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RESEARCH ARTICLE

Oncogenic and Wild-type Ras Play Divergent Roles in the Regulation of Mitogen-Activated Protein Kinase Signaling Amy Young 1 , 2 , David Lou 1 , 3 , and Frank McCormick 1

Research. on September 22, 2020. © 2013 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst October 25, 2012; DOI: 10.1158/2159-8290.CD-12-0231

http://cancerdiscovery.aacrjournals.org/

JANUARY 2013�CANCER DISCOVERY | 113

Authors’ Affi liations: 1 Helen Diller Family Comprehensive Cancer Center; 2 Biomedical Sciences Graduate Program; and 3 Division of Hematology and Oncology, Department of Medicine, University of California San Francisco, San Francisco, California Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). Corresponding Author: Frank McCormick, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, 1450 3rd Street HD371, San Francisco, CA 94143. Phone: 415-502-1710; Fax: 415-502-1712; E-mail: [email protected] doi: 10.1158/2159-8290.CD-12-0231 ©2012 American Association for Cancer Research.

ABSTRACT H-Ras, K-Ras, and N-Ras regulate cellular growth and survival and are often acti-vated by somatic mutation in human tumors. Although oncogenic lesions occur in a

single Ras isoform within individual tumors, it is unclear whether the remaining wild-type isoforms play supporting roles in tumor growth. Here, we show that oncogenic and wild-type Ras isoforms play inde-pendent and nonredundant roles within the cell. Oncogenic Ras regulates basal effector pathway sig-naling, whereas wild-type Ras mediates signaling downstream of activated receptor tyrosine kinases (RTK). We show that both are necessary for exponential growth of Ras-mutant cell lines. Furthermore, we show that oncogenic Ras desensitizes signaling from EGF receptor (EGFR). Depletion of oncogenic Ras with siRNA oligonucleotides relieves this negative feedback, leading to the hyperactivation of EGFR and wild-type Ras signaling. Consistent with this model, combining oncogenic Ras depletion with EGFR inhibition potently increases cell death.

SIGNIFICANCE: The results of this study highlight a novel role for wild-type Ras signaling in cancer cells harboring oncogenic RAS mutations. Furthermore, these fi ndings reveal that therapeutically targeting oncogenic Ras signaling alone may be ineffective owing to feedback activation of RTKs, and suggest that blocking upstream RTKs in combination with downstream effector pathways may be benefi cial in the treatment of Ras-mutant tumors. Cancer Discov; 3(1); 112–23. ©2012 AACR. See related commentary by Hayes and Der, p. 24.

INTRODUCTION

H-Ras, K-Ras, and N-Ras are the founding members of the Ras family of small GTPases, and regulate cell growth, differ-entiation, and survival ( 1, 2 ). Ras GTPases function as binary molecular switches, cycling between inactive GDP-bound and active GTP-bound states. In the basal state, Ras is predomi-nantly GDP bound. Activated receptor tyrosine kinases (RTK) recruit guanine nucleotide exchange factors (GEF) to promote the exchange of bound GDP for GTP on nearby Ras mol-ecules. In its GTP-bound state, Ras interacts with downstream effectors to activate signaling pathways important for cell growth and survival, including the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) path-ways ( 3, 4 ). GTPase-activating proteins (GAP) facilitate the hydrolysis of bound GTP to GDP, returning Ras to its basal, inactive conformation and terminating downstream signal-ing. Ras, therefore, acts as a sensor of extracellular growth cues to ensure that signaling output through downstream effector pathways are of the appropriate intensity and duration.

The aberrant hyperactivation of Ras plays a causal role in human cancer, and an estimated 30% of human tumors har-bor oncogenic somatic mutations in HRAS , KRAS , or NRAS

( 5, 6 ). Oncogenic RAS alleles differ from their wild-type coun-terpart by a single missense point mutation that results in an amino acid substitution typically at position 12, 13, or 61 ( 6, 7 ). These substitutions impair the rate of GAP-mediated GTP hydrolysis and consequently deregulate Ras signaling ( 5–7 ). Oncogenic mutation in any one of the 3 RAS genes is thought to be suffi cient to constitutively activate down-stream signaling and confer independence from upstream growth cues ( 8 ). Although the remaining 2 wild-type Ras isoforms remain subject to regulation by GAPs and GEFs, the signaling contribution of the wild-type Ras isoforms in this context has been largely unexamined.

