The K-Ras effector p38γ MAPK confers intrinsic resistance to tyrosine kinase inhibitors by stimulating EGFR transcription and EGFR dephosphorylation

Mutations in K-Ras and epidermal growth factor receptor (EGFR) are mutually exclusive, but it is not known how K-Ras activation inactivates EGFR, leading to resistance of cancer cells to anti-EGFR therapy. Here, we report that the K-Ras effector p38γ MAPK confers intrinsic resistance to small molecular tyrosine kinase inhibitors (TKIs) by concurrently stimulating EGFR gene transcription and protein dephosphorylation. We found that p38γ increases EGFR transcription by c-Jun-mediated promoter binding and stimulates EGFR dephosphorylation via activation of protein-tyrosine phosphatase H1 (PTPH1). Silencing the p38γ/c-Jun/PTPH1 signaling network increased sensitivities to TKIs in K-Ras mutant cells in which EGFR knockdown inhibited growth. Similar results were obtained with the p38γ-specific pharmacological inhibitor pirfenidone. These results indicate that in K-Ras mutant cancers, EGFR activity is regulated by the p38γ/c-Jun/PTPH1 signaling network, whose disruption may be a novel strategy to restore the sensitivity to TKIs.

Epidermal growth factor receptor (EGFR) 3 is a trans-membrane protein-tyrosine kinase and plays a critical role in promoting cell proliferation. In response to the ligand EGF, the receptor dimerizes, leading to autophosphorylation of multiple tyrosine residues on its intracellular domain (1). This in turn activates downstream proliferative pathways, such as Ras/ MAPKs and PI3K/AKT (1). EGFR is activated via overexpression and amplification in colon, pancreas, lung, and breast cancers and via mutations in lung and brain tumors. Inhibiting EGFR is therefore considered to be an important anti-cancer strategy (1). EGFR can be inhibited by small molecule tyrosine kinase inhibitors (TKIs) that compete for the ATP-binding site of the catalytic domain (2) and by anti-EGFR antibodies that block the extracellular ligand-binding domain (3). Although anti-EGFR therapy has great potential, resistance remains a major obstacle for its successful clinical application (4).
K-Ras mutations occur in up to 50% of colon cancers, 95% of pancreatic cancers, and 25% of lung cancers (5). Such mutations are invariably associated with the intrinsic resistance to anti-EGFR therapy (4,6). Recent studies show that mutant K-Ras also causes acquired resistance to anti-EGFR treatment (7). However, the mechanisms by which K-Ras mutation confers the resistance are largely unknown (1,4,6). Mutant K-Ras drives malignant initiation and progression through effector pathways downstream of EGFR (1,5). But K-Ras can also directly regulate EGFR activity and endogenous EGFR is still required for K-Ras tumorigenesis (8 -10). These results together suggest an active mechanism by which mutant K-Ras inactivates EGFR therapeutic target activity while still depending on its intrinsic oncogenic potential for K-Ras dependent malignant growth and progression. This potential mechanism could be exploited to develop novel strategies to overcome resistance to anti-EGFR therapy in K-Ras mutant cancers.
p38␥ MAPK (gene name: MAPK12), a member of the noncanonical p38 protein family, has a unique PDZ motif on its C terminus (11). p38␥ promotes K-Ras invasive and transforming activity in intestinal epithelial cells (12)(13)(14). Through its PDZ motif, p38␥ binds, phosphorylates, and thus activates proteintyrosine phosphatase H1 (PTPH1) (13,15). PTPH1 in turn catalyzes tyrosine dephosphorylation of key signaling molecules, such as EGFR and estrogen receptor (15)(16)(17). Moreover, p38␥ interacts with the transcription factor c-Jun, through which it is recruited to AP-1 target gene promoters, leading to AP-1-dependent gene expression and malignant progression (14,18,19). Importantly, p38␥ is overexpressed in K-Ras mutant colon cancer cells (14,15) and in several types of primary human tumors (13,18,20), and increased p38␥ predicts a poor clinical prognosis (21,22). Here, we tested the hypothesis that activated p38␥ mediates K-Ras signaling to confer the intrinsic resistance to TKIs through its interaction and activation of c-Jun and PTPH1. Our results show that upon K-Ras mutation, activated p38␥ stimulates EGFR gene transcription and protein tyrosine dephosphorylation through increased complex formation with c-Jun and PTPH1. Together, these lead to elevated levels of non-phosphorylated EGFR, which is unresponsive to TKIs but is still proliferative. Further analyses demonstrate that disrup-tion of the p38␥/c-Jun/PTPH1 signaling network can restore the sensitivity to EGFR TKIs.

