Cancer Research SABCS EMT and Cancer Progression and Treatment
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smakman, N.
Right arrow Articles by Kranenburg, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smakman, N.
Right arrow Articles by Kranenburg, O.
[Cancer Research 66, 5403-5408, May 15, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

KRASD13 Promotes Apoptosis of Human Colorectal Tumor Cells by ReovirusT3D and Oxaliplatin but not by Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand

Niels Smakman1, Diana J.M. van den Wollenberg3, Sjoerd G. Elias2, Takehiko Sasazuki2, Senji Shirasawa4, Rob C. Hoeben3, Inne H.M. Borel Rinkes1 and Onno Kranenburg1

1 Department of Surgery, University Medical Center Utrecht; 2 Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, the Netherlands; 3 Virus and Stem Cell Biology Laboratory, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands; and 4 Research Institute, International Medical Center of Japan, Toyama, Shinjuku-ku, Tokyo, Japan

Requests for reprints: Onno Kranenburg, Department of Surgery, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, the Netherlands. Phone: 31-30-253-9729; Fax: 31-30-254-1944; E-mail: o.kranenburg{at}azu.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Colorectal tumors frequently contain activating mutations in KRAS. ReovirusT3D is an oncolytic virus that preferentially kills tumor cells with an activated Ras pathway. Here we have assessed the contribution of endogenous mutant KRAS in human colorectal cancer cell lines to ReovirusT3D replication and to tumor cell oncolysis. In addition, treatment combinations involving ReovirusT3D, oxaliplatin, and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) were tested for their efficacy in tumor cell killing. The mutation status of KRAS did not predict the sensitivity of a panel of human colorectal cancer cell lines to ReovirusT3D. Virus replication was observed in all cell lines tested regardless of KRAS status and was not affected by deletion of endogenous mutant KRASD13. However, deletion of KRASD13 or p53 did reduce apoptosis induction by ReovirusT3D whereas deletion of ß-catenin{Delta}S45 had no effect. Likewise, KRASD13- or p53-deficient cells display reduced sensitivity to oxaliplatin but not to death receptor activation by TRAIL. Finally, the treatment of colorectal cancer cells with ReovirusT3D combined with either oxaliplatin or TRAIL resulted in a nonsynergistic increase in tumor cell killing. We conclude that oncolysis of human tumor cells by ReovirusT3D is not determined by the extent of virus replication but by their sensitivity to apoptosis induction. Oncogenic KRASD13 increases tumor cell sensitivity to activation of the cell-intrinsic apoptosis pathway without affecting ReovirusT3D replication. (Cancer Res 2006; 66(10): 5403-8)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the search for alternative effective anticancer drugs that selectively target tumor cells over normal cells, an arsenal of oncolytic viruses has been identified (1). Whereas most of these viruses have been genetically modified to attain tumor selectivity, some display inherent selectivity for transformed or tumor cells. One such virus is ReovirusT3D, which exploits signaling pathways activated by oncogenic RAS (2), the most frequently activated oncogene family in human cancer. ReovirusT3D-induced cell killing is greatly enhanced in cells overexpressing mutant HRASV12 (2, 3). One of the tumor types that frequently (~35%) harbor activating mutations in the KRAS gene are colorectal carcinomas (4, 5). Colorectal tumors are the second leading cause of cancer-related deaths worldwide and response rates to conventional chemotherapeutics are low. Thus, ReovirusT3D may be an attractive additive therapeutic in the treatment of colorectal cancer tumors.

