ASMA = α-smooth muscle actin; CDK = cyclin-dependent kinase; COX = cyclo-oxygenase; ER = estrogen receptor; ESA = epithelial-specific
antigen; HMEC = human mammary epithelial cell; HPV = human papillomavirus; hTERT = catalytic subunit of human telomerase; PD = population
doubling; Ral-GEF = Ral guanine nucleotide exchange factor; TDLU = terminal ductal–lobular unit.
Available online http://breast-cancer-research.com/contents/7/4/171
Normal human mammary epithelial cells (HMECs) have a finite life
span and do not undergo spontaneous immortalization in culture.
Critical to oncogenic transformation is the ability of cells to overcome
the senescence checkpoints that define their replicative life span and
to multiply indefinitely – a phenomenon referred to as immortalization.
HMECs can be immortalized by exposing them to chemicals or
radiation, or by causing them to overexpress certain cellular genes or
viral oncogenes. However, the most efficient and reproducible model
of HMEC immortalization remains expression of high-risk human
papillomavirus (HPV) oncogenes E6 and E7. Cell culture models
have defined the role of tumor suppressor proteins (pRb and p53),
inhibitors of cyclin-dependent kinases (p16INK4a, p21, p27 and p57),
p14ARF, telomerase, and small G proteins Rap, Rho and Ras in
immortalization and transformation of HMECs. These cell culture
models have also provided evidence that multiple epithelial cell
subtypes with distinct patterns of susceptibility to oncogenesis exist
in the normal mammary tissue. Coupled with information from distinct
molecular portraits of primary breast cancers, these findings suggest
that various subtypes of mammary cells may be precursors of
different subtypes of breast cancers. Full oncogenic transformation
of HMECs in culture requires the expression of multiple gene
products, such as SV40 large T and small t, hTERT (catalytic subunit
of human telomerase), Raf, phosphatidylinositol 3-kinase, and Ral-
GEFs (Ral guanine nucleotide exchange factors). However, when
implanted into nude mice these transformed cells typically produce
poorly differentiated carcinomas and not adenocarcinomas. On the
other hand, transgenic mouse models using ErbB2/neu, Ras, Myc,
SV40 T or polyomavirus T develop adenocarcinomas, raising the
possibility that the parental normal cell subtype may determine the
pathological type of breast tumors. Availability of three-dimensional
and mammosphere models has led to the identification of putative
stem cells, but more studies are needed to define their biologic role
and potential as precursor cells for distinct breast cancers. The
combined use of transformation strategies in cell culture and mouse
models together with molecular definition of human breast cancer
subtypes should help to elucidate the nature of breast cancer
diversity and to develop individualized therapies.
More than 80% of adult human cancers are carcinomas,
tumors originating from malignant transformation of epithelial
cells. However, much of our understanding of oncogenic
transformation comes from fibroblast transformation systems.
Breast cancer is the second leading cause of cancer-related
deaths among women in the USA . The vast majority of
breast cancers are carcinomas that originate from cells lining
the milk-forming ducts of the mammary gland (for review ).
Deliberate transformation of these cells provides a practical
window into human epithelial oncogenesis. Malignant
transformation represents a complex multistep process in
which genetic, environmental, and dietary factors together are
thought to alter critical cell growth regulatory pathways
resulting in uncontrolled proliferation, which is a hallmark of
tumorigenesis [3,4]. Understanding the nature of these
cellular pathways is of central importance in cancer biology.
The growth of normal human mammary epithelial cells
(HMECs), which include luminal, myoepithelial and/or basal
cells (described below), is tightly controlled. These cells grow
for a finite life span and eventually senesce (for review [5-7]).
Both cell culture and mouse models have provided evidence
that essential initial steps in tumorigenesis involve the loss of
senescence checkpoints and immortalization, which allow a
cell to grow indefinitely and to go through further oncogenic
steps, resulting in fully malignant behavior. In addition, cell
culture model systems have identified a number of genes
whose alterations are involved in HMEC immortalization and
thereby have provided significant insights into the biology of
early breast cancer [5,7,8]. Use of oncogene combinations
has allowed researchers to create cell culture models of full
HMEC transformation, thereby illuminating the process of
Mammary epithelial cell transformation: insights from cell
culture and mouse models
Goberdhan Dimri1, Hamid Band2and Vimla Band1
1Division of Cancer Biology, Department of Medicine, ENH Research Institute, and Robert H Lurie Comprehensive Cancer Center, Feinberg School of
Medicine, Northwestern University, Evanston, Illinois, USA
2Division of Molecular Oncology, Department of Medicine, ENH Research Institute, and Robert H Lurie Comprehensive Cancer Center, Feinberg
School of Medicine, Northwestern University, Evanston, Illinois, USA
Corresponding author. Vimla Band, email@example.com
Published: 3 June 2005
This article is online at http://breast-cancer-research.com/content/7/4/171
© 2005 BioMed Central Ltd
Breast Cancer Research 2005, 7:171-179 (DOI 10.1186/bcr1275)
Breast Cancer Research July 2005 Vol 7 No 4Dimri et al.
