Tumor-specific silencing of COPZ2 gene encoding coatomer protein complex subunit ζ 2 renders tumor cells dependent on its paralogous gene COPZ1.

Abstract

Anticancer drugs are effective against tumors that depend on the molecular target of the drug. Known targets of cytotoxic anticancer drugs are involved in cell proliferation; drugs acting on such targets are ineffective against nonproliferating tumor cells, survival of which leads to eventual therapy failure. Function-based genomic screening identified the coatomer protein complex ζ1 (COPZ1) gene as essential for different tumor cell types but not for normal cells. COPZ1 encodes a subunit of coatomer protein complex 1 (COPI) involved in intracellular traffic and autophagy. The knockdown of COPZ1, but not of COPZ2 encoding isoform coatomer protein complex ζ2, caused Golgi apparatus collapse, blocked autophagy, and induced apoptosis in both proliferating and nondividing tumor cells. In contrast, inhibition of normal cell growth required simultaneous knockdown of both COPZ1 and COPZ2. COPZ2 (but not COPZ1) was down-regulated in the majority of tumor cell lines and in clinical samples of different cancer types. Reexpression of COPZ2 protected tumor cells from killing by COPZ1 knockdown, indicating that tumor cell dependence on COPZ1 is the result of COPZ2 silencing. COPZ2 displays no tumor-suppressive activities, but it harbors microRNA 152, which is silenced in tumor cells concurrently with COPZ2 and acts as a tumor suppressor in vitro and in vivo. Silencing of microRNA 152 in different cancers and the ensuing down-regulation of its host gene COPZ2 offer a therapeutic opportunity for proliferation-independent selective killing of tumor cells by COPZ1-targeting agents.

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Tumor-specific silencing of COPZ2 gene encoding
coatomer protein complex subunit ζ2 renders tumor
cells dependent on its paralogous gene COPZ1
Michael Shtutmana,b,1, Mirza Baiga, Elina Levinaa, Gregory Hurteaua, Chang-uk Lima,b, Eugenia Broudea,b,
Mikhail Nikiforovc, Timothy T. Harkinsd, C. Steven Carmacke, Ye Dinge, Felix Wielandf, Ralph Buttyana,
and Igor B. Roninsona,b,1
aCancer Center, Ordway Research Institute, Albany, NY 12208;bTranslational Cancer Therapeutics Program, Department of Pharmaceutical and Biomedical
Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208;cDepartment of Cell Stress Biology, Roswell Park Cancer
Institute, Buffalo, NY 14263;d454 Life Sciences, a Roche Company, Branford, CT 06405;eWadsworth Center, New York State Department of Health,
Albany, NY 12208; andfBiochemie Zentrum Heidelberg, Ruprecht-Karls-University, D-69120 Heidelberg, Germany
Edited* by George R. Stark, Lerner Research Institute NE2, Cleveland, OH, and approved June 14, 2011 (received for review March 9, 2011)
Anticancer drugs are effective against tumors that depend on the
moleculartargetofthedrug.Knowntargetsofcytotoxicanticancer
drugsare involvedin cellproliferation; drugsactingon such targets
are ineffective against nonproliferating tumor cells, survival of
which leads to eventual therapy failure. Function-based genomic
screeningidentifiedthe coatomerproteincomplexζ1 (COPZ1)gene
as essential for different tumor cell types but not for normal cells.
COPZ1 encodes a subunit of coatomer protein complex 1 (COPI)
involved in intracellular traffic and autophagy. The knockdown of
COPZ1, but not of COPZ2 encoding isoform coatomer protein com-
plex ζ2, caused Golgi apparatus collapse, blocked autophagy, and
induced apoptosis in both proliferating and nondividing tumor
cells. In contrast, inhibition of normal cell growth required simulta-
neous knockdown of both COPZ1 and COPZ2. COPZ2 (but not
COPZ1) was down-regulated in the majority of tumor cell lines
and in clinical samples of different cancer types. Reexpression of
COPZ2 protected tumor cells from killing by COPZ1 knockdown,
indicating that tumor cell dependence on COPZ1 is the result of
COPZ2 silencing. COPZ2 displays no tumor-suppressive activities,
but it harbors microRNA 152, which is silenced in tumor cells con-
currently with COPZ2 and acts as a tumor suppressor in vitro and
in vivo. Silencing of microRNA 152 in different cancers and the
ensuingdown-regulationofitshostgeneCOPZ2offeratherapeutic
opportunity for proliferation-independent selective killing of tu-
mor cells by COPZ1-targeting agents.
cancer targets|genetic suppressor elements
D
a primary goal of current cancer pharmacology. Target-
specific agents are effective against tumors that depend on the
function of the gene targeted by the drug, e.g., trastuzumab in
breast cancers that overexpress human epidermal growth factor
receptor 2 (1), gefitinib in tumors with EGFR mutations (2), and
all-trans retinoic acid in acute promyeolocytic leukemia, a disease
thatinvolvesatranslocationofretinoicacidreceptorα(3).Tumor
dependence on a gene that is necessary for tumors but not for
normal cells has been termed “oncogene addiction” (4), but there
aregenesthatarerequiredselectivelybytumorcellsbutcannotbe
classified as oncogenes (5). Most existing anticancer drugs act
either on DNA or on protein targets involved in cell proliferation.
