Please cite this article in press as: Lim et al., Divergent Roles for RalA and RalB in Malignant Growth of Human Pancreatic
Carcinoma Cells, Current Biology (2006), doi:10.1016/j.cub.2006.10.023
Current Biology 16, 1–10, December 19, 2006 ª2006 Elsevier Ltd All rights reserved
DOI 10.1016/j.cub.2006.10.023
Article
Divergent Roles for RalA and RalB
in Malignant Growth of Human
Pancreatic Carcinoma Cells
Kian-Huat Lim,1 Kevin O’Hayer,1 Stacey J. Adam,1
S. DiSean Kendall,1,2 Paul M. Campbell,3
Channing J. Der,3,* and Christopher M. Counter1,*
1
Department of Pharmacology and Cancer Biology
Department of Radiation Oncology
2
Department of Medicine
Duke University Medical Center
Durham, North Carolina 27710
3
Lineberger Comprehensive Cancer Center
Department of Pharmacology
University of North Carolina at Chapel Hill
Chapel Hill, North Carolina 27599
Summary
Background: The Ral guanine nucleotide-exchange
factors (RalGEFs) serves as a key effector for Ras oncogene transformation of immortalized human cells. RalGEF is an activator of the highly related RalA and RalB
small GTPases, although only the former has been found
to promote Ras-mediated growth transformation of human cells. In the present study, we determined whether
RalA and RalB also had divergent roles in promoting the
aberrant growth of pancreatic cancers, which are characterized by the highest occurrence of Ras mutations.
Results: We now show that inhibition of RalA but not
RalB expression universally reduced the transformed
and tumorigenic growth in a panel of ten genetically
diverse human pancreatic-cancer cell lines. Despite
the apparent unimportant role of RalB in tumorigenic
growth, it was nevertheless critical for invasion in seven
of nine pancreatic cancer cell lines and for metastasis as
assessed by tail-vein injection of three different tumorigenic cell lines tested. Moreover, both RalA and RalB
were more commonly activated in pancreatic tumor
tissue than other Ras effector pathways.
Conclusions: RalA function is critical to tumor initiation,
whereas RalB function is more important for tumor
metastasis in the tested cell lines and thus argues for
critical, but distinct, roles of Ral proteins during the
dynamic progression of Ras-driven pancreatic cancers.
Introduction
Activating mutations of K-Ras are a hallmark of pancreatic cancer, found in as many as 30% of early-staged,
dysplastic pancreatic lesions and approximately 90%
of all advanced or metastatic tumors [1, 2]. K-Ras encodes a GDP-GTP-regulated binary on-off relay switch
that is associated with the inner face of the plasma membrane. Ras functions as a molecular transmitter that
relays the signal from extracellular stimulus-activated
cell-surface receptors to diverse cytoplasmic signaling
*Correspondence: count004@mc.duke.edu (C.M.C.), cjder@med.
unc.edu (C.J.D.)
networks [3]. Mutations in the K-Ras gene leave the protein in a stimulus-independent, constitutively active
GTP-bound state and thus cause persistent deregulated
signaling, leading to cellular transformation [4].
The best-characterized downstream Ras effectors are
the Raf family of serine and threonine kinases that activate MEK and the ERK mitogen-activated protein kinase
(MAPK) pathway. The importance of this pathway in
Ras-mediated oncogenesis is supported by mutational
activation of B-Raf in human cancers [5]. However,
B-Raf mutations are not found in pancreatic cancers,
suggesting that other effector pathways may be critical
for Ras-mediated pancreatic cancer growth [6]. Consistent with this possibility, we and others found that ERK
activation is not consistently seen in pancreatic cancers
[7, 8]. Similarly, we found that activation of the Akt serine
and threonine kinase, a key downstream target of the
second major class of Ras effectors, the phosphatidylinositol 3-kinases (PI3Ks), was activated infrequently
in pancreatic cancers [8]. Furthermore, mutational
activation of PI3K is also not commonly seen in this
cancer [9]. Thus, other effectors may be more critical
for Ras-mediated development and growth of pancreatic cancers.
Recent studies support an important role for the Ral
guanine nucleotide-exchange factors (RalGEFs) as key
effectors of Ras-mediated growth transformation of
human cells. RalGEFs serve as activators of the highly
homologous Ras-like RalA and RalB small GTPases [3].
