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EMBO reports 4, 8, 800–806 (2003)
doi:10.1038/sj.embor.embor899
AOP Published online: 11 July 2003
RAL GTPases are linchpin modulators of human tumour-cell proliferation and survival
Yuchen Chien & Michael A. White
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Department of Cell Biology, University of Texas
Southwestern Medical Center, 5323 Harry Hines Boulevard,
Dallas, Texas 75390-9039, USA
To whom correspondence should be addressed
Michael A. White Tel: +1 214 648 2861; Fax: +1 214 648 8694;
michael.white@utsouthwestern.edu
Received 2 January 2003; Accepted 12 June 2003; Published online 11 July 2003.
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Abstract
The monomeric RAL (RAS-like) GTPases have been indirectly implicated
in mitogenic regulation and cell transformation. Here, we show that RALA and
RALB collaborate to maintain tumorigenicity through regulation of both
proliferation and survival. Remarkably, this task is divided between these
highly homologous isoforms. RALB is specifically required for survival of
tumour cells but not normal cells. RALA is dispensable for survival, but is
required for anchorage-independent proliferation. Reducing the 'oncogenic
burden' in human tumour cells relieves the sensitivity to loss of RALB. These
observations establish RAL GTPases as crucial components of the cellular
machinery that are exploited by factors that drive oncogenic
transformation.
EMBO reports 4, 8, 800–806 (2003)
doi:10.1038/sj.embor.embor899
AOP Published online: 11 July 2003
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Introduction
RAL (RAS-like) GTPases, as the name implies, were originally
identified on the basis of their sequence similarity to the RAS family of small
GTPases (Chardin, 1988). Two RAL genes, RALA
and RALB, are ubiquitously expressed in humans and produce proteins that
are 80% identical. Interest in the function of RAL proteins in cell regulation
was sparked by the observation that RAL proteins lie in a RAS effector pathway.
RAL GTPases are activated in response to several mitogenic regulatory cascades,
and RAL activation has been implicated as a contributing factor to oncogenic
RAS-induced cellular transformation (Feig et al.,
1996; Reuther & Der, 2000). The
mechanistic contribution of RAL proteins to cell proliferation and
transformation is unclear at present; however, expression of gain-of-function
RAL variants suggests that RAL can affect several mitogenic regulatory
cascades, including Src (Goi et al., 2000),
phospholipase D1 (PLD1; Jiang et al., 1995)
and nuclear factor- B (Henry et al.,
2000). As is generally the case with RAS-family isoforms, it is
unknown at present whether RALA and RALB have non-overlapping, fully
overlapping or partially overlapping functions.
Here, we use loss-of-function analysis to define explicitly the roles
of RALA and RALB in the proliferation and transformation of human cells. We
show that RALA is dispensable for the proliferation of human epithelial cells
and tumour-derived cell lines in adherent cultures; however, RALA is required
for the anchorage-independent proliferation of transformed cells. By contrast,
RALB is required to prevent transformed cells from initiating programmed cell
death. We propose that RAL isoforms collaborate in the maintenance of oncogenic
transformation, mediating both oncogenic proliferation and survival
signals.
Cell-autonomous molecular events that can drive the genesis of cancers
are multifarious and complex. However, the activation of proliferation coupled
with suppression of apoptosis has been aptly described as a "minimal
platform" that supports oncogenic transformation (Evan
& Vousden, 2001; Green & Evan,
2002). The observations described here show that RAL GTPases have the
ability to support both 'legs' of this oncogenic platform. The selective
sensitivity of tumour cells versus normal cells to loss of RALB reveals an
Achilles' heel with potential for exploitation by targeted therapy
approaches.
Results
To assess directly the contribution of RAL GTPases to cell regulation,
we selectively inhibited the expression of RALA and RALB using
small-interfering-RNA (siRNA)-mediated RNA interference (RNAi;
Elbashir et al., 2001; Fig.
1). Inhibiting RALA expression had no noticeable effect on cell
proliferation under standard in vitro culture conditions. By contrast,
inhibition of RALB expression with either of two siRNA duplexes was toxic to
HeLa cells, resulting in the appearance of condensed picnotic nuclei and marked
cell loss by 150 h post-transfection (Fig. 2A). Annexin-V
staining and TUNEL (terminal deoxynucleotidyltransferase-mediated
dUTP–biotin nick-end labelling) 72 h post-transfection showed that
inhibition of RALB activates programmed cell death (Fig.