breast cancer progression [9-11]. Additional insights have
come from mouse models, using transgenic overexpression of
oncogenesis-promoting genes and deletion of tumor
suppressor genes, which often produce breast adeno-
carcinomas that closely resemble human breast cancers.
Studies using cell culture transformation models have pointed
to the existence of HMEC subtypes with distinct suscepti-
bilities to oncogenesis by different oncogenes [5,8].
Remarkably, direct cDNA microarray profiling of human
breast cancers has led to similar insights, identifying multiple
subtypes of human breast cancer with distinct outcomes;
phenotypic and genotypic characteristics of these breast
cancer subtypes point to their possible origin from specific
subtypes of HMECs, such as basal or luminal cells .
Finally, cell culture and mouse model systems have begun to
identify mammary stem cells that may provide progenitors for
oncogenic transformation  and have led to an appreciation
of the microenvironment for oncogenesis [14,15].
Thus, studies conducted over the past several years have
established the importance of HMEC transformation models
to our understanding of the pathways that control normal
mammary cell growth, development, and oncogenesis.
However, many challenges remain, including the identification
of mammary cell subtypes or oncogenic strategies that result
in cancers that resemble naturally occurring human breast
cancers, and translation of new research to devise more
specific diagnostic and treatment strategies for different
subtypes of breast cancer.
Mammary gland and various epithelial cell
The mammary gland consists of a branching ductal system
that ends in terminal ducts with their associated acinar
structures, termed the terminal ductal–lobular units (TDLUs),
together with interlobular fat and fibrous tissue [16,17]. Most
breast cancers arise in the TDLU (Fig. 1). Unlike other
epithelial cancers, such as that of colon, different stages of
breast cancer are not clearly defined. However, it is clear that
benign stages (such as typical and atypical hyperplasia),
noninvasive cancers (such as carcinoma in situ – ductal or
lobular), and invasive cancers (such as invasive ductal or
lobular carcinomas) do exist. Additionally, multiple types of in
situ carcinomas, such as solid, cribiform, papillary and
comedo types, have been reported and it is possible that
these represent tumors originating from different epithelial
Histological examination of TDLU reveals two major types of
cells: inner secretory luminal cells and outer contractile
myoepithelial cells (Fig. 1). In addition to luminal and
myoepithelial cells, there is emerging evidence that basal
cells (presumed to be the progenitor for myoepithelial cells)
and stem cells exist in the TDLU [17,18]. Until recently it was
believed that the vast majority of breast carcinomas arise from
luminal epithelial cells . This was based on the keratin
expression and other phenotypic markers of cultured tumor
cell lines, mostly derived from metastatic lesions .
Unfortunately, the great majority of primary breast tumors
have proved difficult to establish in cultures, either on plastic
or as three-dimensional cultures [5-7,19-21]. However,
recent molecular profiling studies clearly show the existence
of multiple subtypes of breast cancers probably originating
from luminal, basal, and possibly stem cell compartments
 (described below in detail).
Culturing of various epithelial cell subtypes
For more than two decades, various investigators have
attempted to develop cell culture models that lead to isolation
of breast cancer cells resembling those found in human
breast cancers. In order to establish such models, it was
essential to culture normal HMECs. In 1980s, work from
several laboratories showed that normal HMECs could be
cultured in cell culture [22,23] (for review [2,5,7]).
In our laboratory we defined a medium, termed DFCI-1, that
helped us to establish and culture normal and some primary
breast cancers under identical conditions . However, in
general the difficulty in establishing primary tumor cells in cell
culture has persisted. Notably, early cultures derived from
reduction mammoplasty or mastectomy specimens exhibit
considerable heterogeneity (with multiple cell types – luminal,
stem cells, basal and myoepithelial cells) and grow for three
to four passages or about 15–20 population doublings
(PDs), and then senesce (Figs 2 and 3) [5-7]. The senes-
cence in these cells is also termed as M0 stage .
