Download Molecular Consequences Of Silencing Mutant K

Molecular Consequences of Silencing Mutant K-ras in
Pancreatic Cancer Cells: Justification for
K-ras –Directed Therapy
Jason B. Fleming,1 Guo-Liang Shen,1 Shane E. Holloway,1
Mishel Davis,1 and Rolf A. Brekken1,2
1
Division of Surgical Oncology, Department of Surgery, and Hamon Center for Therapeutic
Oncology Research and 2Department of Pharmacology, University of Texas
Southwestern Medical School, Dallas, Texas
Abstract
Mutation of the K-ras gene is an early event in the
development of pancreatic adenocarcinoma and,
therefore, RNA interference (RNAi) directed toward
mutant K-ras could represent a novel therapy. In this
study, we examine the phenotypic and molecular
consequences of exposure of pancreatic tumor cells to
mutant-specific K-ras small interfering RNA. Specific
reduction of activated K-ras via RNAi in Panc-1 and
MiaPaca-2 cells resulted in cellular changes consistent
with a reduced capacity to form malignant tumors.
These changes occur through distinct mechanisms but
likely reflect an addiction of each cell line to oncogene
stimulation. Both cell lines show reduced proliferation
after K-ras RNAi, but only MiaPaca-2 cells showed
increased apoptosis. Both cell lines showed reduced
migration after K-ras knockdown, but changes in
integrin levels were not consistent between the cell
lines. Both cell lines showed alteration of the level of
GLUT-1, a metabolism-associated gene that is
downstream of c-myc, with Panc-1 cells demonstrating
decreased GLUT-1 levels, whereas MiaPaca-2 cells
showed increased levels of expression after K-ras
knockdown. Furthermore, after K-ras RNAi, there was a
reduction in angiogenic potential of both Panc-1 and
MiaPaca-2 cells. Panc-1 cells increased the level of
expression of thrombospondin-1, an endogenous
inhibitor of angiogenesis, whereas MiaPaca-2 cells
decreased the production of vascular endothelial
growth factor, a primary stimulant of angiogenesis in
pancreatic tumors. We have found that silencing
mutant K-ras through RNAi results in alteration of
Received 12/9/04; revised 5/17/05; accepted 6/6/05.
Grant support: NIH grant R21 CA10669 (J.B. Fleming), American Cancer
Society grant CRTG-01-152-01-CCE (J.B. Fleming), National Pancreas Foundation (R.A. Brekken), and Effie Marie Cain Scholarship in Angiogenesis Research
(R.A. Brekken).
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Rolf A. Brekken, Hamon Center for Therapeutic
Oncology Research, University of Texas Southwestern Medical Center, 6000
Harry Hines Boulevard, Dallas, TX 75390-8593. Phone: 214-648-5151; Fax:
214-648-4940. E-mail: [email protected]
Copyright D 2005 American Association for Cancer Research.
tumor cell behavior in vitro and suggests that targeting
mutant K-ras specifically might be effective against
pancreatic cancer in vivo. (Mol Cancer Res 2005;
3(7):413-23)
Introduction
Pancreatic cancer is now the fourth leading cause of cancerrelated death in the United States, and nearly all patients
diagnosed with pancreatic adenocarcinoma die a cancer-related
death (1, 2). Novel therapy is needed. Molecular genetic
analysis has identified mutations in the p53, p16, Smad4 tumor
suppressor genes and the ras proto-oncogene in >80% of
pancreatic cancers (3). Sequence analysis identified activating
ras mutations as the earliest and most common genetic
mutation in pancreas cell transformation and tumor progression
(4). K-ras, H-ras, and N-ras mutations are involved in f25%
to 30% of all human cancers (5); however, activating K-ras
mutations (in codons 12, 13, and 16) are present in nearly all
pancreatic adenocarcinomas (6, 7). These facts, in association
with the central importance of ras signaling for cell function and
survival, highlight that mutant K-ras is an attractive target for
the treatment of pancreatic cancer.
The most developed efforts to target ras for solid tumor
therapy are inhibitors of posttranslational farnesylation (FTI) of
the ras protein, which blocks membrane attachment and effector
pathway signaling; however, studies to date have failed to show
a correlation between enzyme inhibitory activity and clinical
activity, which may explain the limited therapeutic effect of
FTIs in pancreatic cancer trials (8, 9). The specific nature of the
activating K-ras mutations offers hope of an equally specific
therapy against mutant K-ras. Despite encouraging results in
preventing tumor growth in animals (10-12), clinical studies
using antisense DNA oligonucleotides against ras have not
entered large clinical trials (13, 14). One major problem in
designing a sequence-specific anti-ras therapy lies in the fact
that wild-type and mutant ras differ only in a single codon. The
extraordinary sequence specificity of RNA interference (RNAi)
makes it an attractive tool for cancer therapy that may capitalize
on the small difference between mutant and normal K-ras
genes. Brummelkamp et al. (15) used a retroviral RNAi system
to inhibit the expression of mutated K-ras v12 while leaving
other ras isoforms unaffected. These studies showed that small
interfering RNA (siRNA) for K-ras decreases anchorageindependent growth in a pancreatic cancer cell line (Capan-1).
Mol Cancer Res 2005;3(7). July 2005
Downloaded from mcr.aacrjournals.org on June 18, 2017. © 2005 American Association for Cancer Research.
413
414 Fleming et al.
However, the molecular consequences of specific knockdown
of mutant K-ras in human pancreatic cancer cells should be
explored to determine the impact of siRNA on cancer cell
survival and functions critical for tumor growth and metastasis,
such as angiogenesis, metabolism, adhesion, and motility. The
overall hypothesis driving the present work is that RNAi
therapy directed at specific K-ras mutations identified in
pancreatic cancer cells will induce cell death or reverse the
neoplastic cellular phenotype.
Results
Specificity of Mutant K-ras siRNA in Human Pancreatic
Cancer Cells
Transfection of human pancreatic cancer cell lines, Panc-1
and MiaPaca-2, with duplexed oligonucleotides specific for
mutations in the K-ras gene found in either cell line reduced
the expression of K-ras protein (Fig. 1A). The reduction of
gene/protein expression was specific as oligonucleotides with a
single bp alteration (Mismatch; Fig. 1A) or specific for other
targets (Lamin; Fig. 1A) were ineffective at reducing the level
of K-ras expression. Furthermore, exposure of cells to siRNA
constructs encoding other wild-type sequences within the
K-ras open reading frame also inhibited K-ras protein
expression. A dose-dependent decrease in K-ras protein was
observed after exposure of the cells to increasing concentrations of siRNA (25, 50, or 100 nm; Fig. 1B). Additionally, a
temporal decrease in protein expression was observed with
maximal inhibition of K-ras protein levels in MiaPaca-2 and
Panc-1 cells 48 hours after exposure to 100 nm mutant-specific
K-ras siRNA (Fig. 1B). Exposure of Panc-1 cells to siRNA
specific for lamin A/C selectively reduced lamin protein levels,
but exposure of the cells to siRNA specific for wild-type or
mutant K-ras did not alter the level of lamin A/C (Fig. 1C). To
show sequence specificity of the siRNA, Panc-1 and MiaPaca2
cells were treated with the siRNA oligonucleotides encoding
the activating K-ras mutation found in each cell line. The
Panc-1 – specific siRNA reduced the level of K-ras protein in
Panc-1 cells but not MiaPaca2 cells (Fig. 1D). The MiaPaca2specific siRNA was also specific such that it only reduced the
level of K-ras protein in the MiaPaca2 cells (Fig. 1D).
