pRb inactivation in mammary cells reveals common mechanisms for tumor initiation and progression in divergent epithelia.

Abstract

Retinoblastoma 1 (pRb) and the related pocket proteins, retinoblastoma-like 1 (p107) and retinoblastoma-like 2 (p130) (pRb(f), collectively), play a pivotal role in regulating eukaryotic cell cycle progression, apoptosis, and terminal differentiation. While aberrations in the pRb-signaling pathway are common in human cancers, the consequence of pRb(f) loss in the mammary gland has not been directly assayed in vivo. We reported previously that inactivating these critical cell cycle regulators in divergent cell types, either brain epithelium or astrocytes, abrogates the cell cycle restriction point, leading to increased cell proliferation and apoptosis, and predisposing to cancer. Here we report that mouse mammary epithelium is similar in its requirements for pRb(f) function; Rb(f) inactivation by T(121), a fragment of SV40 T antigen that binds to and inactivates pRb(f) proteins, increases proliferation and apoptosis. Mammary adenocarcinomas form within 16 mo. Most apoptosis is regulated by p53, which has no impact on proliferation, and heterozygosity for a p53 null allele significantly shortens tumor latency. Most tumors in p53 heterozygous mice undergo loss of the wild-type p53 allele. We show that the mechanism of p53 loss of heterozygosity is not simply the consequence of Chromosome 11 aneuploidy and further that chromosomal instability subsequent to p53 loss is minimal. The mechanisms for pRb and p53 tumor suppression in the epithelia of two distinct tissues, mammary gland and brain, are indistinguishable. Further, this study has produced a highly penetrant breast cancer model based on aberrations commonly observed in the human disease.

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pRb Inactivation in Mammary Cells Reveals
Common Mechanisms for Tumor Initiation
and Progression in Divergent Epithelia
Karl Simin1, Hua Wu1¤, Lucy Lu1, Dan Pinkel2, Donna Albertson2, Robert D. Cardiff3, Terry Van Dyke1*
1 Department of Genetics, Lineberger Comprehensive Cancer Center, The University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of
America, 2 Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, United States of America, 3 Center for Comparative Medicine,
University of California, Davis, Davis, California, United States of America
Retinoblastoma 1 (pRb) and the related pocket proteins, retinoblastoma-like 1 (p107) and retinoblastoma-like 2 (p130)
(pRbf, collectively), play a pivotal role in regulating eukaryotic cell cycle progression, apoptosis, and terminal
differentiation. While aberrations in the pRb-signaling pathway are common in human cancers, the consequence of
pRbfloss in the mammary gland has not been directly assayed in vivo. We reported previously that inactivating these
critical cell cycle regulators in divergent cell types, either brain epithelium or astrocytes, abrogates the cell cycle
restriction point, leading to increased cell proliferation and apoptosis, and predisposing to cancer. Here we report that
mouse mammary epithelium is similar in its requirements for pRbffunction; Rbfinactivation by T121, a fragment of
SV40 T antigen that binds to and inactivates pRbf proteins, increases proliferation and apoptosis. Mammary
adenocarcinomas form within 16 mo. Most apoptosis is regulated by p53, which has no impact on proliferation, and
heterozygosity for a p53 null allele significantly shortens tumor latency. Most tumors in p53 heterozygous mice
undergo loss of the wild-type p53 allele. We show that the mechanism of p53 loss of heterozygosity is not simply the
consequence of Chromosome 11 aneuploidy and further that chromosomal instability subsequent to p53 loss is
minimal. The mechanisms for pRb and p53 tumor suppression in the epithelia of two distinct tissues, mammary gland
and brain, are indistinguishable. Further, this study has produced a highly penetrant breast cancer model based on
aberrations commonly observed in the human disease.