Suchdrugs killproliferating tumorcells but are ineffective against
the nondividing tumor cell population, which includes growth-
arrested cells that secrete various tumor-promoting factors (6),
dormant cells capable of eventual reentry into cell cycle (7), and
resting tumor stem cells (8). Proliferating and nondividing tumor
cells, however, share common genetic and epigenetic changes,
and some of the tumor-specific targets in proliferating cells also
may be required for the survival of nondividing tumor cells. With
an increase in the number of proteins targeted by drugs in the
preclinical pipeline and the improvement in methods for target-
based de novo drug design (9), identification of additional cate-
eveloping drugs against tumor-specific molecular targets is
gories of tumor-specific targets can guide the development of new
classes of anticancer agents.
Genes playing essential roles in tumor cells can be revealed
through the selection of phenotypically active transdominant ge-
netic inhibitors, gene-derived DNA sequences that block the
function of their cognate gene. These sequences include antisense
cDNAs (10), genetic suppressor elements (GSEs, short cDNA
fragments that express dominant negative protein fragments or
antisense RNA segments) (5, 11, 12), and shRNAs that inhibit
geneexpressionthroughRNAinterference(13–16).Inthepresent
article, we report the identification, through GSE selection, of a
tumor-specific gene target, inhibition of which kills both pro-
liferating and nondividing tumor cells. This gene, coatomer pro-
tein complex ζ1 (COPZ1), encodes one of two isoforms of the ζ
subunit ofcoatomer proteincomplex1 (COPI),a secretoryvesicle
coat protein complex involved in Golgi apparatus and endoplas-
mic reticulum traffic, endosome maturation, and autophagy (17,
18). We show that tumor cell dependence on COPZ1 is caused by
the widespread down-regulation of its isoform coatomer protein
complex ζ2 (COPZ2) in different types of cancer, as a corollary of
the silencing of a tumor-suppressive microRNA (miR), miR-152,
encoded within the COPZ2 gene.
Results
Selection of Growth-Inhibitory GSEs. A GSE library comprising
cDNAfragmentsofthehumantranscriptome(averagelength135
bp) was prepared from a mixture of normalized (reduced-
redundance) cDNA preparations of 18 cell lines derived from
different types of cancer and leukemia (SI Methods). The GSE
library was cloned into a tetracycline/doxycycline-inducible len-
tiviral vector. As recipients for the selection of growth-inhibitory
GSEs, we used four tumor cell lines: MDA-MB-231 (breast
cancer), PC3(prostate cancer), T24 (bladder carcinoma), and
HT1080 (fibrosarcoma), as well as human telomerase reverse
transcriptase (hTERT)-immortalized BJ normal foreskin fibro-
blasts (BJ-hTERT). To enable doxycycline-inducible GSE ex-
pression, the cell lines were modified by the introduction of tTR-
KRAB, a tetracycline/doxycycline-sensitive repressor. Cells
transduced with the GSE library were subjected to selection for
doxycycline-dependent resistance to BrdU suicide, a procedure
that selects for cells carrying growth-inhibitory GSEs (5, 16). The
library-derived cDNA fragments were amplified by PCR from
Author contributions: M.S., E.B., Y.D., R.B., and I.B.R. designed research; M.S., M.B., E.L.,
G.H., C.-u.L., T.T.H., and C.S.C. performed research; M.N. and F.W. contributed new re-
agents/analytic tools; M.S., G.H., C.-u.L., E.B., and I.B.R. analyzed data; and I.B.R. wrote the
paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whomcorrespondence may be addressed.E-mail: shtutmanm@sccp.sc.eduor roninsoni@
sccp.sc.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1103842108/-/DCSupplemental.
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genomic DNA of the unselected and BrdU-selected cells. PCR
products were analyzed by high-throughput sequencing. The
cDNA fragment sequences were matched to their cognate genes
and compared in the unselected and BrdU-selected cells to
identify genes that give rise to putative GSEs enriched by BrdU
suicide selection. Genes targeted by the enriched sequences then
were tested for their role in cell proliferation by testing the effects
on cell growth of synthetic siRNAs targeting the same genes. The
complete results of this analysis will be presented elsewhere.
COPZ1 Knockdown Causes Golgi Apparatus Collapse, Inhibition of
Autophagy, and Tumor-Specific Cell Death Independent of Cell
Proliferation. Among the genes enriched by growth-inhibitory
GSE selection, we investigated in greater detail COPZ1, which
encodes the ζ1 subunit of the coatomer protein complex COPI.
COPZ1 was targeted by seven antisense-oriented and one sense-
oriented putative GSEs, which were enriched by BrdU selection
in all four tumor cell lines but not in immortalized normal BJ-
hTERT fibroblasts. We investigated the role of COPZ1 and other
COPI subunits in the growth of normal and tumor cells by
transfection with different siRNAs that target the genes for these
subunits, reducing their RNA levels by >90% (Fig. S1A). Fig. 1 A
and B shows that the knockdown of COPZ1 by siRNAs obtained
from different sources inhibited the growth of PC3 prostate car-
cinoma cells, as did the knockdown of another COPI component,
COPA. However, no growth inhibition resulted from the knock-
down of COPZ2, which encodes ζ2, the isoform of COPZ1 gene
product. In contrast to PC3, the growth of immortalized normal
BJ-hTERT fibroblasts (Fig. 1 C and D) or normal human prostate
epithelial cells (Fig. 1E) was not inhibited by the knockdown of
COPZ1 or COPZ2 alone. However, normal cell proliferation was
inhibited by simultaneous knockdown of COPZ1 and COPZ2 or
by COPA knockdown (Fig. 1 C–E). The siRNA growth-inhibition
assays were extended to the other COPI subunit genes (four
siRNAsper gene,allfromQiagen),andapaneloftumorcelllines
including PC3, HT1080, MDA-MB-231, T24, HeLa, and in vitro-
transformed BJ-ELR fibroblasts (19). COPZ1-targeting siRNAs
inhibited the growth of all six transformed cell lines but not of BJ-
hTERT, whereas none of siRNAs targeting the other COPI
proteins exhibited tumor selectivity (Fig. S1B). Hence, COPZ1 is
required for cell growth in a wide spectrum of tumor cell lines but
not in normal cells.
