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. 2012 Aug;14(8):690–701. doi: 10.1593/neo.111810

Stromal Cell-Derived Factor 1α Mediates Resistance to mTOR-Directed Therapy in Pancreatic Cancer1,2

Colin D Weekes *, Dongweon Song †, John Arcaroli *, Lora A Wilson *, Belen Rubio-Viqueira †,‡, George Cusatis †, Elizabeth Garrett-Mayer §, Wells A Messersmith *, Robert A Winn *,, Manuel Hidalgo †,‡
PMCID: PMC3432475  PMID: 22952422

Abstract

Purpose

The factors preventing the translation of preclinical findings supporting the clinical development mTOR-targeted therapy in pancreatic cancer therapy remain undetermined. Stromal cell.derived factor 1α (SDF-1α)-CXCR4 signaling was examined as a representative microenvironmental factor able to promote mTOR-targeted therapy resistance in pancreatic cancer.

Experimental Design

Primary pancreas explant xenografts and in vitro experiments were used to perform pharmacodynamic analyses of SDF-1α-CXCR4 regulation of the mTOR pathway. Combinatorial effects of CXCR4, EGFR, and mTOR pharmacologic inhibition were evaluated in temsirolimus-resistant and -sensitive xenografts. Intratumoral gene and protein expressions of mTOR pathway effectors cyclin D1, c-Myc, and VEGF were evaluated.

Results

Baseline intratumoral SDF-1α gene expression correlated with temsirolimus resistance in explant models. SDF-1α stimulation of pancreatic cells resulted in CXCR4-mediated PI3-kinase-dependent S6-RP phosphorylation (pS6-RP) on exposure to temsirolimus. Combinatorial therapy with AMD3465 (CXCR4 small-molecule inhibitor) and temsirolimus resulted in effective tumor growth inhibition to overcome temsirolimus resistance. In contrast, SDF-1α exposure induced a temsirolimus-resistant phenotype in temsirolimus-sensitive explants. AMD3465 inhibited CXCR4-mediated intratumoral S6-RP phosphorylation and cyclin D and c-myc gene expression. Next, CXCR4 promoted intratumoral EGFR expression in association with temsirolimus resistance. Treatment with AMD3465, temsirolimus- and erlotinib-mediated tumor growth inhibition to overcome temsirolimus resistance in the explant model. Lastly, SDF-1α-CXCR4 signaling increased intratumoral VEGF gene and protein expression.

Conclusions

SDF-1α-CXCR4 signaling represents a microenvironmental factor that can maintain mTOR pathway fidelity to promote resistance to mTOR-targeted therapy in pancreatic cancer by a variety of mechanisms such as recruitment of EGFR signaling and angiogenesis.

Introduction

Pancreatic adenocarcinoma remains a devastating disease, with a predicted 5-year survival at the time of diagnosis of only 4% [1]. Oncogenesis occurs through the development of premalignant pancreatic intraepithelial neoplasms (PanIN) that are associated with the sequential acquisition of specific genetic abnormalities [2,3]. The acquisition of K-ras-activating mutations is a key initiator in pancreatic tumorigenesis. K-ras-mediated mTOR (mammalian target of rapamycin) pathway activation is an important component of pancreatic tumor initiation and K-ras-independent maintenance of oncogenesis [4–8]. mTOR integrates signals generated by nutrients and growth factors through the PI3-kinase/Akt pathway and regulates key cellular processes [9]. The mTOR inhibitors, rapamycin and temsirolimus, negatively affect pancreatic cancer cell proliferation [10–12]. Consistent with these in vitro observations, temsirolimus and gemcitabine, in combination, demonstrate significant antitumor effects in preclinical xenograft models [12]. The expression of vascular endothelial cell growth factor (VEGF) has been associated with growth-inhibitory effects of mTOR inhibitors. However, when these data are extrapolated to the clinical setting, mTOR inhibitors unfortunately demonstrate limited clinical activity against pancreatic cancer [13–15]. The surprising lack of clinical benefit in these studies suggests that the presence of alternative survival factors abrogates the effect of inhibiting mTOR alone. Several stromal elements have been implicated in cancer cell survival, including the chemokine stromal cell-derived factor 1α (SDF-1α)/CXCR4 ligand-receptor pair.