K-Ras mutation or EGFR dephosphorylation causes intrinsic resistance to TKIs
To determine whether K-Ras mutation causes the unresponsiveness to TKIs as observed clinically (7), colon cancer cells with and without mutated K-Ras were analyzed for growth inhibition by the Food and Drug Administration-approved TKIs lapatinib (Lap) and gefitinib (Gef). EGFR antibody was not included in the analyses because it inhibits EGFR by a distinct mechanism (3). As shown in Fig. 1A and supplemental Fig. S1A, TKIs have no substantial effects on colony formation of HCT116 human colon cancer cells with mutant K-Ras, but they significantly suppress the growth in HCT116 sublines HKe3 Figure 1. Resistance of K-Ras mutated cancer cells to EGFR TKIs couples both with increased p38␥, c-Jun, and EGFR expression and with decreased EGFR phosphorylation. A, the indicated cells were cultured in the presence and absence of TKIs (Lap (2 M) and Gef (0.125 M)) for about 2 weeks, and the number of colonies formed was manually counted. Results were normalized to DMSO control (mean Ϯ S.D. (error bars), n ϭ 3). B, cells were cultured with the indicated TKIs in the absence and presence of Dox for colony formation (mean Ϯ S.D., n ϭ 3), with the inset showing mutant K-Ras knockdown after Dox addition overnight. C and D, K-Ras WT and MT cancer cells were analyzed by WB (C, p-EGFR detected with anti-p-EGFR/Tyr-1173 antibody and the same for all other p-EGFR unless specified) and band intensities from these cell lines (C, asterisk indicates results from a separate experiment) were measured by Image Quant software (normalized to ␤-actin). Quantitative combined results from C are presented in D (mean Ϯ S.D., n ϭ 4). E, EGFR/Y1173F was stably expressed by retrovirus, and the engineered cells were assessed for colony formation. The bar graph (left and middle) is from three separate experiments (mean Ϯ S.D., n ϭ 3), whereas WB shows EGFR expression and phosphorylation (right, EGFR transfection used as a positive control for p-EGFR/Tyr-1173).

p38␥ MAPK, mutated K-Ras, and resistance to TKIs
and HK2-8, in which the mutant K-Ras allele was selectively disrupted by homologous recombination (23,24). In a similar manner, doxycycline (Dox)-inducible knockdown of mutant K-Ras in LS174T (15,25) increases the sensitivity, whereas a forced expression of a oncogenic K-Ras (G12V) in HKe3 cells led to the unresponsiveness ( Fig. 1B and supplemental Fig. S1B) (22). These results show that K-Ras mutation causes the unresponsiveness to TKIs in cell culture and suggest that dissecting the signaling events in this system may be able to reveal novel mechanisms that could lead to ways to improve the sensitivity to TKIs.
Our previous studies demonstrated elevated levels of p38␥ and c-Jun protein expression in K-Ras mutant cells (14,15). EGFR is an AP-1 target gene (26,27), and its proliferative effects can be inhibited by TKIs through blocking EGFR phosphorylation (2,28). We next investigated whether the hyperexpression of p38␥/c-Jun in K-Ras mutant cells can impact EGFR expression and phosphorylation. The results (Fig. 1, C and D) show increased p38␥, c-Jun, and EGFR protein levels but decreased EGFR phosphorylation in a panel of K-Ras mutant (MT) cells as compared with those with WT K-Ras. Because TKIs prevent EGFR phosphorylation, subsequently blocking activation of downstream proliferative pathways (29,30), the unresponsiveness of K-Ras mutant cells to TKIs may be caused by decreased p-EGFR. To directly test this possibility, a non-phosphorable mutant EGFR (Y1173F) (31) was expressed by retroviral infection, and resultant cells were analyzed for growth inhibition by TKIs. Consistent with the decreased p-EGFR and the TKI insensitivity in K-Ras mutant cells, ectopically expressed EGFR/Y1173F leads to TKI resistance in K-Ras wild-type cells without affecting the K-Ras mutant line ( Fig. 1E and supplemental Fig. S1C). These results indicate that a non-phosphorylated EGFR is sufficient to confer TKI resistance downstream of mutant K-Ras and that one mechanism for the K-Ras mutation-associated resistance may occur through increased EGFR dephosphorylation.