Importantly, alterations in protein kinase R expression (Fig.
1E) or phosphor-PKR (data not shown) were not observed
after K-ras siRNA exposure; this protein is associated with
interferon response to ectopic RNA exposure (16, 17).
Colony Formation, Cell Proliferation, and Apoptosis after
K-ras siRNA
Panc-1 or MiaPaca-2 cells exposed to mutant-specific K-ras
siRNA showed decreased cell proliferation, colony formation,
and alteration in cell cycle proteins (Fig. 2). Both cell lines
showed a 75% reduction in colony formation after treatment
with mutant-specific K-ras siRNA (Fig. 2B) and both cell lines
FIGURE 1. Reduction of the level of K-ras protein in pancreatic tumor
cells with mutant-specific siRNA. A. K-ras protein levels in whole cell
lysates prepared from cells pretreated for 48 hours with siRNA (100 nmol/L)
specific for lamin A/C (Lamin ), wild-type K-ras (WT K-ras ), a mismatch
version of the RNA oligospecific for the activating mutation in either Panc-1
or MiaPaca-2 cells (Mismatch ), mutant K-ras (K-ras ), or Oligofectamine
alone (Control ). B. K-ras protein levels in Panc-1 and MiaPaca-2 cell
lysates after titration of the mutant-specific K-ras siRNA or a time course
of mutant-specific K-ras siRNA (100 nmol/L). h-actin protein levels were
used as a cell lysate loading control. C. The level of lamin in Panc-1 cells
after treatment with siRNA specific for lamin A/C, wild-type K-ras , mutant
K-ras , and Oligofectamine alone was determined by Western blotting.
D. K-ras h-actin protein levels in Panc-1 (P) and MiaPaca2 (M ) cells after
exposure to mutant-specific K-ras siRNA. The siRNA oligonucleotides
used encoded the sequence specific for the mutation found in Panc-1
[K-ras (P) ] or MaiPaca2 [K-ras (M) ] cells. E. The level of PKR protein
assessed by Western blotting in Panc-1 and MiaPaca-2 cell lysates after
titration of mutant-specific K-ras siRNA.
Mol Cancer Res 2005;3(7). July 2005
Downloaded from mcr.aacrjournals.org on June 18, 2017. © 2005 American Association for Cancer Research.
Molecular Consequences of K-ras siRNA
FIGURE 2. Specific reduction of mutant K-ras reduces colony formation and cell proliferation in pancreatic tumor cells. A. The level of K-ras, h-actin, and
selected cell cycle proteins in lysates from Panc-1 and MiaPaca-2 cells was determined by Western blotting at the indicated time point after exposure to
mutant-specific K-ras siRNA (100 nmol/L) or Oligofectamine alone (CTL). B. The colony-forming ability of Panc-1 and MiaPaca-2 cells was assessed after
selective knockdown of mutant K-ras. Top, the number of MiaPaca-2 colonies 14 days after treatment with either Oligofectamine alone (Oligo ) or 100 nmol/L
of mutant-specific K-ras siRNA (siRNA ). Bottom, the mean number of colonies per plate of Panc-1 and MiaPaca-2 cells treated with Oligofectamine alone
(Control ) or 100 nmol/L mutant-specific K-ras siRNA (siRNA ). C. MiaPaca-2 cells were plated in a 12-well plate and 24 hours later were treated with optiMEM (Control ), Oligofectamine alone (Oligo), 100 nmol/L mutant-specific K-ras siRNA (K-ras ), or 100 nmol/L scrambled K-ras oligo siRNA (Scrambled ) as a
specificity control. The number of cells in each well was determined by counting with a hemocytometer 72 hours posttreatment with siRNA (*P < 0.05). D. The
effect of various siRNA treatments on the number of Panc-1 and MiaPaca-2 cells is displayed. Cells were treated with Oligofectamine alone (Control ) or with
siRNA specific for the indicated target and counted with a hemocytometer at the indicated time.
had a reduced capacity to proliferate after K-ras knockdown
(Fig. 2C and D). To determine the molecular changes
associated with decreased cell proliferation, we analyzed the
changes in the level expression of cell cycle proteins by
Western blot analysis. Panc-1 and MiaPaca-2 cells treated with
mutant-specific K-ras siRNA showed a time- and dosedependent decrease in the expression of cyclin D1 and a
concomitant increase in the level of p27cip1/kip 1. Additionally,
Panc-1, but not MiaPaca-2 cells, showed a reduction in the
level of p21waf1 (Fig. 2A). p21waf1 was not detectable in
MiaPaca-2 cells (Fig. 2A).
Apoptosis of both cell lines after exposure to mutant-specific
K-ras siRNA was measured by the use of the DNA-binding dye
Hoechst 33342 (18, 19), which allows for evaluation of nuclear
morphology. Additionally, the level of activation of caspase-3
and caspase-9 was determined by Western blot analysis. These
studies identified condensed nuclei in 30% to 40% of MiaPaca-2
cells as well as a coincident increase in the expression of
activated caspase-3 and caspase-9 after exposure to mutantspecific K-ras siRNA (25 nm; Fig. 3A and B). Panc-1 cells, on
the other hand, did not show an increase in the number of
apoptotic cells nor was there any detectable increase in the
activation of caspase-3 or caspase-9 (Fig. 3A).
Cell Migration after K-ras siRNA
The effect of siRNA-mediated inhibition of mutant K-ras
expression on the migration of MiaPaca-2 and Panc-1 cells
through an extracellular matrix was examined using a modified
Boyden chamber assay (20-22). We observed an f70%
reduction in cell migration in both cell lines after exposure to
mutant-specific K-ras siRNA (100 nmol/L; Table 1; Fig. 4).
Pancreatic cancer cell migration through an extracellular matrix
requires interaction of cell surface integrins with the extracellular matrix. Therefore, we examined the expression of the
fibronectin-binding integrin a5h1 in both cell lines after K-ras
RNAi. Both MiaPaca-2 and Panc-1 cells showed a reduction in
Mol Cancer Res 2005;3(7). July 2005
Downloaded from mcr.aacrjournals.org on June 18, 2017. © 2005 American Association for Cancer Research.
415
416 Fleming et al.
FIGURE 3. Specific inhibition of mutant K-ras induces apoptosis in
MiaPaca-2 cells. A. The level of active caspase-3 and caspase-9 in Panc1 and MiaPaca-2 cells after exposure to mutant-specific K-ras siRNA was
determined by Western blotting. B. Panc-1 and MiaPaca-2 cells were
treated with the indicated concentration of mutant-specific K-ras siRNA
and then stained with the DNA-binding dye Hoechst 33342. The
percentage of cells showing nuclear condensation, consistent with
apoptosis, was determined. Representative data from the MiaPaca-2
cells is shown.
the level of a5h1 integrin by immunocytochemistry (Fig. 5A
and B). The level of a5 integrin was reduced significantly in
Panc-1 cells treated with K-ras siRNA, whereas the level of h1
integrin dropped significantly in the MiaPaca-2 cells treated
with K-ras siRNA. Western blot analysis of whole cell lysates
confirmed the immunocytochemical staining (data not shown).
