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Aldehyde dehydrogenases in cancer: an opportunity for biomarker and drug development?
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Aldehyde dehydrogenases in cancer: an opportunity for biomarker and drug development? Klaus Pors1 and Jan S. Moreb2 Q1 1 2
Institute of Cancer Therapeutics, University of Bradford, Bradford BD7 1DP, UK Hematological Malignancies, PO Box 100278, Gainesville, FL 32610-0278, USA
Aldehyde dehydrogenases (ALDHs) belong to a superfamily of 19 isozymes that are known to participate in many physiologically important biosynthetic processes including detoxification of specific endogenous and exogenous aldehyde substrates. The high expression levels of an emerging number of ALDHs in various cancer tissues suggest that these enzymes have pivotal roles in cancer cell survival and progression. Mapping out the heterogeneity of tumours and their cancer stem cell (CSC) component will be key to successful design of strategies involving therapeutics that are targeted against specific ALDH isozymes. This review summarises recent progress in ALDH-focused cancer research and discovery of small-molecule-based inhibitors.
Introduction The aldehyde dehydrogenase (ALDH) superfamily of enzymes comprises 19 human isozymes with 11 distinct families localised in the cytoplasm, mitochondria or nucleus [1]. ALDHs are known to participate in many physiologically important biosynthetic pathways [2] and the activity of certain ALDHs has been shown to be crucial in the detoxification of specific endogenous and exogenous aldehyde substrates [1,3,4]. Accordingly, many ALDHs have crucial roles in the protection of cellular homeostasis and organism functions against toxic effects from the aldehydes [1]. Aldehydes are detoxified via ALDH-catalysed metabolism resulting in the corresponding carboxylic acids through a pyridine-nucleotide-dependent reaction [5]. ALDH activity, through aldehyde metabolism, is essential for the synthesis of molecules such as retinoic acid, betaine and g-aminobutyric acid, which are important for cell proliferation, differentiation and survival [1]. Abnormal behaviour in ALDH activity linked to specific metabolic pathways has implications for several disease states, including ¨ gren–Larsson syndrome, type II hyperprolinaemia, hyperamSjo monaemia and cancer [4]. High ALDH cellular levels and a relationship to cyclophosphamide resistance were first identified in normal haematopoietic stem cells [6]. The prominence of ALDH
Corresponding authors: Moreb, Pors, K. ([email protected]), J.S. ([email protected])
isozymes over the past five years has been mainly caused by hypothesis of cancer stem cells (CSCs), with high ALDH enzyme activity identified as a marker for stem cells in multiple tissue types. The availability of the AldefluorTM flow cytometry assay has been the main driving technology for this evolving field because it has enabled the use of ALDH enzyme activity to sort live cells that have been shown to have the characteristics of stem cells such as self-renewal, high proliferative potential, clonogenicity and multipotency. Most of the current cancer chemotherapy regimens are used to reduce the bulk of solid tumours before surgery, eradicate cancer cells that might remain after surgery or target malignant metastases. Over the past 20 years much emphasis has been placed on overcoming multidrug resistance (MDR) and progress on new agents overcoming MDR has been encouraging [7–10]. Regardless, owing to poor overall survival (OS) rates associated with chemotherapy, the target and aim of the treatment might be shifting towards overcoming not only MDR but also other pathways essential for the survival of CSCs. In fact, it is becoming apparent that resistance mechanisms in CSCs to current therapies share similarities to those observed in the treatment of MDR cancer cells: (i) CSCs are generally quiescent and therefore resistant to chemotherapy designed to target proliferating cancer cells [11,12]; (ii) CSCs possess enhanced expression of ABC transporters as evident from observations that CSCs can be isolated by Hoechst stain
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1359-6446/06/$ - see front matter ß 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.drudis.2014.09.009
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exclusion via the ABCG2 protein pump [13,14] and (iii) there is evidence for high content of specific ALDH isozymes in CSCs present in different types of cancers that have been correlated to worse prognosis. Such a phenotype usually correlates with a high proliferation index, higher metastatic potential and increased drug resistance in the respective cancer. We will review and summarise the published literature in this arena. We will discuss the role of the different ALDH isozymes (Table 1 provides a list of all ALDH isozymes with their gene bank access code and their chromosomal location) responsible for this high ALDH content, because these could have implications to all future therapies targeting CSCs. In general, these isozymes are widely expressed in different tissues; however, much of the focus in CSC research to date has been on detection of ALDH1 expression and/or activity, but often without clarification of isozyme specificity. In the past there might have been limited or no knowledge of the isozyme specificity of ALDH-targeting antibodies or accuracy of the AldefluorTM assay, which has led to a large body of work suffering from a lack of clear definitions and with scientific questions remaining.
Because evidence of the fundamental biological and clinical importance of ALDH isozyme expression and function is ever increasing, it is clear that scientists engaged in this research field should agree a more rigorous definition when using the terms ‘ALDH activity’ or ‘ALDH expression’. Accordingly, before we discuss ALDH expression and/or activity in cancer or stem cells in more detail we have proposed terminology in this review that we suggest be adopted in the scientific community in unravelling the significance of ALDH isozymes in the future.
ALDH expression Earlier studies often only reported family type such as ALDH1 and not the specific isozyme members such as ALDH1A1, 1A2, 1A3, among others, perhaps in part because of lack of antibody-specific production and/or information. In this review, we have checked methodologies and corresponded with authors of selected studies discussed in this review, and companies now selling the same antibodies or more-specific isozymes, in an attempt to align current knowledge with studies performed in the past. We have also
TABLE 1
The aldehyde dehydrogenase (ALDH) family ALDH isozyme
Genebank accession number
Compartment
Chromosomal localisation
ALDH1A1
NM_000689
Cytoplasmic
9q21.13
ALDH1A2
NM_003888 NM_170696 NM_170697
Cytoplasmic
15q21.3
ALDH1A3
NM_000693
Cytoplasmic
15q26.3
ALDH1B1
NM_000692
Mitochondrial
9p11.1
ADLH1L1
NM_012190
Cytoplasmic
3q21.3
ALDH1L2
NM_001034173
Mitochondrial
12q23.3
ALDH2
NM_000690
Mitochondrial
12q24.2
Related clinical syndromes
Neural tube defects
ALDH3A1
NM_000691
Cytoplasmic
17p11.2
ALDH3A2 (ALDH10)
NM_001031806 NM_000382
Microsomal
17p11.2
ALDH3B1
NM_000692 NM_001030010
Cytoplasmic
11q13
ALDH3B2
NM_000695 NM_001031615
Cytoplasmic
11q13
ALDH4A1
NM_170726 NM_003748
Mitochondrial
1p36
Hyperprolinaemia
ALDH5A1
NM_001080 NM_170740
Mitochondrial
6p22
Succinic semialdehyde dehydrogenase deficiency
ALDH6A1
NM_005589
Mitochondrial
14q24.3
ALDH7A1
NM_001182
Cytoplasmic
5q31
ALDH8A1
NM_170771 NM_022568
Cytoplasmic
6q23.2
ALDH9A1
NM_000696
Cytoplasmic
1q23.1
Polymorphism in treatment-resistant tardive dyskinesia in Japanese schizophrenic patients
ALDH16A1
NM_153329
Cytoplasmic and membranes
19q13.33
Possible association with gout
ALDH18A1
NM_002860 NM_001017423
Mitochondrial
10q24.3
2
Sjo¨gren–Larsson syndrome
Pyridoxine-dependent epilepsy
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consulted a publically available database for nomenclature and functional and molecular sequence information (www.aldh.org) Q2 for accuracy of information. Wherever possible we have highlighted ALDH specificity in the text or in Tables 2 and 3.
