Abstract
Normal stem cells and cancer stem cells (CSCs) share similar properties, in that both have the capacity to self-renew and differentiate into multiple cell types. In both the normal stem cell and cancer stem cell fields, there has been a great need for a universal marker that can effectively identify and isolate these rare populations of cells in order to characterize them and use this information for research and therapeutic purposes. Currently, it would appear that certain isoenzymes of the aldehyde dehydrogenase (ALDH) superfamily may be able to fulfill this role as a marker for both normal and cancer stem cells. ALDH has been identified as an important enzyme in the protection of normal hematopoietic stem cells, and is now also widely used as a marker to identify and isolate various types of normal stem cells and CSCs. In addition, emerging evidence suggests that ALDH1 is not only a marker for stem cells, but may also play important functional roles related to self-protection, differentiation, and expansion. This comprehensive review discusses the role that ALDH plays in normal stem cells and CSCs, with focus on ALDH1 and ALDH3A1. Discrepancies in the functional themes between cell types and future perspectives for therapeutic applications will also be discussed.
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Introduction
Stem cells are defined as having both the ability to self-renew and the capacity to differentiate into mature cells that can repopulate specific tissues and organs [1]. Depending on the type of stem cells, a variety of stem cell specific markers and strategies can be used to isolate and enrich for target cells when used alone or in combination with other markers.
Recently, there has been a growing interest in the cancer stem cell (CSC) hypothesis, since the discovery of CSCs in leukemia by Dr. John Dick in the 1990s [2]. Currently there are two conflicting models of cancer development [1]. The classic “stochastic model” proposes that the cell population within a tumor is heterogeneous, but that all cells have an equal possibility of acquiring mutations and initiating a tumor. In contrast, the “hierarchical model” (upon which the CSC hypothesis is based) proposes that only a distinct and small subset of cancer cells within the population are highly efficient at initiating tumors, whereas the majority of tumor cells are differentiated cells with limited replicative potential. Currently, because the therapies against cancer are based on the stochastic model and not the hierarchical model, along with the fact that the current therapies available for cancer do not cure cancer, the hierarchical model may be the more accurate model [1].
In both normal stem cells and CSCs, there is a need for a universal marker or set of markers that accurately identify and isolate these rare stem cells in order to characterize them and use this information for research and therapeutic purposes. Currently, it would appear that aldehyde dehydrogenase (ALDH) may be eligible as such a marker [3, 4]. Of the vast ALDH families and subfamilies, it has been shown that certain ALDH isoenzymes, such as ALDH1A1 and ALDH3A1, are important for normal stem cells as well as cancer stem cells (CSCs). In particular, ALDH1 has been used as a marker to identify and isolate normal and cancer stem cells, and both ALDH1A1 and ALDH31 have been shown to play important functional roles in stem cells with regards to self-protection, differentiation, and cellular expansion. This comprehensive review will discuss the various ALDH isoenzymes and their properties, with a focus on ALDH1A1 and ALDH3A1. Additionally, we will discuss how ALDH has been utilized in stem cell research, the functional role that ALDH plays in various types of stem cells (normal and malignant), the molecular mechanisms that may be involved, and future perspectives for research and therapy.
The Human Aldehyde Dehydrogenase Superfamily
The human aldehyde dehydrogenase (ALDH) superfamily currently consists of 19 known putatively functional genes [5–7] in 11 families and 4 subfamilies [7] with distinct chromosomal locations. When compared to the human genome, the rat and mouse genome has an additional ALDH gene (Aldh1a7). Although many mammalian ALDH genes have been identified, as well as various alternatively spliced transcriptional variants of human ALDH genes, several of the corresponding proteins have not been fully characterized [7] and can potentially be a vast area of exploration for researchers.
The ALDH enzymes can be found in the cytosol, nucleus, mitochrondria, and endoplasmic reticulum [5, 8]. Depending on the enzyme family and subfamily, the human ALDHs can vary in their enzyme levels, as well as in their tissue and organ distribution [8–10]. The ALDH superfamily of NAD(P)+-dependent multifunctional enzymes catalyze the oxidation of various endogenous and exogenous aldehydes to their corresponding carboxylic acids [5–7, 11]. Aldehydes are highly reactive electrophilic compounds that have a long lifespan, and can play a vital role in physiological processes as well as playing mutagenic, carcinogenic, and cytotoxic detrimental roles [5, 6]. Endogenous aldehydes can be generated by various metabolic processes, including lipid peroxidation, amino acid catabolism, biotransformation of neurotransmitters and carbohydrates [5]; as well as through metabolism of vitamins (retinal to retinoic acid [12]) and steroids [6, 7]. The source of exogenous aldehydes includes the biotransformation of exogenous aldehyde precursors such as xenobiotics and drugs (i.e. ethanol, cyclophosphamide, ifosfamide); as well as environmental aldehydes that are present in industrial applications such as smog, cigarette smoke, and motor vehicle exhaust [5]. In addition to the oxidization of aldehydes, other catalytic functions of the human ALDH superfamily of enzymes include ester hydrolysis (ALDH1A1, ALDH2, ALDH4A1) and nitrate reductase activity (ALDH2) [5, 6]. Some enzymes of the ALDH superfamily also have the capacity for non-catalytic functions, including being able to bind to endobiotics (ALDH1A1) and xenobiotics (ALDH1A1, ALDH1L1, ALDH2), having antioxidant functionalities (ALDH1A1, ALDH3A1), having structural roles (ALDH1A1, ALDH3A1), and being involved in osmoregulation (ALDH7A1) [5, 6] (Table 1).