Alterations in the Angiogenic Balance after K-ras siRNA
Angiogenesis is critical to the formation of metastatic
tumors and increased microvascular activity in primary
pancreatic adenocarcinoma tumors is associated with metastasis
and decreased patient survival (23, 24). Angiogenesis is
regulated tightly by the balance of promoters and inhibitors,
such as vascular endothelial growth factor (VEGF) and
thrombospondin-1 (TSP-1), a primary stimulant and inhibitor
of angiogenesis, respectively (25). The expression of VEGF
and TSP-1 by Panc-1 and MiaPaca-2 cells was examined after
Table 1. Cell Migration after siRNA-Mediated Knockdown
of Mutant K-ras
Cell Line
Control
siRNA
Panc-1
MiaPaca-2
261 F 10.6
347 F 11.9
92 F 3.8*
102 F 8.1*
NOTE: Cells were treated with Oligofectamine (control) or mutant-specific K-ras
siRNA, seeded in a transwell chamber, and allowed to migrate for 48 hours. Cells
that migrated to the underside of the transwell membrane were stained with
hematoxylin and counted under the microscope (100 total magnification). The
mean number F SD of migrated cells per field is displayed.
*P < 0.005, Student’s t test, control versus siRNAi.
FIGURE 4. Knockdown of mutant K-ras reduces pancreatic cell
migration. Panc-1 and MiaPaca-2 cells were treated with Oligofectamine
alone (Control) or 100 nmol/L mutant-specific K-ras siRNA (siRNA) as
indicated in Materials and Methods. The cells were then placed in the
upper chamber of a transwell insert coated with Matrigel and incubated for
48 hours. Cells that migrated across the transwell membrane were stained
with hematoxylin and counted under the microscope (see Table 1).
exposure to mutant-specific K-ras siRNA. The baseline level
of VEGF production in Panc-1 and MiaPaca-2 cells in culture
was 750 and 550 pg/mL, respectively. After K-ras RNAi
treatment, VEGF production was moderately reduced in the
Panc-1 cells but dropped substantially to <100 pg/mL (P <
0.001) within 72 hours after MiaPaca-2 cells were exposed to
mutant-specific K-ras siRNA (Table 2). Alternatively, Panc-1
cells showed a time- and dose-dependent increase of the level
of expression of TSP-1 transcript (Fig. 6C) and protein (Fig.
6A and C) after treatment with K-ras siRNA. TSP-1
expression was absent in the MiaPaca-2 cells at baseline and
after siRNA treatment.
A primary mechanism that controls TSP-1 expression is the
transcription factor and oncogene c-myc (26). Therefore, we
examined the expression of c-myc in Panc-1 and MiaPaca-2
after K-ras siRNA exposure. Protein levels of the c-myc
oncogene were present in both Panc-1 and MiaPaca-2 cells at
baseline; however, 48 hours after siRNA exposure, c-myc
levels were persistently high in surviving MiaPaca-2 cells, but
decreased to below baseline in Panc-1 cells (Fig. 6B). In both
cell lines, the level of c-myc correlated inversely with TSP-1
expression.
Alteration in Cell Metabolism after K-ras siRNA
Tumor cells growing under conditions of normal oxygen
tension show elevated glycolytic rates that correlate with
increased expression of glycolytic enzymes and glucose
transporters, a phenomenon termed the Warburg effect
(27, 28). One example of the Warburg effect is the level of
expression of glucose transporter-1 (GLUT-1). GLUT-1
expression is regulated by c-myc, is expressed at a high level
in many solid tumors, including pancreatic cancer, and is
associated with tumor metastasis (29). The expression of
GLUT-1 was examined in Panc-1 and MiaPaca-2 cells after
exposure the mutant-specific K-ras siRNA (Fig. 7). Consistent
Mol Cancer Res 2005;3(7). July 2005
Downloaded from mcr.aacrjournals.org on June 18, 2017. © 2005 American Association for Cancer Research.
Molecular Consequences of K-ras siRNA
FIGURE 5. Reduction of
mutant K-ras in pancreatic cancer cells alters the level of
integrin expression. Panc-1 (A)
or MiaPaca-2 (B) cells were
treated with Oligofectamine
alone (Control ) or 100 nmol/L
of mutant-specific K-ras siRNA
(siRNA ) for 48 hours. The cells
were then fixed with 4% paraformaldehyde and incubated
with antibodies specific for
either a5 or h1 integrin. Cells
were subsequently incubated
with Cy-3 – conjugated secondary antibodies (red) along with
Hoechst 33342 (blue ) and
images captured as described.
Signal was quantified using
Metamorph software and showed
a statistical reduction in the
expression of a5 integrin in
K-ras siRNA – treated Panc-1
cells and a statistical reduction
in h1 integrin levels in K-ras
siRNA – treated MiaPaca-2
cells.
with the level of c-myc expression in each cell line, MiaPaca-2
cells showed an increase in GLUT-1 protein expression within
48 hours of exposure to 100 nmol/L of K-ras mutant-specific
siRNA; however, transcription and protein expression of
GLUT-1 decreased in Panc-1 cells after exposure to K-ras
siRNA (Fig. 7). The increase in GLUT-1 protein levels in
MiaPaca-2 cells exposed to mutant-specific K-ras siRNA
seems to occur at a posttranscriptional level because the
amount of GLUT-1 transcript does not change after siRNA
treatment.
Discussion
The present study was undertaken to determine the
molecular consequences of silencing mutant K-ras production
in human pancreatic adenocarcinoma cells. The major finding
to result is that specific knockdown of mutant K-ras through
RNAi disrupts the malignant phenotype of pancreatic adenocarcinoma cells by interfering with multiple characteristics
critical to the metastatic program.
Hanahan and Weinberg (30) described the principle
characteristics of neoplastic cells to include unregulated growth,
Table 2. Level of VEGF Expression after siRNA-Mediated Knockdown of Mutant K-ras
Treatment
Time Point
48 h
Panc-1
MiaPaca-2
72 h
Control
siRNA
Control
580 F 73.7
451 F 42
455 F 67.8
132 F 6.7c
723 F 1.0
360 F 45.8
96 h
siRNA
574 F 41.5*
70 F 10.6c
Control
siRNA
805 F 158.5
540 F 87
401 F 98.9*
96 F 5.9c
NOTE: Cells were treated with control or K-ras mutant siRNA in 12-well plates. At the appropriate time point (24 hours before the time point displayed), the cells were
harvested, counted, and reseeded at the same density in the wells of a 96-well plate. Supernatant was collected 24 hours postplating in the 96-well plate for a total time
posttransfection of 48, 72, or 96 hours. VEGF levels (pg/mL) were determined by ELISA. The level of VEGF secreted from the cells was compared at each time point.