The knockdown of other COPI proteins was reported to arrest
cellular traffic, cause a collapse of endoplasmic reticulum and
Golgi apparatus (20), and inhibit the maturation of the auto-
phagosome, an essential step in autophagy (18). To determine if
COPZ1 knockdown has the same effects, we transfected COPZ1,
COPZ2, COPA, and control siRNAs into PC3 cells expressing
LC3,anautophagosomemarkerfusedwithGFP(GFP-LC3)(21).
The knockdown effects were analyzed 72 h later by fluorescence
microscopy based on GFP-LC3 localization and on immunoflu-
orescencestainingforaGolgiapparatusmarker,GM130.COPZ1
and COPA, but not control or COPZ2, siRNAs caused frag-
mentation and disappearance of GM130+Golgi structures (Fig.
2A). Knockdown of COPA and COPZ1 (but not of COPZ2) also
resulted in the accumulation of GFP-LC3+puncta (Fig. 2A) and
of a 43-kD phosphatidilethanolamine-conjugated form of GFP-
LC3 (Fig. 2B), indicative of autophagosome accumulation and
inhibition ofautophagicflux(22).Thesameeffectswere observed
in normal BJ-hTERT fibroblasts upon the knockdown of COPZ1
and COPZ2 together (but not alone) and upon COPA knock-
down (Fig. S2).
Both Golgi apparatus disruption that leads to endoplasmic re-
ticulum stress (23) and the interference with autophagy can be
cytotoxic (24, 25). We have analyzed the ability of COPZ1 and
COPA siRNAs to induce cell death and apoptosis. This analysis
was conducted by flow cytometric assays for membrane perme-
ability, as defined by the uptake of the fluorescent dye propidium
iodide (PI) and by TUNEL staining for apoptotic cells. Trans-
fection with COPZ1 or COPA siRNA into PC3 cells increased the
Fig. 1.
tumor and normal cells. (A) siRNAs targeting COPA, COPZ1,
COPZ2, or no human genes (siCont), obtained from Dhar-
macon (DH) or Qiagen (Q), were transfected into PC3 cells
at the indicated concentrations. Cell numbers were de-
termined 4 d posttransfection by flow cytometry (in three
independent transfections) and are expressed as mean ±
SD. (B) Experiment similar to A carried out with lower
siRNA concentrations, demonstrating dose-dependent in-
hibition, and including a combination of COPZ1 and COPZ2
siRNAs. Cell numbers were measured 8 d posttransfection
(in three independent transfections) and are expressed as
mean ± SD. (C) Effects of the siRNAs in A on the pro-
liferation of BJ-HTERT cells. Cell numbers were measured
7 d posttransfection (in triplicate) and are expressed as
mean ± SD. (D) Effects of COPA, COPZ1, and COPZ2 siRNAs
and a combination of COPZ1 and COPZ2 siRNAs (all at 5-
nM concentrations) on the proliferation of BJ-HTERT cells.
Cell numbers were measured 7 d posttransfection (six
replicates) and are expressed as mean ± SD. (E) Effects of
COPA, COPZ1, and COPZ2 siRNAs and a combination of
COPZ1 and COPZ2 siRNAs (at the same concentrations as in
B on the proliferation of normal human prostate epithelial
cells (HPEC). Cell numbers were measured 8 d post-
transfection (in triplicate) and are expressed as mean ± SD.
Effects of COPZ1, COPZ2, and COPA knockdown on
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number of membrane-permeable (PI+) (Fig. 2C) and apoptotic
(TUNEL+) cells(Fig.2D)relativetocellstransfectedwithcontrol
siRNA. Time-lapse video microscopy showed that cells trans-
fected with COPZ1 siRNA undergo apoptosis (shrinking and
blebbing) either directly or after first rounding up and remaining
rounded for 10–20 h (Movie S1). Although cell rounding followed
by apoptosis superficially resembles mitotic catastrophe (26),
DAPI staining did not show chromatin condensation in the
rounded cells, indicating that these cells were not in mitosis.
Hence COPZ1 knockdown produces the phenotypic effects
expected from COPI inhibition, and these effects—inhibition of
autophagy and the disruption of Golgi apparatus—lead to apo-
ptotic cell death upon COPZ1 knockdown.
Because the COPI complex affects processes distinct from cell-
cycle progression, we asked if COPZ1 knockdown would affect
nondividing tumor cells that are resistant to agents targeting cell
proliferation. For this analysis, we used HT1080 p21-9, a de-
rivative of HT1080 fibrosarcoma cells expressing the cell-cycle
inhibitor protein p21 (CDKN1A) from a promoter inducible by
isopropyl β-D-1-thiogalactopyranoside (IPTG) (27). HT1080 p21-
9 cells were transfected with control siRNA and siRNAs targeting
COPZ1, COPA, or CDC2 (CDK1), an essential mediator of cell-
cycle progression (28). The transfected cells were plated in the
absence or in the presence of IPTG that induces p21 expression
and cell-cycle arrest within 12–16 h. CDC2 siRNA decreased the
number of proliferating cells but had almost no effect on the
growth-arrested cells. In contrast, the knockdown of COPZ1 or
COPA strongly decreased the numbers of both proliferating and
growth-arrested cells (Fig. 2E). Hence, inhibition of COPI kills
both dividing and nondividing tumor cells.