Reovirus-infected cells contain double-stranded viral RNAs that activate the cellular protein kinase R. Activated protein kinase R phosphorylates the translation elongation factor 2{alpha} and thereby inhibits cellular and viral protein synthesis. Overexpression of HRASV12 interferes with this antiviral cellular defense mechanism and allows reovirus replication in otherwise nonpermissive cells (3). It has been suggested that RAS-mediated suppression of this defense mechanism underlies the selective oncolysis of human tumor cells harboring mutant KRAS or an "activated Ras pathway" (3). However, we have recently shown that suppression of endogenous KRASD12 in mouse colorectal carcinoma cells had no effect on reovirus protein synthesis or on virus replication but abrogated ReovirusT3D-induced tumor cell apoptosis (6). Activation of the apoptotic program in virus-infected cells is a natural defense mechanism that limits viral spread in the infected host (7). Alternatively, apoptosis of infected cells may facilitate the release of virus progeny and subsequent dissemination (7). From a therapeutic point of view, the apoptosis-inducing strains of reovirus may be regarded as self-amplifying inducers of tumor cell apoptosis with limited pathogenic side effects. If ReovirusT3D is to be used as an additional therapeutic agent in the treatment of human colorectal cancer, it is imperative that the mechanisms underlying KRAS-dependent human tumor cell killing are fully understood. In the current report, we have investigated how endogenous mutant KRAS in human colorectal carcinoma cells affects ReovirusT3D protein synthesis, ReovirusT3D replication, and cellular sensitivity to ReovirusT3D-induced oncolysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions. The colorectal cancer cell lines HT29, DLD1, SW480, HCT15, and LS174T were purchased from American Type Culture Collection (ATCC, Manassas, VA). The HCT116 cell line and its isogeneic derivatives lacking ß-catenin{Delta}S45 or p53 were kindly provided by Prof. Bert Vogelstein (The Johns Hopkins Medical Institutions, Baltimore, MD) and were described before (8, 9). The HCT116 cells lacking KRASD13 (Hkh2) with their own HCT116 control cells were obtained from Dr. Shirasawa (International Medical Center of Japan, Tokyo, Japan) and were also described before (10). All cell lines were grown in DMEM supplemented with 5% FCS and antibiotics in a 5% CO2-humidified atmosphere.

Materials. Oxaliplatin was obtained from Sanofi-Aventis (Bridgewater, NJ). A bacterial expression vector encoding His-tagged tumor necrosis factor–related apoptosis-inducing ligand (TRAIL; pETdwHisTRAIL114-281) was kindly provided by Dr. Dai-Wu Seol (University of Pittsburgh School of Medicine, Pittsburgh, PA). TRAIL was produced in bacteria and was purified on a Ni2+-agarose column.

ReovirusT3D stock. ReovirusT3D was purchased from ATCC (VR-824). The virus stock was prepared using 911 cells (11). Cells were infected with ReovirusT3D [5 plaque-forming units (pfu)/cell] in DMEM/2% FCS. After initial infection (2 hours at 37°C, 5% CO2), the medium was replaced with normal DMEM containing 10% FCS. The virus was harvested after 48 hours by resuspending the cells in PBS with 2% FCS at a density of ~109/mL and subsequent freeze-thawing for three cycles. The lysates were cleared by centrifugation at 2,000 rpm in a table centrifuge for 10 minutes. The virus was purified by layering the supernatant on a CsCl gradient as described for purification of adenovirus particles (11). To remove the CsCl, reovirus was dialysed against ReovirusT3D storage buffer (10 mmol/L Tris-HCl, 150 mmol/L NaCl, 10 mmol/L MgCl2·6H2O). The final dialysis was done in ReovirusT3D storage buffer containing 5% sucrose. Virus stocks were stored at –80°C and the concentration was determined by performing standard plaque assays on 911 cells (11).

[35S]Methionine labeling. Infected or mock-infected cells were labeled with Redivue [35S]methionine Pro-mix (200 µCi/mL; Amersham, Roosendaal, the Netherlands) for 4 hours 3 days postinfection. Cells were washed with PBS once and total cell extracts were prepared in sample buffer and were run out on long 45-cm SDS-polyacrylamide gels. Gels were then dried and exposed to radiographic film.

Ras assay. The Ras-binding domain of Raf fused to glutathione S-transferase and coupled to glutathione-sepharose was used as an affinity matrix for activated RAS. Anti-KRAS (F234) was purchased from Santa Cruz Technologies (Santa Cruz, CA). The assay was done exactly as described (12).

Viability assay. Cells were plated at a density of 5,000 per well in 96-well plates. Cell viability of infected and mock-infected cells was then analyzed for 6 consecutive days by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Roche Diagnostics, Almere, the Netherlands) according to the instructions of the manufacturer.

Statistical analyses. All statistical analyses were done with SPSS 11.0 (SPSS, Chicago, IL), tests were two-sided, and the cutoff for statistical significance was 0.003 (adjusted for multiple testing by means of the Bonferroni correction).

