Dual Inhibition of MEK and PI3K/Akt Rescues Cancer Cachexia through both Tumor-Extrinsic and -Intrinsic Activities

The Ras/Raf/MEK/ERK pathway, which promotes cell growth and survival, is mutated in nearly 40% of all human tumors. Although inhibition of Ras is an attractive therapeutic strategy, designing specific anti-Ras compounds has been elusive (1). Thus, efforts have shifted to targeting the downstream effectors of Ras, MEK, and ERK. In addition to regulating tumor cell proliferation and survival, the Ras/Raf/MEK/ERK pathway is also known to promote the production of cytokines from malignant cells (2–5). The aberrant production of soluble factors is thought to contribute to systemic changes that are prominent in patients with advanced cancer, including the cancer cachexia syndrome (6). Cancer cachexia is characterized by the loss of skeletal muscle and adipose tissue mass, which cannot be reversed by nutritional intervention (7). More than half of all cancer patients experience cachexia, and cachexia is a significant contributor to mortality from cancer (8, 9). Currently, no effective pharmacologic approach exists to limit cancer cachexia.

Recently, a particularly interesting observation was made in a clinical trial with biliary tract cancer patients treated with the MEK inhibitor selumetinib. In this trial, only 12% of patients had objective responses to therapy, but 80% of the patients treated with MEK inhibition experienced significant weight gain associated with increases in skeletal muscle mass (10, 11). As weight gain is unusual in biliary tract cancer patients given their high incidence of cachexia, these clinical data suggested that MEK inhibition might possess an anticachectic activity.

To investigate the potential that MEK inhibition might serve as an anticachexia therapy, we initiated a preclinical study using the MEK inhibitor MEK162 in an established mouse model of cancer cachexia, the Colon-26 (C-26) tumor model (12, 13). Similar to prior clinical observations in biliary tract cancer patients, results showed that MEK162 preserves skeletal muscle mass. Although effective at maintaining skeletal muscle, we recognize that ultimately, future successful anticancer therapy will need to maximize the protection of host tissues while targeting malignant cells. Because resistance to MEK inhibition is often associated with a compensatory activity on the PI3K/Akt pathway (14), we treated C-26 tumor-bearing mice with a combination of MEK162 and the PI3K/Akt inhibitor buparlisib. Results showed that this combination led to a potent inhibition of both tumor growth and cachexia, resulting from a direct cytotoxic activity on C-26 tumor cells. Together, these results successfully uncouple multiple mechanisms by which MEK inhibition may benefit patients with advanced cancer, including a tumor-extrinsic effect that preserves skeletal muscle and a tumor-intrinsic activity that reduces tumor load due to the improved efficacy of PI3K/Akt inhibition.

Male CD2F1 mice 7 to 8 weeks of age were purchased from Charles River Laboratories, housed in conventional conditions, and fed a standard ad libitum rodent chow diet at University Laboratory Animal Services at the Arthur G. James Comprehensive Cancer Center of The Ohio State University (Columbus, OH). C-26 adenocarcinoma cells were maintained in RMPI1640 media containing 5% FBS. Mice were randomized between treatment groups and then injected subcutaneously in the right flank with 1 × 10⁶ murine C-26 cells. Animals were monitored daily, with body weight and bidimensional tumor measurements obtained three times weekly using microcalipers. Experiments were terminated when the body condition score of mice in the vehicle treatment group decreased to 1.5 or lower (15), which is consistent with recommendations made by the laboratory animal veterinary staff at The Ohio State University. In the C-26 model, this body condition score is associated with an approximately 20% weight loss. Mice with ulcerated tumors were removed from the study prior to the final endpoint and were excluded from all data analyses. No outliers were excluded from the data analysis. Similar procedures were followed for experiments using Lewis lung carcinoma (LLC) tumors, with the following modifications: (i) LLC tumor cells were maintained in DMEM containing 5% FBS; and (ii) 5 × 10⁵ tumor cells were injected into the right gluteus muscles of C57B/6 mice purchased from The Jackson Laboratory. Both our C-26 and LLC cell lines were obtained through a Material Transfer Agreement with the NCI (Bethesda, MD) in 2001. These cell lines have been maintained in the Guttridge Laboratory and have consistently produced similar cachexia, including in this article, demonstrating that these cells have maintained their identity over time (12, 16). All animal experiments were approved by the Institutional Animal Care and Use Committee of The Ohio State University under protocol 2010A00000177-R1 in accordance with the NIH Guide for Care and Use of Laboratory Animals. In vitro experiments were performed a minimum of three times to ensure veracity of the data.

