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Aldehyde dehydrogenase: Its role as a cancer stem cell


marker comes down to the specific isoform
Paola Marcato, Cheryl A. Dean, Carman A. Giacomantonio & Patrick W.K. Lee
Published online: 01 May 2011.

To cite this article: Paola Marcato, Cheryl A. Dean, Carman A. Giacomantonio & Patrick W.K. Lee (2011) Aldehyde
dehydrogenase: Its role as a cancer stem cell marker comes down to the specific isoform, Cell Cycle, 10:9, 1378-1384, DOI:
10.4161/cc.10.9.15486
To link to this article: http://dx.doi.org/10.4161/cc.10.9.15486

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review

Cell Cycle 10:9, 1378-1384; May 1, 2011; 2011 Landes Bioscience

Aldehyde dehydrogenase

Its role as a cancer stem cell marker comes down


to the specific isoform
Paola Marcato,1 Cheryl A. Dean,1 Carman A. Giacomantonio2 and Patrick W.K. Lee1,3,*
Departments of Microbiology and Immunology, 2Surgery and 3Pathology; Faculty of Medicine; Dalhousie University; Halifax, NS Canada

Key words: cancer stem cells, aldehyde dehydrogenase, aldefluor assay, isoforms, prognosis

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Abbreviations: ALDH, aldehyde dehydrogenase; CSCs, cancer stem cells; EMT, epithelial mescenchymal transition; TICs, tumor
initiating cells; AML, acute myeloid leukemia; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid x receptor; RAREs,
retinoic acid response elements; DEAB, diethylaminobenzaldehyde; ATRA, all-trans-retinoic acid

Recent evidence suggests that enhanced aldehyde


dehydrogenase (ALDH) activity is a hallmark of cancer stem
cells (CSC) measurable by the aldefluor assay. ALDH1A1, one of
19 ALDH isoforms expressed in humans, was generally believed
to be responsible for the ALDH activity of CSCs. More recently,
experiments with murine hematopoietic stem cells, murine
progenitor pancreatic cells and human breast CSCs indicate
that other ALDH isoforms, particularly ALDH1A3, significantly
contribute to aldefluor positivity, which may be tissue and
cancer specific. Therefore, potential prognostic application
involving the use of CSC prevalence in tumor tissue to predict
patient outcome requires the identification and quantification
of specific ALDH isoforms. Herein we review the suggested
roles of ALDH in CSC biology and the immunohistological
studies testing the potential application of ALDH isoforms as
novel cancer prognostic indicators.

cancer stem cells (CSCs) or tumor initiating cells (TICs) and


their presence has now been identified in most cancers.
CSCs are defined by two key characteristics, enhanced tumorigenicity and the capacity for self-renewal/differentiation.6-8 Thus
the isolated CSC population will not only give rise to de novo
tumors in high efficiency, but also recapitulate the tumor with both
CSC and non-CSC populations. Likely due to the feasibility of isolating viable cells for experimentation, CSCs are most commonly
identified by expression of cell surface molecules. These cell surface markers vary depending on the cancer, but in many instances
are the same markers employed to identify the normal stem cells of
the tissue (e.g., CD34 + CD38- is used for isolation of leukemia and
hematopoietic stem cells7,9). Importantly, not all tumor cells identified by certain markers are CSCs. For example, the CD34 + CD38leukemia cell population is only enriched for the CSCs and there
are many CD34 + CD38- cells that are not CSCs. The CSC enrichment achieved using specific markers was highlighted recently by
Ishizawa et al. who reported that TIC frequency that ranged from
1 in 2,500 to 1 in 36,000 in various cancers (pancreatic, lung
and hand and neck cancer),10 jumped to 1/100 to 1/500 in cells
isolated using certain CSC markers (a 10- to 100fold enrichment). Therefore, it seems reasonable to propose that identification of other novel markers will lead to the further refinement
of the CSC population, which is increasingly tumorigenic.
In addition to cell surface marker expression, events based on
the functional characteristics of stem cells are being employed
for CSC isolation. These include the exclusion of vital dyes like
Hoechst stain,11 which is due to increased expression of adenosine triphosphate-binding cassette proteins (ABC transporters)
in stem cells and CSCs, resulting in low-stained population of
cells that can be isolated. A more recent approach uses retention
of a lipophillic fluorescent dye, PKH26,12 which stains quiescent cells, allowing for the isolation of non-dividing cells from a
mixed population of proliferating cells.13 Finally, the focus of this
review, increased aldehydes dehydrogenase (ALDH) activity as
measured by the aldefluor assay, originally used for the isolation
of hematopoietic stem cells, is now commonly used for the isolation of CSCs of many cancers.14,15