ALDH activity ALDH activity is often described in association with the identification and isolation of CSCs using the AldefluorTM assay. This assay [15] relies on the trapping of a negatively charged carboxylate metabolite generated from BODIPY aminoaldehyde (BAA; 1,
Fig. 1), which causes a subset of cells with high levels of ALDH activity to become highly fluorescent. This fluorescent-labelled subpopulation of cells is designated as ‘ALDH bright cells’ and can be detected using flow cytometry and isolated using a cell sorter. Such cells are likely to possess tumour-initiating properties that can constitute CSCs, based on the ability for long-term self-renewal and on a differentially expressed and stable stem cell lineage marker or phenotype. When describing ALDH activity, care must be exercised because there is some confusion in the field; ALDHcontaining subpopulations of cells often are labelled ALDHbr,
TABLE 2
Aldehyde dehydrogenase (ALDH) expression as a biomarker for cancer prognosis Cancer type
Method of detecting ALDH
% ALDH-posa CSC
OS in ALDH posa (n)
OS in ALDH-nega (n)
P value
% of ALDH-pos cases
Breast [47]
IHC for ALDH1A1b AldefluorTM
Average 5%
5 years: 19.8% (24)
5 years: 58.7% (122)
0.0459
19
Breast [47]
IHC
Average 5%
5 years: 69.59% (102)
5 years: 84.55% (243)
0.000675
30
0.0337
34
b
Inflammatory breast [48]
IHC for ALDH1A1 AldefluorTM
3–5%
5 years: 25% (16)
5 years: 54% (37)
Acute myeloid leukaemia (AML) [49]
AldefluorTM
Median: 14.98% of CD34+ cells
NAc
NAc
AML [51]
AldefluorTM
0.12% of the CD34+ cells had intermediate ALDH activity (ALDHint)
Detectable CD34+CD38 ALDHint cells predictive of relapse from CR
AML [50]
AldefluorTM
Median 0.5% (range 0.01–16.00)
57.5% (40)d
82% (28)
0.029
59
Prostate [23]
IHC for ALDH1A1 AldefluorTM
>10% considered high ALDH
5 years: 65%e
5 years: 90%e
0.0093
20
Rectal [58]
IHC for ALDH1A1b
NA
NAf
Oesophageal [33]
IHC for ALDH1A1b AldefluorTM
Used labelling index: high versus low
HR 3.4 Increased risk of death/relapse (HR 3.87)
Oesophageal [52]
IHC ALDH1g
NA
5 years OS: 8.3%
5 years OS: 52.2%
Lung [27]
IHC for ALDH1A1
Abundant versus scattered
(n = 20)
(n = 27)
48; 100% in resistant tumours
EFS shorter OS not different Pancreatic [22]
IHC for ALDH1A1b AldefluorTM
Ovarian [29]: serous
IHC ALDH1A1b
Clear cell Gallbladder [53]
IHC, ALDH1A3
32.5
0.03 0.006
NA
0.025
15.2, nuclear staining 42.5
0.053 0.141
Median OS: 14 mosh
18 mos
0.05
34
Median: 20%e
5 years: 37 (34)
5 years: 77 (28)
0.006
54.8
Median: 15%
5 years: 54 (18)
5 years: 81 (19)
0.047
48.6
10% of cells with weak to moderate staining
7.5 mos (16)
11.7 mos (30)
0.005
35
a ALDH-pos (ALDH-positive) cells refers to cells that are identified using AldefluorTM (ALDHbr cells) or immunohistochemistry (IHC) using either ALDH1A1 or 1A3-specific antibodies (see below for exception). ALDH-neg (ALDH-negative) cells are either not identified owing to poor fluorescence signal using AldefluorTM assay (defined as ALDHdim) or stained positively with ALDH1A1/1A3-specific antibodies. b The studies reported on ALDH1 expression using clone 44/ALDH1 antibody from BD Biosciences or ALDH1 antibody (ab52492; Abcam); however both are specific to ALDH1A1. c Results show that patients with ALDHbr subpopulations of AML cells were more likely to relapse after short remission. d ALDH+ terminology was used in the study: ALDH+ AML if frequency contained 0.36% ALDHbr cells, whereas cells below the threshold were determined ALDH AML. The values are the survival in each group during the observation period of 21.5 months. e The median was used to distinguish patients with low versus high ALDH-expressing tumours. We estimated the 5-year OS from the Kaplan–Meier curves given in the reference. f High ALDH expression predicted shorter disease-free survival (DFS) and poor outcome (P = 0.009). g ALDH1 antibody ab51028 is no longer available in Abcam’s product range, so no information available on the isozyme. h The 25% survival rate was 28.4 months (mos) in patients in primary tumours stained with ALDH1A1-specific antibody versus 46 mos in patients with ALDH1A1-negative specimens.
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TABLE 3
Aldehyde dehydrogenase (ALDH) isozymes identified in different types of cancer and their functional relevance
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ALDH isozyme
Methods of detection
Cancer type
Cancer-related function
Co-existing ALDH isozymes
ALDH1A1a [39]
AldefluorTM, IHC, spectrophotometry, HPLC
All cancer types
Biomarker for CSC, drug resistance
Other ALDH1 family members, ALDH3A1
ALDH1A2 [65]
IHC, Western blot
Prostate
Reduced expression in prostate cancer in comparison with normal tissue. Retinoid metabolism
ALDH1A1 and ALDH1A3
ALDH1A2 [63]
Mass spectrometry, PCR and Western blot
Acute myeloid leukaemia (AML)
High expression and resistance to Ara-C
Not determined
ALDH1A3 [53]
IHC
Gallbladder
Poor prognosis, high expression in advanced disease
Not determined
ALDH1A3 [66]
DNA methylation microarray
Bladder
Decreased expression and aggressive clinicopathological characteristics
Not determined
ALDH1A3 [35]
DNA methylation
Glioblastoma
Hypermethylation associated with better prognosis
Not determined
ALDH1A3 [41]
AldefluorTM, qRT-PCR
Melanoma
Cell cycle arrest, apoptosis and decreased viability
ALDH1A1
ALDH1A3 [67]
Genome-wide microarrays, IF and qPCR, IHC
Breast
Correlation to tumour grade and metastasis
ALDH1A1, 2, 4A1, 6A1, 7A1, 18A1
ALDH1A3 [68]
RT-PCR
Prostate
Androgen-responsive, retinoic acid biosynthesis
Not determined
ALDH1B1 [119]
IHC
Colon, lung, breast and ovary
Biomarker for colon cancer
ALDH1A1
ALDH2 [69]
Western blot, TaqMan low density array, PCR
K562 leukaemia cell line
Drug resistance, cell proliferation
ALDH1A1, ALDH1A2, ALDH3A1, ALDH7A1, ALDH8
ALDH3A1 [70–72]
Western blot
Breast, lung, liver and glioblastoma
Drug resistance, CSC, cell proliferation
ALDH1A1
ALDH3B1 [73]
Microarrays and RT-PCR
Breast cancer cell line
Tyrosine metabolism, downregulation by PGGc
Not determined
ALDH3B1 [74]
IHC
Colon, lung, breast and ovarian
Protective against oxidative stress
Not determined
ALDH4A1 [76]
Microarrays and Northern blot
Glioblastoma (U373MG, cell line)
Induced by overexpression of p53, protective against oxidative stress
Not determined
ALDH5A1 [77]
Northern blot
Hepatoma (cell line)
Not known
ALDH1b and ALDH2b
ALDH5A1 [78,79]
Microarrays, RT-PCR
Renal cell carcinoma (RCC) and HEK293 (cell lines)
Possible regulation by HNF4a, deregulated in RCC
Not determined
ALDH5A1 [80]
New-generation sequencing, RT-PCR, Western blot
Breast DCIS (cell lines)
Differentially overexpressed
Not determined
ALDH6A1 [81]
RT-PCR, enzyme activity, Western blot
Normal breast epithelium versus MCF-7 cancer cell line
RA metabolism in MTSV1.7 versus MCF-7 cell lines
Not determined
ALDH7A1 [82]
qRT-PCR
Renal cell, RCC4 (cell line)
Regulated by pVHLd, does not affect cell growth or motility
ALDH1A1
ALDH7A1 [83]
Western blot, AldefluorTM
Prostate (cell line)
Knockdown impairs migration and reduces metastases in animal model
Not determined
ALDH10 (ALDH3A2) [120]
Microsatellite markers and DNA PCR
Oesophageal SCCA with del 17p13.3-p11.1
LOHe in this specific patient population
ALDH3b
a
Given there are many studies on ALDH1A1, we refer here instead to a recent review describing this isozyme in cancer and CSCs. No isozyme specificity reported. c PGG: 1,2,3,4,6-penta-O-galloyl-beta-D-glucose. d VHL: von Hippel–Lindau gene. e LOH: loss of heterozygosity. b
4
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H3C
REVIEWS
H
O
N +
O
N
B
H
2, DEAB
N F O H 1 Active molecule, aldefluor assay
O Cl H
5, Chlorpropamide
CH3
3, Acetaldehyde
S N
H O H N N S O O
H
F
H3C
CH3 n 4 (n = 1-8)
O
S
S
N
OH
O
OH
OH
HO
N CH3
6, Disulfiram OH
H
O
S
OH OH H
HO
7, Pargyline
O
10, Gossypol
HO HO
O OH
O
H N
O
8, Daidzin
O S
O
CH3
O HO
O
O CH3
9, CVT-10216
O
N
O N 11
IC50 [µm] ALDH1A1: 0.02 ALDH2: 82 ALDH3A1: 7.7
O
13 O N IC50 [µm] ALDH1A1: 2.0 ALDH2: 0.05 ALDH3A1: 18
12
F
14 (benzimidazole) O
N
IC50 [µm] ALDH1A1: 12 ALDH2: >100 ALDH3A1: 0.3
O
Br
O N S O
N N
N
O N S N
O
S
O O
O
15 (fused benzothienopyrimidin-4-one) 11-13 (indole-2,3-diones)
N
N
ALDH1A1
O
O Cl
OCH3
S
N H
N
Cl
N H
N
N
O
O OH 16, Seco-CBI analogue
OCH3
OH
O OH
17, Seco-CBI analogue
18, Seco-CBI analogue Drug Discovery Today
FIGURE 1
Known chemical modulators of aldehyde dehydrogenases (ALDHs) and their chemical structures.
ALDH(bri), ALDH(br), ALDH(high), ALDHhiSSClo or simply ALDH+ or ALDH-positive. Stem cell markers are often labelled CD44+, CD133+, among others but, given that ALDH expression also commonly occurs in normal tissues, the ALDH+ terminology is
confusing. Accordingly, we believe it is more appropriate to use ALDHbr to describe subpopulations with stem cell characteristics; this is also in line with the protocol for using the AldefluorTM assay. In studies where ALDH expression originates from analysis of a
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–
N
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heterogenous cell population (e.g. a primary sample or a cell line with no isolation of CSCs or side population) or where the information in the published articles is ambiguous we will use ALDHpositive to describe results.