MEK162 and buparlisib were provided by Novartis, Inc. as lyophilized stocks and resuspended in DMSO to a concentration of 100 mmol/L for in vitro experiments. For in vivo experiments, the inhibitors were resuspended at a concentration of 100 μg/mL in 1% carboxymethyl cellulose and 0.5% Tween 80, with MEK162 given at a dose of 30 mg/kg and buparlisib given at 25 mg/kg. These dosages are consistent with previous clinical trials (17, 18). Vehicle-treated animals received 1% carboxymethyl cellulose and 0.5% Tween 80. All treatments were administered via oral gavage in a volume of 200 μL daily. Chemical structures of the inhibitors are available in refs. 19 and 20.

To generate a line of tumor cells resistant to MEK162, C-26 cells were seeded in 6-well plates at 1 × 10⁶ cells per well overnight. Cells were then treated with MEK162 beginning at 1 μmol/L for 3 days and increased to 30 μmol/L incrementally over a 2-week period. MEK162 concentration was increased when cell confluence increased. Resistance of C-26 cells to MEK162 was validated using an MTT assay. The subsequent MEK162-resistant C-26 cell line was designated “C-26R.” Prior to implantation in vivo for therapeutic studies, C-26R cells were maintained in the presence of MEK162 (30 μmol/L) to ensure maintenance of MEK162 resistance. Similar to our parental line studies, 1 × 10⁶ C-26R cells were injected into the right flank of randomized groups of mice. For our in vivo C-26R studies, our animal initial numbers were based upon our findings in our first experiments utilizing our parental line. However, due to the aggressive nature of our resistant tumor cells, we repeated these experiments to ensure that our findings were accurate. After achieving the same results, we collapsed our two datasets.

Muscle cross-sectional area was determined in the gastrocnemius muscle. Ten-micron sections were cut from muscles frozen in liquid nitrogen–cooled isopentane. Three sections representing the entire length of the muscle were selected for H&E staining, and images were acquired using an Olympus BX51 bright field microscope. The Olympus Microsuite Pathology software was used to determine individual muscle fiber CSA by manual outlining with software assistance. Results from all three sections from each animal were averaged prior to statistical analysis. Across all experiments, an average of 316 fibers per section and 930 total fibers per animal were analyzed.

Messenger RNA was isolated from snap-frozen quadriceps muscle using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. Total RNA was reverse transcribed to cDNA using M-MLV Reverse Transcriptase (Life Technologies) according to the manufacturer's instructions. Real-time PCR was performed on an Applied Biosystems StepOnePlus instrument using SYBR Green Mix (Bio-Rad). GAPDH was used as the housekeeping gene. Primer sequences appear in Supplementary Table S1.