Introduction
Aberrant retinoblastoma 1 (pRb) pathway activity, resulting
from defects in pRb itself, cyclin-dependent kinase inhibitor
2A (p16INK4a), cyclin D1 (CCND1), or cyclin-dependent kinase
4 (CDK4), is observed in the majority of human sporadic
cancers (Marshall 1991; Weinberg 1995; Sherr 1996; Ortega et
al. 2002). This pathway is commonly altered early in cancer
development, indicating an ability to predispose cells to
tumorigenesis. However, whether the mechanism(s) is similar
among cell types is not known. Examination of pRb
inactivation in specific cell types in vivo has been technically
challenging due to the apparent functional compensation or
redundancy among pRb, retinoblastoma-like 1 (p107), and
retinoblastoma-like 2 (p130) in many cell types of the mouse
(Luo et al. 1998; Robanus-Maandag et al. 1998; Dannenberg et
al. 2000; Sage et al. 2000). Thus, genetic inactivation of the Rb
gene alone, either by conditional deletion (Marino et al. 2000)
or by the generation of chimeric mice harboring pRb-
deficient cells (Maandag et al. 1994; Williams et al. 1994)
yields only medulloblastomas, pituitary, and thyroid tumors.
We have begun to systematically examine the role of
retinoblastoma protein family (pRbf) inactivation in multiple
cell types of the mouse by dominant expression of T121, a
truncation mutant of simian virus 40 (SV40) T antigen that
inactivates all three pRb-related proteins (DeCaprio et al.
1989; Dyson et al. 1989; Ewen et al. 1989; Stubdal et al. 1997;
Sullivan et al. 2000). In this report we determine the role of
pRb inactivation in mammary adenocarcinoma predisposi-
tion, establish a role for p53 inactivation in subsequent
mammary adenocarcinoma progression, and, together with
our previous studies, provide a comprehensive comparison of
these mechanisms in distinct epithelial lineages.
pRb plays a critical role in eukaryotic cell cycle progres-
sion, when cells exit G0 or G1 and enter S phase, thereby
acting as a crucial negative regulator of cellular proliferation
and neoplasia (Sherr and McCormick 2002). In quiescent or
early G1-phase cells, pRb is hypophosphorylated and asso-
ciates with specific members of the E2F transcription factor
family, converting them to active transcriptional repressors
(Hamel et al. 1992; Weintraub et al. 1992). Gene repression is
Received September 19, 2003; Accepted December 10, 2003; Published
February 17, 2004
DOI: 10.1371/journal.pbio.0020022
Copyright: ? 2004 Simin et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Abbreviations: aCGH, microarray comparative genomic hybridization; BAC,
bacterial artificial chromosome; Brca1, breast cancer 1; cCGH, chromosome
comparative genomic hybridization; CCND1, cyclin D1; CCNE1, cyclin E1; CDK2,
cyclin-dependent kinase 2; CDK4, cyclin-dependent kinase 4; CDK6, cyclin-
dependent kinase 6; CGH, comparative genomic hybridization; c-myc, myelocyto-
matosis oncogene; LOH, loss of heterozygosity; MMTV, murine mammary tumor
virus; p16INK4a, cyclin-dependent kinase inhibitor 2A; PCNA, proliferating cell
nuclear antigen; pRb, retinoblastoma 1; pRbf, retinoblastoma protein family; PTEN,
phosphatase and tensin homolog; SV40, simian virus 40; v-erb-b2, erythroblastic
leukemia viral oncogene homolog 2; v-Ha-ras, Harvey rat sarcoma viral oncogene;
WAP, whey acidic protein; Wnt-1, wingless-related MMTV integration site 1
Academic Editor: Chris Marshall, Institute for Cancer Research
*To whom correspondence should be addressed. E-mail: tvdlab@med.unc.edu
¤Current address: Phenomix Corporation, La Jolla, California, United States of
America
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P PL Lo oS S BIOLOGY

Page 2

also mediated by pRb and p130 recruitment of histone
deacetylase to promote formation of inhibitory nucleosomes
(Brehm et al. 1998; Luo et al. 1998; Magnaghi-Jaulin et al.
1998). The many proteins found in association with pRb
suggest other regulatory mechanisms may also be involved
(Morris and Dyson 2001), although the biological potential
for most of these interactions remains yet unproven. Cell
cycle progression from G to S phase occurs when complexes
of D-type cyclins/CDK4/CDK6 phosphorylate pRb, thereby
derepressing E2Fs to direct transcription of DNA-replication
machinery and nucleotide biosynthesis genes (Dyson 1998).