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Introduction: Cancer Stem Cells


Metastasis, not the initial tumor formation, is the primary cause
of cancer mortality. As a consequence, much of current cancer
research is focused on deciphering the metastatic process, in
the hopse that increased understanding will lead to more accurate prognoses and effective treatments. Many factors influence
the ability of a cancer to metastasize. These include the tumor
microenvironment and the interplay of surrounding cells and
chemokines, the vascularization of the tumor, tumor immune
evasion strategies and epithelial mescenchymal transition (EMT,
the transformation of the cancer cells from an adhesive to motile
phenotype).1-5 Further, in recent years, it is becoming increasingly evident that a particular sub-population of tumor cells
plays a potentially critical role. These cells are commonly called

*Correspondence to: Patrick W.K. Lee; Email: patrick.lee@dal.ca


Submitted: 03/11/11; Accepted: 03/15/11
DOI: 10.4161/cc.10.9.15486

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REVIEW

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review

Figure 1. The role of ALDH in retinoic acid signaling. Retinol (vitamin


A) is taken up by cells and is oxidized by retinol dehydrogenases to
yield product retinal. Retinal is oxidized by cytoplamic ALDH enzymes
resulting in retinoic acid. Retinal is the preferred substrate of ALDH
isoforms ALDH1A1, ALDH1A2, ALDH1A3 and ALDH8A1 (and ALDH1A7 in
rodents). Retinoic acid diffuses into the nucleus and initiates transcription of genes with RAREs via activation of heterodimers of RAR and
RXR. Depending on the cellular context, this can lead to differentiation,
apoptosis and/or cell cycle arrest. Addition of DEAB, a specific ALDH
inhibitor blocks retinoic acid production and signaling.

responsible for oxidizing aldehydes to carboxylic acids.37,38


Aldehydes are generated by a wide variety of metabolic processes
and most commonly arise from the oxidative degradation of
membrane lipids (lipid perioxidation), but can also arise from
amino acid, carbohydrate and neurotransmitter catabolism.
While many aldehydes play a critical role in physiological processes like vision, neurotransmission and embryonic development, most are cytotoxic and need to be detoxified, as reviewed
by Marchitti et al.37
In addition to their important function in aldehyde detoxification, ALDHs perform other functions including ester hydrolysis,
serving as binding proteins for various molecules (e.g., androgen and cholestorol) and potentially function as antioxidants
by NAD(P)H production, ultra violet light absorption and/or
hydroxyl radical scavenging.37-40 Finally, a few of the isoforms
(ALDH1A1, ALDH1A2, ALDH1A3 and ALDH8A1) function
in retinoic acid (RA) cell signaling via RA production by oxidation of all-trans-retinal and 9-cis-retinal.38,41-43 This function in
particular has been linked to the stemness characteristics of
CSCs.44 Therefore, as discussed below, increasing evidence indicates that ALDH may be more than just a CSC marker and have
a potential functional role in CSC biology.
Retinoic Acid-Mediated Cell Signaling
Induced by ALDH

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e.and thereby modulates a wide
RAB
induces
gene
transcription
variety of biological processes like cell proliferation, differentiao
no
di
s
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r
i
b
ut
e
.and apoptosis. As generated by ALDHs,
Using the aldefluor assay, Cheung et al. wereD
the
first
to t
show
tion,
cell
cycle
arrest
ALDH Activity as a Universal CSC Marker

45-49

that it is possible to isolate the leukemia stem cells based on the


increased ALDH activity.16 The researchers detected a population of ALDH+ acute myeloid leukemia (AML) cells in 14 of
43 patient samples. The ALDH+ AML cells in most cases coexpressed CD34 + (previously identified leukemia stem cell
marker) and engrafted significantly better than the ALDH- AML
cells in immunocompromised mice. The same year, Ginestier et
al. (2007) showed it was possible to isolate ALDH+ cells from
breast cancers that had the tumorigenic and self-renewal properties of CSCs.14 Their groundbreaking work showed the potential
applicability of quantifying ALDH activity in solid tumors. In
the following years, ALDH activity would be used successfully
as a CSC marker for many cancers including lung, liver, bone,
colon, pancreatic, prostate, head and neck, bladder, thyroid,
brain, melanoma and cervical.17-34 With the exception of one
recent study for melanoma,35 increasing evidence suggests that
ALDH activity is a universal CSC marker. However, as discussed
below, the cause of ALDH activity as measured by the aldefluor
assay in the various tissues and cancers may differ. Importantly,
identification of specific ALDH isoforms prevalent in certain
cancers may have critical prognostic applicability.36
Function of Aldehyde Dehydrogenases
The ALDH enzymes are a family of evolutionarily conserved
enzymes comprised of 19 isoforms that are localized in the
cytoplasm, mitochondria or nucleus. Generally, ALDHs are