Following euthanasia, tumors were removed and formalin fixed overnight before undergoing processing and paraffin embedding. For our initial experiments with the parental tumor cells, tumors were sectioned at 4 μm. Slides were deparaffined, rehydrated, then stained with anti-phospho-p42/p44 (phospho-ERK, Cell Signaling Technology #4370) using the Dako Autostainer Immunostaining System, with Vulcan Fast Red or Biocare Romulin AEC chromogens and counterstained with Richard Allen hematoxylin. Tumors were analyzed in a blinded fashion by a board-certified pathologist (Dr. Alton Brad Farris, Emory University, Atlanta, GA). For all additional experiments, tumors were sectioned at 5 μm and stained using a Bond Rx autostainer (Leica). Slides were heated for 15 minutes at 65°C and then automated software dewaxed, rehydrated, performed antigen retrieval, blocked, incubated with primary antibody, detected (DAB), and counterstained using Bond reagents (Leica). Samples were then removed from the machine, dehydrated through ethanol and xylenes, mounted and coverslipped. The following antibodies were diluted in Leica antibody diluent: anti-phospho-pERK (Thr202/Tyr204; Cell Signaling Technology #4370), anti-pAKT (Thr308, Cell Signaling Technology #9275), CD3 (Dako A0452), cleaved caspase-3 (Cell Signaling Technology #9661), or Ki67 (Abcam ab16667). Quantification of cells positive for CD3, cleaved caspase-3, and Ki67 was performed using the Vectra Automated Quantitative Pathology Imaging System and the InForm Software (PerkinElmer).

Immunoblots on tumor cells were performed as previously described and probed with antibodies specific for pERK (Thr202/Tyr204, Cell Signaling Technology #4370) (21), ERK (Cell Signaling Technology #4695), pAKT (Thr308, Cell Signaling Technology #9275), and AKT (Cell Signaling Technology #9272). Western blotting on muscle tissues was conducted as previously described using pERK (Thr202/Tyr204, Cell Signaling Technology #4370) or LC3B (Cell Signaling Technology #2775), fluorescently conjugated secondary antibodies, and a Li-Cor Odyssey imaging system (13). β-Actin (CST #4967) or α-tubulin (Sigma T5168) were used as loading controls.

Phenotypic analysis of the myeloid-derived suppressor cell (MDSC) and T lymphocyte populations in splenocytes from mice was conducted using standard methods. Briefly, 10⁶ splenocytes were resuspended in 100 μL of FACS buffer (PBS + 5% FBS) and incubated with fluorochrome-labeled antibody targeting murine CD11b (BD Biosciences #557686), GR1 (BD #553127), CD4 (BD #552775), CD8 (BD #552877), or appropriate isotype control antibody (BD Biosciences) for 1 hour at 4°C. Cells were washed, resuspended in 1% formalin, and analyzed via flow cytometric analysis on a LSRII flow cytometer (BD Biosciences).

Antibody depletion of CD4⁺ and CD8⁺ cells were conducted as described previously (22). Briefly, rat anti-mouse CD8 (clone 2.43) and rat anti-mouse CD4 (clone GK1.5) antibodies were purchased from Bio X Cell. For depletion experiments, 100 μg of antibody was injected intraperitoneally on days −3, −1, +1, +3, and every 4 days thereafter in relation to the tumor injection (on day 0). Purified rat IgG was used as a control (Sigma). Depletion of CD4⁺ and CD8⁺ cells was confirmed by flow cytometric analysis of lymphocytes obtained from splenocytes at the study endpoint.

C26 cell lines were seeded in 96-well plates in triplicate and allowed to adhere overnight. Fresh media containing MEK162, buparlisib, or both were added to each well at various concentrations (5 and 10 μmol/L of MEK162, and 2.5 and 5 μmol/L of buparlisib), and cells were incubated for 72 hours. At this time, the percentage of cell growth was evaluated using the MTT assay (ATCC 30-1010K) and quantified by determining the optical density at 595 nm using a Bio-Rad iMark microplate reader.