Currently, no curative treatments for Ras-mutant cancers are available ( 9 ). Efforts to develop drugs that specifi cally block the activity of oncogenic Ras have been unsuccess-ful, although this remains an active area of investigation ( 10, 11 ). Clinical studies show that oncogenic KRAS mutations predict resistance to EGF receptor (EGFR) inhibitors; hence, the use of RTK inhibitors in this subset of cancers is usually contraindicated ( 12 ). Developing drugs that inhibit signaling pathways downstream of Ras has thus been a key focus of clinical development. Small-molecule compounds targeting the MAPK and PI3K effector pathways are currently under clinical investigation, as these pathways have been well char-acterized and shown to be critical in mediating Ras-driven tumorigenesis ( 9 , 13 , 14 ). However, the response to MAP–ERK kinase (MEK) inhibitors varies across Ras-mutant tumors, and clinical effi cacy is thought to be limited by feedback activation of the PI3K pathway ( 14–17 ). Furthermore, Raf kinase inhibi-tors paradoxically activate MAPK signaling in cells harboring oncogenic RAS mutations, thereby precluding the use of these inhibitors in the treatment of RAS-mutant cancers ( 18–20 ). These fi ndings highlight the complexity of effector pathway regulation and feedback mechanisms downstream of onco-genic Ras. Continued investigation of these signaling processes will be required to design effective targeted therapeutics.




114 | CANCER DISCOVERY�JANUARY 2013 www.aacrjournals.org

Young et al.RESEARCH ARTICLE

In this study, we examine the regulation of MAPK and PI3K signaling in cancer cells with oncogenic RAS mutations. We use 3 cancer cell models, each of which harbors a homozygous RAS mutation: the T24 bladder cancer cell line ( HRAS G12V), the MIA PaCa-2 pancreatic cancer cell line ( KRAS G12C), and the RD rhabdomyosarcoma cell line ( NRAS Q61H; Catalog of Somatic Mutations in Cancer, Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom). By deplet-ing either the single oncogenic Ras isoform or the remaining 2 wild-type Ras isoforms, we delineate independent roles for oncogenic and wild-type Ras. Our studies uncover an unex-pected role for wild-type Ras isoforms in mediating growth factor signaling and sustaining the proliferation of cancer cells harboring oncogenic RAS mutations. Furthermore, we show that oncogenic Ras regulates basal MAPK signaling and negatively regulates RTK signaling. Thus, although depletion of oncogenic Ras impairs basal signaling, the concomitant relief of negative feedback allows for compensatory activation of RTKs and wild-type Ras-mediated downstream signaling as a secondary driver of cell survival and proliferation.

RESULTS

Growth Factor Signaling Can Enhance Effector Pathway Signaling in Cancer Cell Lines Harboring Oncogenic RAS Mutations

Oncogenic mutations in RAS are thought to confer growth factor independence and constitutive activation of down-stream signaling pathways. However, acute stimulation with EGF can still further enhance MAPK and PI3K signaling in cancercells harboring oncogenic mutations in HRAS , KRAS , or NRAS , as evidenced by increased extracellular signal–regulated kinase (ERK) and Akt phosphorylation, respectively ( Fig. 1A ). Although the oncogenic Ras isoform is constitutively GTP bound, EGF stimulation induces GTP loading on the wild-type Ras isoforms. This fi nding suggests that cancer cells with onco-genic RAS mutations have not saturated the capacity to activate downstream signaling, but that heightened activity can be achieved through activation of wild-type Ras isoforms by RTKs.

Wild-type Ras Regulates Growth Factor Signaling To directly test the requirement for wild-type Ras in growth

factor signaling, cells were transfected with siRNA to deplete the expression of the 2 wild-type RAS isoforms. The cells were then serum starved overnight, and signaling was measured before and after acute stimulation with EGF ( Fig. 1B ). Indeed, siRNA-mediated depletion of the wild-type Ras isoforms attenuates the EGF-induced phosphorylation of ERK ( Fig. 1B and C ). These fi ndings have been confi rmed in additional cell lines and with several siRNA oligonucleotides (Supplemen-tary Figs. S1A–S1C and S2). These results show that the wild-type Ras isoforms are required for the growth factor–induced activation of MAPK signaling in cancer cells with oncogenic RAS mutations. siRNA-mediated depletion of the wild-type Ras isoforms also attenuates the EGF-induced phosphor-ylation of Akt in MIA PaCa-2 and RD cells, indicating that growth factor–induced PI3K signaling is partially mediated by wild-type Ras in these cell lines ( Fig. 1B ). Interestingly, depletion of the oncogenic Ras isoform results in a more robust induction of EGFR, ERK, and Akt phosphorylation in

response to EGF stimulation ( Fig. 1B and C ). The increase in signaling correlates with enhanced GTP loading on the wild-type Ras isoforms (Supplementary Fig. S1A and S1B), again highlighting a critical role for wild-type Ras in mediating growth factor signaling and suggesting that oncogenic Ras negatively regulates EGFR signaling, as we will discuss below.