Combinations of ReovirusT3D, oxaliplatin, and TRAIL were used to investigate their main and combined effects in killing HCT116 cells. We defined the measure of effect as percentage viable cells at day 3 compared with the initiation of the experiment, which was ascertained for each treatment regimen (control, ReovirusT3D, oxaliplatin, TRAIL, ReovirusT3D + oxaliplatin, ReovirusT3D + TRAIL, and oxaliplatin + TRAIL). First, we compared the effect of each combined regimen with each of the two individual treatment strategies as well as the control samples by means of a Student's t test. This was done to evaluate any added value of a combination strategy above the individual treatment strategies in killing HCT116 cells. Interactive effects were investigated on a multiplicative scale by log-transforming the data. For this, we estimated the effect of each individual treatment strategy compared with the control and added both effects to form an additivity reference. The ratio of the observed combination strategy effect and this additivity reference was used to judge interaction (i.e., an interaction ratio of 1 suggests additivity, a ratio of <1 antagonism, and a ratio of >1 synergy). Linear regression was used to test any deviation from the additivity reference for statistical significance (R2 change test after introducing interaction terms to the model already containing indicator variables for the two individual treatment strategies).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The sensitivity of colorectal cancer cells to ReovirusT3D does not correlate with the presence of active KRAS. The notion that reovirus replication and oncolysis of tumor cells depends on RAS signaling is largely based on the use of nonepithelial cells overexpressing mutant RAS or growth factor receptors (2, 3, 13). Therefore, we first assessed whether the sensitivity of human colorectal carcinoma cells to ReovirusT3D correlates with the presence of endogenous active KRAS. The mutation status of the KRAS and BRAF genes in a panel of human colorectal cancer cell lines was analyzed by PCR and sequence analysis and was in line with previous analyses in all cases. Next, KRAS activity was measured in the same panel of serum-starved cell lines. High levels of constitutive KRAS activity were detected in LS174T (KRASG12D), DLD1 (KRASG13D), SW480 (KRASG12V), and HCT116 (KRASG13D) cells (Fig. 1A ). HCT15 cells also harbor a KRASG13D allele, but constitutive KRAS activity was markedly lower than in the other cell lines expressing mutant KRAS (Fig. 1A). HT29 cells with two wild-type KRAS alleles (but carrying BRAFV600E) displayed the lowest level of constitutive KRAS activity, as expected (Fig. 1A). None of the cell lines harbored mutations in the NRAS or HRAS genes at codon 12, 13, or 61 (not shown) and mutant BRAF was only found in HT29.


Figure 1
View larger version (15K):
[in this window]
[in a new window]
  		Figure 1. KRAS activity does not correlate with cellular sensitivity to ReovirusT3D. A, LS174T, HT29, DLD1, HCT15, SW480, and HCT116 cells were serum starved overnight and KRAS activity was assessed by the RAS activity assay. The "active" lane shows the amount of KRAS that was precipitated from the cell lysates using the Ras-binding domain of Raf as an affinity matrix for active RAS, immobilized on glutathione sepharose. Ten percent of the input in the activity assays was also used to directly assess the amount of KRAS in the cell lysates. Western blot analysis using a KRAS-specific antibody was done to assess the levels of KRAS in both sets of samples. B, the same cell lines were seeded in 96-well plates (5,000 per well) and were subsequently infected with ReovirusT3D at a multiplicity of infection of 20 pfu/cell or were mock infected using ReovirusT3D storage buffer. The relative number of viable cells was then assessed over time by standard MTT assays. C, the reduction in cell viability induced by ReovirusT3D over time is plotted relative to untreated control cells.

 
Next, we assessed whether constitutive KRAS activity would correlate with cellular sensitivity to oncolysis by ReovirusT3D. All cell lines were infected with ReovirusT3D (20 pfu/cell) and the relative number of viable cells was assessed over a period of 6 consecutive days. All cell lines, except LS174T, showed a reduction in cell viability following infection with ReovirusT3D. However, the sensitivity to oncolysis varied considerably between the cell lines tested, from LS174T cells being completely resistant, to HT29, HCT15, DLD1, and SW480 showing intermediate sensitivity, and HCT116 cells being extremely sensitive (Fig. 1B). Surprisingly, neither the level of active KRAS protein nor the presence of mutant KRAS alleles correlated with tumor cell sensitivity to ReovirusT3D-induced oncolysis (Fig. 1A-C).