Structure of the mammary gland. Terminal ductal–lobular unit (TDLU),
composed of ductal cells, is the unit thought to be the origin of most
breast cancer. The stroma is composed of fatty tissue (adipocytes) and
fibroblasts. Also shown are the two primary types of cells in normal
ducts: outer contractile myoepithelial and inner columnar luminal cells.
A putative progenitor/stem cell is also indicated.
Terminal ductal-lobular unit
However, in some cases (not always) an occasional
homogenous cell population emerges that continue to grow
further for 30-60 PDs (Figs 2 and 3) [5-7] before senescence
occurs (also called agonescence, described below) .
This process of emergence of cells that are able to proliferate
for extended periods is also known as self-selection; before
selection the cells are termed preselection cells, whereas
those that emerge after selection are called postselection
cells. The keratin profile of preselection cells (K-5, K-6, K-7,
K-14, K-17, K-18 and K-19 positive) [8,19,26] suggests the
existence of both luminal and basal (myoepithelial) cells.
However, postselection cells generally exhibit a loss of
expression of K-19 but retain the expression of all other keratins
[8,18,25]. These cells also express α-smooth muscle actin
(ASMA), suggesting that these may be of myoepithelial origin.
Further development of cell sorting techniques and chemically
defined media have helped in culturing of luminal and progenitor
epithelial cells [14,27] (described below in detail).
It has also been reported that postselection cells lose the
expression of p16INK4a, a cyclin-dependent kinase (CDK)
inhibitor [24,25], and gain expression of cyclo-oxygenase
(COX)-2, a gene that is thought to be involved in
tumorigenesis . As both of these genes are implicated in
oncogenesis, it is conceivable that loss of p16 or gain of
COX-2 expression may make these cells more susceptible to
transformation, although it is unclear whether the loss of p16
and gain of COX-2 occur de novo during self-selection or
represent selection of a minor population of cells with pre-
existing high COX-2 and low p16 expression. Notably, p16-
negative and COX-2-positive cells could be detected using
immunohistochemistry in normal mammary tissue [28,29].
Immortalization of various HMEC subtypes in
As alluded to above, normal mammoplasty-derived HMECs
exhibit a limited life span, which is followed by replicative
senescence. Replicative senescence acts as a strong tumor
suppressor mechanism and
immortalization of human cells [30-33]. A major determinant
of replicative senescence is the enzyme telomerase, which
maintains the length of telomere ends [30,31]. Most somatic
cells express little or no telomerase, resulting in telomere
shortening with successive cell divisions, which eventually
elicits a senescence checkpoint [30-32]. A senescence-like
phenotype can also be induced by a variety of nontelomeric
signals such as DNA-damaging agents, adverse cell culture
conditions, and overexpression of certain oncogenes [30,32].
The tumor suppressor protein p53 and its target gene
product p21, and p16INK4aplay a crucial role in senescence
Available online http://breast-cancer-research.com/contents/7/4/171
Establishment of mammary epithelial cells from reduction
mammoplasty/mastectomy specimens. The tissue is chopped, digested
with collagenase and hyaluronidase, and plated in medium as organoids.
Over a week or so, multiple types of epithelial cells and fibroblasts
emerge; fibroblasts are removed by differential trypsinization (fibroblasts
are loosely attached), remaining epithelial cells grow for 10–15
population doublings (PDs) followed by senescence of the majority of
cells. Occasionally, an homogenous population of cells emerges that
continue to proliferate for an additional 30–60 PDs, and eventually these
cells also senesce (this step is referred to as agonescence).
(Reduction mammoplasty or mastectomy)
Chop and digest
cells and fibroblasts
Homogeneous epithelial cells
Morphological heterogeneity of cells before and after selection.
(a–d) Two views of mammary epithelial preselection cells (original
magnifications: panels a and c, 40×; panels b and d, 100×). Cells
shown in panel a grow as compact clusters and are relatively uniform,
whereas cells in panel b grow more dispersed and exhibit different
types of cells (small and large). (e,f) Views of postselection human
mammary epithelial cells with relatively uniform morphology (original
magnifications: panel e, 40×; panel f, 100×).
(a) (c) (e)
(b) (d) (f)
induced by telomeric as well as nontelomeric signals [30-33].
Much of our knowledge about senescence comes from
studies conducted in human fibroblasts [30-34]. Only
recently have we begun to elucidate the mechanisms of
senescence in epithelial cells, in particular in HMECs .