*P < 0.02, Student’s t test.
cP < 0.001, Student’s t test.
Mol Cancer Res 2005;3(7). July 2005
Downloaded from mcr.aacrjournals.org on June 18, 2017. © 2005 American Association for Cancer Research.
417
418 Fleming et al.
FIGURE 6. Reduction of mutant
K-ras in pancreatic cancer cells results
in decreased angiogenic potential. A.
The level TSP-1 and h-actin protein was
determined at various time points after
treatment of Panc-1 and MiaPaca-2
cells with 100 nmol/L mutant-specific
K-ras siRNA. Cell lysates from treated
cells, conditioned media from human
umbilical vascular endothelial cell (EC ),
or recombinant TSP-1 (rTSP ) were
separated by nonreducing SDS-PAGE,
transferred to nitrocellulose, and probed
with a monoclonal antibody specific for
human TSP-1. B. The level of K-ras,
c-myc, TSP-1, and h-actin in Panc-1 and
MiaPaca-2 cells treated with various
concentrations of mutant-specific K-ras
siRNA was determined by Western blot
analysis. C. The level of mRNA encoding TSP-1 in Panc-1 and MiaPaca-2
cells treated with the indicated concentration of mutant-specific K-ras siRNA
was determined by reverse transcriptionPCR analysis. The level of glyceraldehyde-3-phosphate dehydrogenase
mRNA was used as a loading control.
mRNA isolated from human umbilical
vascular endothelial cell or Oligofectamine alone – treated pancreatic tumor
cells (C) were used as controls.
decreased apoptosis, angiogenesis induction, and the ability of
cells to migrate and metastasize to distant organs. Accordingly,
therapy that reverses these characteristics represents a potentially powerful treatment option. However, the accumulated
molecular alterations and signaling abnormalities found in
tumor cells make it unlikely that a single therapy directed of
any one distinct mutation will reverse the neoplastic phenotype
in toto. Additionally, the pattern of mutation in individual
tumors, and clonal populations within each tumor, often
encompasses multiple distinct genetic alterations so that a
single therapy may be effective in only a subset of the tumor
cell population. These therapies would likely be ineffective
against the tumor as a whole. However, the unique and nearly
universal presence of activating mutations in the gene encoding
K-ras in pancreatic adenocarcinoma cells and the ability to
target these mutations with specificity through siRNA-mediated
RNAi offer an opportunity to achieve a potentially broad-based
potent therapy for this disease.
The synergy between the c-myc and ras pathways in
malignant transformation of epithelial cells has been noted
previously (31-33). Recently, c-myc – induced mammary tumorigenesis was observed to proceed through a preferred
secondary oncogenic pathway involving K-ras2 over other ras
species (34). Furthermore, Yeh et al. (35) described that the
molecular interaction between ras and c-myc results in the
stabilization and accumulation of the normally short-lived
c-myc, which promotes transformation and survival (36-39). It
is also known that activated ras protects cells from apoptosis,
which is a normal consequence of c-myc accumulation (40).
Increased levels of c-myc activity promote an angiogenic switch
(38), growth, protein synthesis, and mitochondrial function
(41). Seventy percent of malignant tumors show increased
levels of c-myc expression, and Panc-1 and MiaPaca-2 cells fall
within this group. When K-ras was silenced in Panc-1 and
MiaPaca-2 cells, the observed cellular phenotypic changes in
proliferation and apoptosis, cell adhesion and migration,
angiogenic profile, and metabolism can potentially be explained
by changes in c-myc – controlled genes.
The genetic profile of Panc-1 and MiaPaca-2 cells represent
>90% of the mutations found in human pancreatic adenocarcinoma tumors. In addition to mutant K-ras, both cell lines
possess p53 mutations and homozygous deletion of p16ink4a.
Fifty percent of human pancreatic tumors possess an interrupted
Smad/transforming growth factor-h signaling pathway (3). In
our study, Panc-1 cells retain transforming growth factor-h
signaling via the Smad4 mechanism, but MiaPaca-2 cells lack a
functional transforming growth factor-h receptor 2. Transforming growth factor-h signaling is known to promote
degradation of c-myc protein levels, which protects cells from
the deleterious effects of c-myc overexpression and offers a
potential explanation for the different responses of Panc-1 and
MiaPaca-2 cells to K-ras siRNA (42, 43).
The balance between cancer cell production and excretion of
proangiogenic (e.g., VEGF) and antiangiogenic (e.g., TSP-1)
factors defines the ability of neoplastic cells to recruit new
vasculature and progress to metastasis (26). Oncogenic ras has
been shown repeatedly to promote VEGF secretion from
neoplastic cells (44) and both Panc-1 and MiaPaca-2 produce
high levels of VEGF in culture. Ablation of K-ras activity
resulted in marked reduction in VEGF production from
MiaPaca-2 cells and a more modest reduction from Panc-1
cells. These results confirm that there are multiple oncogenic
pathways capable of regulating VEGF levels and suggest that
c-myc does not directly control VEGF production in MiaPaca-2
cells. However, it is conceivable that c-myc is an important
component driving VEGF production in the Panc-1 cells because
Mol Cancer Res 2005;3(7). July 2005
Downloaded from mcr.aacrjournals.org on June 18, 2017. © 2005 American Association for Cancer Research.
Molecular Consequences of K-ras siRNA
the level of VEGF does drop after c-myc levels are reduced by
K-ras RNAi (45). Previous work suggests that c-myc represses
the expression of TSP-1 (46). Furthermore, H-ras has been
shown to repress TSP-1 expression and increase tumor
angiogenesis (26). K-ras might serve a similar function in
Panc-1 cells as loss of K-ras results in increased TSP-1 levels
coincident with the reduction of c-myc protein levels. In
MiaPaca-2 cells, c-myc levels were persistent after K-ras
reduction. We were unable to detect TSP-1 at the mRNA level
or at the protein in whole cell lysates or in ethanol-precipitated
tumor cell – conditioned media (data not shown). If the changes
in VEGF and TSP-1 levels after K-ras knockdown in Panc-1 and
MiaPaca-2 cells are taken together, they predict that the
molecular changes after specific knockdown of mutant K-ras
will potentially result in an antiangiogenic phenotype.
Differences in the level and activity of c-myc after
knockdown of K-ras in Panc-1 and MiaPaca-2 cells potentially
explains the alterations in cell survival and integrin expression
we observed between the two cell lines. c-myc promotes cell
cycle progression through sequestration of p27 and repression
p21 and p15 (40, 47). Accordingly, an increase in p27 and p21
was observed in Panc-1 cells 24 hours after reduction in K-ras
FIGURE 7. Reduction of mutant K-ras alters the expression
of the c-myc – regulated metabolism gene glucose transporter 1
(GLUT-1 ). Panc-1 (A) or MiaPaca-2 (B) cells treated with
Oligofectamine (Control ) or mutant-specific K-ras siRNA were
evaluated for the expression of
GLUT-1 by immunocytochemistry. The level of GLUT-1 expression was quantified by the use of
Metamorph software as described. C. The level of mRNA
encoding GLUT-1 in Panc-1 and
MiaPaca-2 cells after exposure
for the indicated time to 100
nmol/L mutant-specific K-ras
siRNA was determined by reverse transcription-PCR. D. The
level of GLUT-1 and h-actin
protein expression in Panc-1
and MiaPaca-2 cells after exposure to mutant-specific K-ras
siRNA was determined by Western blot analysis of whole cell
lysates.