Of the vast ALDH families and subfamilies, it has been shown that the ALDH enzymes that are involved in normal stem cells as well as cancer stem cells (CSCs) include the ALDH1 family (ALDH1A1, 1A2, 1A3, 1L1, 1L2), ALDH2*2 (with an association between alcoholism and alcohol-induced cancer risk), ALDH3A1 (highly protective for normal stem cells and cancer cells, with a role in hormone-dependent tumors), ALDH4A1 (through p53 and DNA damage), and ALDH7A1 (putatively involved in the regulation of cell cycle) [5], all of which are located in various chromosome locations [10] (Table 1). In particular, ALDH1 has been used as a marker to identify and isolate normal and cancer stem cells, and both ALDH1A1 and ALDH3A1 have been shown to play an important functional role in stem cells. Therefore, for the remainder of this review article, the focus will be on these two ALDH isoenzymes and their potential functional role in normal and cancer stem cells in terms of self-protection, differentiation, and/or expansion of stem cell populations.
ALDH as a Marker for Normal and Cancer Stem Cells
Evolution of ALDH as a Tool in Normal and Cancer Stem Cell Research
As an enzyme, it is intuitive to study the activity of ALDH using enzyme kinetics, where the rate of reduction of NAD+ substrate to NADH by ALDH present in cell lysates can be measured at 37°C at a wavelength of 340 nm [13–15]. Another common method to study ALDH in normal and cancer stem cells has been by immunoblotting [13, 14, 16]. Although immunoblotting does not measure the activity of ALDH, it does measure protein levels present in the cells. Unfortunately, there are a couple of pitfalls to these methods. Firstly, both the enzyme kinetics and immunoblotting methods require the endogeneous ALDH enzymes to be released from the cells, which would require lysis of the cells [13, 14]. It may be more useful to study the activity of the ALDH enzyme in viable cells. Secondly, there may be difficulties associated with the use of immunoblotting to determine protein levels of ALDH as there is significant cross-reactivity between antibodies against different ALDH isoforms due to a high degree of amino acid sequence conservation [9, 17]. Additionally, ALDH enzymes contain reactive cysteine active sites and are relatively unstable enzymes, making immunoblotting a difficult method to pursue as the enzymes may be inactivated after cell lysis [9]. Although it has been important to study the enzyme kinetics and protein levels of ALDH in the initial stages of ALDH research, a more efficient and suitable approach was necessary in this field. Thus currently, the “gold standard” of studying the activity of ALDH (more specifically ALDH1) in viable cells has been the use of flow cytometry and fluorescent substrates for ALDH1 [18–24] .
A decade and a half ago, Jones, et al. reported for the first time that intracellular ALDH1 activity could be measured in viable cells. They found that dansyl aminoacetaldehyde (DAAA), a fluorescent aldehyde, could be used in flow cytometry to isolate and enrich for viable human hematopoietic stem cells (HSCs) and leukemic SCs (mouse and human) based on their ALDH1 activity [25]. Hydrophobic DAAA can diffuse freely across cell membranes, so cells with ALDH1 activity can oxidize DAAA into dansyl glycine. Dansyl glycine is negatively charged at physiological pH, and therefore is not able to exit the cells, thus causing the cells with dansyl glycine to become fluorescent [25]. As a negative control, cells were also incubated with 4-(diethylamino)benzaldehyde (DEAB), a specific ALDH1 inhibitor [25, 26]. Jones, et al. hypothesized that there were two advantages to using a technique that could detect ALDH1 activity levels within viable cells. The first advantage was that it could isolate and enrich for CD34+ primitive HSCs that also expressed high levels of cytosolic ALDH1. Secondly, having a method that could also isolate viable tumor cells that are resistant to alkylating agents such as cyclophosphamide and express high levels of ALDH1 could be advantageous for studying drug resistance in tumor cells [25]. Unfortunately, there were some drawbacks to this technique [27]. The DAAA fluorescence was excited by UV emissions, which could be mutagenic for cells that would be isolated and used for downstream applications. In addition, the emission spectra of DAAA overlapped with other fluorochromes, making this technique difficult to combine with analysis of other stem cell markers [25, 27].
Four years after the Jones, et al. publication, Storms, et al. developed a more straightforward and efficient strategy for isolating primitive HSCs using the fluorescent substrate BODIPY aminoacetaldehyde (BAAA) [27], more commonly known now as the Aldefluor® Assay [19, 21, 22, 24, 28]. The BAAA strategy is similar to the DAAA one in that cells expressing ALDH1 will uptake uncharged ALDH substrate BAAA by passive diffusion and then convert BAAA into negatively-charged BODIPY-aminoacetate (BAA-). BAA- is then retained inside cells, causing the subset of cells with a high ALDH activity (ALDHhi) to become highly fluorescent. The ALDHhi or ALDH+ subset in human cancer cells can be determined by the criteria of the sorting gates. Populations in the top 10–20% have been considered ALDHhi, whereas populations in the bottom 10–20% can be considered ALDHlow. These ALDHhi populations can be distinguished easily and specifically via comparison to the DEAB negative control (a specific inhibitor of ALDH) [19, 24, 27] (Fig. 1). It is of note, however, that although the Aldefluor® assay works well in various human models [19, 21, 24, 29], there is controversy as to whether or not the assay is appropriate in the murine HSC model. Some groups have reported using the Aldefluor® assay to successfully identify functional murine HSCs, while others have not been able to do so [29–32]. This will be discussed further later in this review.