The senescence associated with the ‘selection’ phase in
HMEC cultures is accompanied by classic features of senes-
cence, such as flat morphology, presence of vacuoles, and
positive staining for senescence-associated β-galactosidase
(SA-β-gal), a marker of senescence . The block in cell
proliferation at this stage is dependent on the pRb/p16
pathway [24,35], because the human papillomavirus (HPV)
oncogene E7, which binds and inactivates pRb, can over-
come the M0/selection stage . Similarly, a constitutively
active p16-insensitive CDK4 mutant can overcome the M0
stage . Thus, senescence of preselection cells appears
to be telomere independent. At the end of their replicative life
span, postselection HMECs exhibit senescence as well as
cell death with a high level of genomic instability. This
phenomenon is termed as agonescence, as opposed to
replicative senescence . Most importantly, unlike rodent
cells, human HMECs derived from reduction mammoplasties
or from milk do not exhibit spontaneous immortalization and
thus provide suitable models of human cell transformation.
Immortalization of HMECs in culture is characterized by their
continuous growth beyond the agonescence checkpoint. It is
thought that immortalization is an early step in human cancer,
and continued proliferation of immortal cells allows the
accumulation of additional genetic changes that promote
malignant and metastatic behavior.
Stampfer and Bartley  presented initial evidence that
HMECs could be immortalized in cell culture using benzo(a)-
pyrene; however, the immortalization was a rare event in this
case. Similar to carcinogen-induced immortalization, we found
that γ-radiation induced the transformation of HMECs relatively
infrequently [5,8,39]. In general, most viral oncogenes
(including SV40 T antigen, adenovirus E1A and E1B, polyoma
T antigen) have not proven very efficient as immortalizing
genes for human cells . While the introduction of the
SV40 T antigen into breast tumor tissue-derived epithelial
cells gave rise to immortal cell lines, SV40-transfected cells go
through a long crisis period, and emergence of immortal cells
is rare . Over the past several years, our studies have
defined a system to immortalize human HMECs efficiently and
reproducibly, using the urogenital carcinoma-associated HPV
oncogenes E6 and E7 [5,8,36].
Comparison of early (preselection) and late-passage (post-
selection) cultures revealed that different HMEC subtypes
exhibit a remarkably distinct susceptibility to E6 or E7, or their
combination . One HMEC subtype was exclusively
immortalized by E6 but not by E7; such cells predominated
the late-passage cultures but were rare at early passages.
Surprisingly, a second cell type, present only in early
passages of tissue-derived cultures, showed extension of life
span and infrequent immortalization by E7 alone. Finally, E6
and E7 together were required to immortalize fully a large
proportion of preselection HMECs .
Human milk is an easily available source of relatively pure
HMECs that are thought to be differentiated luminal cells
[2,19]. However, these cells can be cultured for only a limited
number of passages (typically two to three passages, or five
to nine PDs), which has precluded their detailed biochemical
study [2,18]. Most of the work on milk cells has been carried
out in Taylor-Papadimitriou’s laboratory and has demon-
strated that these cells can be immortalized by SV40 T
antigen . Interestingly, neither E6 nor E7 alone could
induce the immortalization of milk-derived HMECs, whereas a
combination of E6 and E7 was effective .
The reproducibility and relatively high efficiency with which E6
(in postselection HMECs) or E6 and E7 combined can induce
immortalization of human HMECs have therefore yielded a
practical approach to elucidate the biochemical mechanisms of
HMEC immortalization. In recent years, using Yeast Two-hybrid
analysis, we identified several novel targets of the E6 oncogene
in HMECs. These targets represent novel mediator of HMEC
immortalization . These include ADA3 (alteration/deficiency
in activation 3), a novel coactivator of p53 and steroid
receptors (estrogen receptor [ER] and retinoic acid receptor)
[42-44]; E6 targeted protein 1 (E6TP1), a novel GTPase
activating Rap small G protein; and protein kinase N (PKN), an
effector for Rho small G protein . We recently found that
MamL1, a human homolog of the Drosophila mastermind gene
and a known coactivator for Notch , also interacts with E6
(I Bhat, V Band, unpublished data). These studies have
implicated the p53, Notch, ER, Rho, and Rap signaling
pathways in early transformation of human HMECs. Consistent
with these analyses, we have shown that expression of mutant
p53  or activated Rho (X Zhao, V Band, unpublished data)
induces immortalization of HMECs. Furthermore, several
studies support a role for p53 mutations as an early event in
breast cancer . Taken together, these studies demonstrate
that E6 is the most efficient immortalizing gene for
postselection HMECs and that E6 immortalizes the HMECs by
concurrently altering multiple biochemical pathways. Future
studies will need to address the precise role played by these
novel oncogene targets in early breast cancer.