All data are displayed as mean ± SEM. For experiments comparing MEK162 treatment to vehicle treatment, differences between group means were determined using a two-tailed Student t test. Experiments assessing treatment with MEK162, buparlisib, and MEK162 + buparlisib in combination were analyzed utilizing one-way ANOVA with Tukey HSD post hoc when appropriate, with the exception of tumor growth and body weight. The rate of tumor growth was determined using a mixed-effects regression model on log-transformed volumes (exponential growth model). Random effects for intercept and slope by mouse were included, and slopes were compared using linear contrasts. Body weights were analyzed using a linear model with fixed effects for group, time, and the group by time interaction. A first-order autoregressive covariance structure for the residual error was used, and P values were adjusted using the Bonferroni method. Degrees of freedom were determined by the Kenward–Roger method (23). Log transformations were used when appropriate to eliminate the effects of heteroscedasticity and are noted in the figure legends of transformed data. All analyses were performed in SAS v9.4 (SAS Institute). P values less than 0.05 were considered significant.

Biliary cancer patients display a high incidence of cachexia, but patients treated with MEK inhibition regained body weight in the form of lean mass (10, 11). This suggested that MEK might be a therapeutic target in cancer cachexia. To examine the impact of MEK inhibition on cachexia, we utilized the established C-26 cancer cachexia model. CD2F1 mice bearing palpable C-26 tumors were dosed with a MEK1/2-specific inhibitor, MEK162. Daily treatment for 16 days with MEK162 had a pronounced effect at preventing tumor-induced body weight loss as compared with vehicle-treated animals (Fig. 1A). To assess whether this activity of MEK162 was derived from the protection of lean mass, hindlimb muscles from tumor-bearing mice were analyzed. Results showed that MEK162-treated mice had larger tibialis anterior (TA), quadriceps (QUAD), and gastrocnemius (GAST) muscle masses compared with vehicle controls (Fig. 1B). In addition, the increase in muscle weight was concomitant with an increase in GAST muscle fiber size (Fig. 1C and D and Supplementary Fig. S1A). Furthermore, MEK162 treatment reduced the expression of a panel of established muscle atrophy genes in QUAD, including E3 ligases of the ubiquitin proteasome system (MuRF1, Atrogin-1, and Musa1) and genes associated with autophagy (Atg5 and Bnip3; Fig. 1E). Similar protection against muscle atrophy was seen in a second established cancer cachexia model, the LLC model. LLC tumor-bearing mice treated with MEK162 had significantly larger GAST muscle fiber cross-sectional areas compared with vehicle-treated tumor-bearing mice (Supplementary Fig. S1B–S1D).

Significantly, when non-tumor–bearing CD2F1 mice were treated with a similar dosing regimen of MEK162, total body weight and muscle mass remained unchanged, demonstrating that MEK162 does not function simply as an anabolic agent to increase skeletal muscle mass (Supplementary Fig. S1E and S1F). These data, coupled with our previous work demonstrating that our C-26 tumor-bearing mice do not exhibit decreased food intake compared with mice without tumors (24), suggest that a mechanism other than increased food intake is responsible for the anticachexia effect of MEK162. Comparison of muscle weights from C-26 tumor-bearing mice to non-tumor–bearing controls demonstrates that MEK162 provides a nearly complete rescue of muscle mass for all three of the muscles tested (Supplementary Fig. S1G).

A common problem plaguing cancer cachexia research is that although many compounds appear to be anticachectic, their ability to rescue muscle wasting actually results from a direct effect on tumors and not a protection of host tissues (25–27). As such, any treatment that is effective at reducing the tumor load would be expected to have a downstream benefit of rescuing muscle wasting. When we examined changes in tumor volume in our mice treated with MEK162, we found that MEK inhibition exhibited a modest effect on the rate of C-26 tumor growth, although this was not statistically significant (Fig. 2A). Nevertheless, MEK162 treatment reduced ERK activity in C-26 tumors (Fig. 2B), demonstrating its on-target activity. Furthermore, MEK162 significantly reduced serum levels of IL-6 (Supplementary Fig. S2A), which in this model has been shown to be tumor-derived and functions as a major driver of cachexia (28–30). These data suggested that the anticachectic activity of MEK162 is associated with some effects on the tumor. However, upon examining ERK activity within skeletal muscle, we also observed that MEK162 treatment was effective in reducing tumor-induced increases in ERK phosphorylation (Supplementary Fig. S2B), implying that a portion of the anticachectic effect of inhibiting MEK could be tumor-extrinsic.