2B). This was partially reversed by the broad-specificity caspase
inhibitor zVAD-FMK. Previous work suggested that inhibition of RAL function, by
the expression of dominant inhibitory RAL variants to block RAL activation, or
by expression of a minimal RAL-binding domain (RBD) to block RAL–effector
interactions, is not toxic in a variety of cell types (Goi
et al., 1999; Henry et al.,
2000; Jullien-Flores et al.,
2000; Moskalenko et al., 2002;
Rosario et al., 2001). These apparently
conflicting observations may be a consequence of selective inhibition of RALB
function by siRNA versus inhibition of both RALA and RALB by the expression of
dominant interfering molecules. Consistent with this, we found that
siRNA-mediated inhibition of RALA and RALB together reversed the cell-death
phenotype seen on loss of RALB alone (Figs 2A,3). This result suggests that RALA and RALB have antagonistic
functions in the regulation of cell survival.
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Figure 1
Small-interfering-RNA-mediated inhibition of RAL isoform expression.
The indicated cell lines were transfected with small interfering RNAs (siRNAs)
that were designed to selectively target RALA or RALB. Whole-cell lysates were
prepared 72 h post-transfection and equivalent amounts of total protein were
analysed by SDS–polyacrylamide gel electrophoresis for the indicated
proteins. Extracellular-signal-regulated protein kinase 1/2 (ERK1/2) was used
as a loading control. Similar results were obtained using two independent siRNA
sequences for both RALA and RALB. HMECs, human-mammary epithelial cells;
HMEC-hTERT, human diploid mammary epithelial cell line immortalized by hTERT
expression; PrECs, primary human-prostate epithelial cells; RAL, RAS-like.
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Figure 2
RALB is required for cell survival. (A) HeLa cells were
transfected with the indicated small interfering RNAs (siRNAs) and incubated in
the presence or absence of 50  M zVAD-FMK. Ninety-six hours
post-transfection, cells were fixed and stained with DAPI
(4',6-diamidino-2-phenylindole) to visualize nuclei. Representative
fields of view are shown for each treatment. (B) HeLa and SW480 cells
were transfected as described above. Seventy-two hours post-transfection, cells
were labelled by TUNEL (terminal deoxynucleotidyltransferase-mediated
dUTP–biotin nick-end labelling) to detect fragmented DNA, or with annexin
V to detect surface phosphatidyl serine. The percentages of annexin-V-positive
and TUNEL-positive cells were quantified by microscopic observation. Bars
indicate s.e.m.s for three independent experiments. Panels showing TUNEL
labelling from a representative experiment are shown below the graphs. RAL,
RAS-like.
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Figure 3
Tumour-derived cell lines are sensitized to RALB-dependent survival
pathways. DNA content in propidium-iodide-treated cells was analysed by
fluorescence-activated cell-sorting (FACS) 96 h after transfection with the
indicated siRNAs. Asynchronous, proliferating, adherent cell cultures were
used, except where indicated. MCF7 and SW480 cell lines are aneuploid and give
multiple peaks. DNA fragmentation results in a shift of the population of
events towards reduced signal intensities (hypodiploid). Inhibition of RALA
and/or RALB expression was verified by western blotting. The data shown are
representative of three independent experiments. HMECs, human-mammary
epithelial cells; PrECs, primary human-prostate epithelial cells; RAL,
RAS-like.
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To explore the broad-spectrum contribution of RALB to cell survival,
we inhibited RALB expression in two other tumour-derived cell lines, MCF7
(human breast adenoma) and SW480 (human colorectal carcinoma), as well as in
non-cancerous primary human-prostate epithelial cells (PrECs), non-cancerous
primary human-mammary epithelial cells (HMECs), and in non-cancerous,
telomerase-immortalized normal HMECs (HMEC-hTERT; Herbert
et al., 2002). Similar to HeLa cells, SW480 cells responded to
inhibition of RALB expression by the induction of programmed cell death, as
observed by microscopic examination (not shown), by TUNEL (Fig.