In addition to viral oncogenes, alterations in the expression of
cellular genes can also help to overcome senescence and
promote HMEC immortalization. Among the cellular genes,
we recently reported that Bmi-1, a member of the polycomb
group of transcriptional repressors, could immortalize
postselection HMECs . Although the detailed mechanism
of immortalization induced by Bmi-1 remains to be explored,
Bmi-1 does not appear to immortalize these cells by down-
regulating the INK4a/ARF locus. Interestingly, recent studies
have implicated Bmi-1 in stem cell function and renewal
Breast Cancer Research July 2005 Vol 7 No 4Dimri et al.
[49,50], suggesting that Bmi-1 could function as a potential
breast cancer stem cell marker . Another study showed
that ZNF217, a zinc finger protein that is overexpressed in
breast cancers, can promote immortalization of postselection
HMECs . Furthermore, introduction of hTERT also
induces immortalization of
Interestingly, induction of telomerase has been documented
early after E6 was introduced into HMECs , although the
cause and effect relationship between telomerase induction
and E6-induced immortalization continues to be debated.
Recently, the E6 and E6–AP binding protein NFX-91 was
implicated in E6-mediated induction of telomerase .
postselection cells .
Cell culture models of full transformation of
The ability of researchers to establish normal HMECs and to
induce their reproducible immortalization has provided
momentum for further efforts to define the nature of
biochemical alterations that can lead to full oncogenic
transformation. As we and others have demonstrated,
HMECs immortalized by most currently known procedures
(such as E6 or E6 plus E7, mutant p53, Bmi-1 and hTERT)
are preneoplastic and do not grow in an anchorage-
independent manner or produce tumors when implanted in
immune-deficient mice [5,8]. Weinberg and colleagues 
recently established a multistep model of full HMEC
transformation in cell culture by serial introduction of SV40
large T and small t, hTERT, and activated Ras (Fig. 4). It was
shown that introduction of the SV40 large T, which binds and
inactivates p53 and pRb, abolished senescence, whereas
hTERT was needed to promote immortalization . Notably,
these studies showed an essential role for the SV40 small t,
which inhibits protein phosphate 2A . HMECs
transformed by this method
independence and produced poorly differentiated carcinoma
(but not adenocarcinoma) when implanted in nude mice .
Further dissection of the role of small t revealed the
importance of the downstream targets of phosphatidylinositol
3-kinase, Akt1 and Rac1, and direct activation of these
pathways could fully substitute for small t in the transfor-
mation assays . A recent refinement of the transformation
in cell culture scheme suggests that perturbation of p53,
pRb, protein phosphate 2A, telomerase, Raf, and Ral guanine
nucleotide exchange factor (Ral-GEF) pathways are required
for the full tumorigenic conversion of normal human cells .
The requirement in terms of modulating Raf and Ral-GEF
pathways is cell type specific; HMECs require activation of
Raf, phosphatidylinositol 3-kinase and Ral-GEFs, whereas
human fibroblasts require the activation of Raf and Ral-GEFs
. Thus, serial use of viral and/or cellular genes is
beginning to unravel the various combinations of genetic
lesions that can convert a completely normal mammary
epithelial cell into a fully tumorigenic one.