In an attempt to determine whether the anticachectic activity of MEK inhibition could be uncoupled from its tumor effect, we set out to produce a C-26 tumor line that was resistant to MEK162. To produce this line, we continually cultured C-26 tumor cells in increasing concentrations of MEK162 until tumor cells became resistant to its cytotoxicity and ERK activity could no longer be inhibited (Fig. 2C and D). We named these resistant cells C-26R, to distinguish them from the C-26 parental line. Similar to resistance in vitro, the growth of implanted C-26R tumor cells in mice was nearly indistinguishable between vehicle- and MEK162-treated mice (Fig. 2E and Supplementary Fig. S2C). In addition, compared with the pronounced effect that MEK162 had on reducing circulating IL-6 levels from parental C-26 tumors (Supplementary Fig. S2A), serum IL-6 from MEK162-treated C-26R tumor-bearing mice was actually higher than vehicle-treated C-26R mice (Supplementary Fig. S2D), providing further evidence that we had established a cell line resistant to MEK162.

Several distinguishing features were observed with the C-26R cell line. In culture, the morphology of these cells appeared different than C-26 parental cells, exhibiting a greater number of cell extensions that were longer (Supplementary Fig. S2E). C-26R cells tended to have slower rates of tumor growth in vivo compared with C-26 cells (Fig. 2F), yet mice bearing these tumors exhibited profound cachexia, reaching our endpoint criteria 3 days faster than parental C-26 tumor-bearing mice (Fig. 2G). Consistent with this high level of cachexia, serum IL-6 levels of vehicle-treated C-26R tumor-bearing mice were nearly five times higher than from mice bearing C-26 parental tumors (Supplementary Fig. S2A and Fig. 2D).

Importantly, despite the aggressive nature of C-26R tumors, treatment with MEK162 decreased tumor-induced body weight losses compared with vehicle-treated mice (Fig. 3A). Consistent with this preservation of body weight, C-26R tumor-bearing mice treated with MEK162 also exhibited significantly larger TA, QUAD, and GAST muscle masses compared with vehicle-treated mice (Fig. 3B). In addition, MEK162 treatment resulted in larger GAST muscle fiber size (Fig. 3C and D and Supplementary Fig. S3A). Moreover, although MEK162 treatment only tended to decrease expression of proteasome genes associated with muscle atrophy, the drug significantly lowered expression of genes associated with the autophagy system in QUAD, suggesting that MEK162 decreases activation of the autophagy pathway in skeletal muscle (Fig. 3E). To confirm this finding, we probed for the autophagy-associated protein microtubule-associated protein light chain 3 (LC3). The conversion of LC3 from LC3-I to LC3-II is a commonly used surrogate marker of autophagosome formation (31). Compared with muscle from control, non-tumor–bearing mice, muscles from mice bearing C-26R tumors demonstrated increased LC3-II (Fig. 3F and G). In contrast, muscles from mice treated with MEK162 demonstrated reduced LC3-II conversion, suggesting that MEK inhibition reduces autophagosome formation and thus prevents atrophy by decreasing autophagic activity.

Although MEK inhibition appears effective as an anticachexia treatment, ultimately, the most efficient cachexia care will require a therapy that can both limit the wasting of skeletal muscle and reduce the tumor. In many cases, tumors treated with MEK inhibition as a monotherapy develop resistance due to compensatory activation of other pro-oncogenic signaling pathways, such as the PI3K/Akt pathway (14). In line with this notion, C-26 parental cells treated with MEK162 exhibited a robust activation of Akt activity (Fig. 4A). From this result, we hypothesized that treatment of C-26 tumor-bearing mice with a combination of MEK162 and an inhibitor of PI3K/Akt would be more efficacious than either drug alone. This combination of MEK162 with buparlisib is currently in clinical trials for both advanced melanoma and advanced solid tumors (NCT02159066 and NCT01363232) and thus represents a plausible clinical therapy.