p38␥ stimulates EGFR transcription through c-Jun-mediated binding to the EGFR promoter and c-Jun is required for K-Ras-dependent growth and for TKI resistance
Results ( Fig. 1, C and D) also show increased EGFR protein expression in K-Ras mutant cells. Whereas EGFR is frequently overexpressed in human cancers, the responsible mechanisms are largely unknown (1,32). Having previously demonstrated that p38␥ increases AP-1-dependent MMP9 transcription through interacting with and activating c-Jun (14,19), we determined whether p38␥ stimulates EGFR expression in collaboration with c-Jun, which may also contribute to the resistance to TKIs. The results (Fig. 2, A-C) show that p38␥ overexpression in normal rat intestinal epithelial cells (IEC-6) (12) stimulates c-Jun and EGFR mRNA and protein expression, whereas p38␥ depletion from K-Ras mutant colon cancer HCT116 and SW480 cells has an opposite effect. A forced c-Jun expression in HCT116 cells increases the growth and elevates EGFR protein levels without impacting p38␥ expression (Fig. 2D), indicating its oncogenic activity downstream of p38␥ and upstream of EGFR. Consistent with this notion, c-Jun depletion in K-Ras mutant cells reduces colony formation and decreases EGFR protein expression without affecting p38␥ levels (supplemental Fig. S2, A-C). Of great interest, EGFR knockdown in these cells still inhibits growth without affecting p38␥ and c-Jun protein levels (supplemental Fig. S2, A-C). These results indicate that intrinsic EGFR in K-Ras mutant cells is still capable of driving the malignant growth despite the resistance of these cells to TKIs, as observed in patients with K-Ras mutant cancer (1). Together with the ability of p38␥ to promote K-Ras oncogenic activity (12)(13)(14) and with the increased levels of p38␥, c-Jun, and EGFR in K-Ras mutant cells (Fig. 1, C and D), the data support an essential role of the p38␥/c-Jun/EGFR transcription axis in K-Ras-dependent growth.
Previous studies showed that p38␥ is both cytoplasmic and nuclear, whereas its phosphorylated form is predominantly localized in the nucleus (33)(34)(35). Because phosphorylated p38␥ protein is up-regulated in K-Ras mutant cells (15), we examined whether K-Ras mutation triggers p38␥ nuclear translocation, thus increasing its interaction with c-Jun. Cell fractionation analyses show that there is increased p38␥ nuclear accumulation in K-Ras mutant cells relative to their K-Ras wild-type counterparts (Fig. 3A). These increases in p38␥ also correlate with elevated c-Jun in the nucleus, as revealed by cell fractionation and co-localization analyses ( Fig. 3A and supplemental Fig. S3A). These results indicate that p38␥ and c-Jun may collaborate to promote K-Ras oncogenesis through their enhanced nuclear activities.
We next used ChIP assays (14) to determine whether p38␥ and c-Jun cooperate to stimulate EGFR transcription through their binding to the EGFR promoter. Of great interest, p38␥ is only significantly recruited to the EGFR promoter at an AP-1 site in K-Ras mutant, but not in K-Ras wild-type, cells, whereas c-Jun binds the same location independent of K-Ras mutation status (Fig. 3, B and C). Consistent with a mediating role for c-Jun in the p38␥/AP-1 promoter DNA binding (14), p38␥ immunoprecipitates from K-Ras mutant cells consistently contain higher levels of c-Jun (Fig. 3B). These results indicate that K-Ras mutation results in constitutive binding of p38␥ to the EGFR promoter through its increased interaction with c-Jun. Furthermore, c-Jun depletion from K-Ras mutant cells not only decreases the growth and down-regulates EGFR expression, but also increases the growth-inhibitory activity of TKIs (supplemental Figs. S2 (A and C) and S3B). These results together indicate that K-Ras mutation can directly trigger p38␥ binding to the EGFR promoter through its increased nuclear translocation in a c-Jun-dependent manner and that increased c-Jun in K-Ras mutant colon cancer cells is, at least in part, responsible for elevated EGFR expression and the unresponsiveness to TKIs.