We next examined whether effector pathway signaling could be suppressed by blocking RTK-mediated activation of wild-type Ras. The EphA2 RTK negatively regulates Ras/MAPK signaling upon stimulation with its ligand ephrin-A1 ( 21, 22 ). We thus used ephrin-A1 to block wild-type Ras activation. Ephrin-A1 attenuates EGF-induced activation of MAPK signaling and coincides with reduced GTP loading on wild-type Ras isoforms (Supplementary Fig. S3A). Impor-tantly, wild-type Ras isoforms are required to mediate this effect, given that ephrin-A1 has no effect on ERK signaling in RD cells depleted of wild-type Ras compared with a robust suppression of ERK signaling when the oncogenic N-Ras iso-form is depleted (Supplementary Fig. S3B and S3C). Taken together, these data show that effector pathway signaling can still be modulated through wild-type Ras even in the presence of an oncogenic Ras isoform.

Oncogenic Ras Regulates Basal MAPK Signaling Depletion of oncogenic Ras results in a subtle yet repro-

ducible reduction in the basal levels of ERK phosphorylation in serum-starved cells ( Fig. 1B and D ) and in cells asynchro-nously growing in media supplemented with 10% serum ( Fig. 2A and B ), indicating that oncogenic Ras is critical in regulating basal MAPK signaling. In contrast, depletion of oncogenic Ras increases basal Akt signaling in T24 and RD cells ( Fig. 2A , Supplementary Fig. S2), highlighting a diver-gence in the regulation of basal MAPK and PI3K signaling downstream of oncogenic Ras.

Intriguingly, depletion of the wild-type Ras isoforms mod-estly increases basal ERK phosphorylation in MIA PaCa-2 and RD cells under conditions of serum starvation or asyn-chronous growth in media supplemented with 10% serum ( Figs. 1B and 1D; 2A and 2B ). Several studies show that wild-type Ras can antagonize the transforming potential of its oncogenic counterpart in vitro and in vivo ( 23–25 ). Thus, the relief of antagonism on oncogenic Ras may serve as one potential mechanism by which basal ERK signaling increases in cells depleted of wild-type Ras. Depleting oncogenic Ras in combination with the 2 wild-type Ras isoforms prevents this increase in basal ERK phosphorylation, suggesting that the increased ERK phosphorylation observed under conditions of wild-type Ras depletion is indeed driven by oncogenic Ras (data not shown). These data are consistent with a model in which the wild-type Ras isoforms antagonize basal oncogenic Ras signaling to the MAPK pathway.

Wild-type and Oncogenic Ras Differentially Regulate Cell Proliferation

The results discussed above highlight unique roles for wild-type and oncogenic Ras in the regulation of effector pathway signaling: The oncogenic Ras isoform regulates basal MAPK signaling, whereas the wild-type Ras isoforms regulate growth factor signaling. Because Ras regulates cellular growth and survival, we next tested the requirement for the oncogenic and





Regulation of Signaling by Wild-type and Oncogenic Ras RESEARCH ARTICLE

Figure 1. Wild-type Ras mediates growth factor–induced activation of MAPK signaling in cancer cells harboring oncogenic RAS mutations. A, cancer cell lines with oncogenic mutations in HRAS , KRAS , and NRAS were serum starved overnight before acute stimulation with EGF for 5 minutes. Levels of active GTP-bound Ras were determined by a Ras–GTP pulldown assay, and lysates were subjected to immunoblot analysis. B, cells were transfected with the indicated siRNAs for 48 hours and placed in serum-free medium overnight. Signaling was measured before and after 5 minutes of EGF stimulation. Lysates were subjected to immunoblot analysis. K-Ras protein levels are below the detection limit of the assay for the RD cell line. C, quantifi cation of the fold-change in EGF-induced ERK phosphorylation (p-ERK) from the immunoblot analyses in B are depicted in histograms. Values are normalized to samples transfected with non-silencing siRNA. D, quantifi cation of basal ERK phosphorylation levels from the immunoblot analyses in B are depicted in histograms. Values are normalized to samples transfected with non-silencing siRNA. p-EGFR, phosphorylated EGFR; p-ERK, phosphorylated ERK.