Like most human solid tumors, breast cancers harbor
frequent alterations in the pRb pathway, including CCND1
overexpression in 45% (Buckley et al. 1993), p16INK4Aloss in
49% (Geradts and Wilson 1996), and pRb loss in 6% of breast
tumors (Geradts and Wilson 1996). In the Rb-deficient mouse
mammary gland, p107 and/or p130 may play overlapping or
compensatory roles, as they do during embryonic develop-
ment, given that pRb is dispensable for normal mammary
development and mammary tumor suppression. pRb-defi-
cient embryonic stem cells participate in normal mammary
gland formation in chimeric mice (Maandag et al. 1994), and
donor pRb?/?mammary precursor cells transplanted into
wild-type mice can populate a normal mammary gland
without evidence of neoplasia, even after multiple pregnan-
cies (Robinson et al. 2001).
The interplay between pRb signaling and the tumor
protein p53 pathway is also critical to the understanding of
breast cancer biology. Since the pRb pathway is defective in a
majority of human tumors and the p53 gene is mutated in
about half of them, including approximately a fifth of
sporadic breast cancers (Nigro et al. 1989; Greenblatt et al.
1994), these aberrations often coexist. Whether loss of these
tumor suppressor pathways collaborate in tumorigenesis is
also cell type-specific. In a brain epithelial tumor model, we
previously demonstrated that, in the absence of pRbf
function, inactivation of p53 significantly decreases apoptosis
and accelerates tumor growth in vivo (Symonds et al. 1994).
However, in astrocytic brain tumors induced by pRbf
inactivation, tumor progression is not accelerated by reduced
p53 activity; rather, the phosphatase and tensin homolog
(PTEN) regulates the apoptosis, and reduction in its function
accelerates tumor growth (Xiao et al. 2002).
In this report, we extend our analysis of pRb function in
vivo and examine the consequence of pRbfloss specifically in
mammary epithelium. These studies serve not only to provide
insight into the cell specificity of tumor suppression
mechanisms, but also to model the stepwise evolution of
breast adenocarcinomas that harbor defects in this pathway.
Results
Generation of Mice with Inducible pRbfDeficiency in
Mammary Cells
Seven founder mice were generated in which the T121gene
was regulated by the whey acidic protein (WAP) transcrip-
tional signals (Figure 1; see Materials and Methods). Of these,
two founder animals died spontaneously of unknown causes,
while the transgenic progeny of the third line died
prematurely, also of unknown cause (Figure 2A). The extent
to which the transgene contributed to these deaths was not
investigated further; however, ectopic transgene expression
was detected in several tissues (data not shown). Character-
ization of female mice of the four remaining lines is the focus
of this report.
T121Is Expressed in Lactating Mammary
Western immunoblotting analyses of mammary gland
extracts demonstrated that this tissue expresses T121protein
at the expected size in all four lines (Figure 2B). T121
expression in lines 1 and 2 was only revealed following
immunoprecipitation using an anti-T-antigen antibody prior
to Western blot analysis, indicating lower levels of T121(right
panel in Figure 2B). A survey of select tissues showed that
detectable expression was restricted to the mammary gland in
lines 1–3, while expression was more widespread in the higher
expressing line 4 (data not shown) and included brain and
kidney expression. As expected, T121expression was induced
by lactation with highest levels observed 5 d postpartum
(Figure 2C). Southern blot analyses indicate that mice in line
3, which was used as a representative line for extensive
characterization, harbor approximately ten copies of the
transgene at a single insertion site (data not shown).
Impact of RbfInactivation in Mammary Epithelium
Representative histological analysis of lactating mammary
glands (day 1) from single-pregnancy females of the line 2
founder (F0) and a line 3 F1mouse shows that the impact of
Rb perturbation is severalfold. Compared to an age- and
parity-matched control tissue, the normal architecture of the
lactating mammary tissue is disturbed. In contrast to normal
tissue where acini consist of a single layer of secretory
epithelia with milk-filled lumen (Figure 3A), transgenic
animals have a lower density of acini (Figure 3K), consistent
with atrophy, and are often atypical (Figure 3I). T121-positive
mammary epithelial cells were associated with abnormalities
(Figure 3B, 3F, and 3J). The line 2 F0animal was mosaic for
Figure 1. Diagram of the WAP-T121Transgene and Protein
The fragment consists of the 2.4 kb WAP promoter (hatched) and the
mutant SV40 T-antigen coding region (white box) containing two
deletions, the 196-bp amino-terminal deletion, which abolishes small
t antigen production, and the dl1137 deletion, which truncates T
antigen. Both the J domain and the LXCXE domain are required for
pRb family inactivation (see Materials and Methods).
