Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis.
ABSTRACT Human breast cancers are heterogeneous, both in their pathology and in their molecular profiles. This suggests the hypothesis that breast cancers can initiate in different cell types, either breast epithelial stem cells or their progeny (transit amplifying cells or committed differentiated cells). In this respect, breast cancer could be viewed as being similar to haematological malignancies for which an analogous model has been proposed. Drawing such parallels might help to unravel the molecular nature of the initiating events in breast cancer and might have substantial clinical implications.
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ABSTRACT: Triple-negative breast cancer (TNBC) has poor prognostic outcome compared with other types of breast cancer. The molecular and cellular mechanisms underlying TNBC pathology are not fully understood. Here, we report that the transcription factor BCL11A is overexpressed in TNBC including basal-like breast cancer (BLBC) and that its genomic locus is amplified in up to 38% of BLBC tumours. Exogenous BCL11A overexpression promotes tumour formation, whereas its knockdown in TNBC cell lines suppresses their tumourigenic potential in xenograft models. In the DMBA-induced tumour model, Bcl11a deletion substantially decreases tumour formation, even in p53-null cells and inactivation of Bcl11a in established tumours causes their regression. At the cellular level, Bcl11a deletion causes a reduction in the number of mammary epithelial stem and progenitor cells. Thus, BCL11A has an important role in TNBC and normal mammary epithelial cells. This study highlights the importance of further investigation of BCL11A in TNBC-targeted therapies.Nature Communications 01/2015; 6:5987. · 10.74 Impact Factor
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ABSTRACT: Glioblastoma multiforme (GBM) is the most common human primary brain malignancy and has a dismal prognosis. Aggressive treatments using maximal surgical resection, radiotherapy, and temozolomide result in median survival of only 14.6 months in patients with GBM. Numerous clinical approaches using small molecule inhibitors have shown disappointing results because of the genetic heterogeneity of GBM. The epithelial to mesenchymal transition (EMT) is a crucial biological process occurring in the early development stages of many species. However, cancer cells often obtain the ability to invade and metastasize through the EMT, which triggers the scattering of cells. The hepatocyte growth factor (HGF)/MET signaling pathway is indicative of the EMT during both embryogenesis and the invasive growth of tumors, because HGF potently induces mesenchymal transition in epithelial-driven cells. Activation of MET signaling or co-overexpression of HGF and MET frequently represents aggressive growth and poor prognosis of various cancers, including GBM. Thus, efforts to treat cancers by inhibiting MET signaling using neutralizing antibodies or small molecule inhibitors have progressed during the last decade. In this review, we discuss HGF/MET signaling in the development of diseases, including cancers, as well as updates on MET inhibition therapy.OncoTargets and Therapy 01/2014; 7:1933-44. · 1.34 Impact Factor
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ABSTRACT: Estrogen receptor positive breast cancers have high recurrence rates despite tamoxifen therapy. Breast cancer stem/progenitor cells (BCSCs) initiate tumors, but expression of estrogen (ER) or progesterone receptors (PR) and response to tamoxifen is unknown. Interleukin-6 (IL-6) and interleukin-8 (IL-8) may influence tumor response to therapy but expression in BCSCs is also unknown.BMC Cancer 09/2014; 14(1):733. · 3.32 Impact Factor
Human breast carcinomas have hetero-
geneous pathologies and molecular
profiles, and in this respect it is plausible
that breast cancer might be more similar
to haematological malignancies than to
other common epithelial cancers. Indeed,
unlike colon cancer or pancreatic cancer,
in which in virtually all tumours mutation
within a single pathway has a dominant
role during tumour progression1-3, in breast
tumours no single dominant pathway or
histological presentation has emerged4.
Characterization of the chromosomal
aberrations, gene mutations and gene
expression profiles of breast tumours has
shown that breast tumorigenesis does not
necessarily progress in a stepwise linear
fashion from well-differentiated to poorly
differentiated tumours5–7. Human breast
tumours have been historically categorized
into approximately 18 subtypes according
to the histological features of the primary
tumour at the time of diagnosis4. Features
used in the classification of breast tumours
include lesion size, cellular arrangement
patterns and the presence of necrosis, as
well as cellular features such as nuclear
grade and mitotic index. Clinical responses
of patients to therapy reveal that although
this method of tumour classification has
prognostic value, considerable heterogene-
ity in response to therapy still exists8. As a
result there has been considerable effort in
the breast cancer research community to
identify biomarkers that more accurately
predict patient outcome (see Brenton et al.
for a review9).
The classification of human breast
tumours on the basis of histological
criteria is confounded by a number of
factors. These include scoring subjectivity,
regions of distinct morphologies within a
given tumour and a general inexactitude
in the classification of breast tumours.
This is exemplified by the observation
that 60–70% of all breast tumours are
ultimately classified as invasive ductal
carcinoma not otherwise specified (IDC
NOS), also designated breast carcinoma of
no special type (breast carcinoma NST).
Another difficulty in the categorization
and understanding of the origins of breast
cancer has been the bewildering number
of genes that are associated with the
disease10 — it is uncommon for different
tumours to share mutations in the same
gene (with the exception of TP53 and
PIK3CA (phosphatidylinositol 3-kinase,
catalytic, α-polypeptide)). Also, there
is a lack of understanding of the target
cells involved. Unlike the haematopoietic
system, the cellular hierarchy that is
present in the mammary epithelium is still
only partially understood, and so it has
been difficult to relate a given mutation
or cancer cell property to a specific type
of breast epithelial cell. This situation is
exemplified by the fact that breast cancer
cell lines are commonly used as a model in
breast cancer research, yet until recently
there has been little regard as to the type of
breast cell (for example, luminal or basal)
Recent gene expression profiling by
microarray analysis has offered a new
way to classify human breast tumours11–14.