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Figure 2. Oncogenic Ras regulates basal MAPK signaling. A, cells were transfected with the indicated siRNAs for 72 hours. Levels of active GTP-bound Ras were determined by a Ras–GTP pulldown assay, and lysates were subjected to immunoblot analysis. B, quantifi cation of ERK phosphorylation levels from the immunoblot analyses in A are depicted in histograms. Values are normalized to samples transfected with non-silencing siRNA.

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wild-type Ras isoforms in maintaining cellular proliferation. siRNA-mediated depletion of either the oncogenic or wild-type Ras isoforms reduces the proliferative capacity of cells asyn-chronously growing in media supplemented with 10% serum ( Fig. 3 ). In all 3 cell lines tested, depletion of the oncogenic Ras isoform more potently inhibits cell growth than does depletion of the wild-type Ras isoforms. Despite sustaining steady-state ERK signaling, depletion of the 2 wild-type Ras isoforms also impedes the growth of each of the 3 cell lines, although not as drastically as depletion of the oncogenic isoform. This observa-tion suggests that wild-type Ras might regulate cellular growth through additional pathways, whose roles are independent from maintaining steady-state ERK activation. Several studies show that transient activation of ERK signaling promotes cell pro-liferation, whereas sustained ERK proliferation promotes differen-tiation ( 26 ). Thus, we postulate that the growth defect observed in cells depleted of wild-type Ras may result from a defect in the growth factor–mediated transient activation of ERK and additional Ras effector pathways. Taken together, these data

show that the differential regulation of basal MAPK signaling by wild-type and oncogenic Ras translates into independent and nonredundant roles in the regulation of cell growth, and that both are required to maintain optimal growth rates.

Oncogenic Ras Negatively Regulates EGFR Sensitivity

As mentioned above, silencing oncogenic Ras expression sensitizes cells to EGF stimulation ( Fig. 1B and C ), showing that oncogenic Ras negatively regulates EGFR sensitivity. It has been shown that pharmacologic MEK inhibition similarly sensitizes cells to EGF stimulation ( 17 , 27–29 ). One mecha-nism by which MEK inhibition sensitizes EGFR signaling is by relieving an inhibitory ERK-mediated phosphorylation of EGFR at residue T669 ( 30–32 ). To examine whether silencing oncogenic Ras achieves the same effect, a time-course study of EGF stimulation was conducted in cells depleted of onco-genic Ras or treated with the MEK inhibitor U0126. Consistent with prior studies, pharmacologic MEK inhibition reduces






Figure 3. Wild-type and oncogenic Ras differentially regulate cell proliferation. Cells were transfected with various combinations of siRNAs targeting either the oncogenic or wild-type Ras isoforms. Two days after transfection, cells were counted and seeded in triplicate at equal densi-ties in complete medium. Cell counts were obtained every 24 hours using a Coulter particle counter. An initial cell count was obtained at the time of seeding to confi rm equal seeding densities. Values represent the fold-change in cell count relative to the 48-hour timepoint. NS, non-silencing control; H1, H2, H3, 3 independent siRNAs targeting HRAS ; K1, K3, 2 independent siRNAs targeting KRAS ; N1, N2, N3, 3 independent siRNAs targeting NRAS .

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basal phosphorylation of ERK and EGFR T669, and potenti-ates the EGF-induced phosphorylation of EGFR at the acti-vating Y1068 residue, resulting in enhanced and sustained Akt signaling (Supplementary Fig. S4). Oncogenic Ras deple-tion produces a comparatively modest reduction in EGFR T669 and basal ERK phosphorylation, but more robustly amplifi es EGFR signaling, suggesting additional mechanisms by which oncogenic Ras attenuates EGFR signaling ( Fig. 4A ). It should be noted that sustained siRNA-mediated oncogenic Ras depletion (72 hours) may allow time for rewiring of the EGFR–MAPK axis, compared with short-term MEK inhibi-tion (1 hour), and may explain the differences observed in the differential sensitivity to EGF stimulation. Nevertheless, these results show that depletion of oncogenic Ras sensitizes cells to the acute activation of EGFR signaling.

To directly test whether ectopic expression of oncogenic Ras is suffi cient to attenuate EGFR signaling, HBL100 cells stably expressing GFP or oncogenic H-Ras, K-Ras, or N-Ras were serum starved overnight before acute stimulation with EGF. As expected, expression of oncogenic Ras increased basal levels of ERK phosphorylation. In addition, we observed a striking suppression of EGF-induced phosphorylation of EGFR, Akt, and ERK in cells expressing the ectopic onco-genic Ras isoforms when compared with the GFP-expressing control ( Fig. 4B ). The results support our observations that oncogenic Ras negatively regulates EGFR sensitivity.