Mol Cancer Res 2005;3(7). July 2005
Downloaded from mcr.aacrjournals.org on June 18, 2017. © 2005 American Association for Cancer Research.
419
420 Fleming et al.
and c-myc, whereas no such changes are observed in MiaPaca2 cells after K-ras siRNA. Cell proliferative and apoptotic
pathways are coupled because induction of the cell cycle
sensitizes the cell to apoptosis. The apoptotic pathway is
suppressed in the presence of appropriate survival factors.
Oncogenic K-ras acts as a survival signal and suppresses
c-myc – induced apoptosis (48). However, inhibition of K-ras
allows c-myc to trigger caspase-related apoptosis as was
observed in the MiaPaca-2 cells. Therefore, Panc-1 cells, with
decreased c-myc levels, showed no evidence of increased
apoptosis after siRNA-mediated reduction of K-ras.
Tumor cell migration and interaction with the extracellular
matrix is critical for metastasis. Reduction of mutant K-ras
levels resulted in decreased migration of both Panc-1 and
MiaPaca-2 cells consistent with the results of Yan et al.
(49, 50). Expression of h1 integrin promotes metastasis of rasmyc – transformed fibroblastoid cell tumors in animal studies
(51), and the interaction between integrin a5h1 and fibronectin
promotes the survival of growth-arrested cancer cells at sites of
distant metastasis (52). The integrin a5 promoter contains a
CCAAT/enhancer binding protein binding site and CCAAT/
enhancer binding protein overexpression represses integrin a5
expression (53). Furthermore, ras activity can regulate the
expression of CCAAT/enhancer binding proteins (54, 55),
which might explain, at least in part, the reduction of integrin
a5 levels in Panc-1 cells after K-ras siRNA (Fig. 5). This is
supported by proteomic analysis of myc-regulated proteins that
identified integrin h1 and other adhesion molecules as targets of
c-myc regulation (56). Another study showed that N-myc
overexpression results in decreased h1 levels and increased
apoptosis (57), which is similar to the results we obtained in
MiaPaca-2 cells after reduction of K-ras levels.
Normal nontransformed mammalian cells use oxygen to
generate energy, whereas cancer cells rely on glycolysis for
energy and are, therefore, less dependent on oxygen. This
results in increased survival of tumor cells in the hypoxic
microenvironment of the tumor (28). Increased reliance on
glycolysis is often accompanied by an increase in the rate of
glucose transport (58). A significant increase in GLUT-1
mRNA has been described in tumors of the esophagus, colon,
pancreas, and lung (59, 60), whereas GLUT-1 protein
expression in gastric adenocarcinoma is associated with
metastasis and decreased patient survival (61). Studies in
fibroblasts overexpressing c-myc have shown that c-myc
regulates the expression of GLUT-1 and other genes critical
to glucose metabolism (29). Our results suggest that the level of
GLUT-1 is differentially regulated in Panc-1 and MiaPaca-2
cells after reduction of K-ras, which likely reflects the alternate
regulation of c-myc in these cells.
Our findings are another example of the complexity of
multistage process of carcinogenesis and the ‘‘bizarre’’
molecular circuitry of cancer cells (62). The results, taken
together, show that in cells addicted to mutant K-ras, in this
case Panc-1 and MiaPaca-2 cells, the maintenance of the
malignant phenotype is dependent on the activity of an
oncogene that is also crucial for the development of the initial
tumor, as suggested by Weinstein (62). We conclude that the
development of mutant-specific K-ras siRNA as a potential
therapeutic is justified.
Materials and Methods
Cell Culture
The human pancreatic cancer cells lines, Panc-1 and
MiaPaca-2 (CRL-1469 and CRL-1420, American Type Culture
Collection, Manassas, VA), were maintained in culture with
DMEM (Invitrogen, Carlsbad, CA) supplemented with 10%
FCS (Gemini Bio-Products, Woodland, CA) at 37jC in a
humidified atmosphere of 5% CO2.
K-ras RNAi
Using the described mutations identified in the MiaPaca-2
(G12C) and Panc-1 (G12D) cell lines, RNA oligonucleotides
were synthesized by the Center for Biomedical Inventions
(University of Texas Southwestern, Dallas, TX). The
following oligonucleotide sequences (sense/antisense) were
used: for wild-type K-ras, 5 V-GTTGGAGCTGGTGGCGTAG3V/5 V-CAACCTCGACCACCGCATC-3V; for MiaPaca-2 mutant
K-ras , 5 -V GTTGGAGCTTGTGGCGTAG-3V/5V-CAACCTCGAACACCGCATC-3V; and for Panc-1 mutant K-ras ,
5V-GTTGGAGCTGATGGCGTAG-3V/5 -V CAACCTCGACTACCGCATC-3V, scrambled 5 -V CGAAGUGUGUGUGUGUGGC-3V/5V-GCCACACACACACACUUCG-3 V. Sense and
antisense oligonucleotides were resuspended to 50 Amol/L in
diethyl pyrocarbonate – treated water, mixed, heated for
2 minutes at 90jC, and annealed for 1 hour at 37jC. Annealed
oligos (20 Amol/L) were aliquoted and stored at 80jC until use.
For RNAi Panc-1 (f3.5 104) and MiaPaca-2 (f4.5 4
10 ), cells were incubated in a 12-well plate (BD Falcon,
Bedford, MA) overnight (14-16 hours), washed, and incubated
in Opti-MEM (Invitrogen), and exposed to duplexed siRNA
oligonucleotides in the presence of Oligofectamine (Invitrogen) for 6 hours. Cells were incubated with duplexed siRNA
oligonucleotides under two different conditions: increasing
siRNA concentrations (25, 50, or 100 nmol/L) for 24, 48, and
72 hours each or 100 nmol/L for 48, 72, or 96 hours. Six
hours posttransfection, the media was changed to DMEM with
10% FCS. At the indicated time point, the supernatant or cells
from three identical wells were harvested and pooled for
analysis.
Cell Proliferation, Colony Formation, and Apoptosis
Analysis
Cell proliferation was assessed by seeding Panc-1 or
MiaPaca-1 cells into 12- or 24-well plates on day 1 and
exposing to K-ras or control siRNA (100 nmol/L) for 6 hours
on days 0 and 2. Representative wells were counted on the days
indicated. Cell proliferation was also assessed using a MTS
assay (data not shown). Briefly, cells were plated into 12-well
plates (3.5 104-4.0 104 per well) and exposed to siRNA
(25, 50, or 100 nmol/L) for 6 hours on days 1 and 2. Cells were
harvested on day 3 and reseeded (2 104/well) in a 96-well
plate for 48 hours before the MTS assay, which was done
according to the instructions of the manufacturer (Promega,
Madison, WI). Colony formation was assessed as follows:
enhanced green fluorescent protein – transfected Panc-1 and
MiaPaCa-2 cells were treated with Oligofectamine or 100
nmol/L mutant-specific K-ras siRNA for 6 hours on days
1 and 2. Cells were harvested on day 3 and reseeded in
Mol Cancer Res 2005;3(7). July 2005
Downloaded from mcr.aacrjournals.org on June 18, 2017. © 2005 American Association for Cancer Research.