Classifying them according to the levels of
mRNA expression of specific genes typi-
cally identifies at least five reproducible
subtypes: luminal A, luminal B, ERBB2,
basal and normal-like. Correlating this
type of classification system with the tradi-
tional method based on tumour histology
has revealed that some tumours that are
classified according to their morphology
correlate with a particular gene expres-
sion subset, whereas others do not11,15,16.
This new approach of categorizing breast
tumours represents a paradigm shift in
how we consider the origins and catego-
rization of breast cancer. Indeed, gene
expression patterns of luminal and basal
cells in vivo were used to establish the defi-
nition of the original molecular portraits
of breast cancers11,17. However, in order
to make sense of this new classification
system it is important to understand the
cellular hierarchy that is present in the nor-
mal mammary epithelium. For example,
some human breast tumours are classified
as being luminal A and others luminal B,
but is there a normal cellular counterpart
of a luminal A or a luminal B cell, and if so,
what is it?
Mammary epithelial cell hierarchy
The human mammary gland is a com-
pound tubulo-alveolar gland that consists
of two general lineages of epithelial cells:
luminal cells and myoepithelial cells
(FIG. 1a). Electron microscopic studies have
Molecular heterogeneity of breast
carcinomas and the cancer stem
John Stingl and Carlos Caldas
Abstract | Human breast cancers are heterogeneous, both in their pathology
and in their molecular profiles. This suggests the hypothesis that breast
cancers can initiate in different cell types, either breast epithelial stem cells
or their progeny (transit amplifying cells or committed differentiated cells).
In this respect, breast cancer could be viewed as being similar to
haematological malignancies for which an analogous model has been
proposed. Drawing such parallels might help to unravel the molecular nature
of the initiating events in breast cancer and might have substantial clinical
NATURE REvIEwS | cancer?
vOlUmE 7 | OCTOBER 2007 | 791
© 2007 Nature Publishing Group
Nature Reviews | Cancer
Loss of TP53 and BRCA1?
Ductal epithelial cell
Steroid receptor+ cell
Ductal epithelial cell
Steroid receptor+ cell?
long identified heterogeneity within the
mammary epithelium, with a number of
cells having an undifferentiated morpho-
logy. Among these are a small number of
basally positioned small undifferentiated
electron-lucent cells (small light cells) that
may represent a mammary gland stem cell
(maSC). The study of the stem cells in the
human mammary epithelium has been
hampered by the lack of a validated in vivo
xenotransplantation assay that can be used
to detect human mammary repopulating
units (mRUs). However, in vitro colony
assays have been used to detect progenitor
cells within the mammary gland, includ-
ing progenitors that have the ability to
generate progeny of multiple lineages18–20.
These multilineage progenitors, which are
perceived to reside in a suprabasal position
in the ducts of the mammary glands, have
a keratin (K) 19+K14+EpCAmhighCD49f+m
UC1–SSEA-4high phenotype and are thought
to represent maSCs because they generate
multilineage ductal lobular outgrowths
in reconstituted basement membrane
Surrogate stem cell assays that are
based on the ability of cells to generate
clonal ‘mammospheres’ under anchor-
age-independent culture conditions have
also identified cells that appear to have
properties of maSCs (multilineage dif-
ferentiation potential and self-renewal)22.
These mammospheres are highly enriched
in multilineage progenitors, which suggests
that a subpopulation of these progeni-
tors could be a candidate for the maSC.
Unfortunately, the relatively short time
period over which these various in vitro
approaches can maintain proliferation (~3
weeks) makes it difficult to conclude that
the candidate cells identified are maSCs,
as opposed to multilineage progenitors
that are at an intermediate position in the
developmental hierarchy. Recent advances
in xenotransplant systems offer the means
to more definitively identify a human
maSC candidate that is defined by its ability
to generate clonal multilineage outgrowths
over extended periods of time in primary
and secondary in vivo assays23–25.
In addition to multilineage progenitors,
two types of luminal-restricted progenitors
and one type of myoepithelial-restricted
progenitor have been identified18–20.
luminal-restricted progenitors are perceived
to reside in the luminal cell compartment of
the ducts as they express the luminal marker
mucin 1 (mUC1)19,20 and can be subdivided
into two categories, those that are K19+K14–
and those that are K19–K14– (REF. 20).
Unfortunately, the respective contributions
of these progenitors during the development
and maintenance of the mammary ductal
lobular epithelium is not well understood.
A non-clonogenic luminal epithelial cell
population can also be identified19. These
cells have an EpCAm+mUC1+CD49f– phe-
notype. myoepithelial cells can be isolated
on the basis of an EpCAmlowmUC1–CD49f+
Figure 1 |?proposed?epithelial?cell?hierarchy?and?the?respective?cellular?phenotypes?present?
in?the?human?and?mouse?mammary?glands.?Arrows indicate the potential target cells of oncogenic
mutations of TP53 and BRCA1 (breast cancer 1) in the human and Wnt and Erbb2 in the mouse.
er, oestrogen receptor; Pr, progesterone receptor.