To test whether oncogenic Ras negatively regulates the sensitivity of additional RTKs, cells depleted of oncogenic Ras were treated with a panel of growth factors. Depletion of oncogenic Ras heightens the sensitivity to additional growth factors in a cell line–dependent fashion (Supplementary Fig. S5), suggesting that oncogenic Ras may engage a program to globally desensitize signaling from upstream RTKs.

Combining Oncogenic Ras Depletion with EGFR Inhibition Enhances Cell Death

Although several small-molecule compounds targeting MEK are currently under clinical investigation, effi cacy may be limited by feedback activation of EGFR–PI3K signaling ( 14–17 ). Similarly, recent studies show that colorectal cancer cells harboring BRAF V600E mutations are unresponsive to the small-molecule RAF inhibitor PLX4032 (vemurafenib) owing to feedback activation of EGFR, and that combining RAF and EGFR inhibition improves effi cacy ( 33, 34 ). Our results indi-cate that feedback activation of EGFR may also be a mecha-nism of resistance to therapies targeting either oncogenic Ras or oncogenic Ras-driven MAPK signaling. We hypothesized that combining oncogenic Ras depletion with EGFR inhibi-tion would block feedback EGFR activation. To test this idea, siRNA-transfected cells were serum starved overnight and pre-treated with the EGFR inhibitor erlotinib or vehicle control [dimethyl sulfoxide (DMSO)] before acute stimulation with EGF. Consistent with earlier results, depletion of oncogenic Ras markedly sensitizes cells to acute stimulation with EGF and results in increased downstream signaling. Treatment with erlotinib predictably blocks EGFR phosphorylation but notably attenuates EGF-stimulated GTP loading on the wild-type Ras isoforms, and abrogates the phosphorylation of both ERK and Akt ( Fig. 5 ). Thus, blocking the activation of EGFR prevents the hypersensitivity to EGF stimulation induced by oncogenic Ras






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Figure 4. Oncogenic Ras negatively regulates EGFR sensitivity. A, cells were transfected with the indicated siRNAs for 48 hours and placed in serum-free medium overnight. Signaling was measured before and after EGF stimulation. Lysates were subjected to immunoblot analysis. Lysates derived from T24, MIA PaCa-2, and RD cells were probed with H-Ras, K-Ras, or N-Ras antibodies, respectively. B, HBL100 cells stably expressing GFP or oncogenic H-Ras, K-Ras, or N-Ras were serum starved overnight, and signaling was measured before and after 5 minutes of EGF stimulation. Levels of active GTP-bound Ras were determined by a Ras–GTP pulldown assay, and lysates were subjected to immunoblot analysis. Endogenous levels of K-Ras are below the detection limit of the assay.






depletion. As shown in prior experiments, depletion of onco-genic Ras increases basal Akt signaling in serum-starved T24 and RD cells. In T24 cells, basal Akt signaling is suppressed by erlotinib, suggesting the signal is primarily driven by an EGFR-dependent autocrine loop. However, erlotinib does not suppress basal Akt signaling in RD cells, indicating that EGFR is not the primary driver of basal Akt signaling in these cells.

To test whether EGFR inhibition and oncogenic Ras deple-tion synergize to induce a more robust growth defect than that shown in Fig. 3 , siRNA-transfected T24 cells were treated with erlotinib or a vehicle control for 72 hours before assaying for growth inhibition or induction of apoptosis. Depletion of oncogenic Ras not only increases the potency of erlotinib ( Fig. 6A ) but also promotes apoptosis to a greater extent than either treatment alone, as assessed by increases in sub-G 1 cell population, annexin staining, and PARP cleavage ( Fig. 6B–D ). In contrast to T24 cells, combining EGFR inhibition with oncogenic Ras depletion shows modest benefi t over single-arm treatment in MIA PaCa-2 and RD cells (Supplementary Fig. S6A–S6C). These results are consistent with our signaling observations, which suggest that EGFR is not a primary driver of basal Akt signaling in either of these cell models, which

likely activate prosurvival Akt signaling through other RTKs ( Fig. 5 and Supplementary Fig. S5). Thus, dual targeting of oncogenic Ras signaling and the appropriate RTK may be benefi cial in the treatment of Ras-mutant tumors.