2B), and by a marked increase in the number of hypodiploid apoptotic
bodies (Fig. 3). Similarly, MCF7 cells were acutely
sensitive to loss of RALB expression. Also consistent with observations in HeLa
cells, MCF7 and SW480 sensitivity to loss of RALB was relieved by co-inhibition
of RALA expression. In contrast with the behaviour of cancer cell lines, loss
of RALB expression did not induce apoptosis in 'normal' human prostate or
mammary epithelial cells (Fig. 3). For both HMECs and
PrECs, less than 1% of the cells were apoptotic in control cultures, and no
differences were seen on inhibition of RALA or RALB alone or together. This
suggests that tumour cells may develop an increased dependency on RALB-mediated
survival pathways relative to non-cancerous, proliferating epithelial cells.
Whereas RALB is dispensable for the survival of HMECs proliferating on
tissue-culture plates, loss of RALB sensitized HMECs to induction of apoptosis
on release from the extracellular matrix (Fig. 3). At
least 48 h of incubation in suspension culture is typically required for most
HMECs to induce anoikis (data not shown). Inhibition of RALB accelerated this
process such that most cells were apoptotic within 16 h (Fig.
3). This was partially rescued by co-inhibition of RALA.
Several reports suggest that RAL GTPases can promote cell
proliferation and oncogenic RAS-dependent transformation (Lu
et al., 2000; Miller et al.,
1997; Urano et al., 1996;
White et al., 1996) and are required for
serum-independent tumour-cell proliferation (Rosario et
al., 2001). The observations described above suggest that RAL
proteins contribute primarily to the regulation of cell survival in human cell
lines and are not limiting for serum-dependent proliferation under standard
culture conditions. To examine the contribution of endogenous RAL GTPases to
oncogenic transformation, we tested the consequences of RAL inhibition on the
anchorage-independent proliferation of human tumour cell lines. Expression of
the minimal RBD of RAL-binding protein 1 (RALBP1), a candidate RAL effector,
inhibits RAL function in cells, presumably through inhibition of the
association of endogenous RAL effectors with activated RALA and RALB (De Ruiter et al., 2001; Jullien-Flores et al., 2000; Moskalenko et al., 2002; Rosario
et al., 2001). As shown in Fig. 4A, and
consistent with observations using RALA and RALB siRNAs, RBD expression does
not significantly interfere with proliferation of these cell lines in adherent
cultures. By contrast, the proliferative ability of RBD-expressing cells was
severely impaired in suspension cultures. BrdU
(5-bromo-2-deoxyuridine)-negative RBD-expressing cells showed no signs of
blebbing or of condensed or picnotic nuclei (as visualized by DAPI
(4',6-diamidino-2-phenylindole) staining), suggesting that the
proliferative defect was a consequence of cell-cycle arrest rather than
apoptosis. Selective inhibition of RALA expression by RNAi resulted in a
similar inhibition of anchorage-independent proliferation to that seen on RBD
expression (Fig. 4B). This last observation suggests that
RALA is responsible for transmitting a positive proliferative signal in tumour
cells that is selectively required in the absence of matrix association.
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Figure 4
RALA is required for anchorage-independent proliferation of
transformed cells. (A) The indicated cell lines were transiently
transfected with MYC–RBD (a fusion of a MYC epitope to the RAL-binding
domain (RBD) of RAL-binding protein 1) or empty vector. Twenty-four hours after
transfection, cells were incubated with BrdU (5-bromo-2-deoxyuridine) for
another 24 h in adherent (attached (Att.)) or suspension (Susp.) cultures. The
percentages of RBD-expressing cells that incorporated BrdU are shown. More than
100 transfected cells were analysed for each experimental group. Transfection
efficiencies ranged from 30% to 50%. Bars represent s.e.m.s for three
independent experiments. An overlay image showing the detection of RBD
expression and BrdU incorporation from a representative experiment is shown.
(B) MCF7 and SW480 cells were transfected with control small interfering
RNAs (siRNAs) that targeted mouse caveolin 1, or with RALA siRNAs. Seventy-two
hours after transfection, cells were incubated with BrdU as described above.
Quantitation of BrdU incorporation was carried out as described in (A).
RAL, RAS-like.
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Telomerase-immortalized HMECs that stably express H-RAS-G12V gain the
ability to proliferate in the absence of matrix association (K. Kaur, J. Shay
and M.W., unpublished data; Fig. 5), and this phenotype
is RAL-dependent (Fig. 5A). We therefore tested the
ability of oncogenic RAS to render cells sensitive to RALB-dependent survival
pathways. As shown in Fig. 5, HMEC–hTERT cells are
resistant to loss of RALB expression, whereas the partially transformed
HMEC–hTERT:H-RAS-G12V cells are sensitive to the loss of RALB, mimicking
the observations from tumour-derived cell lines.