792 | OCTOBER 2007 | vOlUmE 7
© 2007 Nature Publishing Group
In the mouse, cells that can generate
ductal lobular outgrowths in vivo (mRUs)
have also been isolated and enumerated26,27
(BOX 1). The markers used to identify these
mRUs include a lack of expression of hae-
matopoietic (CD45 (also known as PTPRC)
and Ter119) and endothelial (CD31; also
known as PECAm1) markers; and include
the expression of CD24, low levels of Sca-1
and high levels of α6- and β1-integrins
high expression of CD49f (also known as
ITGA6) and CD29 (also known as ITGB1)
by the mRUs suggests that maSCs have a
basal position within the mammary epithe-
lium. This and other data (BOX 1) indicate
that these mRUs meet the functional
definition of an maSC. The transplantation
of donor cells at limiting dilutions into
cleared fat pads provides an estimate of the
frequency of mRUs among total mammary
cells in young adult nulliparous mice to
be about 1 in 1,400 total cells, which cor-
responds to about 1,400 mRUs per mouse
Progenitor cells, which are defined as
any cell with a proliferative capacity, can
also be detected within the mouse mam-
mary epithelium by the use of in vitro
colony assays19,26–29. Interestingly, a large
pool (>10,000 cells) of luminal-restricted
progenitors within the luminal (CD24high
CD49flowCD29low) cell compartment of the
mouse mammary gland has been identified
and these cells have a phenotype (CD24high
CD49flowCD61+CD14+) that is distinct
from that of the stem cells (REFS 26,29; J.S.
and C. watson, unpublished observations).
A population of progenitors in the basal
(CD24+CD49fhighCD29high) cell compart-
ment of the mouse mammary gland
has also been detected by colony assays;
however, the phenotypic profile that allows
their separation from maSCs has yet to be
determined27. Differentiated myoepithelial
cells in the mammary gland can be puri-
fied on the basis of their CD24lowCD49f+
ER and the mammary epithelium
The expression of the oestrogen recep-
tor (ER) is the single most important
predictive marker in anti-oestrogen-based
therapies for the treatment of breast cancer,
but surprisingly the distribution of the ER
among cells of the mammary epithelial
cell hierarchy is poorly understood. Stem
cells in the mouse mammary gland do not
express ER; instead, ER is expressed by
approximately 40% of luminal epithelial
cells31. Fluorescence-activated cell sorting
of mouse mammary epithelial cells using
CD133, CD24 and Sca-1 has revealed
that the luminal cell compartment can
be subdivided into two lineages: those
cells that are enriched for ER expression
(CD133+Sca1+CD24high) and those that
are enriched for milk protein expression
Interestingly, when assayed for pro-
genitor content, the ER+ cell population
is relatively deficient when compared
with their milk-protein-expressing
counterparts. Considering this, the milk-
protein-expressing progenitor cells may
represent alveolar precursors that are
stimulated to divide during pregnancy.
Another study, using CD61 as a marker
of luminal-restricted progenitors, also
observed the CD61– subpopulation as
enriched for ER-expressing cells29. These
observations, along with the observation
that ER-expressing cells are rarely found
to be dividing in both adult mouse and
human mammary tissue, raises the pos-
sibility that ER+ cells represent a relatively
differentiated cell that has a limited
proliferative capacity32. However, the idea
that ER+ cells are terminally differentiated
cells is difficult to resolve considering the
paradoxical observation that ER+ tumours
contain proliferating ER+ cells that are
responsive to anti-oestrogen therapies
such as tamoxifen33. One explanation for
this paradox is that ER expression and cell
proliferation are not mutually exclusive,
and that cellular intermediates that express
ER and have a limited proliferative capac-
ity exist. Another possibility is that there is
a second type of ER+ cell that functions as
a primitive progenitor in the luminal cell
compartment and is difficult to detect in
quiescent tissues34. A recent report in the
literature suggests that this could be the
case, as a cell that expresses ER and retains
its template DNA strand during cell divi-
sion has been identified35.
Asymmetrical DNA segregation dur-
ing cell division is a property normally
ascribed to stem cells36,37. Although
these rare asymmetrically dividing cells
are unlikely to represent true stem cells
because they express ER (and thus are
deficient in generating ductal lobular
outgrowths in vivo31), they might represent
a pool of primitive progenitors with stem
cell properties (for example, self-renewal)
in the luminal cell compartment. Further
purification and characterization of these
cells and other ER-expressing cells will be
required to fully understand the oestrogen
biology of the mammary epithelium and
to fully understand the role of ER as a
predictive marker in breast cancer.
Interpreting gene expression profiles
Classifying human breast tumours accord-
ing to mRNA expression levels has identi-
fied at least five reproducible subtypes of
breast cancer: luminal A, luminal B, ERBB2,
basal and normal-like. A similar, but not
identical, classification system has also
been defined on the basis of protein expres-
sion using tissue microarray analysis38–41.
Because cancer is essentially organogenesis
gone awry, the cellular categories that are
defined by these gene expression profiles
might just reflect the different lineages
and stages of mammary epithelial cell
differentiation that are present in the
normal environment. Unfortunately, the
corresponding cellular equivalents in the
normal epithelium are not known because
gene expression analysis of subtypes of
luminal and basal progenitor and non-
progenitor cells have not been published.