Although earlier studies of Ras transformation of rodent
fibroblasts indicated RalGEFs are not particularly transforming on their own but could cooperate with the MAPK
pathway to promote transformation [10], RalGEF-Ral
activation was recently found to be necessary and sufficient for Ras transformation of a variety of human cell
types [11, 12]. However, despite the fact that RalA and
RalB share 85% amino acid sequence identity, with
100% sequence identity in sequences important for
effector binding [13], inhibition of RalA but not RalB
expression retarded the anchorage-independent soft
agar of immortalized and SV40-T/t-Ag-expressing
human embryonic kidney (HEK) epithelial cells expressing oncogenic H-Ras and three pancreatic cancer-cell
lines, suggesting that RalA may carry the brunt of the
oncogenic Ras signal transmitted by RalGEFs [8]. Moreover, inhibition of RalA, but not RalB also retarded the
tumorigenic growth of the transformed HEK cells, fibrosarcoma, bladder and colon cancer cell lines [8]. This
suggests the intriguing possibility that RalA, as opposed
to RalB, may play a critical role in tumor initiation in Ras
driven cancers such as pancreatic.
Other studies also suggest functional divergence of
RalA and RalB. First, in vesicle transport, a constitutively
activated version of RalA but not RalB promoted basolateral delivery of secretory vesicles in MDCK dog cells
[14]. Second, in cell viability, transient transfection with
short interfering RNA (siRNA) against RalB, but not
RalA, activated programmed cell death in some [15]
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but perhaps not all [8, 16] human tumor-cell lines when
seeded in suspension but not when it was adherent.
Third, in cell proliferation, transient siRNA against
RalA, but not RalB, decreased DNA synthesis of suspended cells [15]. Fourth, in cell migration, transient
siRNA suppression of RalB, but not RalA, reduced
UMUC-3 bladder and DU145 prostate carcinoma-cell
transwell migration in vitro and normal rat kidney migration on plastic [16, 17]. Conversely, ectopic expression
of constitutively active RalA inhibited migration,
whereas expression of constitutively active RalB stimulated migration of the UMAC-3 and DU145 cancer cells
[16]. Because RalA and RalB may serve distinct functions in cell survival and migration, we determined
whether these two highly related GTPases contribute
to distinct facets of the malignant-growth properties of
pancreatic carcinoma cells.
Results
RalA, but Not RalB, Is Required for Transformed
Growth of Pancreatic Cancer-Cell Lines In Vitro
To address whether RalA and RalB are functionally
distinct and contribute to other facets of cancer-cell
growth, we employed a retrovirus vector-based approach to stably express shRNA against RalA, RalB, or
a scramble control into an expanded panel of K-Ras
mutation-positive human pancreatic cancer-cell lines
and then evaluated the consequence on different facets
of the malignant phenotype. We focused on pancreatic
carcinoma because this cancer has the highest rate of
Ras mutations [4] and because continued mutant KRas function is critical for transformed and tumorigenic
growth of pancreatic cancer cells [18, 19].
To determine whether the dependency on RalA and
not RalB for the anchorage-independent growth is a
universal property with K-Ras mutation-positive pancreatic carcinoma cells, the ten K-Ras mutation-positive
human pancreatic cancer cell lines AsPc-1 [20], HPAC
[21], HPAF-II [22], Panc-1 [23], SW1990 [24], Capan-1
[25], Capan-2 [26], CFPac-1 [27], MIA PaCa-2 [28], and
T3M4 [29] were stably infected with a retrovirus encoding RalA shRNA, RalB shRNA, or a scramble control
and confirmed to appropriately knock down expression
of the targeted Ral protein (Figure 1A). Stable suppression of either RalA or RalB in each cell line had no gross
effect on the morphology or the ability of the cells to proliferate in monolayer culture compared that of the
scramble control counterparts (data not shown). However, stable suppression of RalA expression significantly impaired the anchorage-independent growth of
nine of ten cell lines, ranging from a 55% to 90% decrease in colony numbers (Figure 1B and see Figure S1
in the Supplemental Data available with this article
online). Capan-2 cells, the one exception (data not
shown), were very difficult to seed as single cells and
usually aggregated in soft agar, and this may account
for their apparent resistance to RalA shRNA. This was
not an off-target effect because the decrease in transformation by RalA shRNA was overcome by reintroduction of wild-type RalA into RalA knockdown Capan-1
cells (Figure S1). Conversely, RalB activity did not
seem to be required for anchorage-independent growth
in the pancreatic cell lines tested because near-
complete abrogation of RalB protein levels did not affect
their ability to grow in soft agar (Figures 1A and 1B and
Figure S1). In fact, knockdown of RalB in the HPAC cells
resulted in even greater colony numbers. Based on this
comprehensive analysis of pancreatic cancer-cell lines,
we concluded that knockdown of RalA, but not RalB,
reproducibly inhibited anchorage-independent proliferation of human pancreatic-cancer cells in vitro.