ALDH activity as a CSC marker and prognostic indicator
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Leukaemia stem cells were the first to be described in 1994 by Lapidot et al. [16], whereas solid tumour stem cells were first identified in breast cancer by Al-Hajj et al. in 2003 [17]. Since then, CSCs have been described in many other tumours including multiple myeloma [18], colon cancer [19], pancreatic cancer [20– 22], prostate cancer [23,24], lung cancer [25–27], ovarian cancer [28–30], chondro- and osteo-sarcoma [31,32], oesophageal cancer [33], glioblastoma [34,35], medulloblastoma [36], head and neck cancer [37], bladder cancer [38] and melanoma [39–41]. In most reports, ALDHbr activity has been reported in combination with cell surface markers such as CD44+, CD133+ and integrin a2b1high to identify these CSCs. The significance of CSCs in the biology of cancers has been shown by studies demonstrating an increase in the number of these cells, including ALDH-positive CSCs, in the remaining cancer tissue that survived chemotherapy and/or biological treatment [20–23,25,42–46]. The studies also showed that ALDHbr cells are more tumourigenic in vitro and in vivo [39]. Other studies have correlated the presence of ALDHbr CSCs to the prognosis and survival outcomes of patients with certain cancer types [22,23,27,29,33,47–53]. These are summarised in Table 2. In general, the expression of ALDHbr is correlated with worse prognosis with a few exceptions, such as in melanoma [40,54]. With regard to ovarian cancer, one study by Chang et al. showed [55] that high expression of ALDH1A1 (the study reported ALDH1 expression using clone 44/ALDH1 antibody from BD Biosciences) was a favourable prognostic indicator. However, meta-analysis for seven studies published on the subject (total patients studied was 1258) confirmed that ALDH1-positive expression [>20% staining of cells by immunohistochemistry (IHC)] in ovarian cancer correlates with worse OS (P = 0.005) as well as a trend for worse disease-free survival (DFS; P = 0.052) [30]. The reason for such contradiction is not clear, but it could be related to the cell origin of the cancer and degree of maturation [55,56]. In pancreatic cancer it has been noted that the primary tumour could be ALDH1A1-negative but the metastases ALDH1A1-positive (the study reported ALDH1 expression using clone 44/ALDH1 antibody from BD Biosciences) [22]. ALDH is one of the 15 markers included in an immunohistochemical signature that was shown to be predictive of eight year OS in breast cancer [57]. Overall, the presence of ALDH-positive CSCs correlates with the presence of other prognostic features including cancer histology type [26,27,52] and, in some instances, multivariate analysis showed that ALDH positivity is independently predictive of the prognosis [47,48,58].
ALDH isozymes as targets for cancer drug development High ALDH expression has been shown to be involved in drug resistance to conventional cytotoxic drugs such as cyclophosphamide and analogues [59–61], doxorubicin [62], cisplatin [28,62], arabinofuranosyl cytidine (Ara-C) [63], temozolomide and taxanes [28,64]. This resistance might not only have serious implications for the treatment of CSCs but also strategies focused on the 6
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development of ALDH-specific biomarkers and inhibitors. As we have discussed already, much CSC research has focused on ‘ALDH 1’ because isozymes from this subfamily have been studied in relation to drug resistance in cancer for more than two decades. In addition, the AldefluorTM assay has been promoted as measuring the enzymatic activity of ALDH. ALDH1 specificity is in part related to the use of diethylaminobenzaldehyde (DEAB; 2, Fig. 1), a presumed specific inhibitor of ALDH1 [6], in the control arm of the AldefluorTM assay. Although ALDH1A1 is often attributed to being the marker of stem cells, emerging information suggests that other ALDH isozymes also have key roles in specific tissues. Understanding the function and contribution of these ALDH isozymes is a pressing task. It is important to characterise the pattern of ALDH isozyme expression to be able to target them in all future therapies specifically aiming to eliminate the CSCs. In this context, further research is warranted to identify which of the 19 isozymes are contributing to the high AldefluorTM activity observed in CSCs and which contribute to tumourigenic and metastatic potential. The issues will be: (i) how specific the target should be (i.e. one ALDH isozyme versus several) and (ii) the potential side-effects, because these isozymes are widely expressed in normal tissues. Table 3 summarises the different ALDH isozymes identified in different malignancies and their functional significance [35,39,41,53,63, 65–83]. ALDH has also been proposed as a therapeutic target per se [11,84–86], however selective druggability appears to be a daunting task for two principal reasons: (i) the aforementioned wide distribution of ALDHs in normal tissue, with the highest concentrations most often occurring in the liver and/or kidney [3] and (ii) the oxidative biochemical reaction carried out by ALDHs appears to be somewhat substrate nonspecific. As an example of this, BAA (1, Fig. 1), the main component of the AldefluorTM assay, has been shown to exhibit cross-reactivity with several ALDH isozymes (1A1, 1A2, 1A3, 2, 3A1, 7 and 8) [4,41,69,87] and, hence, is a clear indicator of the difficulties in targeting ALDH subfamilies, let alone specific ALDH isozymes. Regarding selectivity, much encouragement can be derived from progress made in targeting specific human cytochrome P450 (CYP) isozymes, another super family of enzymes that metabolises endogenous molecules and xenobiotics via an oxidative mechanism. However, most of the 57 human CYPs are present in normal tissue, Pors and co-workers demonstrated that CYPs highly expressed in cancer can be exploited for tumour-selective targeting by rationalised small molecule discovery [88–91]. Specifically, duocarmycin natural products have been re-engineered to be bioactivated by CYP1A1 and CYP2W1 in tumour tissue only, releasing ultra-potent duocarmycin metabolites capable of eradicating MDR cancer cells [88] and significantly delaying tumour growth in animal studies [90,91]. The duocarmycins, some of nature’s most remarkable compounds, are further described below.
Progress in design of chemical modulators of ALDH isozymes Understanding the role of ALDH in alcohol metabolism has driven much of the early research behind the discovery of chemical modulators of ALDH. The pharmacophores of these chemical modulators are very diverse in their structural architectures (Fig. 1) and selected compounds are described below – for a recent
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comprehensive review, see [92]. Although these chemical modulators are not very ‘drug-like’, there are some encouraging observations with regard to their ALDH specificity. Early research in this field revealed that genetic polymorphism of ALDH2 leads to accumulation of acetaldehyde (3, Fig. 1) after ethanol consumption, which is known to result in the development of unpleasant physiological effects including facial flushing, nausea and tachycardia [92]. The extension of the aliphatic linker of 3 to provide a range of compounds 4 indicated that some differences in catalytic activity and selectivity could be obtained between human and yeast ALDH1 and ALDH2 isozymes [93,94]. The oral hypoglycaemic agent chlorpropamide (5) has been shown to possess an inhibitory effect that is selective for ALDH2 but not ALDH1A1 or 3A1 [95]. Disulfiram (6) is used as an alcohol-aversive agent in the treatment of alcoholism [92]. After cellular uptake it undergoes complex metabolism to produce metabolites that preferentially inhibit ALDH1A1 over ALDH2 [96]. The monoamine oxidase inhibitor pargyline (7) is activated by CYP2E1 to yield a highly reactive propiolaldehyde that irreversibly inactivates ALDH2 [97]. The isoflavones daidzin (8) and CVT-10216 (9) have been shown to be potent inhibitors of ALDH2 but not ALDH1A1 [98], whereas gossypol (10), a terpenoid aldehyde, acts as a noncompetitive inhibitor and shows more selectivity for ALDH3 than ALDH1 and ALDH2 families [99]. It has been suggested that gossypol could interact with the cofactor binding site, which might underlie the commonality of inhibition of dehydrogenases [99]. The chemical modulators used as inhibitors or pharmacological tools to explore ALDHs have, generally speaking, been discovered by serendipity. Essentially only four ALDH isozymes: ALDH1A1, ALDH1A2, ALDH2 and ALDH3A1, have been studied for their potential as pharmacologically relevant therapeutic targets. It is apparent from the few reports available on chemical modulators, however, that some selectivity can be obtained, but no information is available as to the selectivity across all 19 human isozymes. Nevertheless, recent focused studies on the discovery of chemical modulators of ALDHs have resulted in broad-spectrum and specific inhibitors with in vitro activity. Hurley and co-workers have synthesised small molecules that utilise a cysteine residue within the active site of ALDH1A1, 2 and 3A1 to catalyse the production of a vinyl-ketone intermediate through the formation of a covalent adduct within the active site [100]. Interestingly, efforts from the same group have resulted in encouraging results that indicate that selectivity can be gained from focused studies using computational modelling and in vitro screening assays [101]. Synthetic modifications of an indole-2,3-dione pharmacophore (e.g. 10–12) has resulted in selective targeting of ALDH1A1, 2 and 3A1, respectively (Fig. 1). Other reports [71,102,103] by Hurley’s group include investigations aimed at selective inhibition of ALDH3A1, which could increase chemosensitivity towards cyclophosphamide in ALDH3A1-expressing tumours. A benzimidazole-based analogue 14 was shown to target ALDH3A1 (IC50 = 0.2 mM) but not ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1 or ALDH2 activity [71], whereas a relatively potent benzothienopyrimidin-4-onebased inhibitor 15 was selective for ALDH1A1 (Ki = 300 nM) with in vitro activity against ovarian cancer spheroids [104]. This selective inhibitor was identified from a high-throughput screen of 64,000 compounds performed to identify activators and inhibitors of ALDH1A1.
