Protein 4.1B suppresses prostate cancer progression
Sunny Y. Wong*†‡, Herbert Haack*§, Joseph L. Kissil¶, Marc Barry*?, Roderick T. Bronson**, Steven S. Shen*††,
Charles A. Whittaker*, Denise Crowley*, and Richard O. Hynes*†‡‡
*Howard Hughes Medical Institute, Massachusetts Institute of Technology Center for Cancer Research, Cambridge, MA 02139;†Department of Biology,
Massachusetts Institute of Technology, Cambridge, MA 02139;¶Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA 19104;
and **Department of Biomedical Sciences, Tufts School of Veterinary Medicine, North Grafton, MA 01536
Contributed by Richard O. Hynes, June 12, 2007 (sent for review April 16, 2007)
Protein 4.1B is a 4.1/ezrin/radixin/moesin domain-containing pro-
tein whose expression is frequently lost in a variety of human
tumors, including meningiomas, non-small-cell lung cancers, and
breast carcinomas. However, its potential tumor-suppressive func-
tion under in vivo conditions remains to be validated. In a screen
for genes involved with prostate cancer metastasis, we found that
4.1B expression is reduced in highly metastatic tumors. Down-
regulation of 4.1B increased the metastatic propensity of poorly
metastatic cells in an orthotopic model of prostate cancer. Further-
more, 4.1B-deficient mice displayed increased susceptibility for
developing aggressive, spontaneous prostate carcinomas. In both
cases, enhanced tumor malignancy was associated with reduced
apoptosis. Because expression of Protein 4.1B is frequently down-
of other tumor types, these results suggest a more general role for
Protein 4.1B as a negative regulator of cancer progression to
served N-terminal 4.1/ezrin/radixin/moesin domain. Many of
these proteins link transmembrane glycoproteins such as CD44
to the actin cytoskeleton and have been shown to affect numer-
ous processes, including cell polarization, migration, and prolif-
eration, among other functions (1). Based on sequence homol-
ogy, the Protein 4.1 superfamily of proteins can be further
divided into five subgroups: Protein 4.1 molecules, ezrin-radixin-
moesin (ERM) proteins, talin-related molecules, protein ty-
rosine phosphatase proteins, and novel band 4.1-like 4 (2). Given
their roles in numerous cellular processes, it is not surprising that
some members of these subgroups have also been implicated in
tumor progression. In particular, the ERM-like protein, merlin
(the product of the NF2 gene), is a critical suppressor of
meningiomas and schwannomas (3, 4), and NF2-heterozygous
mice develop a variety of spontaneous and highly metastatic
been shown to enhance metastasis of bone and soft tissue
sarcomas (6, 7).
The Protein 4.1 subgroup, of which 4.1B is a member, includes
at least three additional proteins (4.1G, 4.1N, and 4.1R), and
each member possesses N-terminal 4.1/ezrin/radixin/moesin,
spectrin-actin-binding, and C-terminal domains (2). Inter-
spersed among these highly conserved domains are three unique
regions that likely confer functional specificity to these proteins.
However, although 4.1R has been found to be a regulator of
erythroid cytoskeletal morphology (8), the precise roles of the
other Protein 4.1 subgroup proteins have thus far remained
In a screen for genes involved with prostate cancer metastasis,
we found that 4.1B was down-regulated in highly metastatic
tumor cells. Previous studies have shown that 4.1B, or a trun-
cated form of this protein (known as Deleted in Adenocarci-
noma of the Lung-1), is frequently lost in brain, lung, and breast
cancers, and that overexpression of 4.1B can inhibit the in vitro
rotein 4.1B is a member of the Protein 4.1 superfamily of
proteins, which is characterized by the presence of a con-
growth of tumor cell lines (9–14). In some cases, growth
suppression was associated with increased apoptosis. However,
these results were obtained from overexpression experiments
conducted in vitro, and the putative role of 4.1B as a tumor
suppressor in vivo has yet to be validated. In addition, 4.1B-
deficient mice are healthy and do not develop spontaneous
tumors above background levels (15).