RalA, but Not RalB, Is Required for Establishing
Human Pancreatic Tumors In Vivo
Although growth in suspension is a reliable in vitro assay
for predicting the tumorigenic growth of human tumor
cells, it is also clear that the tumor-forming capacity of
a cell is requires more than simply escape from anchorage dependency of growth. Moreover, although the
aforementioned pancreatic carcinoma cell lines harbor
mutated K-Ras, they differ in other genetic lesions
(e.g., p53, DPC4 and BRCA2 tumor suppressor inactivation, p16 inactivation, and HER2 overexpression), and it
is well established that Ras or effector mutants thereof
can have vastly different effects depending on genotype
[3]. Therefore, to determine whether RalA and not RalB
is required for tumorigenic growth and whether such
growth is influenced by other genetic parameters,
each of the ten different cells line stably expressing
either scramble control shRNA, RalA shRNA, or RalB
shRNA were injected subcutaneously into both flanks
of four immunocompromised mice per cell line. When
compared to scramble control cells, loss of RalA in all
ten pancreatic cancer lines both invariably prolonged
the latency period of tumorigenesis by at least 2-fold
and impeded subsequent tumor growth kinetics (Figures 2A–2C). Again, this was not an off-target effect of
RNAi because restoration of RalA protein level reconstituted the tumorigenic potential of RalA knockdown
Capan-1 cells (Figures 2A and 2B). On the other hand,
loss of RalB had minimal or no measurable effect on tumorigenic growth in vivo (Figures 2A–2C). We therefore
conclude that RalA, but not RalB, is needed to establish
the growth of K-Ras mutation-positive pancreatic
tumors in vivo, independent of other genetic alterations.
RalA and RalB Are Both Required for Invasion
In Vitro
Despite the apparent lack of an involvement for RalB in
tumor growth, transient knockdown of RalB, but not
RalA, was reported to reduce cell migration of three
Ras mutation-negative cell lines by w50% [16, 17].
Thus, although our studies above found that RalB was
dispensable for two different growth parameters, we assessed the possibility that RalB may still be required for
other aspects of malignancy. Nine pancreatic cancer
cells stably expressing shRNA constructs specific to
RalA, RalB, or a scramble version were seeded into invasion chambers containing reconstituted basementmembrane protein matrix. Cells that invaded toward
fetal calf serum containing growth medium were quantitated after 24 hr and expressed as a percentage of
scramble control cells.
Suppression of either RalA or RalB caused a significant reduction in invasion for five cell lines (Capan-1,
HPAF II, T3M4, SW 1990, and MIA-PaCa-2), whereas
suppression of RalB and not RalA reduced invasion for
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Divergent Roles for RalA and RalB in Cancer
3
Figure 1. RalA, but Not RalB, Is Universally Required for Anchorage-Independent Growth of Pancreatic Cancer-Cell Lines
(A) Detection of RalA or RalB by immunoblot analysis in the indicated nine different human pancreatic cancer cells stably expressing a RalA
scramble sequence (scram), RalA shRNA (RalA shRNA), or RalB shRNA (RalB shRNA). Actin serves as a loading control.
(B) Photographs demonstrating anchorage-independent growth of the indicated polyclonal pancreatic cancer cells stably expressing either
a RalA scramble sequence (scram), RalA shRNA (RalA shRNA), or RalB shRNA (RalB shRNA). The bottom labels show the average and variation
of the mean of the percent of the colonies growing in an anchorage-independent fashion compared to scramble controls (normalized to 100%) as
calculated from triplicate plates. Data are from one representative experiment of two independent assays.
two other lines (PANC-1 and CFPac-1). This was not
seen in every cell line in light of the fact that reduction
of RalA enhanced migration of Panc-1 cells and suppression of RalA or RalB had no reproducible effect in
Capan-2 and AsPc-1 cells (Figure 3). Thus, in contrast
to anchorage-independent and tumorigenic growth,
RalB is critical for the invasive properties of the majority
(seven of nine) of pancreatic carcinoma cell lines. However, because RalA was also important for invasion for
five of nine lines, it may also contribute to this malignant
cell phenotype.
RalA and RalB Are Both Required for Metastatic
Growth of RasG12V-Transformed Human Cancer
Cells In Vivo
Because cell migration, invasion, and survival in suspension are all important components of tumor-cell metastasis [30], we speculated that RalB might be important
for pancreatic cancer-cell metastasis. In support of this
possibility, expression of an activated RalGEF protein
was shown to increase the metastatic growth of RasG12Vtransformed murine fibroblasts or spontaneously
transformed hamster fibroblasts cells when injected
into the tail vein [31, 32].
To investigate the separate roles of RalA and RalB
in metastasis, we first determined whether knockdown
of RalA or RalB altered the metastatic potential of
T/t-Ag + hTERT-expressing human embryonic kidney
(HEK) cells transformed by oncogenic H-Ras, given that
these cells have proven to be a reliable but genetically
malleable model of Ras-driven tumorigenesis [33]. These
cells stably expressing an shRNA against RalA, RalB, or
a scramble sequence as negative control [8] were injected into tail veins of four immunocompromised mice
each. The mice were monitored regularly until any of
the three groups of mice manifested signs of respiratory
distress or significant cachexia, at which point all mice
were sacrificed and their internal organs were examined
for evidence of metastasis.