Molecular Consequences of K-ras siRNA
triplicate at a concentration of 2,500 cells per 35 10 mm dish
(Nalge Nunc International, Rochester, NY) in 0.33% top
agarose (SeaKem Agarose, FMC BioProducts, Rockland,
ME). Dishes were incubated at 37jC for 12 days. Colonies
were identified microscopically (Nikon SMZ-1500, Nikon,
Lewisville, TX). Colonies of z0.1 mm in diameter were
counted and expressed as mean number of colonies/well F SD.
Apoptosis was judged by nuclear condensation. Briefly,
Panc-1 (3.5 104) and MiaPaca-1 (4.5 104) cells per well
were seeded into 12-well plates and exposed to siRNA (25, 50,
or 100 nmol/L) for 6 hours on days 1 and 2. On day 4 (72 hours
after siRNA treatment), 0.1 mL of Hoechst 33342 at a
concentration of 2 Ag/mL was added to each well and incubated
at room temperature for 15 minutes. The cells were fixed and
then examined using a Nikon Eclipse TE2000 microscope,
photographed, and analyzed as above.
Cell Migration Assay
The migration of human pancreatic cancer cells through an
artificial extracellular matrix (Matrigel, BD Biosciences, Bedford, MA) was assessed using a modified Boyden chamber
assay. Briefly, Panc-1 (3.0 104) or MiaPaca-2 (4 104) cells
were exposed to 100 nmol/L siRNA for 48 hours, harvested,
counted, and f1 104 cells placed on Matrigel-coated 8 Am
pore polycarbon inserts (BD Biosciences) in a 24-well tissue
culture plate. The cells were incubated for 48 hours at 37jC,
with serum-free media in the upper chamber and media
containing serum (5%) in the lower chamber. Cell migration
was determined by scraping the top of the insert with a Q-Tip
and fixing and staining the bottom/under side of the insert with
hematoxylin (Diff-Quick, Dade Berhring, Inc., New York). The
porous membrane was removed from the insert and placed on a
glass slide under a cover slip. The number of migrated cell per
10 high-powered fields was quantified for each condition in
triplicate and expressed as a mean number of cells per highpowered field.
Reverse Transcription-PCR
Total cellular RNA was harvested from human Panc-1 and
MiaPaca-2 cells using Trizol (Invitrogen) after exposure of the
cells to control or mutant-specific K-ras siRNA. RNA was also
harvested from endothelial cells (human umbilical vascular
endothelial cell, VEC Technologies, Rensselaer, NY) in a
similar manner. After DNase I treatment (Ambion, Austin, TX)
and quantification, cDNA was synthesized using reverse
transcriptase AMV, M-MuLV, or Tth DNA polymerase (Roche
Diagnostics Corp., Indianapolis, IN). PCR was then done using
the following oligonucleotide sequences (sense/antisense; IDT,
Coralville, IA): GLUT-1, 5 V-CAACTGGACCTCAAATTTCATTGTGGG-3 V/5V-CGGGTGTCTTATCACTTTGGCTGG-3 V;
TSP-1, 5 V-CAGAGGCCAAAGCACTAAGG-3V/5V- TGAGTAAGGGTGGGGATCAG-3 V; and glyceraldehyde-3-phosphate
dehydrogenase, 5 V-TCCACCACCCTGTTGCTGTAG-3 V/
5 V-GACCACAGTCCATGCCATCACT-3 V.
Western Blot
Cells were harvested as described above and were lysed in
50 mmol/L Tris-Cl (pH 7.4), 150 mmol/L NaCl, 1% NP40,
1 mmol/L sodium orthovanadate, 5 mmol/L NaF, 20 mmol/L
h-glycerophosphate, and protease inhibitors (Complete,
Roche Diagnostics). Fifty micrograms of protein, as determined by a modified Bradford protein assay (Bio-Rad,
Hercules, CA), were loaded per well onto a precast 4% to
12% polyacrylamide gradient gel (Invitrogen). Proteins were
separated by SDS-PAGE and transferred to an Immobilon-P
membrane (Millipore, Bedford, MA). The membranes were
blocked in 5% nonfat milk and incubated in primary
antibody. Antibodies against the following proteins were
used: K-ras (sc-30), PKR (sc-6282), p21 (sc-756), ERK1 (sc94), and MKP2 (sc-17821) from Santa Cruz Biotechnology
(Santa Cruz, CA); p27 (RB-9019), PARP (Ab-2, RB-1516),
p53 (Ab-6, MS-187), GLUT-1 (Ab-1, RB-078), and TSP-1
(Ab11, MS-1066 and Ab-2, MS-419) from Lab Vision
(Fremont, CA); h-actin (clone AC-40) from Sigma-Aldrich,
Inc. (St. Louis, MO); c-myc (9E10, Developmental Studies
Hybridoma Bank, Department of Biological Sciences,
University of Iowa), integrins a2, a5, h1, and h5
(AB1944, AB1928, AB1952, and AB1926, respectively),
and activated caspase-3 (AB3623) from Chemicon International (Temecula, CA); phospho-ERK1/2, phospho-Raf,
phospho-AKT, cleaved caspase-9, and phospho-PKR from
Cell Signaling Technology (Beverly, MA); and cyclin D1
(SA-260) from BIOMOL (Plymouth Meeting, PA). Mab 133
specific for human TSP-1 was provided generously by
Dr. Joanne Murphy-Ullrich (University of Alabama Birmingham, Birmingham, AL). After incubation with primary
antibody, the membranes were washed in PBS + 0.1%
Tween 20 and incubated with the appropriate peroxidase –
conjugated secondary antibody (Jackson ImmunoResearch
Laboratories, West Grove, PA) followed by development with
chemiluminescence substrate (Pierce Biotechnology, Inc.,
Rockford, IL) and exposure to X-ray film (Kodak, Rochester,
NY). TSP-1 protein used as control was obtained from
human umbilical vascular endothelial cell lysate (Lab Vision),
Dr. Joanne Murphy-Ullrich, and purchased from Protein
Sciences (Meriden, CT).
Immunocytochemistry
Cells were seeded in 12-well plates and treated with
control or K-ras siRNA as described. Forty-eight hours later,
cells (2 104-3 104) were plated onto LabTek II chamber
slides (Nalge Nunc) and incubated at 37jC overnight, washed
with PBS, and fixed in 4% paraformaldehyde (cells were fixed
72 hours after siRNA treatment). The slides were blocked
with 5% FCS/TBST and exposed to primary antibodies at a
concentration of 5 Ag/mL. Antibodies against the following
proteins were used: GLUT-1, integrins a5, h1, and h5. The
cells were counterstained with Hoechst 33342 dye (Molecular
Probes, Eugene, OR) and exposed to 1:500 Cy-3 or FITClabeled secondary antibody (Jackson ImmunoResearch Laboratories, Inc.). Cells were examined using a Nikon E600
upright microscope (Nikon) fitted with a CoolSNAP HQ
camera (Photometrics, Tucson, AZ) and signal quantified
using MetaMorph software. Images to be compared were
captured under identical conditions (e.g., exposure time and
image scaling) and a threshold intensity level was set by
examination of sections stained with secondary alone. The
percentage of pixels reaching the threshold level was
Mol Cancer Res 2005;3(7). July 2005
Downloaded from mcr.aacrjournals.org on June 18, 2017. © 2005 American Association for Cancer Research.