DISCUSSION

The work presented here defi nes distinct roles for onco-genic and wild-type Ras in regulating effector pathway sig-naling in cancer cells harboring oncogenic RAS mutations. We show that oncogenic Ras regulates basal signaling and uncover an unexpected role for wild-type Ras in regulating growth factor signaling. Although oncogenic Ras constitu-tively activates MAPK signaling, acute activation of growth factor receptors can enhance this signaling by stimulating GTP loading on wild-type Ras ( Fig. 7 ). Given that we detect this phenomenon in cell lines harboring oncogenic muta-tions in HRAS , KRAS , or NRAS , we conclude that these are not isoform-specifi c effects, but rather can be generalized to oncogenic and wild-type isoforms as a class. Collectively, these fi ndings uncover an underappreciated role for wild-type Ras signaling in the context of oncogenic RAS mutations.

Figure 5. Erlotinib blocks the feedback activation of EGFR and wild-type Ras signaling. Cells were transfected with the indicated siRNAs for 48 hours, placed in serum-free medium overnight, and treated with either DMSO or 10 μm erlotinib for 1 hour. Signaling was measured before and after 5 minutes of EGF stimulation. Levels of active GTP-bound Ras were determined by a Ras–GTP pulldown assay, and lysates were subjected to immunoblot analysis.

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Figure 6. Combining oncogenic Ras depletion with EGFR inhibition induces cell death. A, T24 cells were transfected with the indicated siRNAs for 48 hours and treated with a serial dilution of erlotinib for 72 hours. Relative cell proliferation values were determined by the CyQUANT Direct Cell Pro-liferation Assay. For each siRNA condition, data are expressed as the fraction of maximal cell growth at 72 hours after drug treatment. B, T24 cells were transfected with the indicated siRNAs for 48 hours and treated with 10 μm erlotinib for 72 hours. Cells were fi xed, stained with PI, and analyzed by fl ow cytometry to determine cell-cycle distribution. C, T24 cells were transfected with the indicated siRNAs for 48 hours and treated with 10 μm erlotinib for 72 hours. Cells were stained with Annexin V-FITC and PI and analyzed by fl ow cytometry to determine the percentage of cells undergoing apoptosis. The fold difference in Annexin V–positive cells is shown relative to the condition in which cells were transfected with non-silencing siRNA and treated with 10 μm erlotinib. D, T24 cells were transfected with the indicated siRNAs for 48 hours and treated with the indicated concentrations of erlotinib for 24 hours. Lysates were subjected to immunoblot analysis.

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Intriguingly, we found that depletion of wild-type Ras increases basal ERK phosphorylation, suggesting that wild-type Ras antagonizes oncogenic Ras signaling. The mechanism by which this antagonism occurs is not fully understood. In the basal state, wild-type Ras is predominantly GDP bound. It is plausible that GDP-bound wild-type Ras inhibits oncogenic Ras signaling by sustaining unique and independent growth-inhibitory signaling pathways. Alternatively, different Ras iso-forms might compete for common regulators, effectors, or proper localization. Prior studies indicate that wild-type Ras can antagonize the transforming potential of its oncogenic

counterpart in a dose-dependent manner in vitro and in vivo ( 23–25 ), and show a strong selective pressure to lose expression of the corresponding wild-type allele of the oncogenic Ras iso-form ( 35–37 ). Whereas these data support a tumor-suppressive role for the wild-type counterpart of the same oncogenic Ras isoform, our studies uncover a novel role for the remaining 2 wild-type Ras isoforms in antagonizing oncogenic Ras signaling.

It is unclear whether the specifi c inhibition of wild-type Ras activity, rather than the outright depletion of wild-type Ras expression, would similarly antagonize oncogenic Ras signaling. Early studies on Ras signaling show that membrane-targeted






Figure 7. Regulation of signaling by wild-type and oncogenic Ras. Oncogenic Ras is constitutively GTP bound and regulates basal MAPK signaling. Growth factor receptors and negative regulatory proteins such as EGFR and EphA2, respectively, modulate wild-type Ras activity and in turn affect downstream signaling. In the basal state, wild-type Ras, which is predominantly GDP bound, inhibits oncogenic Ras signaling. Oncogenic Ras negatively regulates EGFR sensitivity. Suppression of basal MAPK signal-ing, via depletion of oncogenic Ras or pharmacologic inhibition of MEK, relieves this negative feedback and sensitizes cells to EGF stimulation.

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EGFR

EphA2

p120 RasGAP antagonizes the transforming potential of oncogenic Ras in NIH3T3 cells, and that this antagonism is dependent on the catalytic activity of p120 RasGAP ( 38, 39 ). One interpretation of these results is that transformation depends on additive signals from both oncogenic and wild-type Ras, and that the constitutive downregulation of wild-type Ras activity by membrane-targeted p120 RasGAP is suffi cient to inhibit transformation by oncogenic Ras. Another interpre-tation is that wild-type Ras, in a GDP-bound form, directly antagonizes oncogenic Ras signaling. Alternatively, a combi-nation of these mechanisms might be at play.