Although these studies have thus far relied on the use of
known oncogenes, future studies using the cell culture
transformation models with gene libraries should help identify
novel cellular genes that participate at various steps of breast
cancer progression. Vast majority of human breast cancers
are adenocarcinomas, and only a small portion of breast
cancers are poorly differentiated carcinomas. Hence, it
appears that HMEC transformation in culture system is not
optimal because the tumors produced by these transformed
HMECs have usually been poorly differentiated carcinomas
rather than adenocarcinomas. Breast cancer is associated
with overexpression of various cellular proto-oncogenes such
as ErbB2, epidermal growth factor receptor, Src family
kinases, Bmi-1, cyclin D1, cyclin E, CDK4, and other potential
growth regulators. Use of these oncogenes in the multistep
model described above and the use of other HMEC subtypes
(such as luminal cells, potential stem cells, or those derived
from milk) as a starting population may help to achieve full
transformation of HMECs that develop into adenocarcinomas
Available online http://breast-cancer-research.com/contents/7/4/171
Current consensus: normal HMECs can be fully transformed in
definable serial steps. The first step, bypass of senescence, is
achieved by inactivation of p53 and pRb by SV40 large T, human
papillomavirus (HPV) E6 and E7, or by inhibition of p53 and pRb
expression by the RNAi approach (or expression of dominant-negative
mutants in the case of p53). The second step, immortalization, is
achieved through the expression of hTERT. Alternatively, expression of
HPV E6 or overexpression of Bmi-1, mutant p53, or ZNF217 can be
used to induce immortalization of HMECs. The third step, anchorage-
independent growth, can be achieved by SV40 small t mediated
modulation of PI3K and/or other signaling pathways or by
overexpression of activated Rac1 and AKT. The fourth step, full
transformation, requires the introduction of activated H-ras, which can
be substituted by Raf and Ral-GEFs. Although the current model
systems have utilized the serial schemes depicted, other combinations
and/or schemes of oncogene introduction are likely also to be
effective. Adapted from Elenbaas , Zhao , and Rangarajan 
and coworkers. HMEC, human mammary epithelial cell; HPV, human
papillomavirus; hTERT, catalytic subunit of human telomerase; PI3K,
phosphatidylinositol 3-kinase; Ral-GEF, Ral guanine nucleotide
exchange factor; RNAi, RNA interference.
(E6, Bmi-1, mp53)
SV40 Large T
SV40 Small t
in a nude mouse model. Thus, future studies must focus on
developing models that will lead to breast tumors that
faithfully reproduce the pathological characteristics of human
Transgenic mouse models of breast cancers
Mouse models of breast cancers have provided a wealth of
knowledge about the molecular pathways involved in breast
cancers. Initial studies in these models used carcinogens to
induce breast carcinomas . Later studies targeted a wide
variety of genes expressed under either the MMTV (mouse
mammary tumor virus) or the WAP (whey acidic protein)
promoter to target genes to the mammary gland. Importantly,
such studies invariably produced breast adenocarcinomas in
mice that resembled human breast cancers. These include
viral proteins, such as SV40 large T, polyoma virus T antigen
[56-58], or cellular proteins such as c-Myc, ErbB2/neu, cyclin
D1, cyclin E, ERs, mutant p53, c-Ha-ras, and Wnt-1 [59-63].
Recent studies have focused on mouse models with either a
global or a mammary-specific knockout of specific genes to
examine the function of obvious players, such as cell cycle
related proteins and tumor suppressors, either by themselves
or after these deficiencies were combined with transgenic
neu or other oncogenes. For example, cyclin D1-deficient
mice are resistant to mammary carcinomas induced by c-neu/
ErbB2 and Ha-ras but not to those induced by c-Myc or
Wnt-1 . These findings define a pivotal role for cyclin D1
in selective mammary cancers in a mouse model and imply a
functional role for cyclin D1overexpression in a subset of
human breast cancers. In another study, Cre-mediated
deletion of exons 3 and 4 of the mouse Brca2 gene in mice
with a loxP-modified and null Brca2 allele resulted in high
incidence of breast adenocarcinomas . Similarly, the
telomere attrition in aging telomerase-deficient and p53-
mutant mice promoted the development of breast adeno-
carcinomas . Another study showed that loss of Stat5a
delays mammary cancer progression in a WAP-TAg trans-
genic mouse model .
Collectively, these models have defined a role for p53,
pRb, BRCA1/2, cyclins, CDKs, ErbB2, c-Myc, Wnt-1, ER,
and progesterone receptor in mammary cell growth and
development of breast cancers. Finally, these different
oncogenes and the pathways in which they work seem to
target different progenitors or cell types in mammary gland
to develop mammary tumors . For example, the Wnt
signaling pathway targets both luminal and myoepithelial
cells, whereas Neu, H-Ras, and polyoma T antigen target
only luminal epithelial cells . The take-home lesson
here is that the majority of these mouse models result in
tumors that resemble human breast adenocarcinomas
pathologically. The lack of development of adeno-
carcinomas from cells transformed in culture models may
thus reflect the cell type that was used as the starting
normal cell, rather than any peculiarity associated with the
use of mouse as a host.