The chemokine stromal-derived factor 1α (SDF-1α, CXCL12) and its receptor CXCR4 were initially demonstrated to be critical for hematopoiesis and neurogenesis [16]. CXCR7 has also been demonstrated to function as an SDF-1α receptor [17]. Subsequently, SDF-1α has been shown to enhance the metastatic potential of cancer cells through preferential activation of the Akt and mitogen-activated protein kinase pathways in a diverse array of histologic subtypes [18–23]. Importantly, SDF-1α expression has been associated with a poor prognosis in patients with resected early-stage pancreatic cancer [24]. Furthermore, whereas the role of SDF-1α and its ligand CXCR4 in pancreatic cancer remains to be defined, evaluation of a series of PanIN samples has demonstrated an increased frequency of CXCR4 expression associated with PanIN progression [25]. In addition, CXCR4 expression is associated with poor survival in patients with advanced disease states [26]. These clinical observations underscore the potential importance of SDF-1α and CXCR4 in pancreatic cancer development and progression.

Pancreatic cancer represents a relative hypoxic tumor in which the intratumoral vasculature is compressed by the tumor-associated stromal components. Under hypoxic conditions, mTOR signaling can promote VEGF transcription in a hypoxia-inducible factor 1α (HIF-1α)-dependent manner. Both SDF-1α and CXCR4 are HIF-1α target genes that are transcribed in response to hypoxia. Hypoxic damage to the liver results in the recruitment of endothelial precursor cells to promote intrahepatic angiogenesis in response to SDF-1α secretion. SDF-1α-CXCR4 ligand-receptor binding positively regulates VEGF expression under hypoxic conditions, thus providing direct evidence of the interplay between SDF-1α-CXCR4 signaling and mTOR-dependent VEGF regulation. In this article, we present data supporting the hypothesis that CXCR4 propagates secondary intracellular signals that bypass the molecular blockade caused by mTOR inhibitors to promote therapeutic resistance mTOR-targeted therapy. Furthermore, SDF-1α-CXCR4 regulation of VEGF may serve as a potential down mechanism by which persistent mTOR activation induced by CXCR4 signaling promotes resistance to rapalogs. In summary, these data provide a paradigm by which extracellular components may directly regulate the antiproliferative characteristics of mTOR-targeted agents as a direct function of microenvironmental cues.

Materials and Methods

Drugs

Temsirolimus (Pfizer, New York, NY) was dissolved in a proprietary diluent and administered daily by intraperitoneal injection at a dose of 20 mg/kg. AMD3465 is a CXCR4 small-molecule inhibitor that was provided by Genzyme Corporation (Cambridge, MA). AMD3465 was dissolved in phosphate-buffered saline at a pH of 7.4. AMD3465 was administered daily by intraperitoneal injection at a dose of 10 mg/kg. LY294002 was obtained from Sigma-Aldrich (St Louis, MO). Recombinant human stromal cell-derived factor 1α was obtained from ProSpec-Tany Technogene Ltd (Rehovot, Israel). Five micrograms of recombinant human SDF-1α was administered by tail vein injection thrice weekly over a 28-day period.

Pancreas Patient-Derived Explants and Cell Lines

Six-week-old female athymic nude mice (Harlan, Indiana, IN) were used. The research protocol was approved by the Johns Hopkins University Animal Care and Use Committee, and animals were maintained in accordance to the guidelines of the American Association of Laboratory Animal Care. Surgical nondiagnostic specimens obtained by pancreaticoduodenectomy at the Johns Hopkins Hospital were reimplanted subcutaneously to derive human pancreatic cancer explant xenografts. Xenografted tumors were allowed to grow until reaching ∼200 mm3, at which time mice were randomized into treatment groups consisting of control and various experimental groups [27]. Each group contained 8 to 10 tumors that can be evaluated depending on the experiment. Mice were monitored daily for signs of toxicity and were weighed three times per week. Tumor size was evaluated two times per week by caliper measurements using the following formula: tumor volume = (length x width2) / 2. Relative tumor growth inhibition was calculated by dividing the tumor volumes of treated mice by those of control mice (percent growth of treated group/control group for each individual). The temsirolimus-resistant pancreatic cancer cell line HS766T was used in in vitro cell signaling experiments.

Real-time Polymerase Chain Reaction

RNA was isolated from cells or tumor samples using standard procedures of Qiagen RNeasy Mini Kit. Pancreas Xenograft complementary DNA library synthesis was performed with BioRad iScript Reaction Kit using 5x iScript Reaction mix, reverse transcriptase, RNAse-free water, and RNA. Real-time polymerase chain reaction (PCR) was performed using human c-Fos, CXCR4, CXCR7, cyclin D1, c-Myc, EGFR, SDF-1α, VEGF, and 18S primer probes (Applied Biosystems, Foster City, CA) plus TaqMan (Applied Biosystems) in the iQ5 iCycler real-time PCR detection system (Bio-Rad, Hercules, CA). 18S amplification was used as the housekeeping gene. Reaction involved an initial cycle at 50°C for 2 minutes followed by 95°C for 10 minutes. Lastly, 40 cycles at 95°C for 15 seconds and then 60°C for 1 minute. A gene expression score was calculated by taking 2 raised to the difference in Ct between the housekeeping gene and the gene of interest (2ΔCt).