Mice injected with scramble control-treated cells
developed respiratory distress and cachexia at day 32,
and such developments were clear indications of metastasis. Upon necropsy, the mice were found to have
severe destruction and replacement of both lungs by
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Figure 2. RalA Is Required for Tumor Initiation of Pancreatic Cancer Cells In Vivo
(A) Representative subcutaneous flank tumors in mice (top) and resected tumors (bottom) 42 days after Capan-1 cells expressing the described
constructs were injected in mice.
(B) Tumor volume (mm3) and SD versus time (days) of Capan-1 cells stably expressing a Ral scramble sequence (,), RalA-shNA (C),
RalB-shRNA (:), or RalA-shRNA in the presence of siRNA-resistant wild-type RalA (-) injected into the flanks of immunocompromised mice.
(C) Tumor volume (mm3) and standard deviation versus time (days) of the indicated cells stably expressing a Ral scramble sequence (,),
RalA-shNA (C), or RalB-shRNA (:) injected into the flanks of immunocompromised mice.
tumor tissue (Figure 4A). When examined under dissection microscopy, the lung parenchyma of mice injected
with scramble control cells were almost completely occupied with metastatic nodules (Figure 4B). Histological
analysis of ten randomly chosen fields revealed that,
on average, half the lung was occluded with tumor
(Figure 4C).
As would be expected because RalA is needed for the
establishment of tumors, at the time control mice exhibited physiological signs of metastasis, mice injected
with RalA knockdown cells showed normal appearance
and behavior. Upon gross examination of the lungs of
these animals, metastatic nodules were found to be
significantly less in number and also much smaller in
size compared to control animals (Figures 4A). Histologically, there was more than a 2-fold decrease in tumor
occlusion in the lung compared to scramble control cells
(Figures 4B and 4C, 49% versus 18%, p = 0.046).
Perhaps the most interesting observation was that despite loss of RalB expression having no effect on tumor
growth of cells injected subcutaneously [8], the very
same cells injected in the tail vein were nearly incapable
of metastatic tumor growth. Specifically, like RalA
knockdown cells, mice injected with RalB knockdown
cells showed normal appearance and behavior at the
time control mice exhibited physiological signs of metastasis. This was born out at the organ level because
mice injected with cells stably expressing RalB shRNA
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Divergent Roles for RalA and RalB in Cancer
5
Figure 3. Invasion of Pancreatic Cancer Cells Is Dependent on RalA
and RalB Signaling
The indicated pancreatic cancer cells expressing RalA shRNA (gray
bars) or RalB shRNA (black bars) were seeded in invasion chambers
containing reconstituted basement-membrane protein matrix. Cells
that invaded nearly 10% FCS in 24 hr were counted and expressed
as a percentage of scramble control cells. Data are from one representative experiment of two independent assays, and bars indicate
the mean of triplicate wells + SEM.
did not show any macroscopically visible metastatic
nodules in the lungs (Figure 4A). Histological examination of the lungs did reveal some fields with tumor nodules, but this was almost 20-fold less frequent than in
control mice (Figures 4B and 4C, 3% versus 49%, p =
0.005) and 6-fold less than mice injected with RalA
knockdown cells (3% versus 18%, p = 0.017). Thus, in
sharp contrast to subcutaneous tumor growth of transformed HEK cells, where RalB is dispensable, RalB is
critical for metastatic tumor growth of the same cell
line when injected intravenously. These results suggest
that instead of initiating tumorigenic growth, RalB may
play an essential role in the survival of tumor cells in
the bloodstream or in their extravagation into remote
sites during metastasis.
RalA and RalB Are Required for Metastatic Growth
of Two Human Pancreatic Cancer-Cell Lines In Vivo
To address the role of RalA and RalB in a model more
reflective of human cancer, we repeated these experiments by using human pancreatic cancer-cell lines. Unlike subcutaneous tumor growth, most of the pancreatic
lines tested did not grow, or did not reproducibly grow in
a metastatic fashion, at least during the period of observation (up to 4 months, data not shown). Hence, we had
to limit our analysis to two different metastatic human
pancreatic cancer-cell lines, AsPc-1 and CFPac-1,
which were derived from cancerous ascites or liver metastasis [20, 27], respectively and could form tumors
when they were injected into the tail vein of mice.
AsPc-1 scramble control tumor cells readily formed
tumor nodules in the lungs of mice after 4 weeks
(Figure 5A). Histological analysis of the lungs revealed
metastatic nodules overtaking, on average, 82% of the
normal lung tissue (Figures 5B and 5C). Knockdown of
RalA or RalB (Figure 1A) blunted this effect with a noted
decrease in macroscopic nodules and metastatic tumor
growth in the lungs compared to control mice (Figures
5A–5C, 60% versus 82%, p = 0.079; 47% versus 82%,
p = 0.010, respectively). Similarly, knockdown of either
RalA or RalB in CFPac-1 cells visibly reduced the tumor
burden in the lungs when these cells were injected intravenously (Figures 5D and 5E), although this was not statistically significant (p = 0.237 and 0.126, respectively).