The addition of the PI3K/Akt inhibitor buparlisib to MEK162 treatment had a profound effect in reducing the rate of parental C-26 tumor growth and tumor weight compared with single-agent dosing (Fig. 4B and Supplementary Fig. S4A). Staining for MEK and Akt activities in the tumors confirmed on-target effects of MEK162 and buparlisib (Supplementary Fig. S4B).

We next asked whether this significantly improved antitumor response by MEK162 and buparlisib translated to an equally improved outcome on cachexia. Although buparlisib treatment alone reduced the rate of tumor growth, mice lost body weight and skeletal muscle to the same degree as vehicle-treated controls (Fig. 4C–E and Supplementary Fig. S4C and S4D). These data are in agreement with previous reports showing that inhibition of the PI3K/Akt pathway exerts a catabolic effect on skeletal muscle (32, 33). In contrast, the combination of MEK162 and buparlisib spared body weight to a similar extent to MEK162 alone, emphasizing that MEK inhibition is dominant in protecting against cachexia (Fig. 4C and Supplementary Fig. S4C). Consistent with this notion, MEK162 and MEK162 plus buparlisib were equally efficacious in improving GAST muscle fiber cross-sectional area and reducing the expression of muscle atrophy-associated genes in QUAD compared with vehicle-treated mice (Fig. 4D–F and Supplementary Fig. S4D). Taken together, these results revealed that although MEK inhibition alone is effective as an anticachexia therapy, the combination of inhibiting both MEK and PI3K/Akt signaling synergized to provide an added antitumor response.

Next, we sought to determine what might account for the strong antitumor activity of combining MEK162 with buparlisib. Similar to our observations with MEK162 treatment alone, the reduction in tumor mass from MEK and PI3K/Akt inhibition associated with a decrease in circulating IL-6 (Fig. 5A). In addition to being a strong driver of cachexia in the C-26 model, IL-6 contributes to immunologic changes in advanced cancers that promote the differentiation of immunosuppressive cell populations, such as MDSCs, which limit T and NK cell–mediated immune function (34–36). In keeping with this function of IL-6, results showed that the percentage of splenic MDSCs, marked by cell surface markers CD11b and Gr1, declined in response to treatment with either MEK162 or buparlisib and exhibited an even further reduction in mice treated with both compounds (Fig. 5B). We also observed that the decline in MDSCs was inversely proportional to an increase in splenic CD4⁺ and CD8⁺ T lymphocytes in animals treated with MEK162 and buparlisib (Fig. 5C and D).

On the basis of our findings that treatment with the combination of MEK162 was able to reconstitute the systemic adaptive immune system, we were left with two potential mechanisms by which our combination therapy was effectively reducing tumor growth. Either (i) the combinatorial therapy acted by stimulating an antitumor immune response; or (ii) the combinatorial therapy functioned through a direct antitumor cell activity. To test the first of these possibilities, we depleted T-cell lymphocytes using anti-CD4 and anti-CD8 antibodies in C-26 tumor-bearing mice dosed with MEK162 and buparlisib. Results revealed that removing CD4⁺ and CD8⁺ T lymphocytes did not abrogate the antitumor efficacy of MEK and PI3K/Akt inhibition (Fig. 5E and Supplementary Fig. S5A and S5B). These findings were supported by staining tumors with the pan T-cell marker CD3 to identify whether our combination therapy resulted in an increase in T-cell infiltration into tumors. We observed that the increase in splenic CD4⁺ and CD8⁺ T lymphocytes did not reflect a general increase in T cells within tumors from either single-agent or the combination of MEK162 and buparlisib (Supplementary Fig. S5C and S5D). Together, these data supported that the antitumor activity of MEK162 and buparlisib does not result from improved T cell–mediated immunity.