DOI: 10.1371/journal.pbio.0020022.g001
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pRb and p53 in Mammary Tumor Suppression

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T121protein expression with distinct regions of expressing
and nonexpressing cells (Figure 3F), whereas T121expression
in the line 3 animal was in secretory epithelium distributed
throughout the gland (Figure 3J). Increased proliferation,
indicated by proliferating cell nuclear antigen (PCNA)
staining, was also observed in transgenic mammary glands
(Figure 3C, 3G, and 3K), concomitant with increased levels of
apoptosis assayed by TUNEL staining (Figure 3D, 3H, and 3L).
Quantification of T121 expression and apoptosis revealed
higher protein expression levels (see Figure 2B) correlate with
higher percentages of apoptotic cells (Figure 4A). Consistent
with a model for cell-autonomous functioning of T121, the
pattern of abnormalities of morphology, proliferation, and
apoptosis in the mosaic animal mimicked the regionalized
T121expression pattern, and conversely, where T121protein
was absent, the tissue appeared normal.
Figure 3. Mammary-Specific Inactivation
of the pRb Pathway Induces Extensive
Abnormalities
Histologic comparisons of nontransgen-
ic (A–D), mosaic (F0line 2 [E–H]), and
transgenic (F1, line 3 [I–L]) lactating
mammary glands reveals that T121 ex-
pression results in increased prolifera-
tion and apoptosis. Hemotoxylin and
eosin staining shows acini of the normal
lactating gland are composed of a single
layer of secretory epithelial cells (A) with
milk-filled lumen. Consistent with atro-
phy, transgenic animals have a lower
density of acini demonstrated by the
presence of lipid-filled adipocytes (aster-
isk in [K]). Acini composed of T121-
expressing cells are atypical. Many are
collapsed and composed of tall columnar
epithelia of large hyperchromatic cells
with papillary tufting (arrows in [I]).
Transgene-expressing cells have large
pleomorphic nuclei (open arrows in
[G]) as compared to nuclei of nonexpressing cells (arrows in [G]). Staining for T121expression (blue in [B]–[J]) indicates the line 2 F0animal
is mosaic, showing localized expression (F), whereas the transgene expresses throughout the gland of an F1 line 3 animal (J). Increased
proliferation assayed by PCNA staining (red) is also localized in the mosaic founder (G), but found throughout the F1transgenic gland (K).
Similarly, TUNEL staining (brown) demonstrates increased apoptosis in transgenic animals (H and L); moreover, the regionalized apoptosis in
the mosaic gland (H) strongly suggests that transgene expression and not precocious involution is the cause. All samples are from primiparous
females on lactation day 1.
DOI: 10.1371/journal.pbio.0020022.g003
Figure 2. Expression of T121 Protein in
WAP-T121Mice and a Summary of Gross
Phenotypes
As expected, each line showed mam-
mary-specific expression following lacta-
tion induction, while line 4 showed more
widespread expression, with protein de-
tected in brain and kidney. Mice from
the higher-expressing lines 3 and 4 failed
to nurse because of lactation defects.
Mammary glands of adult female mice
from all four lines showed elevated
proliferation and apoptosis. Glands from
line 1 and 2 mice were hyperplastic,
while glands from lines 3 and 4 were
atrophic. Lines 3 and 4 later developed
carcinomas and other neoplasms. T121
protein was detected by Western blot
analysis in lactating mammary glands of
animals from all four lines (B), although
the lower-expressing lines 1 and 2
required immunoprecipitation with
anti-T-antigen antibody prior to West-
ern blot analysis (right panel in [B]).
Brain tumor extract (see Materials and
Methods) was used for a positive control,
and nontransgenic mammary tissue extract was used for a negative control. A timecourse analysis of T121expression (C) shows lactation-induced
expression peaking at 5 d postpartum.