The sensitivity of colorectal cancer cells to ReovirusT3D does not correlate with virus replication. We next tested whether reovirus replication correlated with either the presence of mutant KRAS or with tumor cell killing. Cells were infected with ReovirusT3D and were labeled with [35S]methionine for 4 hours, 3 days following infection. Cell extracts of infected and uninfected cells were then analyzed by SDS-PAGE. ReovirusT3D replicated in all cell lines examined regardless of KRAS activity and regardless of their sensitivity to oncolysis. The most extreme example is the cell line LS174T, which was completely resistant to reovirus-induced cell killing while supporting extensive virus replication (Fig. 2 ). Thus, ReovirusT3D replication can occur in the absence of active KRAS (HT29) and does not necessarily result in the induction of an overt cytopathic effect (LS174T).


Figure 2
View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. ReovirusT3D replication does not correlate with cellular sensitivity to ReovirusT3D. HCT116, SW480, HCT15, DLD1, HT29, and LS174T cells were seeded in 24-well plates and were infected with ReovirusT3D (20 pfu/cell) or were mock infected using ReovirusT3D storage buffer. Three days after infection, cells were labeled with [35S]methionine and cell lysates were analyzed for ReovirusT3D replication. The positions of the reovirus {lambda}, µ, and {sigma} proteins are indicated. All cell lines sustained virus replication irrespective of KRAS mutation status or KRAS protein activity.

 
KRASD13 deletion does not affect ReovirusT3D replication but reduces cellular sensitivity to oncolysis. To further assess the correlation between mutant KRAS, ReovirusT3D replication, and sensitivity to oncolysis, we used the HCT116 cell line and its isogeneic derivative Hkh2 from which the KRASG13D allele has been deleted through homologous recombination (10). We first tested KRAS activity in HCT116 and Hkh2 cells and found that KRAS was constitutively active in HCT116 but not in Hkh2 cells, as expected (Fig. 3A ). HCT116 and Hkh2 cells were then infected with ReovirusT3D and viral protein synthesis was examined 24 and 48 hours postinfection. Figure 3B shows that KRASD13 deletion had no effect on ReovirusT3D protein synthesis. We next assessed whether KRAS deletion would affect ReovirusT3D propagation. HCT116 and Hkh2 cells were infected and viral progeny was quantified 4 days postinfection. Hkh2 cells produced ~50-fold more virus than HCT116 cells (Fig. 3C). Finally, we assessed the capacity of ReovirusT3D to induce oncolysis in both cell lines. Cell viability was assessed for 4 consecutive days following infection. We found, as above, that HCT116 cells were very sensitive to oncolysis by ReovirusT3D as viability rapidly dropped between 2 and 3 days postinfection (Fig. 3D). Hkh2 cells, however, were more resistant and viability dropped ~24 hours later, between 3 and 4 days postinfection. Importantly, the delay in oncolysis was highly reproducible and was not the result of delayed viral protein synthesis or virus production (Fig. 3B and C). The delay in oncolysis provides a possible explanation for the fact that KRAS-deleted cells produce 50-fold more infectious ReovirusT3D particles. By inference, KRAS-deleted Hkh2 cells lose viability when exposed to a much higher dose of ReovirusT3D than the HCT116 cells as infectious virus particles are continuously being produced during the course of the experiment.


Figure 3
View larger version (12K):
[in this window]
[in a new window]
  		Figure 3. Deletion of KRASD13 reduces oncolysis but not virus replication. A, HCT116 and Hkh2 cells were serum starved and KRAS activity was assessed by the RAS activity assay. The "active" lane shows the amount of KRAS that was precipitated from the cell lysates using the RAS-binding domain of Raf. The "lysate" lane contains 10% of the input lysates and is shown as a reference. B, HCT116 and Hkh2 cells were infected with ReovirusT3D or were mock infected and viral protein synthesis was assessed 24 and 48 hours after infection. C, HCT116 and Hkh2 cells were infected with ReovirusT3D or were mock infected. Four days after infection, when most cells in both cultures have died, the virus yield was assessed by plaque assays on 911 cells. D, HCT116 and Hkh2 cells were infected with ReovirusT3D or were mock infected and the relative number of viable cells was then assessed over time by standard MTT assays. E, HCT116 and Hkh2 cells were pretreated for 2 hours with z-VAD (100 µmol/L) or with solvent (DMSO) for 2 hours before infection with ReovirusT3D or mock infection. Two days after infection, cell viability was assessed as above and this is expressed as percent viability relative to mock-infected, DMSO-treated control cells.

