Molecular Consequences of Silencing Mutant K-ras in Pancreatic … · Molecular Consequences of Silencing Mutant K-ras in Pancreatic Cancer Cells: Justification for K-ras–Directed

Molecular Consequences of Silencing Mutant K-ras inPancreatic Cancer Cells: Justification forK-ras–Directed Therapy

Jason B. Fleming,1 Guo-Liang Shen,1 Shane E. Holloway,1

Mishel Davis,1 and Rolf A. Brekken1,2

1Division of Surgical Oncology, Department of Surgery, and Hamon Center for TherapeuticOncology Research and 2Department of Pharmacology, University of TexasSouthwestern Medical School, Dallas, Texas

AbstractMutation 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

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)

IntroductionPancreatic cancer is now the fourth leading cause of cancer-

related 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-rasv12 while leaving

other ras isoforms unaffected. These studies showed that small

interfering RNA (siRNA) for K-ras decreases anchorage-

independent growth in a pancreatic cancer cell line (Capan-1).

Received 12/9/04; revised 5/17/05; accepted 6/6/05.Grant support: NIH grant R21 CA10669 (J.B. Fleming), American CancerSociety grant CRTG-01-152-01-CCE (J.B. Fleming), National Pancreas Founda-tion (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 ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Requests for reprints: Rolf A. Brekken, Hamon Center for TherapeuticOncology Research, University of Texas Southwestern Medical Center, 6000Harry Hines Boulevard, Dallas, TX 75390-8593. Phone: 214-648-5151; Fax:214-648-4940. E-mail: [email protected] D 2005 American Association for Cancer Research.

Mol Cancer Res 2005;3(7). July 2005 413

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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.

ResultsSpecificity of Mutant K-ras siRNA in Human PancreaticCancer 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 concen-

trations 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 MiaPaca2-

specific 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 afterK-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 tumorcells with mutant-specific siRNA. A. K-ras protein levels in whole celllysates 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 mismatchversion of the RNA oligospecific for the activating mutation in either Panc-1or MiaPaca-2 cells (Mismatch ), mutant K-ras (K-ras ), or Oligofectaminealone (Control ). B. K-ras protein levels in Panc-1 and MiaPaca-2 celllysates after titration of the mutant-specific K-ras siRNA or a time courseof mutant-specific K-ras siRNA (100 nmol/L). h-actin protein levels wereused as a cell lysate loading control. C. The level of lamin in Panc-1 cellsafter treatment with siRNA specific for lamin A/C, wild-type K-ras , mutantK-ras , and Oligofectamine alone was determined by Western blotting.D. K-ras h-actin protein levels in Panc-1 (P) and MiaPaca2 (M ) cells afterexposure to mutant-specific K-ras siRNA. The siRNA oligonucleotidesused 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 proteinassessed by Western blotting in Panc-1 and MiaPaca-2 cell lysates aftertitration of mutant-specific K-ras siRNA.

Fleming et al.

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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 dose-

dependent 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 siRNAwas 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 mutant-

specific 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 siRNAThe 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 extracel-

lular 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

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, andselected cell cycle proteins in lysates from Panc-1 and MiaPaca-2 cells was determined by Western blotting at the indicated time point after exposure tomutant-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 afterselective knockdown of mutant K-ras. Top, the number of MiaPaca-2 colonies 14 days after treatment with either Oligofectamine alone (Oligo ) or 100 nmol/Lof 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 opti-MEM (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 aspecificity control. The number of cells in each well was determined by counting with a hemocytometer 72 hours posttreatment with siRNA (*P < 0.05). D. Theeffect of various siRNA treatments on the number of Panc-1 and MiaPaca-2 cells is displayed. Cells were treated with Oligofectamine alone (Control ) or withsiRNA specific for the indicated target and counted with a hemocytometer at the indicated time.

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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 h1integrin 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 siRNAAngiogenesis 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

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 siRNATumor 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

FIGURE 3. Specific inhibition of mutant K-ras induces apoptosis inMiaPaca-2 cells. A. The level of active caspase-3 and caspase-9 in Panc-1 and MiaPaca-2 cells after exposure to mutant-specific K-ras siRNA wasdetermined by Western blotting. B. Panc-1 and MiaPaca-2 cells weretreated with the indicated concentration of mutant-specific K-ras siRNAand then stained with the DNA-binding dye Hoechst 33342. Thepercentage of cells showing nuclear condensation, consistent withapoptosis, was determined. Representative data from the MiaPaca-2cells is shown.