421
422 Fleming et al.
determined for each target and for 4 V,6-diamidino-2-phenylindole in each image. The signal intensity/pixel number for
4 V,6-diamidino-2-phenylindole was used to normalize the
specific signal for each target antibody.
VEGF Production
After exposure of Panc-1 (f3.0 104) or MiaPaca-2 (f5
104) cells to 100 nmol/L mutant-specific K-ras siRNA or
Oligofectamine alone (for 24, 48, and 72 hours), the cells were
harvested, counted, reseeded, and cultured in DMEM with 10%
FCS without siRNA for 24 hours for a total of 48, 72, and 96
hours after initial siRNA exposure. The supernatants were
harvested and stored at 80jC. The concentration of human
VEGF was determined using the human VEGF immunoassay
kit (R&D Systems, Minneapolis, MN).
Acknowledgments
We thank Dr. Latha Shivakumar for technical assistance; Drs. John Minna and
Helene Sage, members of our laboratory, and David Shames for helpful
discussions and support; and Dr. Joanne Murphy-Ullrich for providing TSP-1 and
anti – TSP-1 antibodies for confirmation of our results.
References
1. Jemal A, Clegg LX, Ward E, et al. Annual report to the nation on the status of
cancer, 1975 – 2001, with a special feature regarding survival. Cancer 2004;
101:3 – 27.
2. Schmidt CM, Powell ES, Yiannoutsos CT, et al. Pancreaticoduodenectomy: a
20-year experience in 516 patients. Arch Surg 2004;139:718 – 27.
3. Rozenblum E, Schutte M, Goggins M, et al. Tumor-suppressive pathways in
pancreatic carcinoma. Cancer Res 1997;57:1731 – 4.
4. Lemoine NR, Jain S, Hughes CM, et al. Ki-ras oncogene activation in
preinvasive pancreatic cancer. Gastroenterology 1992;102:230 – 6.
5. Downward J. Targeting RAS signalling pathways in cancer therapy. Nat Rev
Cancer 2003;3:11 – 22.
6. Li D, Xie K, Wolff R, Abbruzzese JL. Pancreatic cancer. Lancet 2004;363:
1049 – 57.
7. Leach SD. Mouse models of pancreatic cancer: the fur is finally flying! Cancer
Cell 2004;5:7 – 11.
8. Sebti SM, Adjei AA. Farnesyltransferase inhibitors. Semin Oncol 2004;31:
28 – 39.
9. Saad ED, Hoff PM. Molecular-targeted agents in pancreatic cancer. Cancer
Control 2004;11:32 – 8.
10. Kita K, Saito S, Morioka CY, Watanabe A. Growth inhibition of human
pancreatic cancer cell lines by anti-sense oligonucleotides specific to mutated
K-ras genes. Int J Cancer 1999;80:553 – 8.
11. Wickstrom E. Oligonucleotide treatment of ras-induced tumors in nude mice.
Mol Biotechnol 2001;18:35 – 55.
12. Aoki K, Ohnami S, Yoshida T. Suppression of pancreatic and colon cancer
cells by antisense K-ras RNA expression vectors. Methods Mol Med 2004;106:
193 – 204.
gene expression is associated with pancreatic cancer invasiveness and MMP-2
activity. Surgery 2004;136:548 – 56.
21. Duxbury MS, Ito H, Benoit E, Ashley SW, Whang EE. CEACAM6 is a
determinant of pancreatic adenocarcinoma cellular invasiveness. Br J Cancer
2004;91:1384 – 90.
22. Albo D, Berger DH, Tuszynski GP. The effect of thrombospondin-1 and
TGF-h1 on pancreatic cancer cell invasion. J Surg Res 1998;76:86 – 90.
23. Reinmuth N, Parikh AA, Ahmad SA, et al. Biology of angiogenesis in
tumors of the gastrointestinal tract. Microsc Res Tech 2003;60:199 – 207.
24. Takeda A, Stoeltzing O, Ahmad SA, et al. Role of angiogenesis in the
development and growth of liver metastasis. Ann Surg Oncol 2002;9:610 – 6.
25. Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev
Cancer 2003;3:401 – 10.
26. Watnick RS, Cheng Y-N, Rangarajan A, Ince TA, Weinberg RA. Ras
modulates Myc activity to repress thrombospondin-1 expression and increase
tumor angiogenesis. Cancer Cell 2003;3:219 – 31.
27. Warburg O. On the origin of cancer cells. Science 1956;123:309 – 14.
28. Warburg O. On respiratory impairment in cancer cells. Science 1956;124:
269 – 70.
29. Osthus RC, Shim H, Kim S, et al. Deregulation of glucose transporter 1 and
glycolytic gene expression by c-Myc. J Biol Chem 2000;275:21797 – 800.
30. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57 – 70.
31. Land H, Parada LF, Weinberg RA. Tumorigenic conversion of primary
embryo fibroblasts requires at least two cooperating oncogenes. Nature 1983;304:
596 – 602.
32. Leder A, Pattengale PK, Kuo A, Stewart TA, Leder P. Consequences of
widespread deregulation of the c-myc gene in transgenic mice: multiple
neoplasms and normal development. Cell 1986;45:485 – 95.
33. Sinn E, Muller W, Pattengale P, Tepler I, Wallace R, Leder P. Coexpression of
MMTV/v-Ha-ras and MMTV/c-myc genes in transgenic mice: synergistic action
of oncogenes in vivo . Cell 1987;49:465 – 75.
34. D’Cruz CM, Gunther EJ, Boxer RB, et al. c-MYC induces mammary
tumorigenesis by means of a preferred pathway involving spontaneous Kras2
mutations. Nat Med 2001;7:235 – 9.
35. Yeh E, Cunningham M, Arnold H, et al. A signalling pathway controlling
c-Myc degradation that impacts oncogenic transformation of human cells. Nat
Cell Biol 2004;6:308 – 18.
36. Felsher DW, Bishop JM. Reversible tumorigenesis by MYC in hematopoietic
lineages. Mol Cell 1999;4:199 – 207.
37. Felsher DW, Bishop JM. Transient excess of MYC activity can elicit
genomic instability and tumorigenesis. Proc Natl Acad Sci U S A 1999;96:
3940 – 4.
38. Pelengaris S, Littlewood T, Khan M, Elia G, Evan G. Reversible activation of
c-Myc in skin: induction of a complex neoplastic phenotype by a single
oncogenic lesion. Mol Cell 1999;3:565 – 77.
39. Jain M, Arvanitis C, Chu K, et al. Sustained loss of a neoplastic phenotype by
brief inactivation of MYC. Science 2002;297:102 – 4.