The Aldefluor® Assay. To assess ALDH1 activity, cells can be labeled using the ALDEFLUOR® Assay Kit (StemCell Technologies, Vancouver, BC) as per the manufacturer’s protocol. Essentially, cells expressing ALDH1 right will uptake uncharged ALDH substrate (BODIPY-aminoacetaldehyde [BAAA]) by passive diffusion and then convert BAAA into negatively-charged BODIPY-aminoacetate (BAA-). BAA- is then retained inside cells, causing the subset of ALDHhi cells to become highly fluorescent. The addition of cold assay buffer (as provided by the manufacturer) prevents the ATP-binding cassette (ABC) transporters to pump out the BAA- substrate of the cells. As a negative control left, diethylaminobenzaldehyde (DEAB), a specific ALDH1 inhibitor, is used to quench the activity of ALDHhi cells, preventing the cells to become fluorescent. Figure adapted from the Aldagen’s Information Sheet on the Aldefluor® Assay (www.stemcell.com)
Recent novel studies have demonstrated that ALDH1 could potentially be used as a target for stem cell labeling and imaging in vivo, which may be useful and attractive for tracking and identifying normal and cancer stem cells because it does not require purification or manipulation of cells ex vivo [33]. Two novel radiotracers were synthesized to target ALDH1–N-formylmethyl-5-[*I]iodopyridine-3-carboxamide([*I]FMIC), and 4-diethylamino-3- [*I]iodobenzaldehyde ([*I]DEIBA), which could be labeled with 123I and 124I for SPECT (single positron emission computed tomography) and PET (positron emission tomography) imaging, respectively. It was expected that the ALDH1 would oxidize [*I]FMIC into the corresponding polar acid, thus rendering the acid to be trapped within the cell, and imaged via SPECT. Similar to DEAB, [*I]DEIBA was synthesized as an inhibitor of ALDH1 that could be imaged via PET. These novel radiotracers were tested on cell lines that were positive (K562 human leukemia cells, L1210-CPA cells) or negative (L1210) for ALDH1 activity. Although the study did show the successful conversion of radiolabeled aldehyde to an acid form by ALDH1, cells that expressed high ALDH1 did not retain the compounds, rendering these two radiotracers unsuitable for stem cell and cancer stem cell imaging. When performing various studies to determine why the acids were not retained, it was determined that the acid products converted by ALDH1 were not polar enough to be retained within the cells. Thus, if researchers would like to use radiotracers that target ALDH1 to study normal and cancer stem cells in vivo, they must design a substrate that can convert into a sufficiently polar acid to be retained within the cells [33].
ALDH as a Marker for Normal Stem Cells
Stem cells are defined as having both the ability to self-renew, and the capacity to differentiate into mature cells that can repopulate specific tissues and organs [1]. Depending on the type of stem cells, a variety of stem cell specific markers and strategies can be used to isolate and enrich for target cells when used alone or in combination with other markers. For example, hematopoietic stem cells (HSCs) can be identified by CD34 and/or CD133 [34], while neural stem cells can be identified by the surface antigen Lewis X or CD133 [22]. However, it would appear that high ALDH1 activity, in addition to the presence of ATP-binding cassette (ABC) transporter G2 (ABCG2) and high telomerase activity, may be universal stem cell markers, as they are found in stem cells isolated from most tissues [22].
It was first demonstrated about two decades ago that HSCs are highly enriched for ALDH1, while less primitive cells express lower levels of ALDH1 [16]. This study marked the start of a burgeoning interest in using ALDH1 as a way to identify and isolate stem cells such as neural stem cells [22, 35], HSCs [16, 27, 29, 32, 34, 36–42], adipose-derived adult stem cells [43], and myogenic precursor cells [44]. Stem cells that have been identified and isolated using the Aldefluor® assay have been used in the area of regenerative medicine, including for acceleration of tissue repair from liver damage [42], enhancement of vascular recovery by exerting trophic effects on cardiac tissue repair following acute myocardial infarction [40], and improvement of perfusion and increased blood vessel density in ischemic limbs [36]. Additionally, HSCs have been identified and isolated based on their high ALDH1 phenotype and used to engraft into patients following bone marrow transplantation [31]. ALDH isoenzymes such as ALDH1A1 and ALDH3A1 also play a functional role in normal stem cells in the context of self-protection, differentiation, and expansion [8, 21, 31, 45], and these topics will be discussed in detail later in this review.