 
The cytopathic effect of reovirus on susceptible cells and tissues is due to apoptosis induction (7). Therefore, we ascertained the apoptotic nature of ReovirusT3D-induced HCT116 and Hkh2 cell death. To this end, HCT116 and Hkh2 cells were incubated with the pancaspase inhibitor z-VAD, a widely used inhibitor of apoptotic, but not necrotic, cell death or virus replication. Figure 3E shows that, as expected, z-VAD prevented oncolysis of both HCT116 and Hkh2 cells by reovirus.

From the above experiments, we conclude that KRAS promotes ReovirusT3D-induced tumor cell apoptosis but does not facilitate virus replication. These results argue against a model in which activated KRAS stimulates human tumor cell oncolysis by facilitating virus replication.

KRASD13 and p53, but not ß-catenin{Delta}S45, modulate tumor cell sensitivity to ReovirusT3D oncolysis. Multiple genetic changes underlie the progressive development of colorectal carcinomas. These include activating mutations in KRAS, deregulated Wnt signaling (inactivation of the tumor suppressor APC either through oncogenic activation of ß-catenin or through epigenetic silencing of SFRP genes), and loss of the tumor suppressor p53 (14). HCT116 cells express not only oncogenic KRASD13 but also oncogenic ß-catenin{Delta}S45 and wild-type p53 (8, 9). To assess the possible contribution of p53 and mutant ß-catenin to colorectal cancer cell killing by ReovirusT3D, we used HCT116-derived cell lines in which either mutant ß-catenin{Delta}S45 or p53 was deleted through homologous recombination (8, 9). Each mutant cell line was compared with the control HCT116 cell line obtained from the same laboratory. All cell lines were infected with ReovirusT3D and viral protein synthesis and sensitivity to oncolysis were assessed as above. Virus replication was not affected in any of the mutant cell lines (Fig. 4A ). However, deletion of p53, like deletion of KRASD13, but not deletion of ß-catenin{Delta}S45, delayed tumor cell killing by ReovirusT3D by ~24 hours (Fig. 4B). Thus, neither KRASD13, ß-catenin{Delta}S45, nor p53 affects ReovirusT3D replication in human HCT116 colorectal cancer cells. Tumor cell oncolysis through apoptosis induction, however, is stimulated by KRASD13 and p53, but not by ß-catenin{Delta}S45.


Figure 4
View larger version (19K):
[in this window]
[in a new window]
  		Figure 4. Deletion of p53, but not ß-catenin{Delta}S45, reduces ReovirusT3D-induced oncolysis but not virus replication. A, HCT116 cells and isogeneic derivatives lacking either p53 or ß-catenin{Delta}S45 were infected with ReovirusT3D or mock infected and viral protein synthesis was assessed 48 hours after infection. All cell lines sustained virus replication irrespective of p53 or ß-catenin status. B, HCT116 cells and isogeneic derivatives lacking either p53 or ß-catenin{Delta}S45 were infected with ReovirusT3D or mock infected and the relative number of viable cells was then assessed over time by standard MTT assays as above.

 
KRASD13 deletion reduces colorectal cancer cell sensitivity to oxaliplatin but not to TRAIL. p53-deleted HCT116 cells are relatively resistant not only to ReovirusT3D (this report) but also to standard chemotherapeutics used in the adjuvant treatment of colorectal cancer: 5-fluorouracil (5-FU) and oxaliplatin (8, 15). Furthermore, it has recently been shown that KRAS-deleted HCT116 cells are relatively resistant to 5-FU (16). Therefore, we tested whether KRASD13 deletion affected HCT116 cell sensitivity to oxaliplatin. Figure 5A (top) shows that KRAS-deleted HCT116 cells show reduced sensitivity to oxaliplatin when compared with HCT116 cells. Thus, KRAS-deletion reduces cellular sensitivity to genotoxic stress induced by both 5-FU and oxaliplatin. Apoptosis may ensue from activation of the intrinsic cascade (in response to genotoxic stress) or via activation of death receptor ligands (extrinsic cascade). Reovirus-induced apoptosis may require both pathways (17–19). We tested whether KRAS deletion affected apoptosis induction by TRAIL, a ligand for death receptor 4/5 that induces apoptosis in several human cancer cell lines. Figure 5B shows that KRAS-deleted cells are not resistant to TRAIL-induced apoptosis. In fact, we consistently observed that KRAS-deleted cells were slightly more sensitive to TRAIL-induced apoptosis than the control HCT116 cells.