40. Donaldson TD, Duronio RJ. Cancer cell biology: Myc wins the competition.
Curr Biol 2004;14:R425 – 7.
41. O’Connell BC, Cheung AF, Simkevich CP, et al. A large scale genetic
analysis of c-Myc-regulated gene expression patterns. J Biol Chem 2003;278:
12563 – 73.
14. MacKenzie MJ. Molecular therapy in pancreatic adenocarcinoma. Lancet
Oncol 2004;5:541 – 9.
42. Feinberg MW, Watanabe M, Lebedeva MA, et al. Transforming growth
factor-h1 inhibition of vascular smooth muscle cell activation is mediated via
Smad3. J Biol Chem 2004;279:16388 – 93.
43. Frederick JP, Liberati NT, Waddell DS, Shi Y, Wang XF. Transforming
growth factor h-mediated transcriptional repression of c-myc is dependent on
direct binding of Smad3 to a novel repressive Smad binding element. Mol Cell
Biol 2004;24:2546 – 59.
15. Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2002;2:243 – 7.
44. Rak J, Yu JL. Oncogenes and tumor angiogenesis: the question of vascular
‘‘supply’’ and vascular ‘‘demand.’’ Semin Cancer Biol 2004;14:93 – 104.
16. Gitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular
antiviral immunity in human cells. Nature 2002;418:430 – 4.
45. Knies-Bamforth UE, Fox SB, Poulsom R, Evan GI, Harris AL. c-Myc
interacts with hypoxia to induce angiogenesis in vivo by a vascular endothelial
growth factor-dependent mechanism. Cancer Res 2004;64:6563 – 70.
13. Yoshida T, Ohnami S, Aoki K. Development of gene therapy to target
pancreatic cancer. Cancer Sci 2004;95:283 – 9.
17. Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. Activation of
the interferon system by short-interfering RNAs. Nat Cell Biol 2003;5:834 – 9.
18. Telford WG, King LE, Fraker PJ. Comparative evaluation of several DNA
binding dyes in the detection of apoptosis-associated chromatin degradation by
flow cytometry. Cytometry 1992;13:137 – 43.
19. Moore A, Donahue CJ, Bauer KD, Mather JP. Simultaneous measurement of
cell cycle and apoptotic cell death. Methods Cell Biol 1998;57:265 – 78.
20. Ito H, Duxbury M, Zinner MJ, Ashley SW, Whang EE. Glucose transporter-1
46. Sears R, Nuckolls F, Haura E, Taya Y, Tamai K, Nevins JR. Multiple rasdependent phosphorylation pathways regulate Myc protein stability. Genes Dev
2000;14:2501 – 14.
47. Hipfner DR, Cohen SM. Connecting proliferation and apoptosis in
development and disease. Nat Rev Mol Cell Biol 2004;5:805 – 15.
48. Kauffmann-Zeh A, Rodriguez-Viciana P, Ulrich E, et al. Suppression of
c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature
1997;385:544 – 8.
Mol Cancer Res 2005;3(7). July 2005
Downloaded from mcr.aacrjournals.org on June 18, 2017. © 2005 American Association for Cancer Research.
Molecular Consequences of K-ras siRNA
49. Yan Z, Chen M, Perucho M, Friedman E. Oncogenic Ki-ras but not
oncogenic Ha-ras blocks integrin h1-chain maturation in colon epithelial cells.
J Biol Chem 1997;272:30928 – 36.
56. Shiio Y, Donohoe S, Yi EC, Goodlett DR, Aebersold R, Eisenman RN.
Quantitative proteomic analysis of Myc oncoprotein function. EMBO J 2002;21:
5088 – 96.
50. Yan Z, Deng X, Chen M, et al. Oncogenic c-Ki-ras but not oncogenic c-Haras up-regulates CEA expression and disrupts basolateral polarity in colon
epithelial cells. J Biol Chem 1997;272:27902 – 7.
51. Brakebusch C, Wennerberg K, Krell HW, et al. h1 integrin promotes but is
not essential for metastasis of ras-myc transformed fibroblasts. Oncogene 1999;
18:3852 – 61.
52. Korah R, Boots M, Wieder R. Integrin a5h1 promotes survival of growtharrested breast cancer cells: an in vitro paradigm for breast cancer dormancy in
bone marrow. Cancer Res 2004;64:4514 – 22.
53. Corbi AL, Jensen UB, Watt FM. The a2 and a5 integrin genes: identification
of transcription factors that regulate promoter activity in epidermal keratinocytes.
FEBS Lett 2000;474:201 – 7.
54. Zhu S, Oh HS, Shim M, Sterneck E, Johnson PF, Smart RC. C/EBPh
modulates the early events of keratinocyte differentiation involving growth arrest
and keratin 1 and keratin 10 expression. Mol Cell Biol 1999;19:7181 – 90.
55. Zhu S, Yoon K, Sterneck E, Johnson PF, Smart RC. CCAAT/enhancer
binding protein-h is a mediator of keratinocyte survival and skin tumorigenesis
involving oncogenic Ras signaling. Proc Natl Acad Sci U S A 2002;99:207 – 12.
57. van Golen CM, Soules ME, Grauman AR, Feldman EL. N-Myc overexpression leads to decreased h1 integrin expression and increased apoptosis in
human neuroblastoma cells. Oncogene 2003;22:2664 – 73.
58. Flier JS, Mueckler MM, Usher P, Lodish HF. Elevated levels of glucose
transport and transporter messenger RNA are induced by ras or src oncogenes.
Science 1987;235:1492 – 5.
59. Yamamoto T, Seino Y, Fukumoto H, et al. Over-expression of facilitative
glucose transporter genes in human cancer. Biochem Biophys Res Commun 1990;
170:223 – 30.
60. Ogawa J, Inoue H, Koide S. Glucose-transporter-type-I-gene amplification
correlates with sialyl-Lewis-X synthesis and proliferation in lung cancer. Int J
Cancer 1997;74:189 – 92.
61. Kawamura T, Kusakabe T, Sugino T, et al. Expression of glucose transporter1 in human gastric carcinoma: association with tumor aggressiveness, metastasis,
and patient survival. Cancer 2001;92:634 – 41.
62. Weinstein IB. Cancer. Addiction to oncogenes—the Achilles heel of cancer.
Science 2002;297:63 – 4.
Mol Cancer Res 2005;3(7). July 2005
Downloaded from mcr.aacrjournals.org on June 18, 2017. © 2005 American Association for Cancer Research.
423
Molecular Consequences of Silencing Mutant K-ras in
Pancreatic Cancer Cells: Justification for K- ras−Directed
Therapy
Jason B. Fleming, Guo-Liang Shen, Shane E. Holloway, et al.
Mol Cancer Res 2005;3:413-423.
Updated version
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://mcr.aacrjournals.org/content/3/7/413
This article cites 61 articles, 18 of which you can access for free at:
http://mcr.aacrjournals.org/content/3/7/413.full.html#ref-list-1
This article has been cited by 23 HighWire-hosted articles. Access the articles at:
/content/3/7/413.full.html#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from mcr.aacrjournals.org on June 18, 2017. © 2005 American Association for Cancer Research.