ALDH as a Marker for Cancer Stem Cells
Cancers are heterogeneous, multicellular entities that arise when an epigenetic or genetic change occurs in normal cells, thus disrupting normal cellular homeostasis [46, 47]. These alterations then favor the cancer cell’s uncontrolled proliferation, differentiation, migration, and extracellular matrix (ECM) metabolism, while restricting apoptosis, cellular polarity, and ECM stability. These changes are due to the acquisition of six main malignant capabilities: self-sufficiency in growth signals, insensitivity to inhibitory growth signals, sustained angiogenesis, evasion of apoptosis, unlimited replicated potential, and tissue invasion and metastasis [46, 47].
Growing evidence suggests that the cells responsible for initiating and maintaining cancer are in fact “cancer stem cells” (CSCs). Although the CSC hypothesis was first proposed approximately 150 years ago [48–50], technological advances in the area of rare cell identification and isolation have lead to a resurgence of interest in this area. CSCs were first identified in acute myeloid leukemia (AML) by Dr. John Dick’s group in 1994. They observed that CD34+CD38- leukemia-initiating cells were able to engraft into severe combined immunodeficient (SCID) mice and recapitulate the original tumor population as seen in AML patients [2]. The first report of the existence of CSCs in solid tumors came in 2003, when tumor-initiating breast cancer cells were identified and isolated from primary tumors and pleural effusions of breast cancer patients based on a CD44+CD24- phenotype [51]. The tumor-initiation capacity of CD44+CD24- cells was tested in non-obese diabetic (NOD)/SCID mice, and it was observed injection of only 100 CD44+CD24- breast cancer stem-like cells formed tumors, whereas injection with CD44-CD24+ breast stem-like cancer cells did not result in the formation of tumors [51]. Subsequent studies have shown that CD44+CD24- breast cancer cells display an increased expression of stem cell markers, the ability to self-renew, an enhanced capacity for in vitro mammosphere formation and invasion, expression of higher levels of anti-apoptotic proteins, and the ability to recapitulate a heterogeneous tumor population [51–55]. CSCs have also been identified in several other cancer types, including various forms of leukemias as well as solid tumors of the liver, pancreas, brain, colon, prostate, and other organs based on various surface antigens [51, 56–65] (Table 2).
More recently, the activity of cytosolic ALDH1 has also been shown to be a reliable marker of CSCs in several types of solid tumors, including tumors of the head and neck, lung, liver, pancreas, cervix, ovaries, breast, prostate, colon, and the bladder regions [20, 24, 28, 63, 64, 66–77]. It has also been shown that high activity of ALDH1 is associated with poor prognosis in breast, bladder and prostate cancer patients [24, 70–72, 74, 78]. In a study of 577 breast cancer patients, it was shown that patients with ALDH1-positive tumors had a lower overall survival compared to patients with ALDH1-low tumors [24]. Similarly, in two independent studies analyzing 163 and 269 primary prostate cancer patient samples (respectively), it was shown that patients with high ALDH1A1 expression correlated with lower overall survival [70, 71], Gleason score, and pathologic stage [70]. Furthermore, it has been observed that ALDH1 is a marker of normal and malignant human mammary stem cells [24], as well as normal and malignant human colon stem cells [28]. Interestingly, when tracking the colon stem cell population from the normal epithelium to the mutant epithelium to adenoma progression, the number of cells that express high ALDH1 increased, as well as being distributed further up the crypt from the bottom of the crypt under normal conditions [28]. It has also been shown that cancer stem cells in adenoid cystic carcinoma [73], prostate cancer [70, 76], head and neck squamous cell carcinoma [68], lung cancer [75], pancreatic adenocarcinoma [71], cervical carcinoma [66], and bladder cancer [72] that highly express ALDH1 are highly tumorigenic and have enhanced stem cell characteristics in vitro and in vivo compared to cells with low ALDH1 activity. For example, Ginestier, et al. demonstrated that when 50,000 ALDH1- breast cancer cells were transplanted into humanized cleared mammary fat pads of NOD/SCID mice, no tumor was formed, but when as few as 500 ALDH1+ cells were injected, tumors were formed in 40 days, indicating that ALDH+ breast cancer stem-like cells are highly tumorigenic [24]. In addition, previous work from our laboratory indicates that stem-like breast cancer cells can be identified by an ALDH1hiCD44+ phenotype, and that these cells are significantly more metastatic than ALDH1lowCD44- cells in vitro and in vivo [19]. Other groups have found that breast, bladder, and prostate cancer stem cells with a high ALDH1 activity appear to display more aggressive characteristics, may mediate metastasis, and are associated with a poor prognosis in cancer patients [24, 71, 72, 76, 79].
There have been conflicting reports in terms of whether or not ALDH1 is a predictor of poor or favorable prognosis in ovarian cancer patients. A study by Deng et al. in 2010 showed that similar to other cancers, ovarian cancer patients with high ALDH1 had a shorter disease-free and overall survival times compared to those with low ALDH1 by using a tissue array of serous ovarian cancers collected from patients [69]. Conversely, a study by Chang et al. in 2009 showed that high ALDH1 expression in ovarian carcinoma cells may have favorable prognostic value for ovarian cancer patients [80]. Deng et al. suggested that this discrepancy may be due to the fact that different ovarian epithelial tumors histotypes have unique molecular backgrounds and biological behaviors. Thus, the prognostic value of ALDH1 expression in ovarian cancer may be histotype-specific, and further investigation in large scale independent studies are necessary [69].
