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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on the most common grade seen in each section. See SI Fig. 13
for an example of this scoring approach.
Proliferation and Apoptosis. Zinc-fixed prostate lobes from
TRAMP mice and formalin-fixed lymph nodes from mice im-
St. Louis, MO) for proliferation and/or by TUNEL for apoptosis
titation was performed on grade- and age-matched sections (22
or 26 weeks old) by counting the number of positively stained
per section imaged at ?20 magnification, except for grade 5
sections, which were imaged at ?40 magnification. Grade 4
sections were all derived from the ventral lobes of the prostate,
whereas grade 5 sections were all derived from dorsolateral
(Improvision, Lexington, MA), and quantitation were blindly
performed by using four to five independent prostate samples
Microarrays and Bioinformatics.Forgeneexpressionanalyses,total
RNA was extracted by RNeasy Midi kit (Qiagen, Valencia, CA)
to human U133A chips (Affymetrix, Santa Clara, CA). Data
were analyzed by dChip software (Harvard Medical School,
Boston, MA) (37) and Gene Pattern (The Broad Institute,
Cambridge, MA) (38). The entire MIAME-compliant data set
can be downloaded from National Center for Biotechnology
Information GEO (www.ncbi.nlm.nih.gov/geo/index.cgi), acces-
sion no. GSE7930. Oncomine analyses were performed August
2006 by using Oncomine 3.0 (www.oncomine.org) (39).
Statistics. Statistics were performed by using an unpaired Stu-
dent’s t test (www.physics.csbsju.edu/stats/Index.html), except in
the case of TRAMP tumor incidence data, which were assessed
by chi square (www.psych.ku.edu/preacher/chisq/chisq.htm). All
error bars shown are standard error.
We thank Dr. J.H. McCarty (M. D. Anderson Cancer Center, Houston,
TX) for anti-4.1B antibodies; the Developmental Studies Hybridoma
Bank (University of Iowa, Iowa City, IA) for anti-cytokeratin 8 anti-
bodies; Dr. A. Bai (Massachusetts Institute of Technology) for TRAMP
mice; the Massachusetts Institute of Technology BioMicro Center; and
the Massachusetts Institute of Technology Division of Comparative
Medicine. This work was supported by National Institutes of Health
Grant R01CA17007 (to R.O.H.); the Virginia and D.K. Ludwig Fund for
Cancer Research; the Prostate Cancer Foundation; National Cancer
Institute’s Integrative Cancer Biology Program Grant U54-CA112967
(to R.O.H.); the Howard Hughes Medical Institute, of which R.O.H. is
an Investigator; a National Institute of General Medical Sciences Pre-
doctoral Training Grant (to S.Y.W.); and a David H. Koch Research
Fellowship from the Center for Cancer Research.
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