In this study, we show that loss of 4.1B promotes metastasis in
an orthotopic xenotransplant model of prostate cancer. In
addition, by using the transgenic adenocarcinoma of the mouse
prostate (TRAMP) tumor model, we observed that 4.1B-
deficient mice developed aggressive, spontaneous carcinomas at
and that these tumors often metastasized to local lymph nodes.
In both models, loss of 4.1B was associated with reduced
apoptosis. Combined with clinical data showing that 4.1B ex-
pression is down-regulated in four independent studies of human
prostate cancer, these results provide in vivo evidence that 4.1B
acts as a negative regulator of tumor progression.
We used a technique known as surgical orthotopic implantation
cell line, into immunodeficient mice (16). SOI involves grafting
solid, s.c. tumor-derived tissue into the mouse prostate and
models many of the initial steps of metastasis, including de-
adhesion of malignant cells from the primary tumor, intravasa-
in vivo passaging of PC-3 cells using SOI yielded PC3-pMicro-1
cells, from which PC3–#78 cells and PC3–#82 cells were sub-
sequently derived, following an additional passage in vivo (re-
ferred to as pMicro-1, #78, and #82 cells, respectively) (Fig. 1A
and Materials and Methods). Further characterization of these
three cell lines using SOI revealed that #82 cells formed
orthotopic prostate tumors that displayed increased metastasis
Author contributions: S.Y.W., H.H., and R.O.H. designed research; S.Y.W., H.H., and D.C.
performed research; J.L.K. contributed new reagents/analytic tools; S.Y.W., M.B., R.T.B.,
S.S.S., C.A.W., and R.O.H. analyzed data; and S.Y.W. and R.O.H. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Abbreviations: ERM, ezrin-radixin-moesin; shRNA, short-hairpin RNA; SOI, surgical ortho-
topic implantation; TRAMP, transgenic adenocarcinoma of the mouse prostate.
Data deposition: The data reported in this paper have been deposited in the Gene
Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE7930).
‡Present address: University of California, San Francisco, CA 94158.
§Present address: Cell Signaling Technology, Danvers, MA 01893.
?Present address: Department of Pathology, Brigham and Women’s Hospital, Boston,
††Present address: Helicos BioSciences Corporation, Cambridge, MA 02139.
‡‡To whom all correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
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to the draining paraaortic/lumbar lymph nodes, to the more
distal pararenal lymph nodes, and to the lung, relative to tumors
derived from #78 cells (Fig. 1 B–D). These differences were seen
despite the fact that these cells exhibited similar rates of prolif-
eration in vitro (data not shown) and formed primary tumors of
roughly the same size [supporting information (SI) Fig. 6].
Tumors derived from pMicro-1 cells exhibited an intermediate
Given the significant differences in metastatic potential be-
tween #78 and #82 cells, we decided to perform gene expression
analyses on tumors derived from these cells. We found that
Protein 4.1B was among the genes most significantly down-
regulated in highly metastatic #82 cells (SI Fig. 7). We validated
this result at the protein level by performing Western blot
analysis for 4.1B on our derived cell lines, the original starting
population of PC-3 cells (parental) obtained from American
Type Culture Collection (Manassas, VA) (Fig. 1E), and PC-3M
cells, a highly metastatic subline independently derived by
Kozlowski et al. (18) (Fig. 1F). Consistent with our gene
expression results, 4.1B protein levels were specifically down-
regulated in highly metastatic #82 and PC-3M cells, relative to
poorly metastatic #78 and parental cells. Protein 4.1B was also
present at an intermediate level in pMicro-1 cells. However,
expression of the related proteins, 4.1G and 4.1N, was un-
changed (SI Fig. 8).