K-Ras mutation increases the binding of EGFR with both p38␥ and PTPH1, which promotes EGFR dephosphorylation and TKI resistance
EGFR can be dephosphorylated by several phosphatases, including protein-tyrosine phosphatase TCPTP (36) and receptor-type protein-tyrosine phosphatase-(RRTO) (37). The consequences of such EGFR dephosphorylation for sensitivities to TKIs, however, have not been explored. We previously demonstrated that PTPH1 specifically decreases EGFR phosphory-p38␥ MAPK, mutated K-Ras, and resistance to TKIs lation at Tyr-1173, but not Tyr-1068 and that this increases breast cancer sensitivity to TKIs through disruption of an EGFR inhibitory complex with the estrogen receptor (17). In this study, the growth-inhibitory activity of TKIs was positively correlated with PTPH1 protein levels in EGFR immunoprecipitates (17). Because the EGFR dephosphorylating activity of PTPH1 requires its interaction with and phosphorylation by p38␥ (15), we next determined whether K-Ras mutation alters the binding of EGFR with PTPH1 and its activator p38␥. The results (Fig. 4A) show that EGFR immunoprecipitates contain higher levels of both PTPH1 and p38␥ proteins in K-Ras mutant cells as compared with those in K-Ras wild-type cells. Further analyses (Fig. 4B) show that ectopically expressed EGFR binds PTPH1 and p38␥, but not the C-terminal deleted p38␥⌬4 or the non-phosphorable p38␥/AGF mutant. These results indicate that EGFR binds PTPH1 and p38␥ by a mechanism that depends on both the p38␥ C terminus and p38␥ phosphorylation. An enhanced binding of EGFR to the p38␥-PTPH1 complex may play an important role in decreasing the levels of p-EGFR in K-Ras mutant cells (Fig. 1, C and D).
Consistent with this premise, overexpression of p38␥, but not its p38␥/AGF mutant, stimulates both PTPH1 expression