COPZ1 Dependence of Tumor Cells Results from COPZ2 Down-
Regulation in Cancers. To understand why COPZ1 is the only
COPI subunit that is selectively required by tumor cells, we used
quantitative RT-PCR (qRT-PCR) to measure mRNA levels of
different COPI component proteins in four tumor cell lines and
BJ-hTERT(Fig.3A),inaseriesofisogenicBJfibroblastlinesthat
includes normal immortalized BJ-EN, partially transformed BJ-
ELB, and fully transformed BJ-ELR (19) (Fig. 3B), and in two
samplesofnormalmelanocytesandfourmelanomacelllines(Fig.
3C). COPA, COPB1, COPB2, and COPZ1 were expressed at
comparable levels in all the cell lines. Strikingly, the expression of
COPZ2 was almost undetectable in all the tumor cell lines and
was strongly decreased in BJ-ELB and BJ-ELR cells relative to
BJ-hTERTcells. We analyzedthe expression ofCOPZ1,COPZ2,
and several other COPI subunits in a panel of normal human
tissues (Table S1) and expanded the analysis of COPZ2 and
COPZ1 mRNA expression to additional tumor and leukemia cell
lines(TableS2).COPZ1expressionwasrelativelyuniformamong
the normal and tumor tissues. COPZ2 showed comparable ex-
pression levels among normal tissues (with the lowest levels ob-
served in the thymus, spleen, and ovary) (Table S1), but COPZ2
levels were drastically decreased in 10 tumor cell lines; the only
tumor line that maintained COPZ2 expression (Table S2) was
a relatively benign WM 793 melanoma (29). We also analyzed the
data on COPZ1 and COPZ2 gene expression in microarray
studies included in the Gene Expression Omnibus (GEO) data-
base, comparing normal, benign, and malignant tissues of differ-
ent cancers. COPZ1 expression showed no significant tumor-
specific changes, but in several studies COPZ2 expression was
decreased significantly in association with carcinogenesis or tu-
mor progression. In particular, COPZ2 was strongly down-regu-
lated in superficial bladder carcinoma relative to normal
urothelium (Fig. 4A), in metastatic prostate cancer relative to
primary cancers and benign prostatic tissues (Fig. 4B, two stud-
ies), in colorectal adenoma relative to normal mucosa (Fig. 4C),
and in malignant melanoma relative to benign nevi or normal
melanocytes (Fig. 4D). Hence, COPZ2 down-regulation is a
broad and general event in different forms of cancer.
If COPZ1 and COPZ2 isoforms can substitute for each other,
COPI complexes should remain functional if either COPZ1 or
COPZ2isexpressed.Indeed,theknockdownofCOPZ1orCOPZ2
alone had no effect on normal BJ-hTERT or human prostate ep-
ithelial cells that express both genes, but simultaneous knockdown
of COPZ1 and COPZ2 drastically inhibited the growth of these
cells,causing Golgi apparatuscollapseand inhibition ofautophagy
(Fig. 1 C–E and Fig. S2). To determine whether tumor cell sensi-
tivity to COPZ1 knockdown results from the loss of COPZ2 in
tumor cells, we asked if reexpression of COPZ2 in PC3 cells would
protect them from killing by COPZ1 siRNA. FLAG-tagged
COPZ1 and COPZ2 proteins were introduced into PC3 cells by
lentiviral transduction, leading to very high expression levels (Fig.
5A). COPZ1 or COPZ2 overexpression had no effect on PC3 cell
growth in culture or on the size range of tumors formed by PC3
xenografts in nude mice (Fig. 5B). Transduction with COPZ1 or
COPZ2 also had no effect on in vitro growth of HeLa and MDA-
Fig. 2.
and cell death. (A) PC3 cells expressing the autophagosome marker GFP-LC3
were transfected with control siRNA or siRNAs targeting COPA, COPZ1, or
COPZ2 and were analyzed by fluorescence microscopy for GFP fluorescence
(green), indirect immunofluorescence staining for Golgi marker GM130 (red),
and nuclear DNA staining with DAPI 72 h posttransfection with the indicated
siRNAs. (Scale bars: 10 μM.) (B) GFP-LC3 electrophoretic mobility of the cells in
A analyzed by immunoblotting with anti-GFP antibody. (C) Changes in the
number of membrane-permeable (PI+) PC-3 cells upon transfection with
control siRNA or siRNAs targeting COPA or COPZ1, as determined by flow
cytometry (mean ± SD, triplicate measurements). (D) Changes in the number
of apoptotic (TUNEL+) PC-3 cells upon transfection with control siRNA or
siRNAs targeting COPA or COPZ1, as determined by flow cytometry (mean ±
SD, triplicate measurements). (E) Effects of COPZ1, COPA, and CDC2 siRNAs
on cell number of proliferating and growth-arrested HT1080 cells. HT1080
p21-9 cells with IPTG-inducible expression of the cell-cycle inhibitor p21 were
transfected with the indicated siRNAs. Transfected cells were pIated in the
absence (proliferating) or in the presence (arrested) of 50 μM IPTG, in tripli-
cate; cell numbers were determined 5 d later. Data are shown as mean ± SD.
Effects of COPZ1 knockdown on Golgi apparatus, autophagosomes,
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MB-231 cell lines. Fig. 5C shows the effects of COPA siRNA,
COPZ1 siRNA, and COPZ2 siRNA on PC3 cells transduced with
the insert-free vector or with the vectors expressing COPZ1 or
COPZ2. COPZ2 siRNA did not inhibit cell growth, whereas
COPA siRNA inhibited the proliferation of all three cell pop-
ulations. COPZ1 siRNA inhibited the proliferation of cells trans-
duced with the insert-free vector, but overexpression of either
COPZ1 or COPZ2 rendered cells resistant to COPZ1 knockdown
(Fig. 5C). These results demonstrate that tumor cell dependence
on COPZ1 is a consequence of tumor-specific COPZ2 silencing.