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the lipophilic RA can function in paracrine or endocrine manner by diffusing into neighbouring cells or the nucleus (Fig. 1).
Once inside the nucleus, RA binds to heterodimers of retinoic
acid receptor (RAR) and retinoid x receptor (RXR). Activated
receptor complexes then bind to retinoic acid response elements
(RAREs), which are regulatory sequences in target genes and
induce transcription. Lists of known RA target genes, including
the HOX and RAR genes, have been compiled.50,51
Retinoic acid production by ALDH can induce differentiation of hematopoietic and neural stem cells.52 Conversely, inhibition of ALDH by diethylaminobenzaldehyde (DEAB) expands
hematopoietic stem cells.53 Based on the ability of retinoic acid
to induce differentiation, all-trans-retinoic acid (ATRA) is used
to treat acute promyelocytic leukemia (APL).54-56 ATRA treatment of APL patients induces differentiation of immature leukemia blasts into terminally differentiated granulocytes, leading
to a clinical remission in approximately 90% of patients. Since
the successful application of ATRA in the treatment of APL,
its effects are being studied in other cancers. In general, ATRA
appears to induce apoptosis and differentiation in a number of
cancers.57-60 Ginestier et al. (2009) recently showed that addition
of ATRA to cultured breast cancer cells induced their differentiation in vitro (evidenced by reduced tumorsphere formation
frequency).44 Conversely, DEAB addition increased their tumorsphere formation frequency. Based on their results, the authors
suggested that ATRA could be a novel breast cancer therapeutic,
by inducing the differentiation of breast CSCs.

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ALDH and Chemotherapeutic Resistance of CSCs


CSCs, like normal stem cells, are relatively resistant to radiation treatment or commonly used chemotherapeutics like gemcitabine, temozolomide, carboplatin, etoposide, fluorouracil,
paclitaxel, daunorubicin and mitoxantrone and cyclophosphamide.11,61-65 It is believed that the resistance of CSCs to currently
used chemotherapeutics is a major contributing factor in cancer
recurrence and later metastasis development.
There are several suggested mechanisms for the apparent resistance of CSCs to current anti-cancer therapies. First, CSCs are
similar to slow-growing normal stem cells, which are generally in
the quiescent state and therefore resistant to drugs designed to
target fast-growing cancer cells.8 Second, it has been reported that
resistance of glioblastoma CSCs to irradiation is due to increased
activation of the DNA damage checkpoint response.66 Third, the
observation that CSCs can be isolated by Hoechst stain exclusion
is likely due to enhanced expression of transporters that efflux the
stain.67 These transporters also efflux chemotherapeutic drugs, a
common cause of chemotherapeutic resistance seen in cancer.68
Finally, with the isolation of CSCs based on increased ALDH
activity it is possible that resistance to chemotherapeutic agents is
a result of aldehyde dehydrogenase-specific activity that metabolizes these drugs. For example, in hematopoietic stem cells,
ALDH1A1 in particular and ALDH3A1 to a certain degree,
are known to metabolize and detoxify chemotherapeutic agents
such as cyclophosphamide.69 In a retrospective analysis of breast
cancer patients samples, ALDH1A1 expression was found to be
predictive of tumor responsiveness to cyclophosphamide treatment.70 In support of this potential role for ALDH in CSC resistance to chemotherapeutics, CSC enrichment was observed in
colorectal cancer xenograft tumors after cyclophosphamide treatment, and this correlated with enhanced ALDH1A1 expression
and enzymatic activity.65