It would be interesting to determine, for
example, whether luminal A tumours,
which have a much better prognosis and
Box 1 | Identification of mouse mammary stem cells
In 1998 it was demonstrated through serial transplantation of retrovirally marked mouse mammary
epithelial cells that a single cell can fulfil the functional definition of a mouse mammary gland stem
cell; that is, a cell that can generate both the ductal and lobular components of the mammary tree
(complete with both luminal and myoepithelial cell lineages) and can self-renew116. More recent
studies using fluorescence-activated cell sorting and transplantation of phenotypically distinct
cell types at limiting dilutions into epithelium-divested (cleared) fat pads of syngeneic recipient
mice has revealed that cells that can generate ductal lobular outgrowths in vivo (mammary
repopulating units or MRUs) can be prospectively isolated and enumerated26,27. The markers used
to identify these MRUs include a lack of expression of haematopoietic (CD45 and Ter119) and
endothelial (CD31) markers and include the expression of CD24, low levels of Sca-1 and high
levels of α6- and β1-integrins. The clonal origin of the epithelial outgrowths has been verified by
transplantation of single, visually confirmed cells into recipient animals26,27, as has the self-renewal
properties of these cells by serial transplantation into cleared fat pads of secondary recipients26,27.
Thus, these MRUs meet the functional definition of a mammary gland stem cell.
NATURE REvIEwS | cancer?
vOlUmE 7 | OCTOBER 2007 | 793
© 2007 Nature Publishing Group
higher levels of ER expression than luminal
B tumours12–14,16,42,43, are composed of
relatively well-differentiated cells of the
ER+ lineage, whereas luminal B tumours
have a larger component of primitive ER+
progenitors. This concept is supported by
the fact that GATA3, a member of the gene
cluster that defines luminal A tumours,
is the key transcription factor that deter-
mines luminal cell differentiation from
luminal-restricted progenitors in both
embryonic and post-natal murine mam-
mary glands29,44. Another interpretation of
the gene expression signatures of tumours
is that these represent novel signatures that
do not have a normal cell equivalent, but
instead are the unique gene expression pat-
tern of transformed cells that have evolved
over decades of tumour progression.
methods of classifying human breast
tumours that are based on gene expres-
sion profiles could theoretically allow
the ontological history of the tumour to
be determined. For example, this gene
expression classification strategy could
mean that each of these types of human
breast tumour originates in one of five
different types of cell and that the tumour
generated is a reflection of that cell type.
Indeed, a precedent for this paradigm
— that the biology of a malignant cell is
indicative of its non-transformed cellular
progenitor — has been established for dif-
fuse, large B-cell lymphomas, with one type
expressing genes that are characteristic of
germinal centre (lymphoid-tissue-residing)
B cells and a second type expressing genes
that are normally induced during in vitro
activation of peripheral B cells isolated
from the blood45. However, it could also
mean that the tumours are initiated in a
rare cell type such as a stem cell, with the
specific combination of mutations driving
the malignant clone down a specific dif-
ferentiation pathway that is reflected in the
Another possible scenario is that the
initial mutation occurs in the stem cell,
but subsequent mutations in downstream
non-stem cell progeny are required for the
acquisition of the malignant phenotype. An
mRNA analysis of a tumour cell population
represents the average mRNA levels that
are found in a heterogeneous population
of cells. Although informative, the ultimate
imperative in terms of obtaining a curative
treatment is identifying and targeting the
tumour stem cells. whether or not each
molecular subtype of human breast cancer
is propagated by a tumour stem cell that has
similar properties between all the subtypes
or if each subtype has its own unique
tumour stem cell remains to be resolved.
In the initial landmark paper by Al-Hajj
and colleagues, the human mammary
tumour-initiating cell, as detected by trans-
plantation into immune-deficient recipient
mice, had an EpCAm+CD44+CD24–/low
phenotype in 8 out of 9 tumour specimens
sampled46. Assuming that the in vivo
engraftment followed single-hit engraft-
ment kinetics and was efficient, the fre-
quency of these tumour-initiating cells was
<1% within the EpCAm+CD44+CD24–/low
subpopulation, which itself is just a
subpopulation of the total tumour cell
population. Therefore, the frequency of
tumour-initiating cells is very low among
the total number of cells in the tumour.
Similar observations have been made for
various human tumour types, including
acute myeloid leukaemia and colon, brain
and pancreatic tumours47–50. Owing to the
low frequency of these tumour-initiating
cells, the gene expression profile of these
cells is likely to be masked when tumours
as a whole are analysed for gene expres-
sion. However, it may be that the detection
of human tumour stem cells by transplan-
tation into immune-deficient mice is
inefficient and that the true frequency of
these tumour stem cells within tumours is
higher than originally perceived51. If this
were the case, this could explain why the
signature derived from breast cancer stem
cells may have prognostic value52, although
this is difficult to reconcile with the sig-
nificant molecular heterogeneity of breast
cancers and the difficulty in separating
prognostic signatures from the ER status
of tumours43. Regardless, it is imperative
that strategies to purify tumour stem cells
from each of the five molecular subtypes
of breast cancer be developed such that
meaningful gene expression profiles of
these cells can be obtained.