COPZ2 Down-Regulation in Cancers Is Associated with the Silencing of
a Tumor-Suppressive miRNA Encoded Within COPZ2. Although the
widespread COPZ2 silencing in tumor cells suggests tumor-
suppressive activity, high-level COPZ2 expression had no detri-
mental effect on tumor cell growth in vitro or in vivo, as described
above, indicating that COPZ2 is not a tumor suppressor. How-
ever, the first intron of the COPZ2 gene encodes the precursor of
an miRNA, miR-152 (30). miR-152 was reported to be down-
regulated in several types of cancer (31–34) and to display certain
tumor-suppressive activities (33, 35). We measured miR-152 ex-
pression in different tumor cell lines and found that miR-152, like
COPZ2, was down-regulated in all the lines except WM 793 (the
only tumor line that expressed COPZ2) (Table S2). We analyzed
the effects of miR-152 on tumor cell lines by transfection with two
different miR-152 precursors. miR-152 inhibited the growth of
HeLa and MDA-MB-231 cells but not PC3 cells (Fig. 6A). Stable
miR-152expressioninPC3cellsfromalentiviralvectoralsofailed
toinhibitcellgrowthinculture,butPC3cellsoverexpressingmiR-
152 showed significantly decreased xenograft tumor growth in
nude mice relative to PC3 cells transduced with a control vector
(Fig. 6B). Hence, miR-152 displays both expression changes and
biological activities indicative of a broad-spectrum tumor sup-
pressor, and COPZ2 down-regulation in cancers leads to con-
current silencing of its host gene COPZ2, with the ensuing tumor
cell dependence on COPZ1.
Discussion
The search for tumor-selective molecular targets often involves
the concept of “oncogene addiction” (4), in which a tumor-
specific target is expected to have oncogenic activity. In the
presentstudy,weidentifiedCOPZ1asapromisingtarget,whichis
required by different tumor cell types but is not essential for
normal cells. COPZ1, however, has no known oncogenic activity,
anditsoverexpressiondoesnotaffecttumorcellgrowthinvitroor
in vivo. Instead, the tumor cell requirement for COPZ1 is the
result of the silencing of its isoform COPZ2, which we found to be
broadly down-regulated in different cancers, in vitro and in vivo.
Our results demonstrate that COPZ1 can substitute functionally
for the lack of COPZ2 in tumor cells. This substitution, however,
makes COPI function and survival of tumor cells dependent on
COPZ1, whereas COPZ2 expression in normal cells allows them
to maintain the COPI function and to survive upon COPZ1 in-
Fig. 3.
qRT-PCR analysis of expression of the indicated COPI subunit genes in tumor
cell lines and BJ-hTERT fibroblasts. Expression is presented relative to BJ-
hTERT; data are shown as mean ± SD. (B) qRT-PCR analysis of expression of
the indicated COPI subunit genes in immortalized normal BJ-EN fibroblasts
and their partially transformed (BJ-ELB) and fully transformed (BJ-ELR)
derivatives. Expression is presented relative to BJ-EN. (C) qRT-PCR analysis of
expression of the indicated COPI subunit genes in two normal melanocyte
preparations (NMP) and four melanoma cell lines. Expression is presented
relative to NMP 241.
Down-regulation of the COPZ2 gene in transformed cell lines. (A)
Fig. 4.
ferent tumor types (microarray data from GEO database). P values (student’s
t test) are indicated for significant differences between the groups. (A)
Bladder cancer study (43): normal urothelium (NU) and superficial tumors
(ST). (B) Two prostate cancer studies (44, 45): normal prostate (NP), primary
tumors (PT), and metastatic tumors (MT). (C) Colon cancer study (46): normal
mucosa (NM) and adenocarcinomas (A). (D) Melanoma study (47): normal
melanocytes (NMel), benign nevi (BN), and metastatic melanoma (MM).
COPZ2 expression in normal, benign, and malignant tissues of dif-
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hibition. We suggest a term “isoform addiction” to designate this
type of tumor-cell dependence on a specific protein isoform.
AlthoughCOPZ2isdown-regulatedselectivelyintumorcells,its
overexpression did not inhibit tumor cell growth either in ;vitro or
in vivo. COPZ2 down-regulation in cancers, despite its lack of tu-
mor-suppressive activities,canbeexplained fullyasa consequence
of the selection of cells that have silenced the tumor-suppressive
miR-152harboredwithintheCOPZ2gene.ManymiRNAslocated
within protein-coding genes are linked transcriptionally to the ex-
pressionoftheirhostgenes,becausemiRNAsandtheirhost genes
typically use the same promoter. In the case of miR-152, which is
encodedwithinthefirstintronofCOPZ2,itsexpressioninnormal
tissuescorrelateswiththatofCOPZ2(36),andweshowthatmiR-
152 and COPZ2 are silenced coordinately in different tumor cell
types.Asregulatorsofgeneexpression,miRNAsplayakeyrolein
cancer, with some miRNAs displaying oncogenic and others tu-
mor-suppressive activities (37). miR-152 on chromosome 17 was
shown previously to be down-regulated in clinical samples of
breast cancer (31), endometrial serous adenocarcinoma (where
decreased expression of miR-152 was a statistically independent
riskfactorforoverallsurvival)(32),cholangiocarcinoma (33),and
gastric and colorectal cancers, where low expression of miR-152
was correlated with increased tumor size and advanced primary
tumor stage (34). Furthermore, ectopic expression of miR-152 in
cholangiocarcinoma cells decreased cell proliferation (33), and
miR-152 overexpression in a placental human choriocarcinoma
cell line sensitized the cells to lysis by natural killer cells (35). We
found that miR-152 was silenced in >90% of tumor cell lines de-
rived from different types of cancer and that miR-152 over-
expression inhibited the growth of HeLa cervical carcinoma and
MDA-MB-231 breast carcinoma in vitro and of PC3 prostate
carcinoma in vivo. The molecular mechanism for this very broad
tumor-suppressive activity of miR-152 remains to be investigated.