Molecular classification of breast cancers:
cues from cell culture studies
A vast body of clinical literature indicates that breast tumors
exhibit diverse phenotypes as judged by their distinct clinical
course, pathological features, and responsiveness to various
therapies. However, it has not been clear whether this
diversity reflects cancers arising from distinct subtypes of
HMECs. Consistent with such a possibility, several years ago
we reported the presence of different subtypes of cells in
reduction mammoplasty specimens and in milk that exhibited
differential susceptibility to viral oncogenes [5,8]. Direct
evidence for the conclusions derived from these cell culture
studies was provided by recent work utilizing gene
expression patterns in primary human breast cancers, using
cDNA microarrays. These studies identified distinct gene
expression profiles or molecular portraits based on which
breast tumors could be subclassified into groups that appear
to reflect the original cellular subtypes found in the mammary
gland . Five categories of breast cancers were described
: a basal epithelial-like group, an ErbB2-overexpressing
group, a normal breast epithelial-like group, luminal epithelial
cell type A, and luminal epithelial cell type B. A slightly
different classification was proposed by Sotiriou and
coworkers . The breast tumors were first divided into ER-
positive and ER-negative categories. The ER-negative tumors
were further subgrouped into basal-like 1, basal-like 2, and
ErbB2/neu tumors, whereas ER-positive tumors were
subdivided into luminal-like 1, luminal-like 2, and luminal-like 3
subtypes. Sotiriou and coworkers also re-examined data from
the study by Sorlie and coworkers  and suggested that
luminal-like breast cancer could be classified as luminal A, B,
and C subtypes corresponding to luminal-like 1, luminal-like
2, and luminal-like 3 subtypes.
Interestingly, survival analyses conducted in a subcohort of
patients with locally advanced breast cancer uniformly treated
in a prospective study showed significantly different
outcomes for the patients belonging to the various groups,
with the basal-like subtype correlating with worst outcome,
followed by ErbB2 overexpressing, normal cell type and
luminal cell type groups [12,68]. Interestingly, a significant
difference in outcome for the two ER-positive groups was
also noticed . These studies strongly support the idea
that many of the breast tumor subtypes may represent
malignancies of biologically distinct cell types producing
distinct disease entities that may require different treatment
strategies. Importantly, these analyses provide a strong
rationale for further definition of various mammary epithelial
subtypes and expansion of immortalization and full trans-
formation strategies to derive models that may faithfully
reproduce the histological
encountered in human breast cancers.
and molecular diversity
Do breast cancers arise from stem cells?
Stem cells have enormous replicative potential and capacity
for self-renewal, and give rise to different lineages of cells.
Breast Cancer Research July 2005 Vol 7 No 4 Dimri et al.
Although still a controversial notion, many cancers are
thought to originate from cancer stem cells . This idea
has also attracted a great interest in the field of breast cancer
research, and investigators have begun to examine whether
there are mammary stem cells [13,17,27,70-73]. The cellular
milieu of the mammary gland undergoes significant changes
during pregnancy, lactation, and involution. These include
bursts of proliferation of existing cells during pregnancy,
continued differentiation during lactation, and apoptosis
during involution at the end of the cycle. This cyclical
behavior predicts the presence of a stem cell-like population
in the mammary gland, which would meet the demand of a
pregnancy cycle. The existence of adult mammary epithelial
stem cells has therefore been proposed. Direct evidence for
the existence of such cells has come from clear fat-pad
transplantation, retroviral tagging, and X-chromosome
inactivation studies in rodent model [13,16,17,70-73].
Recently, using various putative stem cell and cell surface
markers, such as sialomucin (Muc), epithelial-specific antigen
(ESA), various cytokeratins, ASMA, and CALLA or CD10,
attempts have been made to identify the mouse and human
mammary epithelial stem cells [13,27,70-73]. Using immuno-
magnetic cell sorting based on surface antigen markers (Muc
and ESA) and subsequent immortalization with E6 and E7,
Gudjonsson and coworkers  separated Muc–/ESA+/
K-19+cells that were able both to self-renew and to give rise
to Muc–/ESA+epithelial cells and ASMA+myoepithelial cells,
thus exhibiting characteristic of breast stem cells. Dontu and
coworkers  isolated undifferentiated mammospheres
from single cell suspensions of HMECs obtained by
mechanical and enzymatic dissociations. Primary mammo-
spheres can be further passaged to generate secondary
mammospheres. Primary as well as secondary mammo-
spheres were highly enriched in early progenitor or stem cells
capable of differentiating along multiple lineages and of self-
renewal. Immunostaining of these mammospheres showed
the presence of CD10, α6integrin and K-5 on early
progenitors, and ESA and K-14 on late progenitor cells .