EphA2 negatively regulates Ras/MAPK signaling, and its expression is induced by high MAPK pathway activity ( 21, 22 ). Remarkably, we show that blocking wild-type Ras activation by stimulating EphA2 with its ligand ephrin-A1 attenuates the EGF-induced activation of downstream signaling in can-cer cells harboring oncogenic RAS mutations (Fig. 7 and Sup-plementary Fig. S3). EphA2 knockout mice are signifi cantly more susceptible to chemically induced skin carcinogen-esis driven by an activating mutation in Hras , showing that EphA2 can exert tumor-suppressive effects despite the pres-ence of oncogenic Ras ( 40 ). In light of our results, it would be interesting to revisit these studies to determine whether the tumor-suppressive effects mediated by EphA2 in this system may result in part from the suppression of wild-type K-Ras and N-Ras signaling.

Several recent studies show that targeted inhibition of a single oncogenic pathway results in the feedback activation of compensatory pathways, thereby limiting the effi cacy of monotherapy ( 33, 34 , 41–45 ). Likewise, our study cautions against targeting oncogenic Ras signaling alone, and indi-cates that combined treatment with the appropriate RTK inhibitor is likely required, owing to the compensatory acti-vation of upstream signaling. Determining the appropriate RTK to target will likely be context dependent and will be contingent upon factors such as tumor type, RTK expression levels, the availability and accessibility of relevant ligands, and whether autocrine RTK signaling is sustained.

The fi nding that oncogenic Ras mediates feedback sup-pression of RTKs may help explain the observation that KRAS -mutant tumors are often resistant to EGFR-based ther-apies and have variable responses to single-agent MEK inhibi-tor treatment ( 12 , 14 , 46 ). We hypothesize that oncogenic mutations in KRAS desensitize cells to EGFR-based therapies by at least 2 mechanisms. The fi rst and most intuitive expla-nation is that oncogenic Ras increases basal effector signaling downstream of EGFR. Second, our data are consistent with an additional model in which oncogenic Ras rewires EGFR signaling dynamics. Indeed, several recent studies show that oncogenic K-Ras desensitizes cells to EGFR activation and inhibition by altering EGFR traffi cking, turnover, and locali-zation ( 29, 30 , 32 , 47–49 ). Suppression of basal MAPK signal-ing, whether by siRNA-mediated depletion of oncogenic Ras or by pharmacologic inhibition of MEK, resensitizes EGFR signaling and primes the receptor for activation. Accordingly, recent studies indicate that treatment with a MEK inhibi-tor alongside an EGFR inhibitor synergistically inhibits the growth of cancer cells harboring oncogenic KRAS mutations ( 29 , 49 ). Taken together, these studies are consistent with our fi ndings and suggest that targeting the MAPK pathway in combination with the appropriate RTK is potentially benefi -cial as well as necessary to effectively treat cancers harboring oncogenic RAS mutations. Importantly, this approach would concurrently block the RTK-mediated activation of wild-type Ras signaling while suppressing the basal levels of MAPK signaling sustained by oncogenic Ras.

METHODS

Cell Culture The T24 cell line was kindly provided by Dr. Osamu Tetsu (University

of California San Francisco, San Francisco, CA); all other cell lines were obtained from the American Type Culture Collection. No addi-tional cell line authentication was conducted by the authors. Cells were maintained in Dulbecco’s modifi ed Eagle’s medium (DMEM) supplemented with 10% FBS and incubated at 37°C in a 5% CO 2 incubator. For stable cell line generation, genes were cloned into pENTR or pDONR 221 vectors (Invitrogen), were transferred into a Gateway-compatible derivative of the pFBNeo retroviral vector using recombination-mediated Gateway technology (Invitrogen), and were transduced into HBL100 cells using retrovirus. All Ras constructs are of human origin and have been described previously ( 2 ).

Cell Stimulation and Drug Treatments For stimulation with recombinant EGF (Invitrogen), cells were

serum starved overnight before acute stimulation with 30 ng/mL EGF for 5 minutes, unless otherwise noted. For analysis of cellular






signaling after U0126 or erlotinib treatment, cells were serum starved overnight and pretreated with either 10 μm of drug or vehicle control (DMSO) for 1 hour before acute stimulation with EGF.

RNA Interference Cells were transfected with 80 nmol/L siRNA (Qiagen) using Lipo-

fectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions. siRNA oligonucleotide sequences are provided in the Supplementary Methods.