Figure 5
View larger version (18K):
[in this window]
[in a new window]
  		Figure 5. Deletion of KRASD13 reduces oncolysis by oxaliplatin but not by TRAIL. HCT116 and Hkh2 cells (lacking KRASD13) were infected with ReovirusT3D or mock infected and were treated with oxaliplatin (A) or TRAIL (B) at the indicated concentrations for 2 days. Cell viability was then assessed by standard MTT assays after 2 days as above. C, HCT116 cells were treated with combinations of ReovirusT3D (10 pfu/cell), oxaliplatin (2 µg/mL), and TRAIL (25 ng/mL) and cell viability was assessed over time. Student's t test: ReovirusT3D + oxaliplatin versus control (P < 0.001), versus oxaliplatin (P < 0.001), versus ReovirusT3D (P < 0.001); ReovirusT3D + TRAIL versus control (P < 0.001); versus TRAIL (P = 0.002), versus ReovirusT3D (P = 0.021); oxaliplatin + TRAIL versus control (P < 0.001), versus oxaliplatin (P = 1.000), versus TRAIL (P = 0.002); cutoff for statistical significance, P = 0.003.

 
Simultaneous activation of the intrinsic and extrinsic pathways of apoptosis induction may lead to synergistic tumor cell killing. Therefore, we investigated how combinations of ReovirusT3D, oxaliplatin, and TRAIL at suboptimal concentrations would affect HCT116 cell viability. Figure 5C shows that the combination of ReovirusT3D with oxaliplatin was significantly more effective in killing HCT116 cells than either compound alone (P < 0.001 on day 3). ReovirusT3D with TRAIL was also significantly more effective in killing HCT116 cells than TRAIL alone (P = 0.002) but not than ReovirusT3D alone (P = 0.021). The combination of oxaliplatin with TRAIL was able to significantly kill HCT116 cells more effective than TRAIL alone (P = 0.002) but not than oxaliplatin alone (P = 1.000). None of the combination treatments produced a statistically significant synergistic effect on tumor cell killing when compared with the respective single treatments. Similarly, it was recently reported that oxaliplatin fails to synergize with FAS-L in the induction of apoptosis in HCT116 cells (20).

Taken together, we propose that mutant KRAS promotes the induction of apoptosis in ReovirusT3D-infected tumor cells by sensitizing these cells to activation of the intrinsic apoptosis cascade that is known to be associated with ReovirusT3D infection (18, 19). Our results do not support a role for KRAS in promoting ReovirusT3D replication.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been proposed that RAS-stimulated ReovirusT3D replication underlies the KRAS dependency of human tumor cell killing by reovirus (3). Although our work subscribes the view that ReovirusT3D oncolysis of tumor cells is facilitated by endogenous mutant KRAS, we propose that a different mechanism underlies this selectivity. We have recently shown that suppression of endogenous mutant KRASG12D in murine colorectal cancer cells does not affect ReovirusT3D replication but abrogates ReovirusT3D-induced apoptosis (6). In the present report, we show that ReovirusT3D can infect and replicate in all human colorectal cancer cell lines tested but that this does not correlate with either RAS/BRAF status or with tumor cell killing. The relative importance of mutant KRAS in determining the sensitivity of human tumor cells to ReovirusT3D is very much dependent on the specific tumor cell under study. We find that although endogenous mutant KRAS can modulate the sensitivity of human tumor cells to ReovirusT3D-induced apoptosis, it is certainly not the only and prime determinant. Additional factors, including p53, clearly play a role in determining cellular sensitivity to oncolysis. It may therefore be expected that the response of individual colorectal tumors to ReovirusT3D as a therapeutic drug will show considerable variation regardless of KRAS status. The safety and efficacy of ReovirusT3D therapy in the treatment of human cancer are currently being evaluated in clinical trials (21).