ALDH1 may not be a suitable CSC marker for all tumor types. A recent study investigated the patterns and levels of ALDH1 expression in 24 types of normal human tissues as well as primary epithelial tumor specimens and epithelial cancer cell lines [69]. From this study, it was determined that ALDH1hi cells can be clearly identified in regions where epithelial stem/progenitor cells are putatively located. Furthermore, it was observed that ALDH1 distribution patterns in normal tissues were distinct, and were classified into three types: 1) tissues with absent or limited ALDH1 expression (i.e. breast and lung); 2) tissue with relatively weak ALDH1 expression (i.e. colon and gastric epitheliums); and 3) tissue with extensive and high ALDH1 expression (i.e. liver and pancreas). Thus, the authors concluded that ALDH1 can be effectively used as a CSC marker in tissue types that normally do not express ALDH1 at a high level (such as breast, lung, colon and gastric epitheliums), but should not be used as a CSC marker in tissue types that normally express a high level of ALDH1 (such as liver and pancreas) [69].
It is clear that ALDH1 can be used as a marker for identifying and isolating normal and cancer stem cells from various tissue sources. However, growing evidence suggests that ALDH1 is not only a putative stem cell marker, but may actually play multiple functional roles that contribute to stem cell self-protection, differentiation and/or self-renewal.
Functional Roles of ALDH in Normal and Cancer Stem Cells
ALDH1 and the Retinoid Signaling Pathway in Normal and Cancer Stem Cells
Retinoid signaling pathways have been implicated in normal stem cells [21, 81–84] and cancer cells [85–88]. Retinoic acid (RA) and its derivatives are involved in many critical physiological processes, including the regulation of gene expression, morphogenesis and development [89–91]. The four distinct families of retinoid dehydrogenases that convert retinol (vitamin A) to RA are alcohol dehydrogenase (ADH), short-chain dehydrogenase/reductase, aldo-keto reductase, and ALDH [89]. Retinol is first oxidized by ADH to retinaldehyde, and this process is reversible. Retinaldehyde is then irreversibly oxidized to RA by cytosolic ALDH1 (human ALDH1A1, ALDH1A2, ALDH1A3). The latter reaction is a tightly regulated process that is tissue-specific, since the oxidation of retinaldehyde to RA is an irreversible reaction, with RA having a potent biological activity [5, 89]. The resulting RA produced can then act on nuclear retinoic acid receptor (RAR)-α, β, γ, and retinoid X receptor (RXR)-α, β, γ, which bind DNA as heterodimers and result in the regulation of gene expression and cell differentiation [89, 92]. The RARs bind all-trans-RA (ATRA) and 9-cis-RA, while the RXRs bind only the 9-cis-RA. Once RA has been synthesized, the RA signaling pathway initiates, whereas the degradation of RA or the cessation of RA synthesis stops RA signaling [89]. Murine retinaldehyde dehydrogenase 1 (Raldh1) has similar tissue-specificity and developmental control as the cytosolic human ALDH1 [92]. Studies by Elizondo, et al. in 2000 and 2009 demonstrated that mouse Raldh1 transcription is under the regulation of a negative feedback mechanism [12, 92]. When there are low intracellular RA concentrations, RARα and CCAAT/enhancer-binding protein (C/EBPβ) transactivate the Raldh1 promoter, thereby increasing the Raldh1 activity to increase the oxidization of retinaldehyde to retinoic acid. As RA levels increase, C/EBPβ mRNA increases, which also increases GADD153 mRNA. A complex of GADD153 and C/EBPβ then forms to decrease DNA binding activity of C/EBPβ to the CCAAT box of the Raldh1 promoter, thereby inhibiting the transactivation of Raldh1. This ultimately results in a decrease in RA synthesis [12, 92] (Fig. 2).
The ALDH1/RA signaling axis. a Retinol is first reversibly oxidized by alcohol dehydrogenase (ADH) into retinaldehyde, where it can then be irreversibly oxidized into retinoic acid (RA) by aldehyde dehydrogenase (ALDH1). RA can then bind to the retinoic acid receptor (RAR) to result in gene expression and cell differentiation. ALDH1 is under the regulation of a negative feedback mechanism. b When endogenous RA concentrations are low, the RAR binds to the retinoic acid response element (RARE), and the CCAAT/enhancer-binding protein-β (C/EBPβ) binds to the CCAAT box. Together, the RAR and C/EBPβ transactivate the Aldh1 promoter, and activates transcription. As the ALDH1 levels increase, this can result in an increase in RA synthesis, as well as cellular protection against cytotoxic drugs. c Conversely, when intracellular RA is high, C/EBPβ increases, which then forms a complex with GADD153. The C/EBPβ – GADD153 complex then decreases the DNA binding activity of C/EBPβ to the CCAAT box of the Aldh1 promoter, thereby inhibiting the transactivation of Aldh1. This results in a decrease in RA synthesis, as well as cellular sensitivity to drugs. Adapted from Duester et al (2003) [89] and Elizondro et al (2009) [92]
ALDH Plays a Self-Protective Role in Normal Stem Cells and CSCs
Given the reported functions of ALDH enzymes, it is not surprising that ALDHs are generally regarded as detoxification enzymes that are critical for protecting organisms against various aldehydes that would be otherwise be harmful to them [5, 6, 11]. This fact is supported by growing evidence that deficiencies and polymorphisms of various ALDH enzymes can lead to clinical phenotypes and diseases [5, 8, 11]. Some examples of these metabolic syndromes and diseases include spina bifida (ALDH1A2) [93], ethanol-induced cancers [94] and hypertension (ALDH2) [95], Sjögren-Larsson syndrome (ALDH3A2) [96], type II hyperprolinemia (ALDH4A1) [97], , γ-hydroxybutyric aciduria (ALDH5A1) [98], and pyridoxine-dependent epilepsy (ALDH7A1) [99] (reviewed in detail in [5, 8, 11]).