Because Protein 4.1B has been previously implicated in tumor
cell migration, adhesion, apoptosis, and growth inhibition in vitro
(9–14), we examined this gene candidate further as a potential
suppressor of prostate cancer metastasis. We began by down-
(shRNAs) against 4.1B (78-DL1–4) did not exhibit apparent
morphological or growth differences in vitro, relative to control
cells expressing either the vector only (78-pSIRISP) or an
irrelevant shRNA against GFP (78-siGFP) (data not shown).
Using SOI, we implanted the shRNA-expressing #78 cells that
had exhibited the two most effective knockdowns of 4.1B
expression (78-DL1 and 78-DL2), as well as the 78-pSIRISP and
78-siGFP control cells. Although the resulting primary tumor
masses in the prostate were roughly equivalent (SI Fig. 10), the
draining paraaortic/lumbar lymph nodes from animals im-
planted with 78-DL1 and 78-DL2 tumors were significantly
larger than nodes from mice implanted with control tumors (Fig.
2A; P ? 0.039). Subsequent analyses also revealed that, of the
two control tumors that exhibited significant lymphatic metas-
tasis in Fig. 2A, one of these tumors (derived from the 78-
pSIRISP cell line) had spontaneously down-regulated 4.1B (SI
Histological examination revealed that paraaortic/lumbar
lymph nodes from mice implanted with 78-DL1 and 78-DL2
tumors were often completely infiltrated with tumor cells (Fig.
2B; n ? 46 nodes examined). However, nodes from animals
implanted with control tumors frequently exhibited only partial,
subcapsular invasion (n ? 42 nodes examined; P ? 0.0001), and
this site was where many tumor cells appeared apoptotic (Fig. 2C
completely replaced by tumor cells (Fig. 2C Lower), as was often
the case with shRNA-expressing tumors. Mice implanted with
tumors expressing shRNAs against Protein 4.1B also developed
somewhat more lung metastases than those implanted with
control tumors, although these differences did not reach statis-
tical significance because of the limited number of animals that
had exhibited systemic spread (SI Fig. 12). Overall, these results
indicate that Protein 4.1B suppresses prostate cancer metastasis,
and down-regulation of 4.1B is sufficient to increase the meta-
static potential of poorly metastatic cells.
We next determined whether loss of Protein 4.1B could also
affect tumor progression in a spontaneous model of prostate
cancer. We obtained 4.1B knockout mice (15) and confirmed the
absence of this protein by performing Western blotting on brain
and prostate lysates from 4.1B?/?or 4.1B?/?animals (Fig. 3A).
We then crossed these animals with TRAMP mice expressing
SV40 in the prostate (19). At 26 weeks of age, palpable carci-
nomas in the prostate were observed in 11 of 19 (58%) of
4.1B?/?;TRAMP?/?mice, compared with 2 of 26 (7.7%) of
4.1B?/?;TRAMP?/?mice (Fig. 3B and Table 1; P ? 0.0002).
Using the system described by Hurwitz et al. (20), we then
performed histopathological grading on both the ventral and
dorsolateral prostate lobes for all animals (see Materials and
Methods). Sections were scored on a scale of 1 to 6 (1, normal
prostate; 6, poorly differentiated adenocarcinoma). Because
tumorigenic prostates often exhibited heterogeneity with regard
to severity, even within the same section, two grades were
assigned for each prostate lobe of each animal: a highest grade
and a predominant grade (SI Fig. 13). Although most mice,
Mass of Lumbar Lymph Nodes (mg) .
Mass of Renal Lymph Nodes (mg)
Number of Lung Micrometastases
Poorly Metastatic Highly Metastatic
#78 pMicro1 #82
p = 0.038
#78 pMicro1 #82 Parental
PC3M #78 #82
p = 0.007
p = 0.064
#78 pMicro1 #82
#78 pMicro1 #82
cation of 4.1B as a protein that is down-regulated in highly metastatic cells. (A)
Highly metastatic #82 cells and poorly metastatic #78 cells were isolated after
(B–D) #82 cells formed orthotopic primary tumors that exhibited an increased
nodes (C), and lung (D) relative to tumors from #78 cells (horizontal bar, mean).