Abbreviations: Adeno-Ca, adenocarcinoma; AP, elevated apoptosis in mammary gland; At, atrophy; dpc, postcoital; FTN, failure to nurse; Hyp,
hyperplastic acini; MG, mammary gland; MIN, mammary epithelia neoplasia; ND, not determined; nt, nontransgenic; pp, postpartum; Pr,
elevated proliferation in mammary gland; pw, post-weaning.
Footnotes:aMosaic founder animal.bAt earlier stages, development defects attributed to atrophy, while MIN and adenocarcinoma were observed
at terminal stages.cApproximately half of progeny died of unknown cause.
DOI: 10.1371/journal.pbio.0020022.g002
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pRb and p53 in Mammary Tumor Suppression

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Role of p53 in Apoptosis
To investigate the impact of germline loss of p53 on
apoptosis levels in Rbf-deficient mammary glands, we mated
line 3 animals to p53 null mice to generate transgenic and
nontransgenic females of distinct p53 genotypes (þ/þ,þ/?,?/?).
Transgene expression was induced by a single pregnancy, and
mammary glands were examined on lactation day 1. As
expected, nontransgenic mammary glands showed no appre-
ciable apoptosis regardless of p53 status (Figure 4B). However,
in transgenic animals, decreased levels of p53 activity were
correlated with lower levels of apoptosis. The mean percent-
age of apoptotic cells in p53 wild-type transgenic glands was
21%; in p53 heterozygous animals, 9%;and in p53 null
animals, 5% (Figure 4B), indicating that 75% of the apoptosis
is p53-dependent. That we could detect haploinsufficiency of
p53 for apoptosis is remarkable, since in the previously
characterized T121-expressing choroid plexus epithelium,
apoptosis levels were the same in p53 heterozygous and
wild-type backgrounds (Lu et al. 2001). This observation
indicates that there is a threshold for p53 levels in eliciting
apoptosis and that either the threshold is different between
cell types or that the absolute functional p53 level is distinct.
Such differences could have significant impact on the
requirements for tumorigenesis.
Figure 4. Reduced p53 Activity Decreases
Apoptosis but Does Not Increase Prolifer-
ation
Representative apoptosis levels of each
mouse line correlate with T121 expres-
sion as indicated by the percentage of
TUNEL positive cells (A). Decreasing
levels of p53 activity correlate with lower
levels of apoptosis in transgenic mam-
mary glands (B). The mean percentage of
apoptotic cells in p53 wild-type trans-
genic glands was 21%; in p53 hetero-
zygous animals, 9%; and in p53 null
animals, 5% (B), indicating that 75% of
the apoptosis is p53-dependent. Apop-
tosis levels are further reduced to 2% in
terminal stage tumors (B, Tumors). The
percentage of PCNA staining cells re-
mains unchanged in p53 heterozygous or
nullizygous animals (C), indicating that
reduction of p53 activity levels had no
significant impact on cell proliferation.
Samples were derived from primiparous
animals on lactation day 1, except as
indicated as tumor samples (B). Trans-
genic animals in (B) and (C) were from
line 3.
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Role of p53 in Proliferation
In two other transgenic mouse models of breast cancer,
where tumors were initiated by activated Harvey rat sarcoma
viral oncogene homolog (v-Ha-ras) (Hundley et al. 1997) or
wingless-related murine mammary tumor virus (MMTV)
integration site 1 (Wnt-1) (Donehower et al. 1995), inactiva-
tion of p53 did not result in a reduction of apoptosis; rather,
loss of p53 was associated with increased proliferation of the
mammary epithelium. To determine whether p53 inactiva-
tion also impacted mammary cell proliferation induced by
Rbfinactivation, glands from primiparous lactating (day 1)
mice were assessed for the expression of nuclear PCNA.
Unlike the tumors initiated by activated Ras or Wnt-1, p53
heterozygosity or nullizygosity had no significant impact on
the level of cell proliferation (Figure 4C). This experiment
indicates that p53 can have distinct mechanisms of action
depending on the nature of the initiating lesion.
pRb Inactivation Predisposes to Tumorigenesis
All females from higher-expressing lines (lines 3 and 4)
failed to nurse pups because of lactation defects and
developed mammary tumors after multiple pregnancies.