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Somewhat unexpectedly, there has been an interesting development in the duocarmycin class of natural products. The duocarmycins are ultra-potent molecules that derive their biological activity from a characteristic sequence-selective alkylation of adenine N3 in the DNA minor groove initiating a cascade of cellular events ultimately leading to apoptosis [105]. Four duocarmycin analogues have progressed into clinical Phase I and II trials, but their haematological toxicity led to severe side-effects and termination of treatment [105]. Nevertheless, owing to the intense potency, unique mechanism-of-action and broad range of activity of the duocarmycins, extensive efforts have been made during the past two decades to find analogues that retain their potency and antitumour activity with potential for clinical progression [105]. Tietze and co-workers have recently demonstrated that alkynylated duocarmycin analogues (e.g. 16) with an altered DNA recognition motif have the potential to target ALDH1A1 as identified using an activity-based protein profiling (ABPP) approach [106,107], a finding that has caused some debate over the true cellular target (DNA versus ALDH1A1) [108,109]. Nevertheless, proteomic analysis supports a duocarmycin metabolite to be covalently linked to a cysteine residue within the active site of the enzyme (17) whereas other analogues completely lacking the DNA recognition motif (e.g. 18) exhibited remarkably low nM cytotoxicity, evidently from the ability primarily to target ALDH1A1; this activity was similar to results achieved using ALDH1A1 siRNA knockdown [107]. Although these molecules are at an early stage of investigation, an opportunity now exists for developing a platform for progression of such molecules to an in vivo stage.
Challenges and opportunities in targeting ALDHs A difficult problem to overcome in cancer treatment is to develop tumour-specific chemotherapeutic agents that do not expose normal tissue to the effects of most clinically used cytotoxic and molecularly targeted agents. This lack of selectivity means that physicians must give suboptimal doses leading to inefficient treatment of the tumour, with high risk of recurrence of drug-resistant secondary tumours or metastases. New directions therefore ought to include agents that directly eradicate CSCs [110] or indirectly by employing strategies that involve agents that can stimulate CSCs to differentiate [12,84–86,111,112]. In this context, the lack of therapeutics that currently can eradicate CSCs is a real problem that needs urgent attention. In view of the high expression of certain ALDH isozymes in CSCs, an opportunity exists to target such cells with ALDH-affinic small molecules. Historically, Q3 ALDH1A1 has been the key ALDH isozyme linked with stem cell populations. However, others are now emerging as potential contributors to ALDH functional activity in CSC populations derived from various cancer tissues. ALDH1A3, in particular, appears to be highly expressed in CSCs of the breast, gallbladder, melanoma and prostate. Understanding the role each of the subfamily 1 isozymes has in cell differentiation, tumour grade and metastasis is essential to unravelling the potential of using these as biomarkers and therapeutic targets for drug development. A recent study revealed the potential in targeting ALDH1A3 present in mesenchymal glioma stem cells (GSCs) but not in proneural (PN) GSCs [34]. ALDH1A3 expression was correlated with high ALDH activity and inhibition of this isozyme in vitro using DEAB (2) attenuated the growth of the more aggressive mesenchymal but not PN GSCs. On
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this basis, it can be postulated that inhibition of ALDH1A3-mediated pathways with a more pharmacologically robust agent than DEAB could provide a clinical candidate to a subset of patients with mesenchymal GSCs. In humans, ALDH7A1 is known to play an important part during lysine catabolism through the NAD-dependent oxidative conversion of aminoadipate semialdehyde (AASA) to its corresponding carboxylic acid: a-aminoadipic acid [113], while it also protects against hyperosmotic stress by generating osmolytes and metabolising toxic aldehydes [114,115]. Mutations in the ALDH7A1 gene can cause pyridoxine-dependent and folic-acidresponsive seizures [116]. ALDH7A1 expression is associated with recurrence in patients with surgically resected non-small-cell lung carcinoma [117] and it has been shown to be highly expressed in prostate cancer [24,83]. Interestingly, knockdown of ALDH7A1 expression has been shown to: (i) inhibit the clonogenic and migratory ability of human prostate cancer cells in vitro; (ii) decrease a2hi/avhi/CD44+ stem/progenitor cell subpopulations in the human prostate cancer cell line PC-3M-Pro4 and (iii) decrease intra-bone growth and inhibit experimentally induced (bone) metastasis [24,83]. In spite of this evidence, it remains a significant challenge to develop an agent that can target ALDH7A1 selectively in prostate cancer without affecting this enzyme expressed in normal tissue. The targeting of ALDHs therefore requires careful attention to be paid to the delivery strategy, otherwise off-target toxicities are to be expected. As outlined in this review, there have been relatively few insights into the synthetic and mechanistic aspects of ALDH inhibitors and, to the best of our knowledge, only one ALDH inhibitor has been tested in vivo. This agent, ‘compound 673’ (no structure was reported) was shown to be nontoxic to normal stem cells whereas, interestingly, it was shown to deplete ovarian cancer cells expressing CSC markers [118]. Furthermore, pre-treatment of tumour cells with 673 significantly reduced tumour initiation and growth rates and the compound was highly
synergistic with cisplatin in vitro and in vivo as assessed by cell growth curves and tumour growth, respectively. Although further studies are clearly required to evaluate this agent, it is tempting to speculate that its therapeutic efficacy comes from targeting the CSC component. The recent results reported by Hurley, Matei and co-workers [104] support the use of an ALDH1A1-specific inhibitor (15) to block ovarian cancer cell proliferation and survival under 3D culture conditions and in combination with cisplatin to achieve a synergistic effect and enhanced cell kill.
Concluding remarks and future directions The presence of specific ALDH isoforms in CSCs and tumourigenesis has started to generate interest in the potential to develop specific inhibitors with clinical potential that can be used to eradicate the CSC component of highly heterogeneous and aggressive tumours. So far, no proven specific inhibitors of any ALDH isozyme beyond the in vitro stage exist. Nevertheless, because most of the current drugs we use for the treatment of cancer are not specific or targeted and because there could be more than one ALDH isozyme expressed in CSCs, then the use of relatively nonspecific inhibitors of ALDH activity, alone or in combination with old drugs, could be justified or even preferred (Fig. 2). Thus, clinical studies might need to proceed testing existing potential inhibitors in the usual established preclinical and clinical trials with the hope that side-effects will be tolerable and give a therapeutic window of opportunity. Although recent progress in smallmolecule-based discovery is encouraging, the use of siRNA [70] might be the most specific inhibitor for any one ALDH isozyme if it can be developed into a clinically applicable targeted gene therapy approach. Currently, several investigators are investing efforts in identifying specific ALDH isozyme inhibitors with potential clinical applications that go beyond just treating cancer, including alcoholism, male contraception and cardiac ischaemia. We predict that, over the next five-to-ten years, there will be serious attempts to arrive at small molecules that can be used to
Is ALDH isozyme expression in CSCs significantly elevated to enable preferential targeting and therapeutic intervention? Benign
CSC
Level of ALDH isozyme expression?
MDR
CSC CSC
I. Standard chemotherapy Untreated CSCs enable recurrence of tumour
II. CSC-targeted therapy
CSC
I+II Tumour loses ability to generate new cells and degenerates 100% tumour eradication? Drug Discovery Today
FIGURE 2
cancer stem cell (CSC)-targeted therapy including aldehyde dehydrogenase (ALDH)-targeting inhibitors in combination with standard chemotherapy could result in a more efficient treatment of heterogeneic tumours, with possible durable remissions and cures. 8
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on the utility in designing inhibitors, either as single- or multitargeting ALDH inhibitors, in the first instance as tools to interrogate ALDH pathways and in the longer term as drug candidates with clinical potential to eradicate the CSC fraction of different cancer types.
Acknowledgement Dr Robert Falconer is thanked for valuable suggestions and proofreading of this manuscript.