Western Blot Analysis

Cells were lysed in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, phosphatase inhibitors (EDTA-free protease inhibitors; Boehringer Mannheim, Petersburg, VA), 20 mM β-glycerol phosphate, 20 mM NaF, and 1 mM NaVO3. Equal amounts of protein (60 µg) were separated on 4% to 12% gradient SDS-PAGE and transferred onto a nitrocellulose membrane at 100 V for 1 hour. Membranes were blocked with 5% milk in TBST (20 mM Tris-HCl pH 7.5, 500 mM NaCl, and 0.5% Tween 20) for 1 hour at room temperature and incubated with appropriate dilutions of primary antibodies (1:1000, Akt [p-Ser473], Akt [p-Thr308], Akt, HIF-1α, and β-actin; 1:5000, p-S6-RP [Ser235/236], S6-RP) from Cell Signaling Technology, (Danvers, MA) and CXCR4 and VEGF from Santa Cruz Biotechnology, Inc (Santa Cruz, CA), in 5% nonfat dried milk in TBST overnight at 4°C. The membrane was washed five times for 5 minutes with TBST and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated goat-anti-rabbit or -mouse secondary antibody for 1 hour at room temperature. After this, the membrane was washed again five times for 5 minutes with TBST and developed using an ECL reagent (VWR International, Radnor, PA).

CXCR4 and CXCR7 Knockdown

HS766T cells were cultured to 80% confluence the day before transfection. The standard protocol using Lipofectamine by Invitrogen (Grand Island, NY) was used to transiently transfect the cells with GIPZ lentiviral short hairpin RNA (shRNA) from Open Biosystems (Lafayette, CO). We used shCXCR7, shCXCR4, and shGIPZ scramble. CXCR4 and CXCR7 quantitative real-time PCR was performed to determine gene transcription repression efficiency of shRNA integration.

Immunohistochemical Staining

Explanted tumor specimens were fixed in 10% buffered formalin and embedded in paraffin. The sections were subsequently dewaxed and rehydrated in graded ethanol. Antigen retrieval was performed by soaking sections in 10 mM sodium citrate buffer (pH 6.0) and heating in a high-power microwave oven for 20 minutes. The sections were incubate in primary antibody targeting either anti-human CXCR4 or anti-human VEGF antibody for 30 minutes at room temperature. After washing, the secondary antibody was added and incubated for an additional 30 minutes. Reactive products were detected with 3,3′-diaminobenzidene and counterstained with hematoxylin for 30 seconds.

Statistical Analysis

To assess the treatment effect, a hierarchical linear regression model was used with random intercepts and fixed slopes to evaluate the 2627 individual tumors from 16 individual patient-derived xenografts treated with temsirolimus versus placebo. On the basis of the results of exploratory plots, the assumption of fixed effects for slopes was deemed reasonable. This model allowed for mouse effects to be nested within patient effects so that the hierarchical structure is fully accounted for. Specifically, we fit the following linear regression model:

log(yijt) = a1ijtimeijt + b2jtimeijt x trtij + βtime2ijt + eijt

where j indexes patient, i indexes mouse, and t indexes time, yijt is the percentage change in tumor volume (compared to day 0) in mouse i, which is a xenograft for patient j at time t, and trtij is treatment (1 = treated, 0 = control) received by mouse i which is a xenograft for patient j. We explored a series of models to determine a good fitting model and found that the previously mentioned model was adequate. An intercept and main effect of treatment were not included in the model that makes two assumptions: 1) at time 0, the change from baseline is zero (which is empirically true); and 2) at baseline, the treated and untreated curves intersect. The model was fit using WinBugs software that estimates models using a Markov chain Monte Carlo estimation procedure. A burn-in of 10,000 iterations was performed, and then the chain was run for an additional 20,000 iterations, of which every 10th iteration was saved for inference. Several chains were run with different starting values to ensure convergence. No convergence issues arose. The fitted model was used to make inferences with respect to the treatment effect and to create fitted regression lines (rescaled on the y axis for clearer interpretation). Point estimates of parameters of interest (e.g., expected difference in tumor volume between treated and control at 28 days) and 95% credible intervals and tail probabilities were used for inference based on the mean of posterior distributions and the 2.5th and 97.5th quartiles of the posterior distribution, respectively.

Pearson correlation was used to determine the correlation of gene expression with tumor growth. All molecular studies were performed at least three times with similar results obtained between replicates. Statistical analysis was performed using Student's t test. Significant associations were defined when P < .05 compared with control.