Because line 4 mice expressed T121in nonmammary tissues,
further characterization focused on line 3. For this line, the
median time following initial transgene induction until a
palpable tumor appeared was 10 mo, and within 16 mo, all
mice developed palpable tumors (Figure 5A). Interestingly,
latency in this line on a BALB/cJ background (see Materials
and Methods) was reduced to a median time of 8.5 mo (p =
0.0077; Figure 5A) indicating the presence of modifier alleles.
The condensed timeframe for tumor development in this
strain will also be valuable for future preclinical studies using
this model. However, all further studies in the current report
were carried out on the original B6D2F1 background.
The median onset for mammary tumors in line 4 was 14 mo
(n = 3; data not shown), which indicates that the transgene
and not its insertion caused tumorigenesis. With two
exceptions, line 3 WAP-T121mice, regardless of p53 status,
developed a single palpable tumor (87% of p53þ/þ, n = 15;
78% of p53þ/?, n = 9). A single mouse with either two or three
palpable tumors was also observed in both p53 þ/þ and þ/?
backgrounds. At least one additional nonpalpable tumor was
visible during necropsy in approximately one-third of all
tumor-bearing mice. While the two lower-expressing lines,
lines 1 and 2, were able to nurse pups and appeared grossly
normal, both had hyperplastic lobular alveoli associated with
increased levels of proliferation and apoptosis. However,
females from low-expressing lines did not develop adenocar-
cinomas after at least four pregnancies and 20 mo of age (line
1, n = 2; line 2, n = 6) (data not shown).
Most terminal stage tumors in either wild-type or p53þ/?
backgrounds were adenocarcinomas (Figure 6A, 6B, and 6E);
however, we also observed four pilar tumors (Figure 6C and
6E) and one spindle cell carcinoma (Figure 6D and 6E).
Terminal-stage mammary adenocarcinomas resembled
poorly to moderately differentiated invasive ductal adeno-
carcinoma in humans. Morphologically, we designate these
tumors as mixed solid and glandular carcinomas with
necrosis and fibrosis. Poorly differentiated solid tumors
(Figure 6A) are composed of nests of epithelial cells with
large pleomorphic nuclei and delicate chromatin patterns
with inverted nuclear:cytoplasmic ratios, while glandular
tumors (Figure 6B) are composed of irregular glands with
varying degrees of differentiation. While most animals had a
single tumor mass, the adenocarcinomas were multifocal,
with solid tumors consisting of subclones of distinct expansile
masses, and with only two exceptions, glandular tumors were
coincident with solid tumors. The adenocarcinomas were
malignant, infiltrating dense, fibrous connective tissue, and
were accompanied by strong peripheral immune response
(Figure 6A).
Mammary Tumor Onset and Growth Are Accelerated by
p53 Reduction
Since 75% of the apoptosis induced by Rbfinactivation was
mediated by p53 and was indeed reduced even in p53þ/?mice,
we investigated the impact of p53 loss on tumor onset and
growth kinetics. Animals harboring either one or two p53 null
alleles were monitored for mammary tumors. As expected, a
Figure 5. Mammary Tumor Onset and Growth Are Accelerated by p53
Reduction
Among line 3 animals, the median time following initial transgene
induction until a palpable tumor appeared was 10 mo, and within 16
mo, all mice developed palpable tumors (red line in [A]). In p53þ/?
transgenic animals (blue line in [A]), mammary tumors were detected
significantly earlier (p , 0.0003) with a median onset of 6 mo. Among
mice with BALB/cJ background (black line in [A]), median mammary
tumor latency (8.5 mo) was significantly shorter (p = 0.0077)
compared to mice of the hybrid BDF1 background strain and
indistinguishable (p = 0.2466) from WAP-T121;p53þ/?mice. Once
palpable, WAP-T121;p53þ/?tumors grew faster than the p53 wild-type
counterparts (B). The average growth rates for p53þ/þ(black solid) and
p53þ/?(dashed) are indicated.
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pRb and p53 in Mammary Tumor Suppression