ALDH1A1 and ALDH3A1 can also offer cellular protection against cytotoxic drugs. It was first observed over two decades ago that hematopoietic and leukemic stem cells with ALDH activity were highly resistant to cyclophosphamide (CP), an alkylating agent [16, 100]. It has also been shown early on that by inhibiting ALDH activity with various inhibitors (such as diethyldithiocarbamate, cyanamide, disulfiram, diethyldithiocarbamate, or ethylphenyl (2-formylethyl) phosphinate), both murine day-12 spleen colony forming cells and pluripotent HSCs were sensitive to mafosfamide, an oxazaphosphorine anticancer agent [101, 102]. Furthermore, when murine HSCs were treated with DEAB, it was shown that the progenitors of various colony forming units (such as granulocyte/macrophage colonies, granulocyte/erythrocyte/ macrophage/megakaryocyte colonies and blast cell colonies) became more sensitive to 4-hydroperoxycyclophosphamide (4-HC) due to the activity of ALDH1 [103]. A possible mechanism for the protection of stem cells from drugs such as 4-HC via ALDH1 may be due to interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) [104]. Moreb, et al. observed that pre-treatment of bone marrow cells with IL-1 and TNF-α for 6 to 20 hours resulted in an increase in ALDH1 mRNA and protein levels [105], and that this ultimately leads cellular protection against 4-HC [104]. Since then, it has been determined that high cytosolic ALDH1A1 or ALDH3A1 activity in normal cells [106, 107], stem cells [44], and cancer stem cells [65, 74] confers resistance to therapy in preclinical model systems. The majority of these studies focused on alkylating agents such as CP and other oxazaphosphorines [13–16, 45, 78, 87, 88, 100–104, 106–110]. Cytosolic ALDH1A1 and ALDH3A1 are able to convert activated cyclophosphamide, 4-HC, to the inactive excretory product carboxyphosphamide [16, 104]. Clinically, 4-HC has been used to purge resident tumor cells ex vivo and treat autologous bone marrow transplantation [104]. However, ALDH1 appears to also be able to confer cellular resistance to drugs other than CPs. For example, it has been found recently that human myoblasts with a high ALDH1 activity can confer hydrogen-peroxide mediated cytotoxicity resistance ex vivo, as well as enhancing cellular viability and promoting engraftment in vivo upon transplantation into the muscle of SCID mice [44].
ALDH1 can also offer drug protection in CSCs. Tanei, et al. identified breast CSCs (using the ALDH1+ CD44+CD24- phenotype) in over 100 breast cancer patients undergoing neoadjuvant chemotherapy consisting of paclitaxel and epirubicin. They observed that the proportion of ALDH1+ tumor cells and ALDH1 expression significantly increased after neoadjuvant chemotherapy, whereas the proportion of CD44+CD24- cells did not, suggesting that the high ALDH1 phenotype is a better predictive marker for chemotherapy resistance compared to CD44+CD24-. Furthermore, it was observed that there was a significant association between ALDH1-high breast cancer tumors and resistance to neoadjuvant chemotherapy with significantly lower pathologic complete response rates compared to ALDH-negative breast tumors [74]. In addition to ALDH1, the estrogen receptor (ER) and Ki67 status are mutually independent predictors of chemotherapy (paclitaxel and epirubicin) response, therefore it may be wise to study ER and Ki67 status along with ALDH1 to predict a patient’s chemotherapy response more accurately [74, 111].
Although no direct association has been reported between high ALDH1 expression and radiation resistance, there have been studies that link high ALDH1 expression to radiation sensitivity [31, 112]. By studying radiation-sensitive versus radiation-resistant cervical carcinoma specimens, Kitahara et al. observed that ALDH1 gene expression was significantly upregulated in the complete response group when compared to the non-responsive group. They concluded that in radiosensitive cervical cancer cells, the high ALDH1 expression level also increased the synthesis of retinoic acid (RA), which induced TRAIL and apoptosis of the cancer cells after radiation. When cervical carcinoma cells were treated with RA before radiation, these cells became radiosensitive [112]. If the cervical carcinoma cells that showed high expression of ALDH1 were CSCs, perhaps another reason for the radiosensitivity was due to the differentiation of these CSCs upon biosynthesis of RA [86], although the authors did not mention the CSC hypothesis in their study. Similarly, it has also been observed that by inhibiting ALDH1 expression via siRNA against ALDH1 or DEAB treatment, murine HSCs show delayed differentiation due to the impairment of retinoid signaling and expand and become radiation resistant [31]. It is likely that the scenario upon which different types of normal or cancer stem cells are resistant versus sensitive to radiation may be species- and/or tissue-specific. Thus, further research is needed to clarify the link between ALDH1 and radiation response in a wide variety of normal and CSC types.