However, these cells also metastasized to the adrenal
glands, and in this organ, the reduction of tumor metastasis when RalB, but not RalA, was knocked down was
almost 40-fold compared to the scramble control cells
(Figures 5G–5I, 2% versus 78%, p = 0.010). Histologically, scramble control and RalA knockdown CFPac-1
cells almost completely destroyed and replaced the
normal architecture of the adrenal glands, whereas the
adrenal glands of mice injected with RalB-shRNAexpressing cells looked completely normal, as shown
by the preservation of the three cortical zones and the
medulla (Figures 5H). Cells would have to transverse
through the systemic circulation after the lungs to reach
the adrenal glands, and hence we speculate that the difference of metastatic potential of RalB knockdown cells
between the lungs and adrenal glands may reflect the
longer period of time in the bloodstream. We concluded
that both RalA and RalB are required for tumor metastasis of these two cell lines when assessed by tail-vein
injection but that RalB may play a more important role
than RalA in this process.
RalA and RalB Are Activated in Human Pancreatic
Adenocarcinoma Samples
To address the importance of RalA and RalB in pancreatic carcinoma cells in the cancer patient, we assayed
the activation status of RalA and RalB as well as the
other two major Ras effector pathways in pancreatic
specimens from patients diagnosed with pancreatic
cancer. For these analyses, we collected two normal
samples with two matched tumor samples that were
harvested from different sites of the surgical specimen
from the same patient as well as two matched normal
samples and one tumor sample from another patient,
and we assayed six normal and five tumor samples
each harvested from eleven other patients.
Activation of K-Ras and Ral proteins, as assayed by
pull-down for the GTP-bound protein followed by immunoblotting for detecting Ras or Ral, was first measured.
Ras-GTP was detected in almost all tumor samples but
not in any of the normal samples, reflecting the high frequency of activating mutation of K-Ras in pancreatic
cancers. RalA was also significantly activated in all tumor samples, although it was slightly activated in normal
pancreatic tissue obtained from one patient (Figure 6).
By stripping the same blot and reincubating it with
RalB antibody, we found that RalB was also activated
in all tumor samples. We note that RalB was more commonly activated in tumor specimens than pancreatic
cancer-cell lines [8], possibly reflecting a need for RalB
expression in vivo. Interestingly, the ratio of K-RasGTP to total K-Ras protein was lower than the ratio of
RalA-GTP or RalB-GTP to total RalA or RalB in some
samples. Why there is discordance between RalA and
K-Ras activation is unclear but may reflect activation
of RalGEFs independent of Ras [34] or by wild-type
Ras. Nevertheless, activation of Ral proteins in the pancreatic cancer cells is presumably mediated to some
extent by oncogenic K-Ras because knockdown of
an oncogenic allele of K-Ras reduces Ral-GTP levels
[8]. On the other hand, activation of the MAPK and
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Figure 4. RalA and RalB Are Required for
Metastatic Growth of Genetically Engineered
Ras-Transformed Human Cells
(A) Representative pictures of lung metastases resulting from intravenous injection of
the HEK-HT-RasG12V cells stably expressing
a Ral scramble sequence, RalA-shRNA, or
RalB-shRNA after excision of the lungs from
the mice. The arrows indicate the metastatic
tumors, which appeared relatively pale
compared to normal lung parenchyma.
(B) Representative histological sections with
H and E staining of the aforementioned lungs
from mice injected with the indicated cells.
‘‘N’’ represents normal lung tissue; ‘‘T’’ represents metastatic tumors.
(C) Average percentage of lung field occupied
by metastatic tumors in mice injected with
the indicated cell lines. Each bar represents
the mean percentage occupied by tumor
tissue plus SD of ten randomly chosen microscopic fields.
PI3K-Akt pathways, as assayed by phospho-ERK1/2
and phospho-Akt, respectively, were infrequently activated and seen in both normal and tumor samples.
Thus, despite the presence of K-Ras activation in these
pancreatic cancer specimens, RalA and RalB, and not
ERK or AKT, were the most commonly activated proteins downstream of Ras. Collectively, these studies
suggest an important role for Ral activation in oncogenic
properties of pancreatic cancers.
Discussion
Recent studies in a mouse model [35] and cell culture
[11, 12] implicate the importance of the RalGEF-Ral effector pathway in mediating Ras oncogenesis. RalGEFs
activate two highly related targets, RalA and RalB.