Table 1. Cell Migration after siRNA-Mediated Knockdownof Mutant K-ras

Cell Line Control siRNA

Panc-1 261 F 10.6 92 F 3.8*MiaPaca-2 347 F 11.9 102 F 8.1*

NOTE: Cells were treated with Oligofectamine (control) or mutant-specific K-rassiRNA, seeded in a transwell chamber, and allowed to migrate for 48 hours. Cellsthat migrated to the underside of the transwell membrane were stained withhematoxylin and counted under the microscope (�100 total magnification). Themean 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 cellmigration. Panc-1 and MiaPaca-2 cells were treated with Oligofectaminealone (Control) or 100 nmol/L mutant-specific K-ras siRNA (siRNA) asindicated in Materials and Methods. The cells were then placed in theupper chamber of a transwell insert coated with Matrigel and incubated for48 hours. Cells that migrated across the transwell membrane were stainedwith hematoxylin and counted under the microscope (see Table 1).

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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.

DiscussionThe 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 adeno-

carcinoma 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,

FIGURE 5. Reduction ofmutant K-ras in pancreatic can-cer cells alters the level ofintegrin expression. Panc-1 (A)or MiaPaca-2 (B) cells weretreated with Oligofectaminealone (Control ) or 100 nmol/Lof mutant-specific K-ras siRNA(siRNA ) for 48 hours. The cellswere then fixed with 4% para-formaldehyde and incubatedwith antibodies specific foreither a5 or h1 integrin. Cellswere subsequently incubatedwith Cy-3– conjugated second-ary antibodies (red) along withHoechst 33342 (blue ) andimages captured as described.Signal was quantified usingMetamorph software and showeda statistical reduction in theexpression of a5 integrin inK-ras siRNA – treated Panc-1cells and a statistical reductionin h1 integrin levels in K-rassiRNA – treated MiaPaca-2cells.

Table 2. Level of VEGF Expression after siRNA-Mediated Knockdown of Mutant K-ras

Treatment Time Point

48 h 72 h 96 h

Control siRNA Control siRNA Control siRNA

Panc-1 580 F 73.7 455 F 67.8 723 F 1.0 574 F 41.5* 805 F 158.5 401 F 98.9*MiaPaca-2 451 F 42 132 F 6.7c 360 F 45.8 70 F 10.6c 540 F 87 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 wereharvested, 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 timeposttransfection 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.



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decreased apoptosis, angiogenesis induction, and the ability of

cells to migrate and metastasize to distant organs. Accordingly,

therapy that reverses these characteristics represents a poten-

tially 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 tu-

morigenesis 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 adenocar-

cinoma 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-hsignaling via the Smad4 mechanism, but MiaPaca-2 cells lack a

functional transforming growth factor-h receptor 2. Trans-

forming 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

FIGURE 6. Reduction of mutantK-ras in pancreatic cancer cells resultsin decreased angiogenic potential. A.The level TSP-1 and h-actin protein wasdetermined at various time points aftertreatment of Panc-1 and MiaPaca-2cells with 100 nmol/L mutant-specificK-ras siRNA. Cell lysates from treatedcells, conditioned media from humanumbilical vascular endothelial cell (EC ),or recombinant TSP-1 (rTSP ) wereseparated by nonreducing SDS-PAGE,transferred to nitrocellulose, and probedwith a monoclonal antibody specific forhuman TSP-1. B. The level of K-ras,c-myc, TSP-1, and h-actin in Panc-1 andMiaPaca-2 cells treated with variousconcentrations of mutant-specific K-rassiRNA was determined by Western blotanalysis. C. The level of mRNA encod-ing TSP-1 in Panc-1 and MiaPaca-2cells treated with the indicated concen-tration of mutant-specific K-ras siRNAwas determined by reverse transcription-PCR analysis. The level of glyceral-dehyde-3-phosphate dehydrogenasemRNA was used as a loading control.mRNA isolated from human umbilicalvascular endothelial cell or Oligofect-amine alone– treated pancreatic tumorcells (C) were used as controls.