p38␥ MAPK, mutated K-Ras, and resistance to TKIs
and EGFR dephosphorylation in association with an enhanced EGFR-PTPH1-p38␥ complex formation (Fig. 4C). To demonstrate whether K-Ras mutation alters PTPH1-mediated EGFR dephosphorylation, p-EGFR proteins were measured using a p-EGFR/Tyr-1173-specific antibody after endogenous PTPH1 was knocked down. Of great interest, PTPH1 depletion significantly increases p-EGFR levels in K-Ras mutant HCT116 cells, but not in the K-Ras-disrupted HKe3 subline (Fig. 5A), indicating that endogenous PTPH is responsible for decreased levels of p-EGFR in K-Ras mutant cells. These results, together with those in Fig. 4 (A and B), indicate that decreased p-EGFR in K-Ras mutant cells (Fig. 1, C and D) is probably caused by enhanced EGFR dephosphorylation by PTPH1 through its increased complex formation with PTPH1 and p38␥. Furthermore, knockdown of p38␥ or PTPH1 increases the growthinhibitory activity of TKIs in K-Ras mutant cells (supplemental Fig. S4, A and B), whereas PTPH1 depletion has no consistent effect in K-Ras wild-type cells (supplemental Fig. S5, A and B). Together, these results demonstrate a causative role of the p38␥-PTPH1 signaling nodule in dephosphorylating EGFR and in resistance of K-Ras mutant cells to TKIs.
To further determine the cooperative role of p38␥ and PTPH1 in EGFR dephosphorylation, a reconstitution experiment was performed in 293T cells by transient co-transfection. The results (Fig. 5B) show that FLAG-p38␥ cooperates with HA-PTPH1 to regulate EGFR phosphorylation and expression in association with increased HA-PTPH1 phosphorylation. Furthermore, the addition of p38␥ protein to Myc-isolated EGFR precipitates in vitro failed to decrease p-EGFR levels as compared with the positive control of HA-PTPH1 (Fig. 5C), indicating that p38␥ is not able to directly dephosphorylate EGFR. These results, together with those in Figs. 1-4, suggest that p38␥ MAPK increases non-phosphorylated EGFR by concurrently enhancing PTPH1-mediated EGFR dephosphorylation and stimulating c-Jun-dependent EGFR trans-activation.
To determine whether an in situ K-Ras mutation activates this dynamic signaling network, mutant K-Ras was re-expressed by Dox removal following its initial knockdown by inducible  B). B and C, the indicated cells were processed for IP/WB (B) and ChIP (C) analysis using the indicated specific antibodies as described (14,19), and p38␥-bound c-Jun was measured from p38␥ precipitates (B). Similar results were obtained in a separate experiment.  ). B, the indicated constructs were transiently expressed in 293T cells, and Myc precipitates were analyzed by WB for EGFR binding to HA-PTPH1 and/or FLAG-p38␥. C, cells were transduced with the indicated adenovirus and EGFR precipitates were analyzed by WB after a 24-h incubation.

p38␥ MAPK, mutated K-Ras, and resistance to TKIs
shRNA (15,25), and cells were then analyzed for protein expression and phosphorylation. Results (Fig. 5D) show that an increase in mutant K-Ras protein stimulates p38␥ expression in Ͻ5 min, leading to decreased p-EGFR and increased total EGFR protein for up to 60 min. These results further indicate that the in situ K-Ras mutation can indeed trigger increased EGFR expression and dephosphorylation through endogenous p38␥ activity.

The p38␥ inhibitor pirfenidone disrupts the EGFR-p38␥-PTPH1 complex, increases EGFR tyrosine phosphorylation, and restores sensitivity of K-Ras mutant cancer cells to TKIs
p38␥ depends on its phosphorylation status to activate both c-Jun and PTPH1 (13)(14)(15), to interact with EGFR (Fig. 4B), and to trigger decreased p-EGFR expression (Fig. 4C). We next explored whether inhibition of p38␥ activity by its pharmacological inhibitor pirfenidone (PFD) (38, 39) impacts EGFR phos-phorylation and TKI sensitivity in K-Ras mutant cancer cells. PFD more significantly inhibits p38␥ in vitro than its family member p38␣ and p38␤ (40). Because of its strong anti-fibrotic effect and relatively non-toxic properties, PFD is approved for the treatment of lung fibrosis (39,41). Treatment of K-Ras mutant cells (colon cancer: HCT116 and SW480; pancreatic cancer: Panc-1 and Mia2PaCa2; and lung cancer: A549) with TKIs alone has no substantial effects on their colony formation. The PFD addition (at a concentration for which PFD was noninhibitory itself) significantly enhanced the growth inhibition by Lap and Gef (Fig. 6A and supplemental Fig. S6A). Further, PFD treatment completely abolishes the EGFR binding of p38␥ in two K-Ras mutant cell lines, leading to decreased p-PTPH1 and increased p-EGFR (Fig. 6B, Input). These results demonstrate a required role for p38␥ activity in PTPH1-mediated EGFR dephosphorylation through a complex formation and suggest that the sensitivity to TKIs in K-Ras mutant cells can be