To test our second hypothetical mechanism for the antitumor effectiveness of our combination therapy, we examined the antitumor activity of MEK162 and buparlisib on the tumor cells themselves. In vitro studies confirmed that tumor cells were the most sensitive to the combination MEK162 and buparlisib (Fig. 6A). When tested in vivo, we observed that combining MEK and PI3K/Akt inhibition was efficacious at reducing the percentage of proliferating tumor cells, as assayed by Ki67 immunostaining (Fig. 6B and C). Tumor cell death also increased, as the percentage of cells positive for cleaved caspase-3 staining increased following dual treatment with MEK162 and buparlisib (Fig. 6B and D). Together, these data strongly suggest that the antitumor effect of combining MEK162 with buparlisib occurs through activities intrinsic to the tumor cell.

Cachexia is perhaps the most notable side effect of advanced cancer, and no pharmacologic agents are currently approved to treat this syndrome. A clinical trial in patients with advanced biliary tract cancer suggested that inhibition of MEK signaling might be an effective means to prevent cancer-induced muscle wasting (10, 11). The preclinical data presented here support these findings in patients and both further expand on a role for ERK activity in muscle as a regulator of cancer cachexia and identify PI3K/Akt as a complimentary pathway that can be targeted to concurrently elicit antitumor efficacy.

Successful treatment of cancer cachexia will require both antitumor efficacy and protection against tumor-induced muscle wasting. Indeed, one of the long-term challenges of developing and implementing anticachexia therapy has been the concern that such therapies can have an unintended effect in promoting tumor growth. Many theoretical strategies of promoting muscle growth, such as stimulating protein synthesis via the PI3K/Akt pathway, have not been viewed as viable options for the treatment of cancer cachexia patients, as these same pathways are often drivers of tumor progression. We propose that inhibiting MEK is an attractive treatment option for cancer cachexia, given its low risk of promoting tumor growth and its effectiveness in preserving lean mass and total body weight in both mice and patients. Our data are consistent with two previous studies that have demonstrated an ability MEK inhibition to prevent muscle wasting induced by cancer (25, 37). In both of these studies, MEK inhibition either led to a decrease in tumor volume or an increase in body or muscle weight of non-tumor–bearing mice, making it difficult to separate the ability of MEK inhibition to prevent cancer cachexia from either direct tumor or muscle effects. In sharp contrast, our data utilizing our tumor cell line resistant to MEK162 demonstrate that MEK162 can directly prevent cancer cachexia without affecting tumor growth and without simply promoting muscle anabolism. This is a significant advance over a number of other interventions that have reduced cachexia in animal models but not convincingly demonstrated that their anticachexia activity was not mediated solely by direct antitumor effects (27, 38–40). Furthermore, the fact that the effectiveness of MEK162 appears to be mediated at least in part through a direct effect on skeletal muscle gives this therapy an added advantage over other potential therapies. Many therapies, such as mAbs, target a single potential driver of cachexia, yet at the present time, no single cause of cachexia has been identified in cancer patients. MEK inhibition may be a better choice, as it could prevent cachexia induced by a number of factors as opposed to targeting one specific soluble factor.

Although MEK inhibitors can elicit antitumor activity, objective responses to these agents are not frequently observed in solid tumors when administered as a monotherapy (11, 41–46). Overall, single-agent MEK inhibitors have been limited as a first-line targeted therapy due to compensatory activation of other oncogenic signaling pathways (42, 47–50). This has necessitated an approach whereby MEK inhibitors are being tested in combination with other agents (51–56). Amongst the most common partners for MEK therapy are inhibitors of the PI3K/Akt pathway (50, 57–59).

Combinatorial therapy is particularly pertinent in the context of cachexia, which will be most effectively treated by both targeting the tumor and preserving skeletal muscle. We tested two hypotheses about how MEK162 and buparlisib concurrently target cachexia and tumor progression. Cell depletion experiments indicated that T cells were dispensable for the efficacy of combined therapy with MEK162 and buparlisib, suggesting that the immune modulatory effects of our combination therapy are not responsible for their antitumor activity. Rather, our data suggest that dual MEK and PI3K/Akt inhibition has an intrinsic impact on the tumor cells themselves by inducing cell death and limiting cell proliferation.