Human breast tumour cell hierarchies
Stem and progenitor cells are believed
to be the initial targets for malignant
transformation. This is because most
mutational events rely on DNA replication
and cell division36 in cells that have the
capacity to generate enough target progeny
such that the probability of obtaining
subsequent genetic mutations becomes
likely, and the self-renewal properties of
such cells can be harnessed to propagate
the malignancy. As previously mentioned,
human mammary tumour-initiating cells
that were isolated from patients had an
EpCAm+CD44+CD24–/low phenotype in
most specimens46,53. The lack of expression
of CD24 among these tumour-initiating
cells suggests that their counterparts in
the non-malignant epithelium are basally
positioned cells, as CD24, like mUC1, is
expressed solely by human luminal epithelial
cells17. The EpCAm+CD24–/low phenotype
is similar to that previously described
for bipotent human mammary epithelial
progenitors18,19,21. Similarly, the observation
that human mammary tumour-initiating
cells are possibly derived from a basal
progenitor/stem cell correlates well with
the observation that mouse maSCs are in
the basal compartment26,27. The question
that now arises is whether or not all breast
tumour-initiating cells are derived from
a basal progenitor/stem cell. The limited
study by Al-Hajj et al. is too small to come
to any definitive conclusion regarding this;
however, some convincing supporting data
can be obtained from cell line studies.
Several studies analysing the gene
expression profiles of breast cancer cell
lines reveal that they can be broadly
divided into luminal subtypes and two
basal cell subtypes54,55. Flow cytometric
analysis of luminal cell lines such as mCF-7
that is based on the expression of CD24
and CD44 reveals that these cell lines do
not have a CD44+CD24–/low subpopulation
of cells56. Considering that cancer cell lines
from various tissues, including breast, have
a stem cell component that is responsible
for maintaining the line57–59, the observa-
tion that not even a small subpopulation
of cells with basal characteristics can be
detected in luminal cell lines suggests
that the tumour stem cells propagating
these luminal breast cell lines is probably
derived from a progenitor in the luminal
The concept that a luminal progenitor
cell could be the cell of origin of a subset
of breast cancers is not really surprising
considering the size of the progenitor cell
pool in the luminal cell compartment in
the mouse. The presence of a large pool
of progenitor cells within the human
luminal cell compartment has also been
described20. Interestingly, in this study
the basal compartment was shown to be
relatively deficient for progenitors. This
distribution of progenitors in the luminal
cell compartment is consistent with the
observation that the highest frequency of
proliferating cells in situ in both rodent60–62
and human63–65 mammary glands is in
cells that have a luminal cell morphology.
A lower frequency of proliferation is
observed in the basal compartment, with
794 | OCTOBER 2007 | vOlUmE 7
© 2007 Nature Publishing Group
little cell division observed in cells with a
differentiated myoepithelial morphology.
Considering the strong link between cell
proliferation and cancer risk66, and the
observation that there is a very large pool
of progenitor cells in the luminal cell
compartment and a relatively small pool of
stem cells in the basal compartment, raises
the question of whether this is why most
human breast cancers have a luminal cell
phenotype. Although it could be argued
that progenitor cells would require more
mutational events to acquire a malignant
phenotype than a stem cell, a substantially
larger pool of these progenitor cells could
compensate for this and thus make luminal
cell-derived tumours possible.
Links with haematopoietic cancers
Several reports have shown that committed
haematopoietic progenitors are potential
targets for malignant transformation
and can function as leukaemia-initiating
cells67–70. For example, in a recent study
by Krivtsov and colleagues71, committed
granulocyte macrophage (Gm) progeni-
tors from mice, which in the normal state
do not possess self-renewal properties,
were transduced to express the mixed
lineage leukaemia–AF9 (mll–AF9)
fusion protein, and the resulting cells were
transplanted into syngeneic recipient mice.
These transduced Gm progenitors were
able to induce acute myeloid leukaemia
in the recipient mice and were able to
undergo self-renewal as demonstrated
by transplantation into secondary recipi-
ents. Interestingly, these mll–AF9 Gm
progenitors retained a gene expression
pattern similar to that of normal Gm
progenitors, rather than haematopoietic
stem cells (HSCs). Evidence implicating
the committed progenitors in leukaemia
in humans comes from studies of acute
promyelocytic leukaemia (APl). APl is
characterized by a 15:17 chromosome
translocation, which results in the fusion
of the promyelocytic leukaemia (PML) and
retinoic acid receptor-α (RARA) genes.
Analysis of the HSC (CD34+CD38–) and
progenitor (CD34+ CD38+) populations by
PCR to detect the PML–RARA fusion gene
reveals that this fusion is not found in the
HSC fraction but only in the more mature
progenitor fraction. This result suggests
that HSCs are not involved in the neoplas-
tic process in APl72.
It is possible that an initial mutation
occurs within a stem cell, with subsequent
mutations occurring in downstream prog-
eny and resulting in a more differentiated
progenitor that functions as a tumour stem
cell (reviewed in REF. 73). Although there
is no direct evidence to date to support
this in human breast cancer, such a situ-
ation has been described in AML1–ETO-
expressing acute myelogenous leukaemia
(Aml)74–76. This fusion transcript can
be detected in the HSC compartment in
patients who are in long-term (>10 years)
remission, but these HSCs are not leukae-
mic as they display normal differentiation
function. Considering that the leukaemic
stem cells in Aml do not express Thy1, a
marker of normal HSCs, it suggests that
the initial mutational event may occur in
the stem cell compartment, and that fur-
ther mutations in downstream progeny are
required for the generation of leukaemic
stem cells77. A similar situation has been
described in human chronic myelogenous
leukaemia (Cml). In Cml, the BCR–ABL
mutation can be detected in HSCs, but this
mutation is not leukaemogenic in itself.
Instead, further downstream mutations
that involve β-catenin signalling and self-
renewal of Gm progenitors are thought to
be responsible for the disease progression78.