reduce aldefluor activity of hematopoietic stem cells, suggesting


that additional factors are contributing to the aldefluor activity
of the cells. Of note, the authors detected expression of ALDH2,
ALDH3A1 and ALDH9A1 in the ALDH1A1-deficient hematopoietic cells suggesting that one or more of these isoforms
are responsible for the aldefluor activity. In another example,
Rovira et al. characterized the aldefluor-positive murine centroacinar and terminal ductal progenitor pancreas cells and found
high levels of ALDH1A1 and ALDH1A7 in these cells (an
ALDH isoform that is absent in humans but present in rodents
and also has retinal oxidation activity).72 Van den Hoogen et al.
used the aldefluor assay to identify prostate CSCs, but reportedly found higher expression of other ALDH isoforms and relatively low expression of ALDH1A1 in prostate cancer cells and
tissues.29 Instead, high expression of ALDH7A1 was found in
prostate cancer cells lines, prostate cancer tissue and matched
bone metastasis samples, suggesting that for prostate cancer,
ALDH7A1 may be contributing to the aldefluor activity of these
cells. Another group reported elevated expression of ALDH3B1
in a high percentage of human tumors (lung, breast, ovarian
and colon).73 Most recently, Chen et al. compared the expression levels of ALDH1A1 and ALDH1B1 in adenocarcinomas
(breast, lung, ovarian and colon cancer) by immunohistochemistry and reported a 5.6-fold higher expression of ALDH1B1
compared to ALDH1A1 in the cancer tissue.74 Notably, 98%
of colon cancer samples were positive for ALDH1B1, leading
the authors to suggest that ALDH1B1 may be the cause of aldefluor activity in colon cancer. It is important to note that while
the above studies show elevated expression of ALDH isoforms
other than ALDH1A1 in cancers, they do not directly prove
that these identified ALDH isoforms are the cause of aldefluor
activity in cancers.
Our recent study with breast cancer patient tumor samples isolated for aldefluor+ and aldefluor- tumor cells shows that at least for
breast cancer, ALDH1A1 expression is not the primary determinant of aldefluor (ALDH) activity.36 Instead we found better correlation with ALDH1A3 (and ALDH2, ALDH4A1, ALDH5A1,
ALDH6A1, ALDH7A1 to a lesser degree). Expression quantification at the mRNA level of all 19 ALDH isoforms in breast cancer
cell lines revealed that ALDH1A3 expression correlated best with
ALDH activity of the cell lines. Only knockdown of ALDH1A3
reduced ALDH activity in all three aldefluor positive breast cancer lines. However, there is indication that other ALDH isoforms
including ALDH1A1 have the potential to promote aldefluor
activity in breast cancer cells if expressed in sufficient levels. For
example, knockdown of ALDH2, which is highly expressed in
aldefluor positive SKBR3 cell line, partially reduced the aldefluor
activity of the cells.
Based on studies such as those mentioned above, it is becoming increasingly clear that the ALDH isoform(s) responsible
for aldefluor activity likely vary depending on cancer type and
tissue/cell of origin. For example ALDH1A1 is predominantly
expressed in the epithelium of testis, brain, eye, liver, kidney and
neural and hematopoietic stem cells,37,75-78 whereas ALDH1A3
is reportedly expressed in the kidneys, salivary glands, stomach,
fetal nasal mucosa and breast.37,42,79,80 An earlier report describes

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Correlating Specific ALDH Isoforms


with Aldefluor Activity in Stem Cells and CSCs
The aldefluor assay quantifies ALDH activity by measuring
the conversion of ALDH substrate, BODIPY aminoacetaldehyde to fluorescent reaction product BODIPY aminoacetate.
Addition of inhibitor DEAB reduces fluorescence, confirming
that ALDH+ cells are correctly identified. This assay was developed by successful isolation of viable hematopoietic stem cells
from human umbilical cord blood15 and was reported to be specific to the ALDH isoform found in high abundance in those
cells, ALDH1A1. However, while individual ALDH isoforms
do have some preferred substrate-specificity they also have crossreactivity, making it likely that the aldefluor assay is detecting
the ALDH activity of one or more of the other ALDH isoforms
expressed in the cells.37
A few recent studies provided some evidence that the ALDH
activity as measured by aldefluor assay is not necessarily due to
ALDH1A1 alone. Levi et al. in 2008, reported that ALDH1A1
expression was not required for hematopoietic and neural stem
cell function.71 Importantly, ALDH1A1 deficiency did not

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Table 1. Summary of Immunohistological studies detecting ALDH prevalence in cancers


ALDH isoform

Cancer

Patient sample size

Prognostic correlation

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Worse outcome/
More aggressive
cancer
*ALDH

Leukemia

ALDH1A1

Bladder

ALDH1A1

Breast

65

No correlation

Publication

Improved outcome/
Less aggressive
cancer

94

216

31

577

14

ALDH1A1

Breast

203

95

ALDH1A1

Breast

381

96

ALDH1A1

Breast

109

ALDH1A1

Breast

639

98

ALDH1A1

Breast

47

36

ALDH1A1

Colon

1420

99

ALDH1A1

Lung

60

ALDH1A1

Ovarian

442

ALDH1A1

Ovarian

439

22

ALDH1A1

Pancreatic

269

27

ALDH1A1

Prostate

40

ALDH1A1

Prostate

163

25

ALDH1A3

Breast

47

36

ALDH2

Breast

47

36

ALDH4A1

Breast

ALDH7A1

Prostate

97

24
X

100

29

47

36

40

29

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*ALDH activity detected using aldefluor assay.