In our study, single-agent treatment with the PI3K/Akt inhibitor buparlisib reduced the rate of tumor growth, yet body weight and muscle mass were similar to vehicle-treated tumor-bearing mice. These data are consistent with results that showed that inhibiting PI3K/Akt signaling has a negative impact on skeletal muscle (32, 33). Our data clearly demonstrate that MEK162 in combination with buparlisib targets C-26 tumor growth more effectively than either agent alone, while its ability to preserve body weight and skeletal muscle was dominant. Thus, MEK inhibition might be an ideal candidate when considering combinatorial therapy with agents that might negatively impact skeletal muscle, such as PI3K/Akt inhibition.

Taken together, our results demonstrate that MEK inhibition appears to provide a unique opportunity to protect skeletal muscle in cachexia. Patients most likely to benefit from this aspect of combined therapy involving MEK inhibitors are those with gastrointestinal cancers, which tend to be associated with cachexia and are often resistant to single-agent therapies. In addition, we envision that single-agent MEK inhibition could improve patient performance status in palliative care situations. Collectively, these data inform the mechanism behind novel applications of agents targeting the MEK pathway for clinical practice.

No potential conflicts of interest were disclosed.

Any opinions, findings, and conclusions expressed in this material are those of the authors and do not necessarily reflect those of the Pelotonia Fellowship Program.

Conception and design: E.E. Talbert, J. Yang, A.B. Farris, M. Bloomston, T. Bekaii-Saab, D.C. Guttridge, G.B. Lesinski

Development of methodology: J. Yang, T.A. Mace, M.R. Farren, A.B. Farris, C.D. Timmers, M. Bloomston, D.C. Guttridge, G.B. Lesinski

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): E.E. Talbert, J. Yang, T.A. Mace, M.R. Farren, A.B. Farris, O. Elnaggar, Z. Che, C.D. Timmers, P. Rajasekera, J.M. Maskarinec, G.B. Lesinski

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): E.E. Talbert, M.R. Farren, A.B. Farris, G.S. Young, Z. Che, C.D. Timmers, T. Bekaii-Saab, D.C. Guttridge, G.B. Lesinski

Writing, review, and/or revision of the manuscript: E.E. Talbert, J. Yang, T.A. Mace, M.R. Farren, A.B. Farris, G.S. Young, C.D. Timmers, M. Bloomston, T. Bekaii-Saab, D.C. Guttridge, G.B. Lesinski

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.D. Timmers, G.B. Lesinski

Study supervision: J. Yang, T. Bekaii-Saab, D.C. Guttridge, G.B. Lesinski

We acknowledge Dr. Patrice Lee (Array Biopharma, Inc.) for input on experiments involving MEK162. We acknowledge the OSU CCC Analytical Cytometry and Biostatistics Shared Resources for assistance with these studies. In addition, we thank the OSU Pathology Core Facility and the OSU Solid Tumor Biology Histology Core and in particular Raleigh Kladney for assistance with immunohistochemistry.

Funding for this work was provided by the William Hall Fund for Liver and Pancreatic Cancer Research (to T. Bekaii-Saab and G.B. Lesinski), The Division of Medical Oncology, Array Biopharma, Inc. (to G.B. Lesinski), a Weiss Postdoctoral Fellowship (to E.E. Talbert), an American Cancer Society Postdoctoral Fellowship, PF-15-156-01-CSM (to E.E. Talbert), and NIHT32CA106196 (to E.E. Talbert), T32CA090223 (to M.R. Farren), and R01 CA180057 (to D.C. Guttridge). This work was also supported by The Ohio State University Comprehensive Cancer Center Pelotonia Fellowship Program (to Z. Che and M.R. Farren).

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.