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target ALDHbr CSCs, but the question remains as to whether this targeting can be achieved in a truly selective fashion with no toxicities to normal tissues. Full characterisation of the heterogeneity of tumours and their CSC component will be crucial for successful design of strategies involving therapeutics and biologics that are targeted against ALDH isozymes. Simultaneously, finetuning drug delivery strategies to target ALDHbr CSCs are also likely to be required to improve on the clinical utility of such agents. We hope that this review can stimulate thought and debate
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101 Kimble-Hill, A.C. et al. (2014) Development of selective inhibitors for aldehyde dehydrogenases based on substituted indole-2,3-diones. J. Med. Chem. 57, 714–722 102 Parajuli, B. et al. (2014) Development of selective inhibitors for human aldehyde dehydrogenase 3A1 (ALDH3A1) for the enhancement of cyclophosphamide cytotoxicity. ChemBioChem 15, 701–712 103 Parajuli, B. et al. (2011) Discovery of novel regulators of aldehyde dehydrogenase isoenzymes. Chem. Biol. Interact. 191, 153–158 104 Condello, S. et al. (2014) b-Catenin-regulated ALDH1A1 is a target in ovarian cancer spheroids. Oncogene http://dx.doi.org/10.1038/onc.2014.1178 105 Ghosh, N. et al. (2009) Chemical and biological explorations of the family of CC-1065 and the duocarmycin natural products. Curr. Top. Med. Chem. 9, 1494–1524 106 Wirth, T. et al. (2012) Duocarmycin analogues target aldehyde dehydrogenase 1 in lung cancer cells. Angew. Chem. Int. Ed. Engl. 51, 2874–2877 107 Wirth, T. et al. (2013) The two faces of potent antitumor duocarmycin-based drugs: a structural dissection reveals disparate motifs for DNA versus aldehyde dehydrogenase 1 affinity. Angew. Chem. Int. Ed. Engl. 52, 6921–6925 108 Tercel, M. et al. (2013) The cytotoxicity of duocarmycin analogues is mediated through alkylation of DNA, not aldehyde dehydrogenase 1: a comment. Angew. Chem. Int. Ed. Engl. 52, 5442–5446 109 Tietze, L.F. and Sieber, S.A. (2013) Duocarmycin analogues without a DNA-binding indole unit associate with aldehyde dehydrogenase 1A1 and not DNA: a reply. Angew. Chem. Int. Ed. Engl. 52, 5447–5449 110 Gupta, P.B. et al. (2009) Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 138, 645–659
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111 Alison, M.R. et al. (2012) Cancer stem cells: in the line of fire. Cancer Treat. Rev. 38, 589–598 112 Yang, T. et al. (2014) Cancer stem cells: constantly evolving and functionally heterogeneous therapeutic targets. Cancer Res. 74, 2922–2927 113 Plecko, B. et al. (2007) Biochemical and molecular characterization of 18 patients with pyridoxine-dependent epilepsy and mutations of the antiquitin (ALDH7A1) gene. Hum. Mutat. 28, 19–26 114 Brocker, C. et al. (2010) Aldehyde dehydrogenase 7A1 (ALDH7A1) is a novel enzyme involved in cellular defense against hyperosmotic stress. J. Biol. Chem. 285, 18452–18463 115 Brocker, C. et al. (2011) Aldehyde dehydrogenase 7A1 (ALDH7A1) attenuates reactive aldehyde and oxidative stress induced cytotoxicity. Chem. Biol. Interact. 191, 269–277 116 Yang, Z. et al. (2014) Clinical diagnosis, treatment, and ALDH7A1 mutations in pyridoxine-dependent epilepsy in three Chinese infants. PLOS ONE 9, e92803 117 Giacalone, N.J. et al. (2013) ALDH7A1 expression is associated with recurrence in patients with surgically resected non-small-cell lung carcinoma. Future Oncol. 9, 737–745 118 Chefetz, I. et al. (2013) Targeting ovarian cancer stem cells using a novel ALDH inhibitor. In Proceedings 104th AACR Annual Meeting, Vol. 54 Abstr. 3731 119 Chen, Y. et al. (2011) Aldehyde dehydrogenase 1B1 (ALDH1B1) is a potential biomarker for human colon cancer. Biochem. Biophys. Res. Commun. 405, 173–179 120 Huang, J. et al. (2000) High frequency allelic loss on chromosome 17p13.3-p11.1 in esophageal squamous cell carcinomas from a high incidence area in northern China. Carcinogenesis 21, 2019–2026
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Aldehyde dehydrogenases in cancer: an opportunity for biomarker and drug development? Klaus Pors1 and Jan S. Moreb2 Q1 1 2
Institute of Cancer Therapeutics, University of Bradford, Bradford BD7 1DP, UK Hematological Malignancies, PO Box 100278, Gainesville, FL 32610-0278, USA
Aldehyde dehydrogenases (ALDHs) belong to a superfamily of 19 isozymes that are known to participate in many physiologically important biosynthetic processes including detoxification of specific endogenous and exogenous aldehyde substrates. The high expression levels of an emerging number of ALDHs in various cancer tissues suggest that these enzymes have pivotal roles in cancer cell survival and progression. Mapping out the heterogeneity of tumours and their cancer stem cell (CSC) component will be key to successful design of strategies involving therapeutics that are targeted against specific ALDH isozymes. This review summarises recent progress in ALDH-focused cancer research and discovery of small-molecule-based inhibitors.
Introduction The aldehyde dehydrogenase (ALDH) superfamily of enzymes comprises 19 human isozymes with 11 distinct families localised in the cytoplasm, mitochondria or nucleus [1]. ALDHs are known to participate in many physiologically important biosynthetic pathways [2] and the activity of certain ALDHs has been shown to be crucial in the detoxification of specific endogenous and exogenous aldehyde substrates [1,3,4]. Accordingly, many ALDHs have crucial roles in the protection of cellular homeostasis and organism functions against toxic effects from the aldehydes [1]. Aldehydes are detoxified via ALDH-catalysed metabolism resulting in the corresponding carboxylic acids through a pyridine-nucleotide-dependent reaction [5]. ALDH activity, through aldehyde metabolism, is essential for the synthesis of molecules such as retinoic acid, betaine and g-aminobutyric acid, which are important for cell proliferation, differentiation and survival [1]. Abnormal behaviour in ALDH activity linked to specific metabolic pathways has implications for several disease states, including ¨ gren–Larsson syndrome, type II hyperprolinaemia, hyperamSjo monaemia and cancer [4]. High ALDH cellular levels and a relationship to cyclophosphamide resistance were first identified in normal haematopoietic stem cells [6]. The prominence of ALDH
Corresponding authors: Moreb, Pors, K. ([email protected]), J.S. ([email protected])
isozymes over the past five years has been mainly caused by hypothesis of cancer stem cells (CSCs), with high ALDH enzyme activity identified as a marker for stem cells in multiple tissue types. The availability of the AldefluorTM flow cytometry assay has been the main driving technology for this evolving field because it has enabled the use of ALDH enzyme activity to sort live cells that have been shown to have the characteristics of stem cells such as self-renewal, high proliferative potential, clonogenicity and multipotency. Most of the current cancer chemotherapy regimens are used to reduce the bulk of solid tumours before surgery, eradicate cancer cells that might remain after surgery or target malignant metastases. Over the past 20 years much emphasis has been placed on overcoming multidrug resistance (MDR) and progress on new agents overcoming MDR has been encouraging [7–10]. Regardless, owing to poor overall survival (OS) rates associated with chemotherapy, the target and aim of the treatment might be shifting towards overcoming not only MDR but also other pathways essential for the survival of CSCs. In fact, it is becoming apparent that resistance mechanisms in CSCs to current therapies share similarities to those observed in the treatment of MDR cancer cells: (i) CSCs are generally quiescent and therefore resistant to chemotherapy designed to target proliferating cancer cells [11,12]; (ii) CSCs possess enhanced expression of ABC transporters as evident from observations that CSCs can be isolated by Hoechst stain
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1359-6446/06/$ - see front matter ß 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.drudis.2014.09.009
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exclusion via the ABCG2 protein pump [13,14] and (iii) there is evidence for high content of specific ALDH isozymes in CSCs present in different types of cancers that have been correlated to worse prognosis. Such a phenotype usually correlates with a high proliferation index, higher metastatic potential and increased drug resistance in the respective cancer. We will review and summarise the published literature in this arena. We will discuss the role of the different ALDH isozymes (Table 1 provides a list of all ALDH isozymes with their gene bank access code and their chromosomal location) responsible for this high ALDH content, because these could have implications to all future therapies targeting CSCs. In general, these isozymes are widely expressed in different tissues; however, much of the focus in CSC research to date has been on detection of ALDH1 expression and/or activity, but often without clarification of isozyme specificity. In the past there might have been limited or no knowledge of the isozyme specificity of ALDH-targeting antibodies or accuracy of the AldefluorTM assay, which has led to a large body of work suffering from a lack of clear definitions and with scientific questions remaining.
Because evidence of the fundamental biological and clinical importance of ALDH isozyme expression and function is ever increasing, it is clear that scientists engaged in this research field should agree a more rigorous definition when using the terms ‘ALDH activity’ or ‘ALDH expression’. Accordingly, before we discuss ALDH expression and/or activity in cancer or stem cells in more detail we have proposed terminology in this review that we suggest be adopted in the scientific community in unravelling the significance of ALDH isozymes in the future.
ALDH expression Earlier studies often only reported family type such as ALDH1 and not the specific isozyme members such as ALDH1A1, 1A2, 1A3, among others, perhaps in part because of lack of antibody-specific production and/or information. In this review, we have checked methodologies and corresponded with authors of selected studies discussed in this review, and companies now selling the same antibodies or more-specific isozymes, in an attempt to align current knowledge with studies performed in the past. We have also
TABLE 1
The aldehyde dehydrogenase (ALDH) family ALDH isozyme
Genebank accession number
Compartment
Chromosomal localisation
ALDH1A1
NM_000689
Cytoplasmic
9q21.13
ALDH1A2
NM_003888 NM_170696 NM_170697
Cytoplasmic
15q21.3
ALDH1A3
NM_000693
Cytoplasmic
15q26.3
ALDH1B1
NM_000692
Mitochondrial
9p11.1
ADLH1L1
NM_012190
Cytoplasmic
3q21.3
ALDH1L2
NM_001034173
Mitochondrial
12q23.3
ALDH2
NM_000690
Mitochondrial
12q24.2
Related clinical syndromes
Neural tube defects
ALDH3A1
NM_000691
Cytoplasmic
17p11.2
ALDH3A2 (ALDH10)
NM_001031806 NM_000382
Microsomal
17p11.2
ALDH3B1
NM_000692 NM_001030010
Cytoplasmic
11q13
ALDH3B2
NM_000695 NM_001031615
Cytoplasmic
11q13
ALDH4A1
NM_170726 NM_003748
Mitochondrial
1p36
Hyperprolinaemia
ALDH5A1
NM_001080 NM_170740
Mitochondrial
6p22
Succinic semialdehyde dehydrogenase deficiency
ALDH6A1
NM_005589
Mitochondrial
14q24.3
ALDH7A1
NM_001182
Cytoplasmic
5q31
ALDH8A1
NM_170771 NM_022568
Cytoplasmic
6q23.2
ALDH9A1
NM_000696
Cytoplasmic
1q23.1
Polymorphism in treatment-resistant tardive dyskinesia in Japanese schizophrenic patients
ALDH16A1
NM_153329
Cytoplasmic and membranes
19q13.33
Possible association with gout
ALDH18A1
NM_002860 NM_001017423
Mitochondrial
10q24.3
2
Sjo¨gren–Larsson syndrome
Pyridoxine-dependent epilepsy
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consulted a publically available database for nomenclature and functional and molecular sequence information (www.aldh.org) Q2 for accuracy of information. Wherever possible we have highlighted ALDH specificity in the text or in Tables 2 and 3.