Oxaliplatin and 5-FU are standard chemotherapeutics in the treatment of metastatic colorectal cancer (22–25). The sensitization of human colorectal tumor cells to ReovirusT3D by mutant KRAS was accompanied by a similar sensitization to oxaliplatin, which kills tumor cells by inducing genotoxic stress and subsequent apoptosis induction. Apoptosis induction by oxaliplatin is accompanied by activation of p53 and is reduced in HCT116 cells from which the genes encoding either p53 or the proapoptotic bcl-2 family member Bax are deleted (15, 26). Thus, oxaliplatin-induced apoptosis occurs through activation of the intrinsic mitochondria-dependent apoptosis pathway. Likewise, ReovirusT3D-induced apoptosis involves activation of the mitochondrial pathway (19, 27) and is reduced in HCT116 cells from which p53 is deleted (this study). ReovirusT3D-induced apoptosis also requires activation of a death receptor-dependent (extrinsic) pathway that is activated by TRAIL (17, 18). In the present study, the reduction in ReovirusT3D-induced apoptosis in KRAS-deleted cells was accompanied by a similar reduction in oxaliplatin-induced, but not TRAIL-induced, apoptosis. Therefore, a general sensitization to apoptosis induction via the intrinsic apoptosis pathway may underlie the KRAS dependency of tumor cell oncolysis by ReovirusT3D. Interestingly, ReovirusT3D-induced apoptosis is associated with decreased expression of DNA repair enzymes, which may promote apoptosis induction through accumulation of DNA damage and activation of the intrinsic pathway of apoptosis induction (28).

Combination of compounds that induce genotoxic stress (such as oxaliplatin) with those that activate death receptors (such as TRAIL) could theoretically generate synergistic antitumor responses. It has previously been shown that ReovirusT3D can synergize with TRAIL in the induction of apoptosis in adenovirus-transformed human embryonic kidney (293) cells (17, 29). In the present study, we have not been able to show synergistic colorectal tumor cell killing by combining ReovirusT3D with either oxaliplatin or with TRAIL. A possible explanation for the lack of synergy could be that ReovirusT3D alone already activates both the intrinsic and the extrinsic pathways that lead to apoptosis induction. Future studies should assess whether or not ReovirusT3D therapy has added value in the combinatorial treatment of colorectal tumors and whether tumor responses depend on the presence of mutant KRAS.


   Acknowledgments
 
Grant support: Wijnand M. Pon Foundation.

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.

We thank Prof. B. Vogelstein for the kind gift of the HCT116 cells lacking p53 or ß-catenin{Delta}S45.

