the same epitope as the AC133 antibody, while the other aptamer, CD133-B19, bound to the extracellular domain of the CD133 protein. These aptamers were efficiently internalised into CD133-positive cancer cells and showed superior penetration of three-dimensional tumour sphere.

Section snippets

Cell lines and cell culture

The cell lines of human origin used in this study were purchased from American type Culture Collection. They are human colorectal cancer HT-29; human hepatocellular carcinoma Hep3B; human glioblastoma multiform carcinoma T98G; human embryonic kidney cells HEK293T; human ductal breast carcinoma, T47D; human lung adenocarcinoma, A549; human ovarian teratocarcinoma, PA-1; human hepatoma, PLC/PRC/5; and human prostate carcinoma, DU145. Cells were grown and maintained in culture with Dulbecco’s

Cell SELEX facilitates the selection of aptamers against cell surface targets

CD133 is a complex pentaspan protein containing two extracellular loops. To effectively select aptamers against only the extracellular portion of the protein, it was necessary to devise a procedure that allowed us to express CD133 so as to preserve its native conformation. To this end, we sought to transiently express the protein on the surface of HEK293T cells. Using Lipofectamine 2000, the C-terminally His-tagged CD133 was transfected into HEK293T cells and allowed to express for 72 h prior to

Discussion

Cancer stem cells (CSCs) are considered to be the root of cancer responsible for cancer recurrence. This model has gained acceptance because it explains radiation- and chemotherapy-resistance [45], and has led to numerous attempts to specifically target this population of cells within the tumour. While there is not one specific marker which defines all CSCs, a number of markers, including CD133, CD44, ALDH, EpCAM and ABCG2 [45], [46], have proven useful for defining the CSC population in solid

Acknowledgements

This work was supported by the Australia–India Strategic Research Fund Grant ST010013 and Victorian Cancer Agency Grant PTCP-02 to WD. Dr. L. Qiao is supported by the Career Development and Support Fellowship Future Research Leader Grant of the NSW Cancer Institute, Australia (Grant ID: 08/FRL/1-04).

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