(E) Western blotting for 4.1B (Upper) or GAPDH loading control (Lower) con-
firmed gene expression analyses indicating that 4.1B was specifically down-
regulated in highly metastatic #82 and PC-3M cells (F).
Derivation of metastatic variant prostate cancer cell lines and identifi-
Wong et al.PNAS ?
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regardless of genotype, developed histologically detectable pros-
tate cancer with a grade of 4 or higher, we invariably found that
4.1B?/?;TRAMP?/?mice developed the highest, least differen-
tiated, and most aggressive grade of prostate cancer (grade 6) at
a significantly increased frequency relative to heterozygotes
[Table 1 and Fig. 4A (P ? 0.003) and SI Fig. 14]. As was seen
in the xenotransplant model, 4.1B?/?TRAMP tumorigenic
prostates also displayed an increased propensity to metastasize
to draining lymph nodes relative to 4.1B?/?TRAMP prostates
(Table 1). The presence of metastatic tumor cells in the nodes
was confirmed by cytokeratin 8 staining (SI Fig. 15). These
results indicate that loss of Protein 4.1B promotes progression of
spontaneous prostate cancer.
We next stained prostate sections for proliferation and apo-
ptotic markers. Because tumorigenesis in the TRAMP model
begins with normal prostate tissue and increases with severity
over time, we reasoned that if proliferative or apoptotic differ-
ences were manifested in the lower graded prostatic tumors, the
overall rate of progression to grade 6 adenocarcinoma would be
affected. Therefore, we began by staining 26-week-old, grade
4-matched prostate sections with Ki67 antibody and TUNEL.
We found no differences between 4.1B?/?or 4.1B?/?TRAMP
prostates for either proliferation or apoptosis, and we also found
no differences in 26-week-old, grade 5-matched sections (SI Fig.
16). However, especially for the 4.1B?/?cohort, these samples
were biased in that many tumors had already progressed to grade
6 and could not be used for this analysis. In other words, what
had been analyzed were the remaining tumorigenic prostates
that had not progressed to the higher grades. To perform a more
unbiased study, we stained grade 4-matched prostate sections
from a younger cohort of 22-week-old TRAMP mice. Although
there was again largely no difference in proliferation (Fig. 4B;
P ? 0.15), we found that 4.1B?/?;TRAMP prostates displayed
significantly lower rates of apoptosis compared with heterozy-
gous TRAMP mice (Fig. 4 C and D; P ? 0.001). These results
suggest that loss of 4.1B protects cancerous lesions from under-
going cell death at 22 weeks, which may facilitate progression to
higher grade, less differentiated, and more invasive tumors by 26
To determine whether down-regulation of 4.1B might con-
tribute to prostate cancer progression in other experimental
systems, we examined the TRAMP-C series of cell lines, which
were derived by Foster et al. (21). Consistent with our xeno-
transplant and spontaneous tumor results, we found that 4.1B
Table 1. Effects of protein 4.1B on tumor progression in a
spontaneous model of prostate cancer
Type of cancer
Grade 6 carcinoma
Palpable grade 6 carcinoma
Lymph node metastasis
Results are given as the number affected from the total number of mice of
each genotype. Percentages are given in parentheses.