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Figure 5
Oncogenic RAS sensitizes mammary epithelial cells to loss of RALB.
(A) HMEC–hTERT:H-RAS-G12V cells were transiently transfected with
MYC–RBD (a fusion of a MYC epitope to the RAL-binding domain (RBD) of
RAL-binding protein 1) or empty vector. Proliferation assays were carried out
as described in Fig. 4B. HMEC-hTERT and
HMEC-hTERT:H-RAS-G12V cells were transfected with the indiciated small
interfering RNAs (siRNAs). Seventy-two hours post-transfection, cells were
stained with DAPI (4',6-diamidino-2-phenylindole) to visualize chromatin
structure. Cells with condensed, picnotic nuclei were scored as apoptotic.
HMEC–hTERT, human diploid mammary epithelial cell line immortalized by
hTERT expression; RAL, RAS-like.
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Discussion
Through selective inhibition of RALA and RALB expression, we have
revealed surprisingly discrete but interlocking contributions of these highly
similar GTPases to the regulation of cell proliferation and survival. We have
shown that RALB is essential for the survival of a variety of tumour-derived
cell lines in culture. However, RALB is not limiting for the survival of
non-cancerous, proliferating epithelial cells. By contrast, RALA seems to be
dispensable for cell proliferation in adherent cultures but is required for
tumour cells to maintain the ability to proliferate in the absence of matrix
association. The coupling of RALA and RALB regulatory function was revealed by
the observation that inhibition of RALA can relieve the sensitivity of tumour
cells to loss of RALB.
In 'normal' cells, there is a tight coupling of the proliferative and
apoptotic machinery, such that increasing the propensity to proliferate
increases sensitivity to apoptosis (Evan & Littlewood,
1998). This observation has led to the hypothesis that oncogenic
transformation minimally requires the acquisition of enhanced proliferative
ability, together with suppression of apoptosis (Evan &
Vousden, 2001; Green & Evan, 2002).
The observations described here suggest that RAL GTPases are crucial components
of oncogenic regulatory pathways, mediating both mitogenic and survival signals
in tumour cells. This task seems to be split between the two isoforms, as RALA
is required for anchorage-independent proliferation, whereas RALB is required
for suppression of apoptosis. This division of responsibility may be the
explanation for the apparent yin and yang relationship between RAL isoforms in
the context of survival signalling. Inhibition of RALA seems to relieve
mitogenic pressure, at least partially reversing oncogenic transformation, and
therefore reducing tumour-cell dependency on RALB survival pathways.
We have not yet characterized the molecular basis of the divergent
contributions of RALA and RALB to cell regulation. Several RAL-interacting
proteins have been identified that may mediate RAL function in cells. These
include phospholipase D1 (PLD1; Jiang et al.,
1995), filamin (Ohta et al.,
1999), the RAC/CDC42 GTPase-activating protein RALBP1 (Cantor et al., 1995; Jullien-Flores et al., 1995; Park
& Weinberg, 1995), and the exocyst subunit Sec5 (Moskalenko et al., 2002; Sugihara
et al., 2002). There is no indication that any of these
proteins can selectively associate with either RAL isoform. However, as
endogenous RAL–effector complexes have not been identified, the
possibility remains that isoform-specific RAL–effector interactions may
occur in cells. Similar to RAS isoforms, most of the sequence variation between
RALA and RALB is in the carboxy-terminal hypervariable domains. These domains
are required for appropriate lipid modification and membrane association
(Reuther & Der, 2000). In the case of RAS
GTPases, the C-terminal hypervariable domains seem to direct
compartmentalization of RAS isoforms to different membrane microdomains
(Roy et al., 1999). This
compartmentalization may contribute to selective effector association among RAS
isoforms by restricting partner availability (Jaumot et
al., 2002; Yan et al., 1998).
A similar phenomenon may contribute to the discrete biological actions of RALA
versus RALB. In addition, partial loss-of-function RAL variants suggest that
there are effectors still to be identified, which may display selective isoform
association.