Very recently, it was discovered that mantle cell lymphoma (MCL) cells can also be identified and isolated using the ALDH1+ phenotype. Importantly, unlike CSCs that have been identified in other malignancies, there have been no surface antigens identified that could isolate and enrich for MCLs and other mature pre-germinal center B cell malignant cells [65]. The ALDH+ MCL cells were observed to be relatively quiescent and resistant to the standard chemotherapeutic agents used for MCL patients, including dexamethasone (anti-inflammatory steroid), etoposide (topoisomerase II inhibitor), and bortezomib (proteasome inhibitor). The authors noted that the observed pan-drug resistance may not be solely due to ALDH1 expression, but rather also due to the protective effects of stem cell quiescence, as well as the expression of other drug resistance proteins such as efflux pumps [65].
Although it would appear that high ALDH1 activity offers cellular protection by conferring drug resistance, thereby enhancing the aggressiveness of CSCs, this may not the case for malignant melanoma, a highly aggressive and drug-resistant cancer [113]. A study by Prasmickaite et al. showed that metastatic melanoma patient biopsies had a substantially large and distinguishable ALDH1+ subpopulation. However, both the ALDH1+ and ALDH1- subpopulations displayed similar aggressive characteristics, in that they were both highly clonogenic in vitro, tumorigenic in vivo, and showed similar drug resistance to dacarbazine and the TRAIL-R2 agonist lexatumumab (anti-melanoma drugs) [113]. Perhaps malignant melanoma is an exception to the observation in other cancers that high ALDH1 contributes to high self-protection and enhanced cell aggressiveness, potentially due to the fact that there have been conflicting opinions regarding the existence of CSCs in this heterogeneous cancer population [113].
ALDH Plays a Role in Expansion and Differentiation of Normal Stem Cells and CSCs
There is a tremendous amount of research effort aimed towards identifying and isolating HSCs because of the need for HSCs to engraft efficiently in bone marrow (BM) transplant recipients. Recently, there have been promising developments in terms of how these HSCs can be expanded ex vivo for clinical use, and it has been suggested that ALDH1 may be valuable for the expansion of normal human stem cells. However, in addition to the controversy of the successful use of the Aldefluor® assay in the murine HSC model, there is also debate as to what the exact functional role of ALDH1A1 is in the murine system. For example, although Levi et al. (2009) did not see a functional impact on hematopoiesis upon manipulation of ALDH1A1 expression [30], others have found that ALDH1A1 can inhibit lymphopoiesis and promote myelopoiesis without impacting erythropoiesis in the murine model [39]. A study by Greene et al. (1998) also suggests that because Hox11 negatively regulates Aldh1, atypical expression of ALDH1 in Hox11-null mice may result in the loss of splenic precursor cells [114]. It has been suggested that one of the reasons for these discrepencies may be due to the fact that the homozygous deletion of the Aldh1a1 gene in vivo in the study by Levi et al. allowed for other pathways to compensate for the loss of the gene function over time [31].
In both the murine [31] and human [21] HSC system, when ALDH1 is inhibited by siRNA targeting or by the addition of DEAB, expansion of the HSC population has been shown to occur in vitro [21, 31]. Inhibition of ALDH1 using DEAB delayed the G0/G1 transition in murine HSCs, resulting in more HSCs in the G0 phase than the G2/S/M phase [31]. Therefore, inhibiting ALDH1 pharmacologically or using molecular targeting may be a way to expand HSCs for use in downstream clinical applications. Retinoic acids such as ATRA are commonly used as differentiation agents in stem cell research [21, 81–84], and have been used to induce remission in acute promyelocytic leukemia (APL) patients by effectively differentiating promyelocytic leukemic cells into neutrophils [85]. It has also been shown in vivo that ALDH1A1 promotes myeloid differentiation in murine HSCs [39]. In murine and human in vitro models, inhibition of ALDH1 using DEAB or siRNA resulted in HSC differentiation and a decrease in cEBPε (an RAR-specific response gene), thus reducing intrinsic retinoic acid [21, 31]. When ATRA was added to DEAB-treated HSCs, differentiation and lineage commitment was promoted in HSCs [21].
Due to the negative feedback mechanisms of ALDH1 and retinoid signaling, one could hypothesize that treating CSCs that have high ALDH1 activity (relative to their normal tissue counterparts) with ATRA could potentially shift the CSCs into a more differentiated state, thereby making them less aggressive. Using GSEA (gene set enrichment analysis) algorithm, Ginestier et al. were able to show that when various breast cancer cell lines were treated with ATRA, the genes that were downregulated were associated with pathways related to stem cell self-renewal programs, WNT signaling, ATK/β-catenin, the carcinogenesis process, metastatic activity, and drug resistance [86]. The results of their study also suggest that ATRA treatment may induce breast CSC differentiation and decrease the CSC population. Conversely, genes that were overexpressed in DEAB-treated breast cancer cell lines were involved in tRNA biosynthesis, which is essential for protein synthesis and cell viability [86]. Similarly, results from our laboratory suggests that treatment of breast CSCs with ATRA results in an enhanced sensitization to chemotherapy and radiation, thus potentially making the CSCs less aggressive [115].