In breast cancer, it has been observed that
CD44+ and CD24+ tumour cells are clonally
related as CD24+ cells, which are presumed
to represent the luminal progeny of CD44+
tumour stem cells, show all the mutations
that are found in CD44+ cells79. However,
CD24+ cells can also undergo further
clonal evolution as they sometimes contain
additional genetic mutations79.
Emerging evidence from studies in
the haematopoietic system shows that
the stem cell compartment in humans is
composed of cells that are variable in their
proliferation and self-renewal properties.
For example, when HSCs are marked by
unique lentiviral integration sites and clon-
ally tracked when implanted into immune-
deficient mice, some HSCs immediately
repopulate a primary host with more
differentiated cells while also undergoing
self-renewal, whereas other HSCs primarily
undergo self-renewal divisions and only
repopulate subsequent serially transplanted
recipients80. An identical heterogeneity
in stem cell self-renewal has also been
described for leukaemic stem cells (lSCs)
in human Aml81. Further complexity
within the lSC compartment is also
observed as lSCs undergo clonal evolu-
tion over time82. Although such detailed
studies using breast tumour tissue have
yet to be carried out, experiments using
normal mouse mammary cells have
shown that mRUs are heterogeneous in
the amount of cleared mammary fat pad
they can fill on transplantation at limiting
dilutions26,30,83. variation in the number
of self-renewal divisions that these mRU
undergo during engraftment is also
observed26. Considering all of this, a
similar heterogeneity in cancer stem cells
within individual breast tumours would
not be surprising. If this were to hold true,
it would have serious implications for
the design of therapies to eradicate these
cells because their heterogeneous nature
may preclude a given therapy successfully
targeting all the cells of the population.
Cellular targets of cancer mutations
In recent years empirical evidence has
challenged the model that breast cancer
progresses in a stepwise linear fashion
from well-differentiated to poorly differ-
entiated tumours5–7. For example, analysis
of the gross genetic mutations that are
present within tumours with different
degrees of differentiation reveals that
the long arm of chromosome 16 (16q) is
almost exclusively lost in well and inter-
mediately differentiated ductal carcinoma
in situ (DCIS), but is rarely lost in poorly
differentiated DCIS. This is despite the
fact that poorly differentiated DCIS con-
tains more genetic aberrations on average.
Similarly, analysis of the invasive compo-
nent that is adjacent to well-differentiated
or poorly differentiated DCIS reveals that
these invasive tumour cells have an almost
identical genetic profile to their respective
DCIS tumours. These results are significant
as they suggest that well-differentiated and
poorly differentiated invasive tumours are
derived from well-differentiated and poorly
differentiated DCIS respectively, and that
these two types of tumour represent two
independent progression pathways.
One potential source of heterogeneity
in the evolution of breast tumours is that
different mammary cells have varying
susceptibilities to malignant transformation.
In vitro studies have certainly demon-
strated that different types of mammary
cell culture have varying susceptibilities
for lifespan extension and immortalization
with different viral oncogenes. For example,
early passage human mammary epithelial
cell cultures are susceptible to lifespan
extension by human papilloma virus
(HPv) E7 (which binds and inactivates
the retinoblastoma protein), whereas late
passage cultures could be immortalized
by E6 alone, which abrogates the p53–p21
checkpoint. Cultures derived from samples
of human milk required both E6 and E7
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© 2007 Nature Publishing Group
for complete immortalization (reviewed in
REF. 84). Unfortunately, the heterogeneity
and the exact constituents of these cultures
preclude any identification of the cells that
are preferentially immortalized in these
experiments. Studies using more defined
populations of cells show that the luminal-
like cells with multilineage potential are
more susceptible to telomerase activation
and immortalization by the Simian virus
40 (Sv40) large T antigen than their
Sequence and cellular context
The cells involved and the sequence of
mutations that occur in the progression
of different breast tumours have been
difficult to determine because the cellular
markers and quantitative functional assays
to identify and detect different subsets
of mammary epithelial cells have only
recently been described. Only now is the
mammary field adopting the methods
long used by the haematopoietic field in
which perturbations in stem and progeni-
tor function are measured by quantitative
in vivo and in vitro assays22,27,86–88.
Studies in mice have clearly shown
that different oncogenes exert their influ-
ence in different cell subpopulations. For
example, enhanced signalling of the Wnt
signalling pathway, either through over-
expression of mouse mammary tumour
virus (mmTv)-Wnt‑1 or gain-of-function
∆89β-catenin mutation, results in tumours
that are composed of both luminal and
myoepithelial cells and an expansion of
the maSC pool27,86,89. when these Wnt‑1
tumours were induced in mice with a
heterozygous Pten background, most of
the Wnt‑1 tumours generated showed a
loss of heterozygosity of the Pten allele in
both the luminal and myoepithelial cells of
the tumour, thereby indicating that a loss
of Pten occurs in cells that have multiline-
age differentiation potential89. This is in
contrast to mmTv-Erbb2 (Neu) mice,
which generate luminal cell-restricted
tumours and display an expansion of the
luminal cell compartment, although the
effect on luminal progenitor cell numbers
and function has yet to be rigorously
studied (REFS 27,89; reviewed in REF. 90).
Interestingly, the tumour cells in these
mmTv-Erbb2 tumours do not express
high levels of Sca-1 (REF. 89) and are ER
negative91,92. This suggests that the target
cell for malignant transformation is the
milk-protein-expressing, steroid hormone
receptor-negative, Sca-1low luminal pro-
genitor cells described by Sleeman and
colleagues30. Consistent with the concept
that these milk-protein-expressing pro-
genitors (alveolar progenitors?) are the
cellular targets for malignant transforma-
tion in mmTv-Erbb2-driven mammary
tumours is the observation that pregnancy
promotes tumorigenesis in this model92.