Received 11/16/05. Revised 2/ 6/06. Accepted 3/ 9/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Biederer C, Ries S, Brandts CH, McCormick F. Replication-selective viruses for cancer therapy. J Mol Med 2002;80:163–75.[CrossRef][Medline]
  2. Coffey MC, Strong JE, Forsyth PA, Lee PW. Reovirus therapy of tumors with activated Ras pathway. Science 1998;282:1332–4.[Abstract/Free Full Text]
  3. Strong JE, Coffey MC, Tang D, Sabinin P, Lee PW. The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus. EMBO J 1998;17:3351–62.[CrossRef][Medline]
  4. Andreyev HJ, Norman AR, Cunningham D, Oates JR, Clarke PA. Kirsten ras mutations in patients with colorectal cancer: the multicenter "RASCAL" study. J Natl Cancer Inst 1998;90:675–84.[Abstract/Free Full Text]
  5. Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer: the "RASCAL II" study. Br J Cancer 2001;85:692–6.[CrossRef][Medline]
  6. Smakman N, van den Wollenberg D, Hoeben RC, Borel Rinkes IHM, Kranenburg O. Sensitization to apoptosis underlies KrasD12-dependent oncolysis of murine C26 colorectal carcinoma cells by Reovirus T3D. J Virol 2005;79:14981–5.[Abstract/Free Full Text]
  7. Clarke P, Debiasi RL, Goody R, Hoyt CC, Richardson-Burns S, Tyler KL. Mechanisms of reovirus-induced cell death and tissue injury: role of apoptosis and virus-induced perturbation of host-cell signaling and transcription factor activation. Viral Immunol 2005;18:89–115.[CrossRef][Medline]
  8. Bunz F, Hwang PM, Torrance C, et al. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J Clin Invest 1999;104:263–9.[Medline]
  9. Chan TA, Wang Z, Dang LH, Vogelstein B, Kinzler KW. Targeted inactivation of CTNNB1 reveals unexpected effects of ß-catenin mutation. Proc Natl Acad Sci U S A 2002;99:8265–70.[Abstract/Free Full Text]
  10. Shirasawa S, Furuse M, Yokoyama N, Sasazuki T. Altered growth of human colon cancer cell lines disrupted at activated Ki-ras. Science 1993;260:85–8.[Abstract/Free Full Text]
  11. Fallaux FJ, Kranenburg O, Cramer SJ, et al. Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors. Hum Gene Ther 1996;7:215–22.[Medline]
  12. Kranenburg O, Verlaan I, Moolenaar WH. Regulating c-Ras function. cholesterol depletion affects caveolin association, GTP loading, and signaling. Curr Biol 2001;11:1880–4.[CrossRef][Medline]
  13. Norman KL, Hirasawa K, Yang AD, Shields MA, Lee PW. Reovirus oncolysis: the Ras/RalGEF/p38 pathway dictates host cell permissiveness to reovirus infection. Proc Natl Acad Sci U S A 2004;101:11099–104.[Abstract/Free Full Text]
  14. Grady WM, Markowitz SD. Genetic and epigenetic alterations in colon cancer. Annu Rev Genomics Hum Genet 2002;3:101–28.[CrossRef][Medline]
  15. Arango D, Wilson AJ, Shi Q, et al. Molecular mechanisms of action and prediction of response to oxaliplatin in colorectal cancer cells. Br J Cancer 2004;91:1931–46.[CrossRef][Medline]
  16. Klampfer L, Swaby LA, Huang J, Sasazuki T, Shirasawa S, Augenlicht L. Oncogenic Ras increases sensitivity of colon cancer cells to 5-FU-induced apoptosis. Oncogene 2005;24:3932–41.[CrossRef][Medline]
  17. Clarke P, Meintzer SM, Gibson S, et al. Reovirus-induced apoptosis is mediated by TRAIL. J Virol 2000;74:8135–9.[Abstract/Free Full Text]
  18. Kominsky DJ, Bickel RJ, Tyler KL. Reovirus-induced apoptosis requires both death receptor- and mitochondrial-mediated caspase-dependent pathways of cell death. Cell Death Differ 2002;9:926–33.[CrossRef][Medline]
  19. Kominsky DJ, Bickel RJ, Tyler KL. Reovirus-induced apoptosis requires mitochondrial release of Smac/DIABLO and involves reduction of cellular inhibitor of apoptosis protein levels. J Virol 2002;76:11414–24.[Abstract/Free Full Text]
  20. McDermott U, Longley DB, Galligan L, Allen W, Wilson T, Johnston P. Effect of p53 status and STAT1 on chemotherapy-induced, Fas-mediated apoptosis in colorectal cancer. Cancer Res 2005;65:8951–60.[Abstract/Free Full Text]
  21. Norman KL, Lee PW. Not all viruses are bad guys: the case for reovirus in cancer therapy. Drug Discov Today 2005;10:847–55.[CrossRef][Medline]
  22. Alberts SR, Horvath WL, Sternfeld WC, et al. Oxaliplatin, fluorouracil, and leucovorin for patients with unresectable liver-only metastases from colorectal cancer: A North Central Cancer Treatment Group Phase II Study. J Clin Oncol 2005;23:9243–9.[Abstract/Free Full Text]
  23. Bismuth H, Adam R, Levi F, et al. Resection of nonresectable liver metastases from colorectal cancer after neoadjuvant chemotherapy. Ann Surg 1996;224:509–20.[CrossRef][Medline]
  24. de Gramont A, Figer A, Seymour M, et al. Leucovorin and fluorouracil with or without oxaliplatin as first-line treatment in advanced colorectal cancer. J Clin Oncol 2000;18:2938–47.[Abstract/Free Full Text]
  25. Martin MJ. Current stage-specific chemotherapeutic options in colon cancer. Expert Rev Anticancer Ther 2005;5:695–704.[CrossRef][Medline]
  26. Boyer J, McLean EG, Aroori S, et al. Characterization of p53 wild-type and null isogenic colorectal cancer cell lines resistant to 5-fluorouracil, oxaliplatin, and irinotecan. Clin Cancer Res 2004;10:2158–67.[Abstract/Free Full Text]
  27. Rodgers SE, Barton ES, Oberhaus SM, et al. Reovirus-induced apoptosis of MDCK cells is not linked to viral yield and is blocked by Bcl-2. J Virol 1997;71:2540–6.[Abstract]
  28. Debiasi RL, Clarke P, Meintzer S, et al. Reovirus-induced alteration in expression of apoptosis and DNA repair genes with potential roles in viral pathogenesis. J Virol 2003;77:8934–47.[Abstract/Free Full Text]
  29. Clarke P, Meintzer SM, Spalding AC, Johnson GL, Tyler KL. Caspase 8-dependent sensitization of cancer cells to TRAIL-induced apoptosis following reovirus-infection. Oncogene 2001;20:6910–9.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smakman, N.
Right arrow Articles by Kranenburg, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smakman, N.
Right arrow Articles by Kranenburg, O.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online