Regardless of this mechanism, however, our finding that miR-152
down-regulation in tumor cells is associated with concurrent si-
lencing of its host gene COPZ2, with the ensuing tumor cell de-
pendence on COPZ1, potentially can be exploited for cancer
therapy.
COPZ1 seems an appealing cancer target, which can be
inhibited either by RNA-targeting agents (such as siRNA) or by
small-moleculeinhibitorsthatwouldaffectζ1encodedbyCOPZ1
preferentially to ζ2, the COPZ2 gene product. The recently
reported solution of the structure of the ζ subunit of COPI (38)
may be useful for structure-based design of small-molecule
COPZ1inhibitors.siRNAorsmall-moleculeinhibitorsofCOPZ1
are expected to be effective against a wide spectrum of cancers in
which COPZ2 has been down-regulated. On the other hand,
COPZ2 expression in normal tissues suggests that such tissues
should not be sensitive to COPZ1 inhibition, as evidenced in our
study by the resistance of BJ-hTERT fibroblasts and normal
prostate epithelial cells to COPZ1 knockdown. The possibility of
achieving an acceptable therapeutic index with COPI-targeting
drugs, even those not selective for COPZ1, is suggested by the
reports that Brefeldin A, a natural compound that interferes with
COPI recruitment to Golgi apparatus (23), showed antitumor
efficacy not only in vitro but also in vivo (39). COPZ1-selective
inhibitors couldbeaseffective asBrefeldin A,withlower systemic
toxicity, especially with the use of selective delivery vehicles, such
as targeted nanoparticles or liposomes.
A special appeal of targeting COPZ1 in cancer therapy (and
potentially other COPI components, using tumor-specific delivery
vehicles)stems from ourfinding that Golgiapparatus collapse and
inhibition of autophagy, which result from COPZ1 or COPA
knockdown, cause cell death not only in proliferating but also in
growth-arrested tumor cells. Nondividing tumor cells are resistant
to agents targeting the process of cell proliferation, as do most of
the existing anticancer drugs. The independence of cell death in-
duced by COPI inhibition from cell-cycle progression is indicated
further by the observation that cells transfected with COPZ1
siRNA die through apoptosis, without undergoing abnormal mi-
tosis (mitotic catastrophe), the common cause of death in cells
treated with conventional anticancer drugs (26, 40). Chemother-
apy-resistant nondividing tumor cells include the damaged/sen-
escent cell population that secretes mitogenic, antiapoptotic,
angiogenic, and proteolytic factors (6), as well as resting tumor
stem cells (8) and dormant cells that can survive for years before
Fig. 5.
sensitivity. (A) Immunoblotting of COPZ1 and COPZ2 proteins in PC3 cells
transduced with control lentivirus or with lentiviral vectors expressing FLAG-
tagged COPZ1 or COPZ2 and probed with FLAG, COPZ1, and COPZ2 anti-
bodies. (B) In vivo growth of PC3 xenograft tumors transduced with a control
lentiviral vector or with vectors expressing COPZ1 or COPZ2. Data shown are
tumor weights (mean ± SD) at the end of the experiment (41 d post-
inoculation). (C) Effects of the indicated siRNAs on the proliferation of PC3
cells transduced with control siRNA (siCont) or vectors expressing COPA,
COPZ1, or COPZ2 (six replicates). Cell numbers were measured 4 d post-
transfection and are expressed as mean ± SD.
Relationship between COPZ1 and COPZ2 expression and siRNA
Fig. 6.
(Dharmacon) on the proliferation of MDA-MB-231, HeLa, and PC3 cells (in triplicate). Cell numbers were measured 7 d posttransfection and are expressed relative
tocellstransfectedwithnegativecontrol.Dataareshownasmean±SD.(B)InvivogrowthofPC3xenografttumorstransducedwithacontrollentiviralvectororwith
a vector expressing miR-152 precursor. (Upper) Tumor weights (mean ± SD) at the end of the experiment (42 d postinoculation). (Lower) Photographs of tumors.
EffectsofmiR-152intumorcells.(A)EffectsoftransfectionwithmiR-152precursorsfromDharmacon(DH)orAmbion(AM)andmiRNAmimicnegativecontrol
Shtutman et al.PNAS Early Edition
| 5 of 6
MEDICAL SCIENCES

Page 6

reentering the cell cycle (7). The failure to destroy resting and
dormant tumor cells is a general cause of tumor relapse after the
initial remission. COPI-targeting therapy potentially could bypass
this problem, with a greater likelihood of achieving the cure.