ALDH activity ALDH activity is often described in association with the identification and isolation of CSCs using the AldefluorTM assay. This assay [15] relies on the trapping of a negatively charged carboxylate metabolite generated from BODIPY aminoaldehyde (BAA; 1,
Fig. 1), which causes a subset of cells with high levels of ALDH activity to become highly fluorescent. This fluorescent-labelled subpopulation of cells is designated as ‘ALDH bright cells’ and can be detected using flow cytometry and isolated using a cell sorter. Such cells are likely to possess tumour-initiating properties that can constitute CSCs, based on the ability for long-term self-renewal and on a differentially expressed and stable stem cell lineage marker or phenotype. When describing ALDH activity, care must be exercised because there is some confusion in the field; ALDHcontaining subpopulations of cells often are labelled ALDHbr,
TABLE 2
Aldehyde dehydrogenase (ALDH) expression as a biomarker for cancer prognosis Cancer type
Method of detecting ALDH
% ALDH-posa CSC
OS in ALDH posa (n)
OS in ALDH-nega (n)
P value
% of ALDH-pos cases
Breast [47]
IHC for ALDH1A1b AldefluorTM
Average 5%
5 years: 19.8% (24)
5 years: 58.7% (122)
0.0459
19
Breast [47]
IHC
Average 5%
5 years: 69.59% (102)
5 years: 84.55% (243)
0.000675
30
0.0337
34
b
Inflammatory breast [48]
IHC for ALDH1A1 AldefluorTM
3–5%
5 years: 25% (16)
5 years: 54% (37)
Acute myeloid leukaemia (AML) [49]
AldefluorTM
Median: 14.98% of CD34+ cells
NAc
NAc
AML [51]
AldefluorTM
0.12% of the CD34+ cells had intermediate ALDH activity (ALDHint)
Detectable CD34+CD38 ALDHint cells predictive of relapse from CR
AML [50]
AldefluorTM
Median 0.5% (range 0.01–16.00)
57.5% (40)d
82% (28)
0.029
59
Prostate [23]
IHC for ALDH1A1 AldefluorTM
>10% considered high ALDH
5 years: 65%e
5 years: 90%e
0.0093
20
Rectal [58]
IHC for ALDH1A1b
NA
NAf
Oesophageal [33]
IHC for ALDH1A1b AldefluorTM
Used labelling index: high versus low
HR 3.4 Increased risk of death/relapse (HR 3.87)
Oesophageal [52]
IHC ALDH1g
NA
5 years OS: 8.3%
5 years OS: 52.2%
Lung [27]
IHC for ALDH1A1
Abundant versus scattered
(n = 20)
(n = 27)
48; 100% in resistant tumours
EFS shorter OS not different Pancreatic [22]
IHC for ALDH1A1b AldefluorTM
Ovarian [29]: serous
IHC ALDH1A1b
Clear cell Gallbladder [53]
IHC, ALDH1A3
32.5
0.03 0.006
NA
0.025
15.2, nuclear staining 42.5
0.053 0.141
Median OS: 14 mosh
18 mos
0.05
34
Median: 20%e
5 years: 37 (34)
5 years: 77 (28)
0.006
54.8
Median: 15%
5 years: 54 (18)
5 years: 81 (19)
0.047
48.6
10% of cells with weak to moderate staining
7.5 mos (16)
11.7 mos (30)
0.005
35
a ALDH-pos (ALDH-positive) cells refers to cells that are identified using AldefluorTM (ALDHbr cells) or immunohistochemistry (IHC) using either ALDH1A1 or 1A3-specific antibodies (see below for exception). ALDH-neg (ALDH-negative) cells are either not identified owing to poor fluorescence signal using AldefluorTM assay (defined as ALDHdim) or stained positively with ALDH1A1/1A3-specific antibodies. b The studies reported on ALDH1 expression using clone 44/ALDH1 antibody from BD Biosciences or ALDH1 antibody (ab52492; Abcam); however both are specific to ALDH1A1. c Results show that patients with ALDHbr subpopulations of AML cells were more likely to relapse after short remission. d ALDH+ terminology was used in the study: ALDH+ AML if frequency contained 0.36% ALDHbr cells, whereas cells below the threshold were determined ALDH AML. The values are the survival in each group during the observation period of 21.5 months. e The median was used to distinguish patients with low versus high ALDH-expressing tumours. We estimated the 5-year OS from the Kaplan–Meier curves given in the reference. f High ALDH expression predicted shorter disease-free survival (DFS) and poor outcome (P = 0.009). g ALDH1 antibody ab51028 is no longer available in Abcam’s product range, so no information available on the isozyme. h The 25% survival rate was 28.4 months (mos) in patients in primary tumours stained with ALDH1A1-specific antibody versus 46 mos in patients with ALDH1A1-negative specimens.
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TABLE 3
Aldehyde dehydrogenase (ALDH) isozymes identified in different types of cancer and their functional relevance
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ALDH isozyme
Methods of detection
Cancer type
Cancer-related function
Co-existing ALDH isozymes
ALDH1A1a [39]
AldefluorTM, IHC, spectrophotometry, HPLC
All cancer types
Biomarker for CSC, drug resistance
Other ALDH1 family members, ALDH3A1
ALDH1A2 [65]
IHC, Western blot
Prostate
Reduced expression in prostate cancer in comparison with normal tissue. Retinoid metabolism
ALDH1A1 and ALDH1A3
ALDH1A2 [63]
Mass spectrometry, PCR and Western blot
Acute myeloid leukaemia (AML)
High expression and resistance to Ara-C
Not determined
ALDH1A3 [53]
IHC
Gallbladder
Poor prognosis, high expression in advanced disease
Not determined
ALDH1A3 [66]
DNA methylation microarray
Bladder
Decreased expression and aggressive clinicopathological characteristics
Not determined
ALDH1A3 [35]
DNA methylation
Glioblastoma
Hypermethylation associated with better prognosis
Not determined
ALDH1A3 [41]
AldefluorTM, qRT-PCR
Melanoma
Cell cycle arrest, apoptosis and decreased viability
ALDH1A1
ALDH1A3 [67]
Genome-wide microarrays, IF and qPCR, IHC
Breast
Correlation to tumour grade and metastasis
ALDH1A1, 2, 4A1, 6A1, 7A1, 18A1
ALDH1A3 [68]
RT-PCR
Prostate
Androgen-responsive, retinoic acid biosynthesis
Not determined
ALDH1B1 [119]
IHC
Colon, lung, breast and ovary
Biomarker for colon cancer
ALDH1A1
ALDH2 [69]
Western blot, TaqMan low density array, PCR
K562 leukaemia cell line
Drug resistance, cell proliferation
ALDH1A1, ALDH1A2, ALDH3A1, ALDH7A1, ALDH8
ALDH3A1 [70–72]
Western blot
Breast, lung, liver and glioblastoma
Drug resistance, CSC, cell proliferation
ALDH1A1
ALDH3B1 [73]
Microarrays and RT-PCR
Breast cancer cell line
Tyrosine metabolism, downregulation by PGGc
Not determined
ALDH3B1 [74]
IHC
Colon, lung, breast and ovarian
Protective against oxidative stress
Not determined
ALDH4A1 [76]
Microarrays and Northern blot
Glioblastoma (U373MG, cell line)
Induced by overexpression of p53, protective against oxidative stress
Not determined
ALDH5A1 [77]
Northern blot
Hepatoma (cell line)
Not known
ALDH1b and ALDH2b
ALDH5A1 [78,79]
Microarrays, RT-PCR
Renal cell carcinoma (RCC) and HEK293 (cell lines)
Possible regulation by HNF4a, deregulated in RCC
Not determined
ALDH5A1 [80]
New-generation sequencing, RT-PCR, Western blot
Breast DCIS (cell lines)
Differentially overexpressed
Not determined
ALDH6A1 [81]
RT-PCR, enzyme activity, Western blot
Normal breast epithelium versus MCF-7 cancer cell line
RA metabolism in MTSV1.7 versus MCF-7 cell lines
Not determined
ALDH7A1 [82]
qRT-PCR
Renal cell, RCC4 (cell line)
Regulated by pVHLd, does not affect cell growth or motility
ALDH1A1
ALDH7A1 [83]
Western blot, AldefluorTM
Prostate (cell line)
Knockdown impairs migration and reduces metastases in animal model
Not determined
ALDH10 (ALDH3A2) [120]
Microsatellite markers and DNA PCR
Oesophageal SCCA with del 17p13.3-p11.1
LOHe in this specific patient population
ALDH3b
a
Given there are many studies on ALDH1A1, we refer here instead to a recent review describing this isozyme in cancer and CSCs. No isozyme specificity reported. c PGG: 1,2,3,4,6-penta-O-galloyl-beta-D-glucose. d VHL: von Hippel–Lindau gene. e LOH: loss of heterozygosity. b
4
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H3C
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H
O
N +
O
N
B
H
2, DEAB
N F O H 1 Active molecule, aldefluor assay
O Cl H
5, Chlorpropamide
CH3
3, Acetaldehyde
S N
H O H N N S O O
H
F
H3C
CH3 n 4 (n = 1-8)
O
S
S
N
OH
O
OH
OH
HO
N CH3
6, Disulfiram OH
H
O
S
OH OH H
HO
7, Pargyline
O
10, Gossypol
HO HO
O OH
O
H N
O
8, Daidzin
O S
O
CH3
O HO
O
O CH3
9, CVT-10216
O
N
O N 11
IC50 [µm] ALDH1A1: 0.02 ALDH2: 82 ALDH3A1: 7.7
O
13 O N IC50 [µm] ALDH1A1: 2.0 ALDH2: 0.05 ALDH3A1: 18
12
F
14 (benzimidazole) O
N
IC50 [µm] ALDH1A1: 12 ALDH2: >100 ALDH3A1: 0.3
O
Br
O N S O
N N
N
O N S N
O
S
O O
O
15 (fused benzothienopyrimidin-4-one) 11-13 (indole-2,3-diones)
N
N
ALDH1A1
O
O Cl
OCH3
S
N H
N
Cl
N H
N
N
O
O OH 16, Seco-CBI analogue
OCH3
OH
O OH
17, Seco-CBI analogue
18, Seco-CBI analogue Drug Discovery Today
FIGURE 1
Known chemical modulators of aldehyde dehydrogenases (ALDHs) and their chemical structures.