In summary, the loss-of-function analysis described here directly
implicates RAL GTPases as crucial components in the maintenance of oncogenic
transformation. The selective dependency of tumour cells on RALB expression
suggests that this GTPase may induce survival pathways that are crucial for
counteracting oncogene-driven apoptotic propensities. Therefore, RALB-dependent
regulatory cascades may represent an Achilles' heel against which to target
future therapeutic strategies.
Methods
Cell culture.
HeLa cells were maintained in DMEM supplemented with 10% FBS (Life
Technologies). MCF7 cells were maintained in RPMI medium 1640 (Invitrogen)
supplemented with 10% FBS. NCI-H1299 cells were cultured in DMEM supplemented
with 5% FBS. PrECs, HMECs and the HMEC–hTERT cell line (a human diploid
mammary epithelial cell line immortalized by hTERT expression; a gift from J.
Shay) were grown in MCDB serum-free medium (Invitrogen) supplemented with 0.4%
bovine pituitary extract (Hammond Cell Tech), 10 ng ml-1
epidermal growth factor (EGF), 5 g ml-1 insulin, 0.5
g ml-1 hydrocortisone, 5 g
ml-1 transferrin and 50 g ml-1
gentamicin (Sigma). MCF7 and NCI-H1299 cells were transfected using Lipofectin
and Lipofectin Plus reagents (Life Technologies) in accordance with the
manufacturer's instructions. HME50–hTERT cells were transfected with
Lipofectamine (Invitrogen). Before transfection, cells were seeded into 35-mm
culture dishes and grown to 80% confluence.
Materials.
pRK5myc and pRK5myc–RBD have been described previously
(Moskalenko et al., 2002). Synthetic siRNAs
against RALA and RALB were designed by standard methods using the following
sense sequences: 5'-GACAGGUUUCUGUAGAAGAdTdT-3' (RALA),
5'-CAGAGCUGAGCAGUGGAAUTdTdT-3' (RALA),
5'-GGUGAUCAUGGUUGGCAGCdTdT-3' (RALB) and
5'-GACUAUGAACCUACCAAAGdTdT-3' (RALB). The following antibodies'
were used: mouse monoclonal anti-RALA and rabbit polyclonal anti-RALB (Becton
Dickinson, Transduction Laboratories); mouse monoclonal anti-BrdU (Becton
Dickinson); and rabbit polyclonal anti-MYC A14 (Santa Cruz Biotechnology).
zVAD-FMK (Enzyme Systems Products) was used at a final concentration of 50
M.
Transfections.
siRNAs (200 pM) were transfected using Oligofectamine (Life
Technologies) in all cell lines. To obtain high transfection efficiencies
(>90%) in HMEC–hTERT cells, cultures were briefly exposed to trypsin
to induce macropinocytosis on apical and lateral surfaces (Hodges et al., 1973). Immediately before
transfection, cultures were incubated in 0.05% trypsin, 0.5 mM EDTA for 60 s.
The reaction was stopped with trypsin inhibitor, followed by a standard
Oligofectamine transfection protocol. Plasmids were transfected using
Lipofectin Plus reagents (Life Technologies) for MCF7 and NCI-H1299, and
Lipofectamine 2000 (Life Technologies) for HME50–hTERT: H-RAS-G12V
cells.
Apoptosis assays.
TUNEL and Annexin-V binding were carried out in accordance with the
manufacturer's instructions (Becton Dickinson, Biosciences). For
fluorescence-activated cell sorting (FACS), cells were collected, fixed with
50% ethanol at 4 °C for 1 h, and stained with propidium iodide at 37 °C
for 30 min. Approximately 10,000 cells were collected for each assay, and were
analysed using Cell Quest software (Becton Dickinson).
Proliferation assays.
Forty-eight hours post-transfection, cells were split and replated
onto glass coverslips and 1%-agarose-coated dishes. BrdU was then added to a
final concentration of 30 M. After another 24-h incubation, cells were
fixed with 3.7% formaldehyde, permeabilized with acetone at -20 °C
for 5 min, and treated with 2 M HCl for 10 min. BrdU incorporation was
visualized using mouse monoclonal anti-BrdU and FITC-conjugated anti-mouse IgG.
Expression of the MYC-tagged RBD was visualized using rabbit anti-MYC A14
polyclonal antibodies and rhodamine-red X-conjugated anti-rabbit IgG.
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Acknowledgements
This work was supported by the National Cancer Institute (grant number
CA71443), the Welch Foundation (grant number I-1414) and a Lung Spore Grant
development award (P50 CA70907).
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