Methods
Poly(A)+RNA, extracted from a mixture of 18 cancer and leukemia cell lines,
was used to prepare normalized cDNA through duplex-specific nuclease
normalization (41), as described in SI Methods. Normalized cDNA fragments
were cloned into a lentiviral vector inducible by tetracycline/doxycycline,
yielding a GSE library of ∼2.6 × 108clones, with an average insert length of
135 bp. The recipient cell lines were transduced first with a lentiviral vector
expressing tTR-KRAB (42) and then with the GSE library. Twenty-five percent
of the transduced cells were used for DNA extraction immediately, and the
rest were subjected to selection for doxycycline-dependent resistance to
BrdU suicide (16), followed by DNA extraction. Library-derived cDNA inserts
were amplified by PCR from genomic DNA using vector specific primers and
subjected to 454 massive parallel sequencing. BLAST analysis was used to
identify genes giving rise to cDNA fragments enriched by GSE selection.
siRNA assays for all the genes were first conducted using siRNAs from
Qiagen (four siRNAs per gene) (Table S3); subsequent assays incorporated
additional siRNAs from Dharmacon (SI Methods). miR-152 precursors were
from Dharmacon and Ambion. siRNA targeting no known genes (Qiagen)
was used as a control. Cell numbers were determined 4–8 d after trans-
fection by flow cytometry or by staining cellular DNA with Hoechst 33342.
Full-length COPZ1 and COPZ2 cDNAs (Open Biosystems) were cloned into
a lentiviral vector, pLenti6-bsd-FLAG, constructed in our laboratory, which
adds a FLAG tag at the C terminus. Retroviral vector expressing GFP-LC3 (21)
was obtained from Addgene. Lentiviral vector expressing miR-152 precursor
was from SBI Bioscience. The recipient cell populations transduced with
these and the corresponding insert-free control vectors were selected with
blasticidin or puromycin before analysis.
Gene and miRNA expression were analyzed by qRT-PCR (primers listed in
Table S4) and, in some cases, by immunoblotting (SI Methods). Flow cyto-
metric assays were used to measure membrane permeability by PI uptake
and for TUNEL assays for apoptosis. Fluorescence microscopy was used for
chromatin staining with DAPI, GFP-LC3 expression, and immunofluorescence
analysis of GM130. Time-lapse phase-contrast microscopy was used for video
analysis of cell death induced by COPI knockdown.
In vivo PC3 xenograft assays were conducted in male NCR nude mice. Mice
were inoculated s.c. with 106cells of each tested PC3 derivative (five mice per
group). Tumor size was measured every 4 d using calipers; tumors were
excised and weighed at the end of the study.
ACKNOWLEDGMENTS. We thank Dr. William Hahn for BJ fibroblast-derived
cell lines. This study was supported by National Institutes of Health Grants
R33 CA95996 and R01 AG028687 (to I.B.R.) and Grant W81XWH-08-1-0070
from the Department of Defense Prostate Cancer Research Program (to M.S.).
1. Garnock-Jones KP, Keating GM, Scott LJ (2010) Trastuzumab: A review of its use as
adjuvant treatment in human epidermal growth factor receptor 2 (HER2)-positive
early breast cancer. Drugs 70:215–239.
2. Yoshida T, Zhang G, Haura EB (2010) Targeting epidermal growth factor receptor:
Central signaling kinase in lung cancer. Biochem Pharmacol 80:613–623.
3. Collins SJ (2008) Retinoic acid receptors, hematopoiesis and leukemogenesis. Curr
Opin Hematol 15:346–351.
4. Weinstein IB, Joe A (2008) Oncogene addiction. Cancer Res 68:3077–3080, discussion
3080.
5. Primiano T, et al. (2003) Identification of potential anticancer drug targets through
the selection of growth-inhibitory genetic suppressor elements. Cancer Cell 4:41–53.
6. RoninsonIB(2003)Tumorcellsenescenceincancertreatment.CancerRes63:2705–2715.
7. Aguirre-Ghiso JA (2006) The problem of cancer dormancy: Understanding the basic
mechanisms and identifying therapeutic opportunities. Cell Cycle 5:1740–1743.
8. Sell S (2006) Potential gene therapy strategies for cancer stem cells. Curr Gene Ther 6:
579–591.
9. Song CM, Lim SJ, Tong JC (2009) Recent advances in computer-aided drug design.
Brief Bioinform 10:579–591.
10. Deiss LP, Kimchi A (1991) A genetic tool used to identify thioredoxin as a mediator of
a growth inhibitory signal. Science 252:117–120.
11. Holzmayer TA, Pestov DG, Roninson IB (1992) Isolation of dominant negative mutants
and inhibitory antisense RNA sequences by expression selection of random DNA
fragments. Nucleic Acids Res 20:711–717.
12. Gudkov AV, et al. (1994) Cloning mammalian genes by expression selection of genetic
suppressor elements: Association of kinesin with drug resistance and cell immortali-
zation. Proc Natl Acad Sci USA 91:3744–3748.
13. Berns K, et al. (2004) A large-scale RNAi screen in human cells identifies new com-
ponents of the p53 pathway. Nature 428:431–437.
14. Silva JM, et al. (2005) Second-generation shRNA libraries covering the mouse and
human genomes. Nat Genet 37:1281–1288.
15. Bernards R, Brummelkamp TR, Beijersbergen RL (2006) shRNA libraries and their use
in cancer genetics. Nat Methods 3:701–706.
16. Shtutman M, et al. (2010) Function-based gene identification using enzymatically
generated normalized shRNA library and massive parallel sequencing. Proc Natl Acad
Sci USA 107:7377–7382.
17. Beck R, Rawet M, Wieland FT, Cassel D (2009) The COPI system: Molecular mechanisms
and function. FEBS Lett 583:2701–2709.
18. Razi M, Chan EY, Tooze SA (2009) Early endosomes and endosomal coatomer are
required for autophagy. J Cell Biol 185:305–321.