Mass of Lumbar Lymph Nodes (mg)
Average % Lymph Node Invasion
(pSIRISP + siGFP) (DL1 + DL2)
siGFP pSIRISP DL1 + DL2
p = 0.039
p < 0.0001
lymph nodes (A), and the percentage node area that had been infiltrated by tumor cells (B) were both significantly increased in mice bearing #78 orthotopic
tumors expressing DL1 and DL2 shRNAs against 4.1B relative to those bearing #78 control tumors. (C) Paraaortic/lumbar lymph nodes from mice bearing #78
tumors expressing DL1 and DL2 were often completely infiltrated by tumor cells, with few apoptotic cells present (Lower). In contrast, nodes from mice bearing
control #78 tumors (Upper) more commonly possessed areas of subcapsular invasion, where apoptotic cells were frequently observed (arrows). In most cases,
the interior regions of these nodes were relatively uninvaded (*). Representative serial lymph node sections stained either by H&E (Left) or for apoptosis (Right)
are shown at ?20 magnification.
WT KO WT TRAMP KO
(Lower) in brain or prostate tissues shows that 4.1B is specifically lost in
knockout animals. (B) 4.1B?/?;TRAMP mice more commonly developed a
(starting from Upper Left and proceeding clockwise) compound, multilobed
lobed carcinomas. These results are tabulated in Table 1.
4.1B?/?mice develop aggressive adenocarcinomas in a spontaneous
www.pnas.org?cgi?doi?10.1073?pnas.0705499104Wong et al.
protein levels were specifically reduced in malignant
TRAMP-C1 cells relative to nonmalignant TRAMP-C3 cells
(Fig. 5A). In addition, we found that 4.1B gene expression was
significantly down-regulated in four studies of human clinical
prostate cancer. For instance, Oncomine analysis of a data set
originally obtained by Lapointe et al. (22) revealed that 4.1B
expression was significantly down-regulated in prostate tumor
samples relative to normal prostate tissues (Fig. 5B; P ? 0.001)
and further down-regulated in lymph node metastases compared
with normal prostate (P ? 0.001). Oncomine analysis also
revealed three additional studies where 4.1B expression was
significantly down-regulated (P ? 0.001) in prostate cancer.
These data sets were originally collected by Welsch et al., Singh
et al., and Yu et al. (23–25) and are displayed in SI Fig. 17.
We have shown here that Protein 4.1B acts as a negative
modulator of the aggressive tumor phenotype in two different in
vivo models of prostate cancer. We found that 4.1B expression
was significantly reduced in highly metastatic human prostate
adenocarcinoma cells, and that down-regulation of 4.1B in
poorly metastatic cells was sufficient to increase their metastatic
propensity in a xenotransplant orthotopic model. Furthermore,
4.1B-deficient TRAMP mice displayed increased susceptibility
for developing invasive, undifferentiated adenocarcinomas rel-
ative to 4.1B-heterozygous TRAMP mice. In both systems,
tumorigenic cells lacking 4.1B displayed reduced apoptosis,
which may have promoted the invasive phenotype.
Our findings are concordant with in vitro studies showing that
overexpression of 4.1B can, in some cases, induce cell death (11,
remain unclear, one study recently showed that overexpression
of 4.1B increases caspase-8 activity in MCF-7 cells (14). Others
have reported that overexpression of 4.1B induces Rac1-
dependent JNK signaling (13). Recent work from our laboratory
has also shown that 4.1B can interact with a potential tumor
suppressor, integrin ?8 (26), and this interaction has been
confirmed in our PC-3 cells (data not shown).
Although 4.1B was originally identified as a protein whose
expression was reduced in human non-small-cell lung carcinomas
(9), subsequent studies have shown that down-regulation occurs
also carcinomas arising from the breast, kidney, and colon/rectum.