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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 mu-tant K-ras alters the expressionof the c-myc – regulated metab-olism gene glucose transporter 1(GLUT-1 ). Panc-1 (A) or Mia-Paca-2 (B) cells treated withOligofectamine (Control ) or mu-tant-specific K-ras siRNA wereevaluated for the expression ofGLUT-1 by immunocytochemis-try. The level of GLUT-1 expres-sion was quantified by the use ofMetamorph software as de-scribed. C. The level of mRNAencoding GLUT-1 in Panc-1 andMiaPaca-2 cells after exposurefor the indicated time to 100nmol/L mutant-specific K-rassiRNA was determined by re-verse transcription-PCR. D. Thelevel of GLUT-1 and h-actinprotein expression in Panc-1and MiaPaca-2 cells after expo-sure to mutant-specific K-rassiRNA was determined by West-ern blot analysis of whole celllysates.



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and c-myc, whereas no such changes are observed in MiaPaca-

2 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 ras-

myc–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 MethodsCell 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 RNAiUsing 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-GTTGGAGCTGGTGGCGTAG-3V/5 V-CAACCTCGACCACCGCATC-3V; for MiaPaca-2 mutant

K-ras , 5 V-GTTGGAGCTTGTGGCGTAG-3V/5V-CAACCTC-GAACACCGCATC-3V; and for Panc-1 mutant K-ras ,

5V-GTTGGAGCTGATGGCGTAG-3V/5 V-CAACCTCGAC-

TACCGCATC-3V, scrambled 5 V-CGAAGUGUGUGUGU-

GUGGC-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. Annealedoligos (20 Amol/L) were aliquoted and stored at�80jC until use.

For RNAi Panc-1 (f3.5 � 104) and MiaPaca-2 (f4.5 �104), 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 (Invitro-

gen) 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 ApoptosisAnalysis

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

Fleming et al.


420



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 AssayThe migration of human pancreatic cancer cells through an

artificial extracellular matrix (Matrigel, BD Biosciences, Bed-

ford, 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 Ampore 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 high-

powered field.

Reverse Transcription-PCRTotal 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. RNAwas 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-CAACTGGACCTCAAATTTCA-TTGTGGG-3 V/5V-CGGGTGTCTTATCACTTTGGCTGG-3 V;TSP-1, 5 V-CAGAGGCCAAAGCACTAAGG-3V/5V-TGAG-

TAAGGGTGGGGATCAG-3 V; and glyceraldehyde-3-phosphate

dehydrogenase, 5 V-TCCACCACCCTGTTGCTGTAG-3 V/5 V-GACCACAGTCCATGCCATCACT-3 V.

Western BlotCells 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 deter-

mined 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 (sc-

94), 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 Interna-

tional (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 Birming-

ham, 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).

ImmunocytochemistryCells 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. Thecells were counterstained with Hoechst 33342 dye (Molecular

Probes, Eugene, OR) and exposed to 1:500 Cy-3 or FITC-

labeled secondary antibody (Jackson ImmunoResearch Labo-

ratories, 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



421



determined for each target and for 4 V,6-diamidino-2-phenyl-

indole 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 ProductionAfter 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).

AcknowledgmentsWe thank Dr. Latha Shivakumar for technical assistance; Drs. John Minna andHelene Sage, members of our laboratory, and David Shames for helpfuldiscussions and support; and Dr. Joanne Murphy-Ullrich for providing TSP-1 andanti –TSP-1 antibodies for confirmation of our results.

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2005;3:413-423. Mol Cancer Res Jason B. Fleming, Guo-Liang Shen, Shane E. Holloway, et al. Therapy

Directed−rasPancreatic Cancer Cells: Justification for K- inrasMolecular Consequences of Silencing Mutant K-

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Molecular Consequences of Silencing Mutant K-ras in Pancreatic … · Molecular Consequences of Silencing Mutant K-ras in Pancreatic Cancer Cells: Justification for K-ras–Directed