the ALDH1A3 isoform expressed in normal breast epithelia42 as


being responsible for RA synthesis in these cells; it is perhaps
not surprising that we found ALDH1A3 expression to be the
predominant determinant of aldefluor activity in breast cancer.36
Our findings suggest that the tissue specificity of the ALDH isoforms may determine their expression in cancers as well.36
Expression of ALDH Isoforms
as Prognostic Indicators in Cancer
Identifying CSCs in patient tumors and quantifying their prevalence could be used to determine the relative aggressiveness of
a cancer and therefore CSCs are potentially prognostic indicators for cancer patients.8 The most common approach is through
the use of putative CSC cell surface markers (such as CD44 +/
CD24-, CD133 + etc.), which has met with varied success.36,81-93
In the last few years ALDH+ is being evaluated as a potential
novel cancer prognostic marker. While the use of the aldefluor
assay to identify cells with ALDH activity works well in the laboratory setting, it is not practical for routine clinical histopathological diagnosis. Cancer prognostics in the clinic are typically
performed using immunohistochemistry of fixed tumor tissue to
detect expression levels of a protein. As such, evaluating ALDH+
for prognostic application requires detection of protein expression at the isoform level (not activity), and, as discussed above,
the ALDH isoform(s) responsible for aldefluor activity is likely
cancer-specific.

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Most ALDH prognostic data thus far is based on ALDH1A1


expression levels as an indicator for cancer patient outcome (Table
1). This has yielded varied results and positive correlation with
ALDH1A1 as a prognostic marker may be dependant on the cancer, although assessing ALDH1A1 expression in combination with
other markers appears to improve the potential prognostic application (Table 2). More recently, investigators are studying the prognostic potential of other ALDH isoforms in various cancers (Table
1). Some of these studies compare the expression levels of multiple
isoforms within the sample set, illustrating that depending on the
isoform that is probed, varying correlations with patient outcomes
are achieved. For example, in our immunohistological analysis of
a panel of breast cancer patient samples, tumor grade and metastasis correlated best with ALDH1A3 levels, followed by ALDH6A1
and ALDH2, but not significantly with ALDH1A1 or ALDH4A1
levels.36 Sine it is highly likely that the ALDH activity detected in
a cancer is due to the combined activity of two or more ALDH
isoforms, the feasibility of using combination probes (for example,
ALDH1A3, ALDH6A1 and ALDH2 combined for breast cancer)
as clinical prognostic application should also be considered.
Concluding Remarks
The existence of CSCs or TICs has become a generally accepted
concept. However, for the full implications of CSCs in cancer
development, progression, prognosis and treatment to be discerned, their accurate identification needs to be achieved. While

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Table 2. Summary of immunohistological studies detecting ALDH+ cells in combination with other CSC markers

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Prognostic correlation
Cancer

ALDH isoform combined


with other markers

Patient
s ample size

Worse outcome/
More aggressive
cancer

Breast

ALDH1A1+CD44+ cytokeratin+

639

98

Breast

ALDH1A1 CD44

47

36

Breast

ALDH1A3+CD44+

47

36

Breast

ALDH2 CD44

47

36

Breast

ALDH6A1+CD44+

47

36

the use of cell surface markers for CSC identification has met
with varying degrees of success, intracellular ALDH activity is
emerging as an important and reliable universal CSC hallmark
applicable for most cancers. However, while measuring ALDH
activity may be an accepted method for the separation of CSC
and non-CSC populations of may cancers, at the protein level,
the ALDH isoform(s) responsible for ALDH activity is likely different and cancer-specific. Precise identification of active ALDH
isoforms for specific cancers would have major diagnostic and
prognostic implications.
Another unresolved puzzle pertains to the precise role(s) of
ALDH in CSCs. In view of the diversity of ALDHs various functions, particularly that of cellular differentiation control, it seems
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ALDH activity can be due to varying isoforms, identifying the
isoform(s) responsible for this activity in a cancer should be a first
step towards this goal.
Acknowledgments

This work is funded by a grant from the Canadian Breast


Cancer Foundation, Atlantic Chapter to Patrick Lee, Carman
Giacomantonio and Paola Marcato.

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