. PTPH1 is only active in decreasing EGFR tyrosine phosphorylation in K-Ras mutant cells and cooperates with mutant K-Ras and p38␥ to promote EGFR dephosphorylation.
A, control cells and cells stably depleted of PTPH1 were analyzed by WB for EGFR expression and phosphorylation (EGFR/Tyr-1173); relative levels of p-EGFR in shPTPH1 versus shLuc cells were normalized to EGFR (55). B, the indicated constructs were transiently transfected into 293T cells, and the resultant cells were analyzed by WB. C, Myc-EGFR protein isolated from transiently transfected 293T cells was incubated in vitro with the indicated proteins, and the mixtures were then analyzed by WB for p-EGFR levels. D, K-Ras mutation in situ activates the p38␥/c-Jun/PTPH1 signaling network, leading to both increased EGFR protein expression and decreased EGFR/Tyr-1173 phosphorylation. Cells were cultured with Dox for 72 h to silence the MT K-Ras. Protein lysates were prepared at the indicated time after Dox removal for K-Ras re-expression and analyzed by WB. The number indicates relative protein amounts over ␤-actin at each time point (normalized to 0 min), and similar results were obtained in a separate experiment.

p38␥ MAPK, mutated K-Ras, and resistance to TKIs
restored by PFD-induced p38␥ depletion from the EGFR-PTPH1 complex. Consistent with this notion, the sensitization effect was further demonstrated in HCT116 xenografts in which systemic PFD administration disrupts the binding of EGFR with both p38␥ and PTPH1, alone and in combination with Lap ( Fig. 6C and supplemental Fig. S6B). The application of Lap, instead of Gef, for the in vivo combination with PFD is due to the fact that Lap was previously tested in colon cancer patients in combination studies (42). These results together demonstrate that PFD can sensitize K-Ras mutant cancers to TKIs by disrupting the EGFR-p38␥-PTPH1 signaling complex.

Discussion
K-Ras mutation has been long known to be associated with resistance to TKIs, and there is an urgent need to identify novel signaling mechanisms that can be used to restore TKI sensitiv-ity (43)(44)(45). p38␥ MAPK promotes K-Ras oncogenesis through activating its signaling network c-Jun and PTPH1 by complex formation (11)(12)(13)(14)(15). Here, our results provide several key pieces of evidence that, together with the previous findings, indicate that the K-Ras effector p38␥ confers resistance to TKIs through its concurrent stimulation of c-Jun-dependent EGFR transcription and PTPH1-catalyzed EGFR dephosphorylation (Fig. 6D). Because expression of EGFR/Y1173F increases the sensitivity to TKIs in K-Ras wild-type but not K-Ras mutant cells (Fig. 1E), in which EGFR silencing still decreases the colony formation (supplemental Fig. 2), these results suggest that endogenous EGFR in K-Ras mutant cells may drive the growth independent of kinase activity. Importantly, inhibiting p38␥ with its pharmacological inhibitor PFD restores the TKI sensitivity of K-Ras mutant cells in vitro and in vivo ( Fig. 6 and supplemental Fig.  S6). Together, these results demonstrate that p38␥ converts  (51), whereas PFD (200 mg/kg in 100 l of water) was given by oral gavage daily for 2 weeks. Changes in tumor volume were monitored every other day (results are means of five tumors Ϯ S.E.). D, an experimental model indicates that K-Ras mutation may confer TKI resistance through p38␥ MAPK-induced concurrent activation of c-Jun-dependent EGFR gene transcription and of PTPH1-induced EGFR dephosphorylation leading to elevated non-phosphorylated EGFR, which may contribute to both K-Ras-dependent growth and unresponsiveness to TKIs.