meningiomas (27), whereas allelic loss of region 18p11.3, where the
4.1B gene resides, was detected in 55% of ductal carcinomas in situ,
as well as in 67% of invasive and metastatic breast cancers (28). In
addition, 4.1B protein expression has been observed to be down-
regulated during tumorigenesis in spontaneous mouse models of
pancreatic and intestinal cancer (29, 30). Consistent with these
findings, we have observed that 4.1B expression is significantly
reduced in four studies of human clinical prostate cancer. We have
also found on Oncomine that 4.1B levels are significantly down-
not shown; see refs. 31 and 32). Even more interestingly, the
Oncomine database revealed that prostate tumors which overex-
pressed the Ets family transcription factor ERG exhibited signifi-
% Apoptag Positive
Grade 4 – 22 Weeks
% Ki67 Positive
Grade 4 – 22 Weeks
Highest Overall Grade
4.1B Het 4.1B KO
p < 0.001
p = 0.15
heterozygous lesions. (A) Each entire prostate was assigned a single highest grade identical to the highest grade assigned to any one constituent lobe. Please
grade 4 TRAMP ventral prostates, but 4.1B?/?TRAMP ventral prostates were significantly less apoptotic relative to grade- and age-matched 4.1B?/?TRAMP
prostates (C). (D) Representative sections stained for apoptotic cells.
Higher grade and less differentiated 4.1B?/?;TRAMP prostate carcinomas likely arise from tumorigenic lesions that exhibit less apoptosis than those of
Wong et al. PNAS ?
July 31, 2007 ?
vol. 104 ?
no. 31 ?
overexpress Ets family transcription factors (SI Fig. 18; P ? 0.001;
see ref. 22). Because recent studies have shown that the genes
encoding ERG and another Ets family transcription factor, ETV1,
are translocated and, consequently, overexpressed in as many as
80% of human prostate cancers, it is important in future work to
determine whether these genes are inversely coregulated (33).
Because Protein 4.1B has been frequently observed to be lost
these findings have led some to propose that loss of this protein
is a critical early step for tumorigenesis (27, 28). However, as one
study examining breast cancer has noted, there were also in-
stances where the chromosomal region 18p11.3 was specifically
lost in invasive tumor foci, but not in regions of ductal carcino-
mas in situ in samples derived from the same patients (28). These
results suggest that down-regulation of 4.1B during either the
early or later stages of tumorigenesis can promote cancer
progression. Our parental PC-3 prostate cells expressed high
levels of 4.1B and loss of this protein was only observed in
aggressive variant cells (PC3–#82 and PC-3M) derived from this
original starting population, suggesting that down-regulation of
4.1B enhanced the later stages of tumor progression to meta-
static disease. Similarly, because genetic ablation of 4.1B in mice
did not increase their predisposition to spontaneous tumor
formation (15), but instead promoted the progression of pros-
tatic tumor lesions already initiated by SV40, this observation
again suggests that loss of 4.1B is important during the later
stages of prostate tumorigenesis. In addition, as we have shown
with the gene expression data set obtained from Lapointe et al.
(22), prostate tumors might down-regulate 4.1B expression
relative to normal tissue early on and then further down-regulate
expression during progression to metastatic disease. Whether
4.1B expression is also correlated with other clinical parameters
for assessing prostate cancer severity, such as Gleason grade,
remains to be seen.
In summary, our results obtained from several independently
derived prostate cancer cell lines, two different in vivo tumor
models, and four human clinical studies of prostate cancer
together suggest that down-regulation of 4.1B is a frequent and
aggressive tumor phenotype. Our findings offer in vivo evidence
that a member of the Protein 4.1 subgroup can act as a
suppressor of tumor progression. Because protein 4.1B expres-
sion is down-regulated in a variety of human cancers, in addition
to those arising from the prostate, these findings suggest that
4.1B may serve a more general role as a negative modulator of
malignancy across a spectrum of tumor cell types.