19. Hahn WC, et al. (1999) Creation of human tumour cells with defined genetic ele-
ments. Nature 400:464–468.
20. Styers ML, O’Connor AK, Grabski R, Cormet-Boyaka E, Sztul E (2008) Depletion of beta-
COPrevealsa role for COP-I incompartmentalizationof secretory compartments andin
biosynthetic transport of caveolin-1. Am J Physiol Cell Physiol 294:C1485–C1498.
21. Fung C, Lock R, Gao S, Salas E, Debnath J (2008) Induction of autophagy during ex-
tracellular matrix detachment promotes cell survival. Mol Biol Cell 19:797–806.
22. Klionsky DJ, Elazar Z, Seglen PO, Rubinsztein DC (2008) Does bafilomycin A1 block the
fusion of autophagosomes with lysosomes? Autophagy 4:849–850.
23. Citterio C, et al. (2008) Unfolded protein response and cell death after depletion of
brefeldin A-inhibited guanine nucleotide-exchange protein GBF1. Proc Natl Acad Sci
USA 105:2877–2882.
24. Platini F, Pérez-Tomás R, Ambrosio S, Tessitore L (2010) Understanding autophagy in
cell death control. Curr Pharm Des 16:101–113.
25. Filimonenko M, et al. (2007) Functional multivesicular bodies are required for auto-
phagic clearance of protein aggregates associated with neurodegenerative disease. J
Cell Biol 179:485–500.
26. Broude EV, et al. (2008) Mitotic Catastrophe in Cancer Therapy. Beyond Apoptosis:
Cellular Outcomes of Cancer Therapy (Informa Healthcare, New York), pp 307–320.
27. Chang BD, et al. (2000) Effects of p21Waf1/Cip1/Sdi1on cellular gene expression: Im-
plications for carcinogenesis, senescence, and age-related diseases. Proc Natl Acad Sci
USA 97:4291–4296.
28. Satyanarayana A, Kaldis P (2009) Mammalian cell-cycle regulation: Several Cdks, nu-
merous cyclins and diverse compensatory mechanisms. Oncogene 28:2925–2939.
29. Kobayashi H, et al. (1994) Variant sublines of early-stage human melanomas selected
for tumorigenicity in nude mice express a multicytokine-resistant phenotype. Am J
Pathol 144:776–786.
30. Rodriguez A, Griffiths-Jones S, Ashurst JL, Bradley A (2004) Identification of mam-
malian microRNA host genes and transcription units. Genome Res 14(10A):1902–1910.
31. Lehmann U, et al. (2008) Epigenetic inactivation of microRNA gene hsa-mir-9-1 in
human breast cancer. J Pathol 214:17–24.
32. Hiroki E, et al. (2010) Changes in microRNA expression levels correlate with clinico-
pathological features and prognoses in endometrial serous adenocarcinomas. Cancer
Sci 101:241–249.
33. Braconi C, Huang N, Patel T (2010) MicroRNA-dependent regulation of DNA meth-
yltransferase-1 and tumor suppressor gene expression by interleukin-6 in human
malignant cholangiocytes. Hepatology 51:881–890.
34. Chen LL, Carmichael GG (2010) Decoding the function of nuclear long non-coding
RNAs. Curr Opin Cell Biol 22:357–364.
35. Zhu XM, et al. (2010) Overexpression of miR-152 leads to reduced expression of hu-
man leukocyte antigen-G and increased natural killer cell mediated cytolysis in JEG-3
cells. Am J Obstet Gynecol 202:592–597, e1–e7.
36. Stuart RO, et al. (2004) In silico dissection of cell-type-associated patterns of gene
expression in prostate cancer. Proc Natl Acad Sci USA 101:615–620.
37. Croce CM (2009) Causes and consequences of microRNA dysregulation in cancer. Nat
Rev Genet 10:704–714.
38. Yu W, Lin J, Jin C, Xia B (2009) Solution structure of human zeta-COP: Direct evidences
for structural similarity between COP I and clathrin-adaptor coats. J Mol Biol 386:
903–912.
39. Sausville EA, et al. (1996) Antiproliferative effect in vitro and antitumor activity
in vivo of brefeldin A. Cancer J Sci Am 2:52–58.
40. Chang BD, et al. (1999) A senescence-like phenotype distinguishes tumor cells that
undergo terminal proliferation arrest after exposure to anticancer agents. Cancer Res
59:3761–3767.
41. Zhulidov PA, et al. (2004) Simple cDNA normalization using kamchatka crab duplex-
specific nuclease. Nucleic Acids Res 32:e37.
42. Wiznerowicz M, Trono D (2003) Conditional suppression of cellular genes: Lentivirus
vector-mediated drug-inducible RNA interference. J Virol 77:8957–8961.
43. Sanchez-Carbayo M, Socci ND, Lozano J, Saint F, Cordon-Cardo C (2006) Defining
molecular profiles of poor outcome in patients with invasive bladder cancer using
oligonucleotide microarrays. J Clin Oncol 24:778–789.
44. Varambally S, et al. (2005) Integrative genomic and proteomic analysis of prostate
cancer reveals signatures of metastatic progression. Cancer Cell 8:393–406.
45. Yu YP, et al. (2004) Gene expression alterations in prostate cancer predicting tumor
aggression and preceding development of malignancy. J Clin Oncol 22:2790–2799.
46. Sabates-Bellver J, et al. (2007) Transcriptome profile of human colorectal adenomas.
Mol Cancer Res 5:1263–1275.
47. Talantov D, et al. (2005) Novel genes associated with malignant melanoma but not
benign melanocytic lesions. Clin Cancer Res 11:7234–7242.
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