However, MUC1, K-18, and ASMA were not expressed in
cells present in mammospheres . Detailed expression
profiling of mammospheres suggests the presence of
additional markers that are upregulated in mammospheres
such as stem cell growth factor, hepatocyte growth factor
antagonist, stem cell growth factor B and apolipoprotein E.
Some markers are exclusively expressed in mammospheres
such as FZD2 (frizzled homolog 2), glypican 4, interleukin-6,
CXCR4 (CXC chemokine receptor), and FGFR1 (fibroblast
growth factor receptor 1). Several genes that are expressed
in mammospheres are also expressed in similar structures
derived from other cell types (such as neurospheres formed
by neural stem cells) .
Thus, culture of human HMECs in mammospheres may
provide a tool with which to isolate and study mammary
epithelial stem cells and their oncogenic susceptibilities.
Based on the above and other related studies [13,17,27], the
candidate mammary stem cells appear to be ESA+, MUC1–,
α6integrin+, and CD10+, and the mammary stem cell niche
appears to be at the suprabasal location within the luminal
cell layer. Further work by other laboratories and adoption of
the schemes employed by Gudjonsson  and Dontu 
and their groups should help in determining the general
feasibility of these novel approaches.
Apart from normal mammary stem cells, the possible
existence of a breast cancer stem cell has been reported in
the literature [74,75]. In a NOD/SCID xenotransplants model,
Al-Hajj and coworkers  used four cell surface markers,
CD44, CD24, ESA and B38.1 (a Breast/ovarian cancer
specific marker), and lineage markers to sort different
populations of breast cells from breast tumor tissues. All mice
injected with Lin–/CD44+/B38.1+/CD24–/low
tumors, whereas none of the mice injected with CD44–/
B38.1–cells developed tumors. Lin–/CD44+/B38.1+fractions
were further subdivided based on ESA expression. When
used in numbers as low as 200, Lin–/ESA+/CD44+/
CD24–/lowcells in xenotransplants generated tumors that
were similar to initial tumors in term of phenotypic hetero-
geneity . The presence of such a population in breast
tumor tissue, which is able to self-renew and differentiate,
supports the stem-cell model of breast tumorigenesis.
Our ability to culture and immortalize normal HMECs has
provided a wealth of knowledge about the behavior of
mammary cells and the genes involved in normal cell growth
and oncogenesis. Characterization of these cells has provided
novel markers that may permit early diagnosis and prognosis of
breast cancers, and has yielded knowledge about potential
precursor cells for breast cancers. Transformation analyses in
cell culture models have also proven important to our
understanding of the multistep nature of breast cancer.
Transgenic mouse models have identified the roles played by
various tumor suppressors, cell cycle proteins, and other proto-
oncogenes in breast cancers. Recent studies using three-
dimensional models have proven useful to our understanding of
the normal and tumor mammary stem cells and the relationship
of microenvironment to epithelial cell growth. Finally, using
gene profiling, we have begun to appreciate that breast
cancers do not originate only from luminal cells but also from
basal and myoepithelial cells, and that there are subtypes of
breast cancers that possibly originate from distinct normal
precursors that have distinct clinical outcomes and may require
different treatment strategies.
However, a number of critical questions remain. What are
breast stem cells and what is their role in breast cancer? Are
myoepithelial cells and basal cells similar or distinct? Why
can we not culture most of the primary breast cancers? How
can we develop transformed breast cells in culture that would
give rise to breast tumors that resemble human breast cancer –
Available online http://breast-cancer-research.com/contents/7/4/171
adenocarcinomas as opposed to poorly differentiated
carcinomas? How do different subtypes of breast cancer
In conclusion, experimental immortalization and trans-
formation models have led to substantial progress in our
understanding of the biology of breast cancer. Future studies
in these model systems should go a long way toward
elucidating the nature of breast cancer heterogeneity and
thus facilitate the development of more individualized
therapies for breast cancer patients.
The author(s) declare that they have no competing interests.
We apologize to many of our colleagues whose original work could not
be cited due to space constrains.
We thank past and present members of our respective laboratories for
their contribution to work published from our laboratories. Work in our
laboratories was supported by the NIH Grants CA94143, CA96844,
CA81076, and DAMD BC010093 (VB); CA 87986, CA 76118, CA
99900, CA99163, and DAMD17-02-1-0303 (HB); and CA 094150
and DAMD17-02-1-0509 (GD). VB and HB gratefully acknowledge the
support of the Duckworth Family Chair in Breast Cancer Research and
Jean Ruggles-Romoser Chair for Cancer Research, respectively.
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