p38␥ MAPK, mutated K-Ras, and resistance to TKIs
K-Ras oncogene signaling to TKI resistance through its dual stimulating activity on EGFR gene expression and protein dephosphorylation (Fig. 6D).
An autocrine mechanism is believed to be responsible for the signaling cross-talk between Ras and EGFR; however, the factors involved have been mostly unidentified (46 -49). Our results suggest that the K-Ras mutation itself simultaneously stimulates EGFR transcription and dephosphorylation by activating the p38␥ signaling network through regulating dynamic protein-protein and protein-DNA interactions. This is supported by enhanced p38␥ binding to the EGFR promoter DNA through interaction with c-Jun and by increased complex formation of EGFR with p38␥ and PTPH1 proteins in K-Ras mutant cells (Figs. 3 and 4). The functional role of this complex is suggested by the fact that the p38␥ inhibitor PFD disrupts the p38␥ interaction with c-Jun/AP-1 DNA (19), suppresses the EGFR binding with p38␥ and PTPH1, and increases the sensitivity of K-Ras mutant cells to TKIs ( Fig. 6 and supplemental Fig. S6). Moreover, depletion of PTPH1 from the ternary EGFR-PTPH1-p38␥ complex increases p-EGFR levels and enhances the sensitivity of K-Ras mutant cells to TKIs ( Fig. 5 and supplemental Fig.  S4B). In addition to elevated p38␥, hyperexpression of c-Jun and EGFR (Fig. 1, C and D) increased p38␥-induced PTPH1 phosphorylation in K-Ras mutant cells, and a phosphorylation-dependent p38␥ interaction with c-Jun and PTPH1 (13-15) may also contribute to the distinct complex formation. These results together reveal a novel mechanism by which the K-Ras oncogene may inactivate EGFR by a p38␥-activated signaling network through its increased interaction with c-Jun/PTPH1/EGFR proteins as well as with the EGFR promoter DNA.
EGFR is a well-established target for cancer therapy (50,51), and our results indicate that levels of EGFR expression and phosphorylation are both important for its therapeutic target activity. The previous studies showed that knock-out of mutant K-Ras by shRNA restores sensitivity to TKIs, but the application potential of this strategy was uncertain (52). Our results show that K-Ras mutation confers unresponsiveness to TKIs through p38␥-induced EGFR gene transcription and EGFR protein dephosphorylation, respectively, via the transcription factor c-Jun and the phosphatase PTPH1 (11)(12)(13)(14)(15) (Fig. 6D). Knocking down each member of the p38␥/c-Jun/PTPH1 network increases the sensitivity of K-Ras mutant cancer cells to TKIs (supplemental Fig. S3B and Fig. 4 (A and B)). Most importantly, the non-toxic p38␥ inhibitor PFD restores the sensitivity of K-Ras mutant cancers in vitro and in vivo ( Fig. 6 and supplemental Fig. S6). These results, together with its inhibitory effects on the p38␥ interaction with c-Jun (19) and on the EGFR association with p38␥ and PTPH1 (supplemental Fig. S6B), further indicate that disruption of the p38␥-activated signaling network (such as by using PFD) may have great application potential to restore the therapeutic response of K-Ras mutant cancers to TKIs.

RNA preparation, quantitative RT-PCR (qRT-PCR), and ChIP
qRT-PCR was carried out as described previously (14). Total RNA was prepared using the TRIzol extraction kit, and qRT-PCR was performed using the Express One-Step SYBER GreenER qPCR kit (Invitrogen). Samples were analyzed by the ⌬⌬Ct method for -fold changes in expression, and the ratios of the individual genes relative to ␤-actin were expressed relative to the respective controls (14,19). For ChIP assays, cells were incubated with 5% formaldehyde, and lysates were then subjected to immunoprecipitation with the indicated antibodies. These precipitated DNAs were used as a template for PCR analysis using primers covering the AP-1 site (14,19,26). An aliquot of each DNA before PCR was included as an input control on the same gel as the PCR products. Other procedures were the same as described (14,19).

Colony formation and animal studies
For colony formation, 200 cells were plated in duplicate in 6-well plates and incubated with TKIs and/or PFD for about 2 weeks. The colonies formed were stained and counted as described (17,22,54). Animal studies were conducted according to the protocol approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee. Briefly, HCT116 cells (2 ϫ 10 6 in 0.1 ml of PBS) were subcutaneously injected into male BALB/c nude mice (Charles River), and therapy with Lapatinib and/or PFD (or DMSO control) was initiated when tumors became palpable. Tumor volume was measured every 2-3 days (22), with representative tumors at sacrifice photographed and shown as the inset (Fig. 6C). Moreover, protein lysates were prepared from tumors and analyzed by WB/IP (19,22).

Statistical analysis
The results were analyzed by Student's t test unless otherwise specified.