Immunoblot Analysis Cells were washed twice in ice-cold PBS and lysed in 1% Triton lysis

buffer [25 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L EDTA, 1 mmol/L EGTA, 20 mmol/L NaF, 1 mmol/L Na 2 VO 4 , and 1 mmol/L dithiothreitol (DTT)] supplemented with a protease inhibitor cocktail (Roche) and cleared by centrifugation. Protein concentrations were determined by the Bio-Rad Protein Assay (Bio-Rad). Equal amounts of protein extracts were resolved using SDS-PAGE (NuPAGE; Invitrogen), transferred to a nitrocellulose membrane, and immunoblotted with primary antibodies. Primary antibodies were detected with secondary antibodies labeled with either IRDye800 (Rockland) or Alexa Fluor 680 (Molecular Probes) and were visualized using a LI-COR Odyssey scanner. A complete list of primary antibodies is provided in the Supplementary Methods.

Ras–GTP Assay Cells were washed twice in ice-cold PBS and lysed in 1% TX100-

TNM lysis buffer (20 mmol/L Tris pH 7.5, 5 mmol/L MgCl 2 , 150 mmol/L NaCl,1% Triton X-100) supplemented with 1 mmol/L DTT, and protease and phosphatase inhibitors (Sigma-Aldrich), and were processed as described above. Equal amounts of protein from each sample were added to 10 μL of packed GST-Raf-RBD beads in 300 to 500 μL of 1% TX100-TNM lysis buffer and rotated at 4°C for 1 to 2 hours. Beads were washed 3 times with 1 mL of cold lysis buffer and boiled in lithium dodecyl sulfate (LDS) sample buffer (Invitrogen).

Cell Proliferation Analysis Forty-eight hours after siRNA transfection, cells were trypsinized

and seeded at equal densities in 12-well plates in triplicate for cell proliferation analysis. Samples were collected immediately at the time of seeding and were harvested at 24-hour intervals thereafter, as indicated. Accurate cell counts were obtained using a Coulter particle analyser.

Erlotinib Growth Inhibition Assay Twenty-four hours after siRNA transfection, cells were trypsinized

and seeded in 96-well plates at 3,500 cells per well. The following day, cells were treated with a serial dilution of erlotinib (6 replicates per drug concentration) and allowed to proliferate for an additional 72 hours. Cell proliferation was measured using CyQUANT Direct Cell Proliferation Assay (Life Technologies). For each siRNA condi-tion, data are expressed as the fraction of maximal cell growth at 72 hours after erlotinib treatment, and dose–response curves were generated using GraphPad Prism software. All assays were conducted at least 3 independent times and representative curves are shown.

Analysis of Cell Cycle and Apoptosis Forty-eight hours after siRNA transfection, cells were treated with

erlotinib or DMSO and harvested 72 hours after drug treatment. For cell-cycle analysis, adherent and fl oating cells were collected, washed once with PBS, permeabilized with ice-cold 70% ethanol, resuspended in PBS, and incubated with RNase A and propidium iodide (PI). For analysis of apoptosis, adherent and fl oating cells were collected, washed with ice-cold PBS, and stained with Annexin V-FITC (BD Pharmingen)

and PI. Cells were sorted using FACSCalibur (Becton, Dickinson and Co.). Ten thousand live cell events were collected per treatment.

Disclosure of Potential Confl icts of Interest No potential confl icts of interest were disclosed.

Authors’ Contributions Conception and design: A. Young, D. Lou, F. McCormick Development of methodology: A. Young, D. Lou Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Young, D. Lou Analysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): A. Young, D. Lou, F. McCormick Writing, review, and/or revision of the manuscript: A. Young, D. Lou, F. McCormick Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Young, D. Lou Study supervision: F. McCormick

Acknowledgments The authors thank A. Balmain, M. Fried, D. Stokoe, and members

of the McCormick Lab for insightful comments and discussion. In addition, the authors thank O. Tetsu for kindly providing the T24 cell line, I. Stowe and T. Yuan for critical review of the manuscript, and J. Galeas, T. Rakhshandehroo, and I.Y. Song for technical assist-ance and advice.

Grant Support This work was supported by a grant from Daiichi-Sankyo Co., Ltd.

Received May 27, 2012; revised October 15, 2012; accepted October 16, 2012; published OnlineFirst October 25, 2012.

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2013;3:112-123. Published OnlineFirst October 25, 2012.Cancer Discovery Amy Young, David Lou and Frank McCormick Regulation of Mitogen-Activated Protein Kinase SignalingOncogenic and Wild-type Ras Play Divergent Roles in the

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