ALDH(bri), ALDH(br), ALDH(high), ALDHhiSSClo or simply ALDH+ or ALDH-positive. Stem cell markers are often labelled CD44+, CD133+, among others but, given that ALDH expression also commonly occurs in normal tissues, the ALDH+ terminology is
confusing. Accordingly, we believe it is more appropriate to use ALDHbr to describe subpopulations with stem cell characteristics; this is also in line with the protocol for using the AldefluorTM assay. In studies where ALDH expression originates from analysis of a
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–
N
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heterogenous cell population (e.g. a primary sample or a cell line with no isolation of CSCs or side population) or where the information in the published articles is ambiguous we will use ALDHpositive to describe results.
ALDH activity as a CSC marker and prognostic indicator
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Leukaemia stem cells were the first to be described in 1994 by Lapidot et al. [16], whereas solid tumour stem cells were first identified in breast cancer by Al-Hajj et al. in 2003 [17]. Since then, CSCs have been described in many other tumours including multiple myeloma [18], colon cancer [19], pancreatic cancer [20– 22], prostate cancer [23,24], lung cancer [25–27], ovarian cancer [28–30], chondro- and osteo-sarcoma [31,32], oesophageal cancer [33], glioblastoma [34,35], medulloblastoma [36], head and neck cancer [37], bladder cancer [38] and melanoma [39–41]. In most reports, ALDHbr activity has been reported in combination with cell surface markers such as CD44+, CD133+ and integrin a2b1high to identify these CSCs. The significance of CSCs in the biology of cancers has been shown by studies demonstrating an increase in the number of these cells, including ALDH-positive CSCs, in the remaining cancer tissue that survived chemotherapy and/or biological treatment [20–23,25,42–46]. The studies also showed that ALDHbr cells are more tumourigenic in vitro and in vivo [39]. Other studies have correlated the presence of ALDHbr CSCs to the prognosis and survival outcomes of patients with certain cancer types [22,23,27,29,33,47–53]. These are summarised in Table 2. In general, the expression of ALDHbr is correlated with worse prognosis with a few exceptions, such as in melanoma [40,54]. With regard to ovarian cancer, one study by Chang et al. showed [55] that high expression of ALDH1A1 (the study reported ALDH1 expression using clone 44/ALDH1 antibody from BD Biosciences) was a favourable prognostic indicator. However, meta-analysis for seven studies published on the subject (total patients studied was 1258) confirmed that ALDH1-positive expression [>20% staining of cells by immunohistochemistry (IHC)] in ovarian cancer correlates with worse OS (P = 0.005) as well as a trend for worse disease-free survival (DFS; P = 0.052) [30]. The reason for such contradiction is not clear, but it could be related to the cell origin of the cancer and degree of maturation [55,56]. In pancreatic cancer it has been noted that the primary tumour could be ALDH1A1-negative but the metastases ALDH1A1-positive (the study reported ALDH1 expression using clone 44/ALDH1 antibody from BD Biosciences) [22]. ALDH is one of the 15 markers included in an immunohistochemical signature that was shown to be predictive of eight year OS in breast cancer [57]. Overall, the presence of ALDH-positive CSCs correlates with the presence of other prognostic features including cancer histology type [26,27,52] and, in some instances, multivariate analysis showed that ALDH positivity is independently predictive of the prognosis [47,48,58].
ALDH isozymes as targets for cancer drug development High ALDH expression has been shown to be involved in drug resistance to conventional cytotoxic drugs such as cyclophosphamide and analogues [59–61], doxorubicin [62], cisplatin [28,62], arabinofuranosyl cytidine (Ara-C) [63], temozolomide and taxanes [28,64]. This resistance might not only have serious implications for the treatment of CSCs but also strategies focused on the 6
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development of ALDH-specific biomarkers and inhibitors. As we have discussed already, much CSC research has focused on ‘ALDH 1’ because isozymes from this subfamily have been studied in relation to drug resistance in cancer for more than two decades. In addition, the AldefluorTM assay has been promoted as measuring the enzymatic activity of ALDH. ALDH1 specificity is in part related to the use of diethylaminobenzaldehyde (DEAB; 2, Fig. 1), a presumed specific inhibitor of ALDH1 [6], in the control arm of the AldefluorTM assay. Although ALDH1A1 is often attributed to being the marker of stem cells, emerging information suggests that other ALDH isozymes also have key roles in specific tissues. Understanding the function and contribution of these ALDH isozymes is a pressing task. It is important to characterise the pattern of ALDH isozyme expression to be able to target them in all future therapies specifically aiming to eliminate the CSCs. In this context, further research is warranted to identify which of the 19 isozymes are contributing to the high AldefluorTM activity observed in CSCs and which contribute to tumourigenic and metastatic potential. The issues will be: (i) how specific the target should be (i.e. one ALDH isozyme versus several) and (ii) the potential side-effects, because these isozymes are widely expressed in normal tissues. Table 3 summarises the different ALDH isozymes identified in different malignancies and their functional significance [35,39,41,53,63, 65–83]. ALDH has also been proposed as a therapeutic target per se [11,84–86], however selective druggability appears to be a daunting task for two principal reasons: (i) the aforementioned wide distribution of ALDHs in normal tissue, with the highest concentrations most often occurring in the liver and/or kidney [3] and (ii) the oxidative biochemical reaction carried out by ALDHs appears to be somewhat substrate nonspecific. As an example of this, BAA (1, Fig. 1), the main component of the AldefluorTM assay, has been shown to exhibit cross-reactivity with several ALDH isozymes (1A1, 1A2, 1A3, 2, 3A1, 7 and 8) [4,41,69,87] and, hence, is a clear indicator of the difficulties in targeting ALDH subfamilies, let alone specific ALDH isozymes. Regarding selectivity, much encouragement can be derived from progress made in targeting specific human cytochrome P450 (CYP) isozymes, another super family of enzymes that metabolises endogenous molecules and xenobiotics via an oxidative mechanism. However, most of the 57 human CYPs are present in normal tissue, Pors and co-workers demonstrated that CYPs highly expressed in cancer can be exploited for tumour-selective targeting by rationalised small molecule discovery [88–91]. Specifically, duocarmycin natural products have been re-engineered to be bioactivated by CYP1A1 and CYP2W1 in tumour tissue only, releasing ultra-potent duocarmycin metabolites capable of eradicating MDR cancer cells [88] and significantly delaying tumour growth in animal studies [90,91]. The duocarmycins, some of nature’s most remarkable compounds, are further described below.
Progress in design of chemical modulators of ALDH isozymes Understanding the role of ALDH in alcohol metabolism has driven much of the early research behind the discovery of chemical modulators of ALDH. The pharmacophores of these chemical modulators are very diverse in their structural architectures (Fig. 1) and selected compounds are described below – for a recent
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comprehensive review, see [92]. Although these chemical modulators are not very ‘drug-like’, there are some encouraging observations with regard to their ALDH specificity. Early research in this field revealed that genetic polymorphism of ALDH2 leads to accumulation of acetaldehyde (3, Fig. 1) after ethanol consumption, which is known to result in the development of unpleasant physiological effects including facial flushing, nausea and tachycardia [92]. The extension of the aliphatic linker of 3 to provide a range of compounds 4 indicated that some differences in catalytic activity and selectivity could be obtained between human and yeast ALDH1 and ALDH2 isozymes [93,94]. The oral hypoglycaemic agent chlorpropamide (5) has been shown to possess an inhibitory effect that is selective for ALDH2 but not ALDH1A1 or 3A1 [95]. Disulfiram (6) is used as an alcohol-aversive agent in the treatment of alcoholism [92]. After cellular uptake it undergoes complex metabolism to produce metabolites that preferentially inhibit ALDH1A1 over ALDH2 [96]. The monoamine oxidase inhibitor pargyline (7) is activated by CYP2E1 to yield a highly reactive propiolaldehyde that irreversibly inactivates ALDH2 [97]. The isoflavones daidzin (8) and CVT-10216 (9) have been shown to be potent inhibitors of ALDH2 but not ALDH1A1 [98], whereas gossypol (10), a terpenoid aldehyde, acts as a noncompetitive inhibitor and shows more selectivity for ALDH3 than ALDH1 and ALDH2 families [99]. It has been suggested that gossypol could interact with the cofactor binding site, which might underlie the commonality of inhibition of dehydrogenases [99]. The chemical modulators used as inhibitors or pharmacological tools to explore ALDHs have, generally speaking, been discovered by serendipity. Essentially only four ALDH isozymes: ALDH1A1, ALDH1A2, ALDH2 and ALDH3A1, have been studied for their potential as pharmacologically relevant therapeutic targets. It is apparent from the few reports available on chemical modulators, however, that some selectivity can be obtained, but no information is available as to the selectivity across all 19 human isozymes. Nevertheless, recent focused studies on the discovery of chemical modulators of ALDHs have resulted in broad-spectrum and specific inhibitors with in vitro activity. Hurley and co-workers have synthesised small molecules that utilise a cysteine residue within the active site of ALDH1A1, 2 and 3A1 to catalyse the production of a vinyl-ketone intermediate through the formation of a covalent adduct within the active site [100]. Interestingly, efforts from the same group have resulted in encouraging results that indicate that selectivity can be gained from focused studies using computational modelling and in vitro screening assays [101]. Synthetic modifications of an indole-2,3-dione pharmacophore (e.g. 10–12) has resulted in selective targeting of ALDH1A1, 2 and 3A1, respectively (Fig. 1). Other reports [71,102,103] by Hurley’s group include investigations aimed at selective inhibition of ALDH3A1, which could increase chemosensitivity towards cyclophosphamide in ALDH3A1-expressing tumours. A benzimidazole-based analogue 14 was shown to target ALDH3A1 (IC50 = 0.2 mM) but not ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1 or ALDH2 activity [71], whereas a relatively potent benzothienopyrimidin-4-onebased inhibitor 15 was selective for ALDH1A1 (Ki = 300 nM) with in vitro activity against ovarian cancer spheroids [104]. This selective inhibitor was identified from a high-throughput screen of 64,000 compounds performed to identify activators and inhibitors of ALDH1A1.