Although RalA and RalB share significant sequence
(85%) and biochemical identity and interact with
common effectors, there is growing evidence for their
distinct roles in promoting Ras-mediated malignant
transformation. However, a rigorous evaluation of RalA
and RalB function in Ras mutation-positive human cancers has not been done. Because 90% of pancreatic
cancers harbor mutant Ras and mutant Ras is critical
for the transformed and tumorigenic growth of this
cancer type, we chose to determine whether RalA and
RalB serve redundant or distinct roles in Ras-mediated
oncogenesis. We now demonstrate that RalA, and
not RalB, is critical for anchorage-independent growth
in vitro and tumorigenic growth in vivo. In striking contrast, RalB, and to a lesser degree RalA, is needed for
cell invasion in vitro in seven of the nine pancreatic cancer-cell lines assayed and for metastatic growth in vivo
as assessed by tail-vein injection of three different
tumorigenic cell lines tested. Finally, we demonstrate
that both RalA and RalB are aberrantly activated in pancreatic tumor tissue. Our results support unique roles for
otherwise highly related Ral GTPases in very distinct
stages of tumor progression and growth.
In regards to RalA, the universal dependency specifically on this protein for transformation and tumorigenesis is particularly poignant given the genetic diversity of
the ten pancreatic carcinoma cell lines. Although all possess mutationally activated K-Ras, they are divergent in
their origins and in their genetic profiles [36]. What cellular phenotype RalA promotes in tumor growth remains
to be determined, but transient knockdown of the protein has been found to decrease the rate of DNA synthesis in a variety of tumor cells placed in suspension [15].
As opposed to RalA, RalB was uniformly dispensable
for transformed and tumorigenic growth. However,
there was a strong dependency on RalB for pancreatic
carcinoma cell invasion in vitro, and transient knockdown of RalB can induce apoptosis of some cells
when put in suspension [15]. As such, perhaps it is not
surprising that a role for RalB in tumorigenesis in vivo
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Figure 5. RalA and RalB Are Required for Metastatic Growth of Human Pancreatic Cancer Cells
Intravenous injection of four mice each with either AsPc-1 cells (A–C) or CFPac-1 cells (D–I) yielded metastatic tumors visible on the lungs
(arrows, A and D) or in representative histological sections stained with H and E (B and E, ‘‘N’’ represents normal lung tissue; ‘‘T’’ represents
metastatic tumors). CFPAC-1 cells also metastasized to the adrenal glands, as observed in the excised gland (G) and histological sections
(H). Average percentage of lung (C and F) or adrenal gland (I) field occupied by metastatic tumors in mice implanted with the indicated cell lines.
Each bar represents the mean percentage occupied by tumor tissue plus SD of ten randomly chosen microscopic fields.
was not uncovered until the cells were put in a similar
situation, such as metastasis, during which cells must
survive in suspension in the circulatory system and
display altered cell-adhesion properties for adhering to
and invading heterologous tissue, etc. Despite the fact
that RalB is important for cell invasion and survival is
a possible lead on the role of RalB in metastasis, this still
must be further defined because knockdown of RalB in
AsPc-1 cells had no effect on cell migration but did
inhibit metastasis. Although our observations support
multiple functions for RalB in facilitating the spectrum
of cellular changes needed for metastatic growth, this
is not to say that RalA plays no role in metastasis.
Knockdown of RalA could suppress cell migration
in vitro, and as one would expect because RalA is
required for tumor initiation, knockdown of RalA also
impeded tumor metastasis but to a lesser extent than
that of RalB. Thus, although both RalA and RalB contribute to malignant processes, they may contribute to
distinct facets and serve nonredundant functions in
pancreatic tumor cell invasion and metastasis.
This division of labor presumably reflects different
pathways engaged by these two nearly identical small
GTPases. How this occurs is still unknown because
both proteins bind to similar effectors [34], but three
models are envisioned. First, RalA and RalB have different subcellular localizations [8, 14], and hence engagement of effectors in different subdivisions of the cell
may underlie their different effects on tumorigenesis.
Evidence for this possibility is supported by recent
observations that spatial differences in Ras localization
results in utilization of distinct effectors [8, 14], and
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Figure 6. RalA and RalB Are Activated in Human Pancreatic Cancer Specimens
Detection of activated GTP-bound K-Ras,
RalA, and RalB via association with effector
protein domains specific for activated
version of these proteins followed by immunoblot analysis with antibodies specific for
K-Ras, RalA, or RalB, respectively in a panel
of 18 matched or unmatched pancreatic
tissue samples obtained from patients.