Another mouse model of Erbb2-induced
tumorigenesis in which an activated
Erbb2 oncogene is expressed under the
endogenous Erbb2 promoter results in
precocious acinar formation, which again
suggests an alveolar progenitor is the target
cell for malignant transformation93.
A bigger question is whether or not
the mmTv-Erbb2 mouse tumour model
accurately mimics ERBB2-overexpressing
tumours in humans. Analysis of ERBB2
levels in a panel of human breast cancer
cell lines reveals that ERBB2 amplifica-
tion is scattered across both luminal and
basal cell lines54. Although microarray
analysis has demonstrated that ERBB2
amplification is typically associated with
ER negativity, there is a subset of ERBB2
tumours that are ER positive. Also, tissue
arrays have demonstrated that ERBB2-
overexpressing tumours typically have a
luminal (mUC1+) phenotype, but a rare
subset of ERBB2 tumours can be composed
of basal cells38. Although these results do
not provide insight into the cell of origin
of human ERBB2 tumours, they do suggest
that ERBB2 amplification can transcend
multiple lineages within the mammary
Basal breast cancers
Approximately 15–21% of human breast
tumours have a basal phenotype: a
phenotype that has been associated with
a poor prognosis11–13,94–96. This probably
represents an oversimplification as basal
tumours are heterogeneous117,118 and their
poor prognosis has not been universally
confirmed97. These basal breast tumours
are characterized as being ER–, PR–,
ERBB2–, epidermal growth factor recep-
tor (EGFR)+ and keratin 6+ and/or 17+.
It has been proposed that basal tumours
are derived from maSCs because the gene
expression profiles of these tumours are
perceived to be similar to that expected of
an maSC98. Basal tumours are not thought
to be derived from differentiated myoepi-
thelial cells because most of these tumours
do not express smooth muscle actin,
which is a functional marker of differenti-
ated myoepithelial cells99. Surprisingly,
basal breast tumours commonly express
luminal-associated keratins 8 and 18 in
addition to basal keratins such as keratin
14 (REF. 99), although it is not clear whether
these tumours are composed of cells that
co-express both basal and luminal keratins
(which might suggest an expansion of a
primitive population of cells) or are com-
posed of two distinct lineages of epithelial
cells. A strong prognostic marker of basal
carcinomas is expression of keratin 6
(REF. 99), a keratin that in both the normal
mouse and human mammary epithelium
is associated with colony-forming cells
in the luminal lineage20,26, although the
distribution of keratin 6 among maSCs is
still unknown. The high level of expres-
sion of keratin 6 among multilineage
progenitor cells in the human mammary
epithelium raises the possibility that these
multilineage progenitors are the targets
for malignant transformation in basal
breast carcinomas. However, as previously
discussed, gene expression profiling of a
tumour cell population unfortunately does
not necessarily permit inferences about the
cell of origin of the tumour.
why are p53 mutations common in
basal breast tumours12, a tumour type that
is perceived to be derived from maSCs?
It has previously been proposed that p53
might serve as a rate-limiting step in
controlling the proliferative lifespan of
maSCs100. In the haematopoietic system,
it has been shown that p53 dosage is
inversely correlated with HSC function101.
The mechanism controlling this is not
known, although it has been speculated
that p53 dosage might influence the
self-renewal rates of HSCs. In the neural
system, an inverse correlation between p53
levels and neural stem cell self-renewal has
been described102. Considering this, it is
tempting to speculate that a similar mecha-
nism occurs in maSCs, where the loss of
p53 increases maSC function (thereby
expanding the maSC pool) and results in
an increased risk of tumour incidence in
these cells. Consistent with the hypothesis
that the loss of p53 regulates maSC func-
tion is the observation that the luminal
and myoepithelial lineage differentiation
markers keratin 19 and smooth muscle
actin are p53 target genes103,104 and that the
loss of p53 in mammary tumours results in
downregulation of these gene products105.
loss of BRCA1 (breast cancer 1) is
synonymous with basal breast cancer,
although the reasons for this are unclear106.
It has been suggested that the loss of
BRCA1 from breast, and indeed ovarian,
tumours is often seen because these tissues
can survive for extended periods in the
796 | OCTOBER 2007 | vOlUmE 7
© 2007 Nature Publishing Group
Nature Reviews | Cancer
subpopulations of cells
and cell sorting
subpopulations of cells
Human breast tumours
absence of BRCA1 (reviewed in REF. 107).
It has been postulated that BRCA1 might
function as an maSC regulator, possibly
through a mechanism in which the loss
of BRCA1 results in the loss of the ability
of maSCs to differentiate108. Certainly,
conditional knockout of Brca1 in the
mammary glands of mice demonstrates a
proliferation defect during pregnancy109,
but whether the deletion of Brca1 is exert-
ing its effect at the level of the maSC or
a progenitor cell remains unknown. The
predominance of p53 mutations in basal-
like breast cancers may also explain why
BRCA1 mutations are predominant in this
type of cancer, as the loss of p53, which
inhibits some apoptotic pathways, may
result in the survival of BRCA1–/– cells.