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Somewhat unexpectedly, there has been an interesting development in the duocarmycin class of natural products. The duocarmycins are ultra-potent molecules that derive their biological activity from a characteristic sequence-selective alkylation of adenine N3 in the DNA minor groove initiating a cascade of cellular events ultimately leading to apoptosis [105]. Four duocarmycin analogues have progressed into clinical Phase I and II trials, but their haematological toxicity led to severe side-effects and termination of treatment [105]. Nevertheless, owing to the intense potency, unique mechanism-of-action and broad range of activity of the duocarmycins, extensive efforts have been made during the past two decades to find analogues that retain their potency and antitumour activity with potential for clinical progression [105]. Tietze and co-workers have recently demonstrated that alkynylated duocarmycin analogues (e.g. 16) with an altered DNA recognition motif have the potential to target ALDH1A1 as identified using an activity-based protein profiling (ABPP) approach [106,107], a finding that has caused some debate over the true cellular target (DNA versus ALDH1A1) [108,109]. Nevertheless, proteomic analysis supports a duocarmycin metabolite to be covalently linked to a cysteine residue within the active site of the enzyme (17) whereas other analogues completely lacking the DNA recognition motif (e.g. 18) exhibited remarkably low nM cytotoxicity, evidently from the ability primarily to target ALDH1A1; this activity was similar to results achieved using ALDH1A1 siRNA knockdown [107]. Although these molecules are at an early stage of investigation, an opportunity now exists for developing a platform for progression of such molecules to an in vivo stage.
Challenges and opportunities in targeting ALDHs A difficult problem to overcome in cancer treatment is to develop tumour-specific chemotherapeutic agents that do not expose normal tissue to the effects of most clinically used cytotoxic and molecularly targeted agents. This lack of selectivity means that physicians must give suboptimal doses leading to inefficient treatment of the tumour, with high risk of recurrence of drug-resistant secondary tumours or metastases. New directions therefore ought to include agents that directly eradicate CSCs [110] or indirectly by employing strategies that involve agents that can stimulate CSCs to differentiate [12,84–86,111,112]. In this context, the lack of therapeutics that currently can eradicate CSCs is a real problem that needs urgent attention. In view of the high expression of certain ALDH isozymes in CSCs, an opportunity exists to target such cells with ALDH-affinic small molecules. Historically, Q3 ALDH1A1 has been the key ALDH isozyme linked with stem cell populations. However, others are now emerging as potential contributors to ALDH functional activity in CSC populations derived from various cancer tissues. ALDH1A3, in particular, appears to be highly expressed in CSCs of the breast, gallbladder, melanoma and prostate. Understanding the role each of the subfamily 1 isozymes has in cell differentiation, tumour grade and metastasis is essential to unravelling the potential of using these as biomarkers and therapeutic targets for drug development. A recent study revealed the potential in targeting ALDH1A3 present in mesenchymal glioma stem cells (GSCs) but not in proneural (PN) GSCs [34]. ALDH1A3 expression was correlated with high ALDH activity and inhibition of this isozyme in vitro using DEAB (2) attenuated the growth of the more aggressive mesenchymal but not PN GSCs. On
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this basis, it can be postulated that inhibition of ALDH1A3-mediated pathways with a more pharmacologically robust agent than DEAB could provide a clinical candidate to a subset of patients with mesenchymal GSCs. In humans, ALDH7A1 is known to play an important part during lysine catabolism through the NAD-dependent oxidative conversion of aminoadipate semialdehyde (AASA) to its corresponding carboxylic acid: a-aminoadipic acid [113], while it also protects against hyperosmotic stress by generating osmolytes and metabolising toxic aldehydes [114,115]. Mutations in the ALDH7A1 gene can cause pyridoxine-dependent and folic-acidresponsive seizures [116]. ALDH7A1 expression is associated with recurrence in patients with surgically resected non-small-cell lung carcinoma [117] and it has been shown to be highly expressed in prostate cancer [24,83]. Interestingly, knockdown of ALDH7A1 expression has been shown to: (i) inhibit the clonogenic and migratory ability of human prostate cancer cells in vitro; (ii) decrease a2hi/avhi/CD44+ stem/progenitor cell subpopulations in the human prostate cancer cell line PC-3M-Pro4 and (iii) decrease intra-bone growth and inhibit experimentally induced (bone) metastasis [24,83]. In spite of this evidence, it remains a significant challenge to develop an agent that can target ALDH7A1 selectively in prostate cancer without affecting this enzyme expressed in normal tissue. The targeting of ALDHs therefore requires careful attention to be paid to the delivery strategy, otherwise off-target toxicities are to be expected. As outlined in this review, there have been relatively few insights into the synthetic and mechanistic aspects of ALDH inhibitors and, to the best of our knowledge, only one ALDH inhibitor has been tested in vivo. This agent, ‘compound 673’ (no structure was reported) was shown to be nontoxic to normal stem cells whereas, interestingly, it was shown to deplete ovarian cancer cells expressing CSC markers [118]. Furthermore, pre-treatment of tumour cells with 673 significantly reduced tumour initiation and growth rates and the compound was highly
synergistic with cisplatin in vitro and in vivo as assessed by cell growth curves and tumour growth, respectively. Although further studies are clearly required to evaluate this agent, it is tempting to speculate that its therapeutic efficacy comes from targeting the CSC component. The recent results reported by Hurley, Matei and co-workers [104] support the use of an ALDH1A1-specific inhibitor (15) to block ovarian cancer cell proliferation and survival under 3D culture conditions and in combination with cisplatin to achieve a synergistic effect and enhanced cell kill.
Concluding remarks and future directions The presence of specific ALDH isoforms in CSCs and tumourigenesis has started to generate interest in the potential to develop specific inhibitors with clinical potential that can be used to eradicate the CSC component of highly heterogeneous and aggressive tumours. So far, no proven specific inhibitors of any ALDH isozyme beyond the in vitro stage exist. Nevertheless, because most of the current drugs we use for the treatment of cancer are not specific or targeted and because there could be more than one ALDH isozyme expressed in CSCs, then the use of relatively nonspecific inhibitors of ALDH activity, alone or in combination with old drugs, could be justified or even preferred (Fig. 2). Thus, clinical studies might need to proceed testing existing potential inhibitors in the usual established preclinical and clinical trials with the hope that side-effects will be tolerable and give a therapeutic window of opportunity. Although recent progress in smallmolecule-based discovery is encouraging, the use of siRNA [70] might be the most specific inhibitor for any one ALDH isozyme if it can be developed into a clinically applicable targeted gene therapy approach. Currently, several investigators are investing efforts in identifying specific ALDH isozyme inhibitors with potential clinical applications that go beyond just treating cancer, including alcoholism, male contraception and cardiac ischaemia. We predict that, over the next five-to-ten years, there will be serious attempts to arrive at small molecules that can be used to
Is ALDH isozyme expression in CSCs significantly elevated to enable preferential targeting and therapeutic intervention? Benign
CSC
Level of ALDH isozyme expression?
MDR
CSC CSC
I. Standard chemotherapy Untreated CSCs enable recurrence of tumour
II. CSC-targeted therapy
CSC
I+II Tumour loses ability to generate new cells and degenerates 100% tumour eradication? Drug Discovery Today
FIGURE 2
cancer stem cell (CSC)-targeted therapy including aldehyde dehydrogenase (ALDH)-targeting inhibitors in combination with standard chemotherapy could result in a more efficient treatment of heterogeneic tumours, with possible durable remissions and cures. 8
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on the utility in designing inhibitors, either as single- or multitargeting ALDH inhibitors, in the first instance as tools to interrogate ALDH pathways and in the longer term as drug candidates with clinical potential to eradicate the CSC fraction of different cancer types.
Acknowledgement Dr Robert Falconer is thanked for valuable suggestions and proofreading of this manuscript.
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target ALDHbr CSCs, but the question remains as to whether this targeting can be achieved in a truly selective fashion with no toxicities to normal tissues. Full characterisation of the heterogeneity of tumours and their CSC component will be crucial for successful design of strategies involving therapeutics and biologics that are targeted against ALDH isozymes. Simultaneously, finetuning drug delivery strategies to target ALDHbr CSCs are also likely to be required to improve on the clinical utility of such agents. We hope that this review can stimulate thought and debate
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Drug Discovery Today Volume 00, Number 00 October 2014