Detection of MAPK and PI3K activation in
the same cell lines by immunoblot analysis
with phosphospecific antibodies for phosphorylated forms of ERK1/2 (P-ERK1/2) and
Akt (P-Akt), respectively. Total K-Ras, RalA,
RalB, ERK1/2, and Akt or actin serve as loading controls. (‘‘N’’ represents normal tissue;
‘‘T’’ represents adenocarcinoma tissue).
a differential association of RalA and RalB with endomembranes, specifically endosomes, has been described [14]. Second, RalA and RalB may bind effectors
with different affinities. In this case, however, it is still unclear as to the extent varied affinities for effectors may
have on Ral signaling. Whereas RalA was found to display greater binding affinity for the exocyst complex
[14], RalB has been argued to preferentially utilize the
exocyst to promote cell motility [17]. Third, although
RalA and RalB share complete sequence identity in the
effector domain and switch I and II sequences (residues
36–56 and 70–87, respectively), they do diverge in sequences immediately upstream of switch II (residues
91–153; 48% identity) [14]. Thus, it remains possible
that RalA and RalB may utilize distinct effectors that
account for their divergent roles in malignant growth.
Studies of Ras transformation in experimental rodentmodel cell systems, together with the identification of
mutationally activated and transforming alleles of BRaf [5] and the p110a subunit of PI3K [9], have argued
that the Raf-MEK-ERK and PI3K-Akt effector pathways
are the most crucial signaling mechanisms for Ras-mediated oncogenesis. It was thus surprising to find that
persistent activation of ERK or Akt was not consistently
seen in the majority of patient tumors. Although a lack of
ERK [7, 8] and Akt [8] activation has been noted in pancreatic cancer-cell lines, the fact that this was born out
in human tumor specimens and that instead RalA and
RalB are consistently activated suggests that aberrant
activation of the RalGEF-Ral pathway may be more important than either the Raf or PI3K effector pathways
in the growth of this cancer type. Indeed, mutational
activation of either B-Raf or p110a is infrequent in pancreatic cancers [6, 9]. Our suggestion that the RalGEFRal pathway is crucial for pancreatic cancer growth emphasizes an emerging concept, in which the oncogenic
functions of Ras will be mediated by distinct, tissuetype-specific, signaling pathways [11, 12]. These results
also argue that anti-Ras strategies that target specific
Ras effector signaling pathways may require cancerspecific approaches. For example, inhibitors of Raf
and MEK are currently under evaluation in phase I and
II clinical trials [37]. These inhibitors may not be the
most effective approach for pancreatic cancer treatment, and instead, approaches to target the RalGEFRal pathway may be needed.
In summary, our study establishes the important contribution of aberrant Ral GTPase activation in multiple
facets of Ras mutation-positive pancreatic tumor-cell
growth. Furthermore, we found that RalA and RalB activation contribute to very distinct facets of tumor-cell progression and growth. Thus, consistent with the dynamic
nature of cancer and the clear involvement of Ras at
many different stages of cancer, it appears that Ral
GTPases are needed through different stages of cancer
progression. Targeting the distinct functions of Ral
GTPases could thus hold promise as a strategy for treating pancreatic cancers.
Experimental Procedures
Human Pancreatic Cancer-Cell Lines
Human pancreatic cancer-cell lines, AsPc-1 [20], HPAC [21], HPAF-II
[22], PANC-1 [23], SW1990 [24], Capan-1 [25], Capan-2 [26], CFPAC1 [27], MIA Paca-2 [28], and T3M4 [29] were purchased from ATCC or
provided by M. Korc and maintained in accordance with ATCC’s
recommendations.
Human Pancreatic Cancer Samples
Matched and unmatched frozen surgical samples of normal human
pancreatic tissues and pancreatic adenocarcinomas were provided
by Dr. A.D. Proia and Dr. D. Tyler (Duke University Medical Center)
and histologically verified to be pancreatic adenocarcinoma by
board-certified pathologists.
Plasmids and Creation of Stable Cell Lines
Retroviruses derived from pSuper-Retro-Puro plasmids encoding
shRNA against RalA, RalB, or a scramble sequence, pBabeBlasti
encoding siRNA-resistant wild-type RalA [8], were used for stable
infection of the indicated cell lines and thus yielded mass
CURBIO 5181
Please cite this article in press as: Lim et al., Divergent Roles for RalA and RalB in Malignant Growth of Human Pancreatic
Carcinoma Cells, Current Biology (2006), doi:10.1016/j.cub.2006.10.023
Divergent Roles for RalA and RalB in Cancer
9
populations of puromycin-resistant cells with approaches previously described [38].
Immunoblotting
The described cell lines were collected from nearly confluent 10 cm
plates and lysed in 150 ml freshly made cold general lysis buffer (25
mM Tris [pH 7.4], 150 mM NaCl, 5 mM EDTA, 1% Triton-X, 1 mg/ml
pepstatin A, 1 mg/ml leupeptin, 1.5 mg/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM Na3VO4, 1 mM NaF, and 1 mM
DTT) for 10 min. To test for activation of Ras and the ERK MAPK,
PI3K, and Ral pathways, we cultured the indicated cells in growth
medium supplemented with 0.5% FBS for 48 hr. Frozen surgical
pancreatic tissue samples were ground with pestle and mortar, incubated on ice with buffer (50 mM Tris [pH 7.5], 200 mM NaCl, 10 mM
MgCl2, 1% NP40, 1% aprotinin, 1 mM PMSF, and 0.5 mM DTT), and
then passed through a 21-gauge needle.