An interesting recent study by Brennan, et al. suggests that a synthetic unmethylated phosphorothioate CpG oligonucleotide 2006 (CpG) was able to activate MCL cells via the toll-like receptor-9 (TLR-9) (which induces plasmacytic differentiation), depleted the quiescent ALDH+ MCL population, and reduced clonogenic growth and tumor formation in vitro and in vivo. Moreover, it was observed that targeting MCLs with the synthetic CpGs resulted in specific sensitivity to bortezomib only. Therefore this preclinical study could potentially translate into the clinic in the future in terms of changing the treatment regime to include the use of synthetic CpGs to activate MCL cells and deplete quiescent ALDH+ cells and/or differentiate them into plasma cells [65].
Future Perspectives and Conclusions
Modulating the ALDH1/retinoic acid signaling pathway could have significant clinical implications. Treating CSCs using ATRA could potentially shift the CSCs into a more differentiated state, thereby making them sensitive to chemotherapy and less aggressive. For example, it has been shown previously that by administering ATRA, as well as 9-cis and 13-cis RA, ALDH1 is downregulated, thereby increasing the sensitivity of various lung cancer cell lines to cyclophosphamide [87]. ATRA has also been used clinically in combination with cyclophosphamide to successfully treat patients with acute promyelocytic leukemia (APL), as ATRA differentiates leukemic promyelocytes into mature neutrophils and results in improved disease-free and overall survival relative to patients were treated with chemotherapy alone [85]. The potential use of ATRA in solid cancer remains less clear. For example, in a small Phase I/II trial of breast cancer, patients with measurable disease or evaluable non-measurable disease were given differing doses of ATRA (70–230 mg/m2/day) on alternating weeks during anti-estrogen treatment. Of the 7 patients with measurable disease, 2 experienced a partial response to the combination therapy of ATRA and Tamoxifen. Of the 18 patients with evaluable, non-measurable disease, 7 experienced a partial response for 6 months or more [116].
Other approaches to targeting ALDH such as inhibiting ALDH1 using pharmacological agents (DEAB) or via molecular targeting (siRNA) has been shown to allow stem cells to be expanded, become radioprotected, and be used in the BM transplantation setting to improve engraftment and recipient survival [21, 31]. Although both the siRNA and DEAB approaches demonstrated similar inhibitory effects on ALDH1 in HSCs [31], DEAB was shown to be non-cytotoxic [104], and thus it may be more plausible for DEAB to be used in the clinical setting as a way to increase the CSC sensitivity to chemotherapy or expand and protect normal HSCs against irradiation. In a pre-clinical model, it has been shown that DEAB restored 4-HC sensitivity to an acute myeloid leukemia cell line that was resistant to cyclophosphamide [117]. Since ALDHs play multiple functional roles, and have different levels and patterns of expression in various cell types, targeted therapies and personalized medicine will most likely be necessary in the context of assessing and/or targeting ALDH levels in the particular tissue of interest. For example, modulation of the ALDH/RA axis for patients may not be useful for patients with liver or pancreatic cancer because of the high intrinsic levels of ALDH in normal liver and pancreatic tissue [69].
It would appear that once CSCs are treated with chemotherapy, ALDH expression levels increase [15, 45, 74], thereby allowing CSCs to acquire the ability to become drug resistant in the future. Could this be an adaptive response in cancer stem cells, thereby negating the benefits of chemotherapy because it increases intrinsic resistance? It would be interesting to see if this is true in cells that are known to express low ALDH levels. Perhaps in the future, this should be taken into consideration when performing preclinical studies using cell lines and/or primary cells from patients [118]. In terms of radiation research, there has been no direct association between ALDH1 and radiation resistance/sensitivity in CSCs. Further research to study the link between ALDH1hi and radiation resistance/sensitivity in a wide variety of normal stem cells and CSC types needs to be further addressed in the future.
Despite the growing evidence that ALDH1 plays multiple functional roles in normal and cancer stem cells, more research is needed. Gaining an enhanced understanding of the pathways associated with ALDH1 and other ALDH isoenzymes together with other important pathways that regulate normal and cancer stem cells will facilitate the development of novel pharmaceuticals to improve the current status of bone marrow transplantation, regenerative medicine, and cancer treatment.
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Acknowledgements
We thank members of our laboratory and our collaborators for their research work and helpful discussions. The authors’ research on ALDH and CSCs is supported by research grants from the Ontario Institute for Cancer Research (#08NOV230), and the Canada Foundation for Innovation (#13199) (to ALA). IM is the recipient of a Canadian Institute of Health Research (CIHR) Strategic Training Program Scholarship and a Translational Breast Cancer Scholarship through the London Regional Cancer Program. ALA is supported by a CIHR New Investigator Award and an Early Researcher Award from the Ontario Ministry of Research and Innovation.
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The authors declare no potential conflicts of interest.
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Ma, I., Allan, A.L. The Role of Human Aldehyde Dehydrogenase in Normal and Cancer Stem Cells. Stem Cell Rev and Rep 7, 292–306 (2011). https://doi.org/10.1007/s12015-010-9208-4
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DOI: https://doi.org/10.1007/s12015-010-9208-4