Synergy in tumour formation has been
observed in Brca1‑null mammary cells
that have also lost Trp53 (REF. 109). Similar
results are observed when another
genomic stability enzyme, Brca2, and
Trp53 are deleted in a mouse mammary
Delineating the mammary epithelial cell
hierarchy is essential for providing a
framework for determining the cellular
targets of breast cancer mutations. In
order to achieve this, better strategies to
identify and purify stem, progenitor and
the differentiated cells of the mammary
epithelium must be developed. This is
particularly important if meaningful gene
expression profiles of these populations
are to be obtained as current protocols to
isolate mouse mammary stem cells result
in purities of 5% at best and even lower
frequencies for human breast tumour stem
cells. Also essential in this process is the
development of a xenotransplantation
assay that supports the clonal growth and
self-renewal of human mRUs. Several
candidate assays look promising24,25 but
remain to be validated. Also integral in
this process is having an understanding of
the limitations of the assays used to study
The strategies used to characterize
normal stem cells are now being applied to
tumour stem cells. A popular approach is
to assay phenotypically distinct subpopu-
lations that are isolated from transgenic
mouse models for the ability to generate
tumours in syngeneic recipients. Although
informative, this approach warrants cau-
tion as some of these transgenic models
rely on promoters that are functional only
in specific, and sometimes undefined,
cellular contexts, and could potentially
force transgene expression into inap-
propriate cell populations. For example,
the commonly used whey acidic protein
(wAP) promoter, which relies on a forced
pregnancy to induce transgene expres-
sion and targets alveolar cells and their
precursors112, might miss entire subsets of
mammary epithelial cells. Similarly, the
commonly used mmTv promoter, which
in young mice targets most mammary
epithelial cells, is expressed in a more
non-uniform mosaic pattern in older
Figure 2 |?Strategy?to?decipher?the?cellular?targets?of?different?oncogenic?mutations?and?
different?cancer?stem?cell?phenotypes.?Breast cancer research should adopt the approach
taken by the haematopoietic field in which cells at specific stages of differentiation within the
cell hierarchy can be isolated by flow cytometry and manipulated ex vivo and then assayed in vitro
and in vivo to determine the influence on progenitor cell and stem cell function (a). This approach
has recently been applied to study the influence of hedgehog signalling components and the
polycomb protein BMI1 on human mammary repopulating unit (MrU) function87. Using such an
approach, tumours could theoretically be reverse-engineered on different cellular backgrounds
to determine which cell subtypes can be transformed with a given combination of genetic muta-
tions and what type of tumour is generated with this cellular and mutation background. A com-
plementary approach would be to isolate cancer-initiating cells from the different molecular
subtypes of breast cancer and to compare the expression profiles of these cancer stem cells with
the putative normal counterpart (b). cFc, colony-forming cells.
NATURE REvIEwS | cancer?
vOlUmE 7 | OCTOBER 2007 | 797
© 2007 Nature Publishing Group
It is without doubt that new strategies
to purify mouse stem and progenitor cells,
beyond what is currently achievable, will be
developed and that accurate gene expression
profiles of these cells will be obtained. As
a result, specific cell differentiation stage
genes that are more suitable to function as
promoters in transgenic mouse mammary
models will be identified. This requirement
for more appropriate promoters is clearly
evident as most transgenic mouse mammary
tumour models result in ER– tumours114, yet
most human mammary tumours are ER+.
In hindsight, the generation of ER– tumours
using wAP-promoter-driven oncogenes is
not surprising considering that milk-protein-
expressing progenitor cells are ER– (REF. 30).
more difficult to explain is why other
promoters do not allow the generation of ER+
tumours. For example, a recent publication
describes the modelling of invasive lobular
carcinoma in mice using K14cre;Cdh1F/
F;Trp53F/F, which results in the recapitulation
of this type of breast tumour histologically.
However, although human lobular cancers are
typically ER+, mouse tumours are apparently
imposed onto an ER– luminal cell lineage115.
An alternative and complementary strategy
to help to resolve these issues is to adopt
the approach taken by the haematopoietic
field (FIG. 2), whereby phenotypically distinct
subsets of cells can be genetically manipulated
ex vivo and assayed for altered stem and
progenitor function. Such an approach will
provide a direct method to help to determine
the cell of origin in human breast cancer.
Determining the cellular targets of dif-
ferent oncogenic mutations and how these
mutations promote the generation of tumour
stem cells will be essential in understanding
what is currently a bewildering disease. Of
particular interest are the pathways, either
acquired or maintained, that regulate tumour
stem cell self-renewal, as the disruption of
these pathways offers a rational therapeutic
target to be tested in human clinical trials.
John Stingl is at the Department of Pathology,
University of Cambridge, Tennis Court Road,
Cambridge CB2 1QP, UK.
Carlos Caldas is at the Cancer Research UK Cambridge
Research Institute, and Department of Oncology,
University of Cambridge, Li Ka Shing Centre,
Cambridge CB2 0RE, UK.
Correspondence to C.C.
Published online 13 September 2007
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J. S. is supported by funds provided by the Breast Cancer
Campaign and C. C. is supported by Cancer Research UK.
The authors would like to thank P. Eirew for critical reading
of the manuscript.
Competing interests statement
The authors declare no competing financial interests.
entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
cD24 | GATA3 | ITGA6 | ITGB1 | MUc1 | PecAM1 | PIK3CA |
PML | PTPrc | RARA | TP53
carlos caldas’s homepage: http://www.cambridgecancer.
NATURE REvIEwS | cancer?
vOlUmE 7 | OCTOBER 2007 | 799
© 2007 Nature Publishing Group