For immunoblot analyses, 100 mg of the lysates were resolved in
a 12.5% SDS-PAGE gel, transferred to PVDF membrane (Millipore),
and incubated with the following antibodies diluted according to the
manufacturer’s recommendation: a-actin (Santa Cruz), a-RalA and
a-RalB (Transduction Laboratory), a-Akt (Cell Signaling Technology), a-phospho(Ser 473)-Akt (New England Biolabs), a-ERK1/2
(K-23, Santa Cruz), a-phospho(Thr 202/Tyr 204)-p42/44 ERK (E10,
Cell Signaling Technology), or a-K-Ras (Santa Cruz). Proteins were
detected with ECL reagent (Amersham Pharmacia Biotech) in accordance with the manufacturer’s protocol.
We assayed levels of endogenous activated Ral-GTP or Ras-GTP
as described previously [39, 40] by incubating cell lysates with
glutathione-agarose bound recombinant glutathione S-transferase
fusion proteins containing the GTP-dependent binding domains
(BD) of the Ral or Ras proteins GST-RalBD or GST-RafBD. Total
Ral or K-Ras, RalA-GTP, RalB-GTP, and K-Ras-GTP levels were
then detected by SDS-PAGE and immunoblotting with the aforementioned anti-RalA, anti-RalB or anti-K-Ras. We determined total
Ral or K-Ras levels by immunoblotting untreated lysates with the
same antibody.
Cell-Proliferation Assay in Monolayer Culture
Cell lines were trypsinized so that single-cell suspensions could be
generated, 104 cells per 6 cm dishes were seeded, and viable (trypan
blue negative) cells were counted daily for 6 days.
Soft-Agar Assay
Five thousand cells per 3 cm plate were suspended in soft agar as
described [11, 41] and colonies greater than 30 cells were scored
after 3–4 weeks. Assays were done in triplicate and three times
independently.
In Vitro Invasion Assay
Log-phase cell lines were incubated in serum-free medium for 24 hr.
Growth-factor-reduced Matrigel invasion chambers (BD BioCoat BD
Matrigel Invasion 24-well Chamber, 8 mm pore, BD Biosciences)
were then rehydrated for 2 hr at 37 C with serum-free medium and
immediately prior to the addition of dissociated cells to the upper
chamber (5 3 104 of Panc1, 105 all others), 10% v/v of FCS was
added to the lower chamber. After 24 hr, Matrigel and uninvaded
cells were removed from the upper chamber with a moistened
cotton swab. Invaded cells on the bottom of the membrane were
fixed and stained with the Diff-Quik kit (Baxter Scientific). After
drying overnight, stained cells were counted under microscopy.
Xenograft Tumorigenesis Assay
Ten million cells of the indicated cell lines were mixed with Matrigel
and injected subcutaneously into both flanks of immunocompromised SCID/Beige mice (Charles River Laboratory) of two mice.
Tumor volumes were determined approximately twice per week
and calculated as 1/2 length2 3 width in unit of mm3. Xenograft
experiments were reviewed and approved by the Duke University
Institutional Animal Care and Use Committee.
Metastasis Assay by Tail-Vein Injection
One million cells of the indicated lines were resuspended in 200 ml
of sterile PBS and injected via tail vein into four mice. Mice were
monitored periodically for signs of weight loss, hair loss, respiratory
distress, and general malaise. Upon detection of any of these
parameters in a single mouse of a treatment group, all mice were
euthanized and necropsied. In mice injected with HEK-HT-RasG12V,
AsPc-1, or CFPAC-1-derived cell lines, this occurred at 32 days,
28 days or 95 days after injection, respectively. Metastasis experiments were reviewed and approved by the Duke University Institutional Animal Care and Use Committee. A Student’s t test was
used for assessing the significance of tumor burden in lung fields
between mice injected with experiment compared to control cells.
Supplemental Data
Supplemental Data include one figure and can be found with this
article online at http://www.current-biology.com/cgi/content/full/
16/24/---/DC1/.
Acknowledgments
We thank members of the Counter, Der, and Cox labs for advice or
assistance and A.D. Proia, and D. Tyler for reagents. This work
was supported by National Institutes of Health grants CA94184
(C.M.C.) and CA42978 (C.J.D.). C.M.C. is Leukemia and Lymphoma
Scholar; K.-H.L. is a Department of Defense Breast Cancer Research
Predoctoral Scholar.
Received: August 21, 2006
Revised: October 3, 2006
Accepted: October 5, 2006
Published: December 18, 2006
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