Materials and Methods
Cells. All derivatives of the human cell line PC-3 (American Type
Culture Collection) were cultured as previously described (17,
34). The metastatic variant cell lines were derived by passaging
PC-3 cells in vivo: The cells were grafted by SOI into the prostate
and allowed to form palpable tumors over a period of 2 to 3
months, after which metastatic cells were isolated from lung
tissues physically dissociated in culture. These cells were ex-
panded in vitro and then reimplanted into mice for additional
rounds of in vivo passage. Implantation of PC-3 cells originally
yielded the pMicro-1 cell line, and subsequent reimplantation of
pMicro-1 cells yielded the PC3–#78 and PC3–#82 cell lines,
which were isolated from separately grafted mice. TRAMP-C1
and -C3 cells (American Type Culture Collection) were cultured
as previously described (21). PC-3M cells were obtained from
I. J. Fidler (M. D. Anderson Cancer Center, Houston, TX) (18).
Plasmids and Antibodies. shRNA sequences were cloned into the
retroviral vector, pSIRISP (W. C. Hahn, Dana–Farber Cancer
Institute, Boston, MA) (35). The plasmids were transfected into
Phoenix cells (American Tissue Culture Collection), and viral
supernatant was used to stably infect PC3–#78 cells expressing
ecotropic receptor (H. Lodish, Massachusetts Institute of Tech-
nology). See SI Materials and Methods for shRNA sequences.
Antibodies for Western blot included rabbit anti-4.1B (26), as
well as rabbit anti-4.1G and anti-4.1N (Protein Express, Chiba,
Japan). Total soluble protein was extracted from frozen tumors
or cells/tissues by using lysis buffer containing Nonidet 40 and
protease inhibitors (Roche, Mannheim, Germany). For immu-
nohistochemistry, zinc- (Becton Dickinson, San Jose, CA) or
formalin-fixed sections were stained with rat anti-cytokeratin 8
(TROMA-1; Developmental Studies Hybridoma Bank, Univer-
sity of Iowa, Iowa City, IA) (36).
Orthotopic Xenograft and Spontaneous Mouse Tumor Models. SOI of
Briefly, cells were injected as s.c. tumors and harvested ?3.5
weeks later to obtain graft material for SOI. The procedure was
performed in accordance with the Massachusetts Institute of
Technology Division of Comparative Medicine animal care
guidelines. Between 2 and 3 months after implantation, mice
were analyzed when moribund. Lymph nodes were removed,
fixed in formalin, and later weighed and sectioned. Lymph node
mass was recorded as the combined fixed mass of all nodes from
a particular site (paraaortic or renal lymph node site) for each
animal. Combined lymph node masses that were ?30 mg could
not be reliably weighed and were given a mass of 10 mg. To
determine percent metastatic invasion of lymph nodes, two
sections at different levels were obtained for each node, percent
invasion was scored blindly by a pathologist (R.T.B.), and the
higher of the two scores was recorded for every individual node.
For spontaneous tumor studies, 4.1B?/?animals were back-
crossed into a C57BL/6 background for two to three generations.
These mice were crossed with TRAMP?/?mice in a pure
C57BL/6 background. In TRAMP studies, individual prostate
lobes were sectioned and blindly graded by a pathologist (M.B.)
(20). A highest grade was assigned based on the area of greatest
pathological severity. A predominant grade was assigned based
Normal Prostate Nodal
Prostate Cancer Metastases
4.1B - Normalized Expression Units
P < 0.001 (Normal vs. Cancer)
P < 0.05 (Cancer vs. Metastases)
P << 0.001 (Normal vs. Metastases)
C1 C3 C1 C3
human clinical prostate cancer. (A) 4.1B protein levels are reduced in malig-
nant TRAMP-C1 cells (C1) relative to nonmalignant TRAMP-C3 cells (C3);
independent replicate samples are shown. (B) 4.1B expression is significantly
down-regulated during human clinical prostate cancer progression, from
normal prostate tissue, to prostate cancer, and finally to prostate cancer
lymph node metastases [data obtained by Lapointe et al. (22) and processed
by Oncomine 3.0]. Three additional human clinical prostate cancer studies
displaying down-regulation of 4.1B can be found in SI Fig. 17.
4.1B is down-regulated in aggressive TRAMP tumor cell lines and in
www.pnas.org?cgi?doi?10.1073?pnas.0705499104Wong et al.