One-way analysis of variance was used to determine treatment effects of therapy relative to control in patient-derived xenograft experiments. Turkey multiple comparison test was used to determine significance of intergroup comparisons.

Results

SDF-1 Gene Expression Is Associated with Temsirolimus Resistance

Previous work by our group demonstrated that a patient-derived pancreas explant xenograft model could be used as a platform for pharmacodynamic-based evaluation of the mTOR inhibitor temsirolimus [27]. This analysis used a hierarchical linear regression analysis to demonstrate that temsirolimus treatment of mice bearing pancreas patient explant xenografts from individual patients resulted in significant tumor growth inhibition, P < .00001 (Figure W1). However, a significant interxenograft variability in temsirolimus tumor growth-inhibitory effects was observed. As such, mechanisms of resistance need to be defined. Sensitivity to temsirolimus did not correlate with intratumoral p70/S6 kinase activity (data not shown).

The chemokine SDF-1α has been demonstrated to activate Akt and ERK upon binding to its receptor CXCR4 [19]. Therefore, we hypothesized that activation of CXCR4 by SDF-1α may provide an alternative stimulatory signal, resulting in mTOR activation to promote resistance to mTOR-targeted therapy. First, we attempted to link baseline CXCR4 and SDF-1α gene expression with temsirolimus growth-inhibitory effects. CXCR4 and SDF-1α mRNA expression was assessed by real-time PCR in baseline tumor specimens before study initiation. Only SDF-1α gene expression, not CXCR4, demonstrated a statistically significant correlation with temsirolimus-induced tumor growth inhibition. Baseline SDF-1α gene expression was inversely related to temsirolimus-induced growth inhibition (Figure 1A). Analysis of SDF-1α gene expression after temsirolimus treatment demonstrated that temsirolimus-resistant patient-derived xenografts retained SDF-1α gene expression in comparison to temsirolimus-sensitive patient-derived xenografts (Figure 1B). These observations may reflect the ability of SDF-1α to bind to either CXCR4 or CXCR7 to propagate signal transduction events.

Figure 1.

Figure 1

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(A) Pearson correlation was used to define the relationship between baseline SDF-1α mRNA expression in the patient-derived pancreas explant xenografts normalized to the lowest expresser to the individual patient-derived pancreas explant xenograft in response to temsirolimus, R = 0.586, P = .01. (B) SDF-1α mRNA expression at baseline and after temsirolimus treatment of both temsirolimus-resistant and -sensitive pancreas explants.

SDF-1α Stimulation of CXCR4 Directly Regulates mTOR Pathway Activation

The subsequent experiments were designed to demonstrate that CXCR4 activation by SDF-1α directly regulates the PI3-kinase/Akt/mTOR pathway. To achieve this goal, the temsirolimus-resistant pancreatic cancer cell line HS766T was stimulated with SDF-1α (100 ng/ml) followed by subsequent PI3-kinase/Akt/mTOR pathway interrogation by Western blot analysis. SDF-1α stimulation of HS766T cells resulted in complete Akt activation, signified by the combined phosphorylation of both threonine 308 (Thr308) and serine 473 (Ser473) Akt residues within 60 minutes (Figure 2A). Similarly, maximal mTOR pathway activation, as measured by S6-ribosomal protein (S6-RP) phosphorylation, in response to SDF-1α stimulation also occurred at 60 minutes. SDF-1α-induced mTOR pathway activation was maintained through 8 hours. Although SDF-1α-induced S6-RP phosphorylation occurs initially within 5 minutes, as a result of Akt Thr308 phosphorylation, maximal SDF-1α-induced mTOR pathway activation required phosphorylation of both Thr308 and Ser473 residues. Persistent S6-RP phosphorylation seemed to be maintained by the Akt Ser473 residue.

Figure 2.

Figure 2

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SDF-1α activation of CXCR4 regulates mTOR pathway activation. The pancreatic cancer cell line HS766T was stimulated with SDF-1α (100 ng/ml) and interrogated for evidence of mTOR pathway activation by Western blot analysis of total CXCR4, total Akt, Akt phosphothreonine 308 (p-AKT [THR308]), Akt phosphoserine 473 (p-AKT [SER473]), total S6 ribosomal protein (t-S6-RP), phosphorylated S6 ribosomal protein (p-S6-RP) as indicated in the individual panels. β-Actin serves as the protein loading control. (A) HS766T cells were serum starved for 18 hours and then stimulated with SDF-1α for 24 hours to demonstrate kinetics of mTOR activation stimulated by SDF-1α exposure. Images are representative experiments performed three times. (B) Cells were exposed to SDF-1α for 60 minutes in the presence of nontargeting shRNA (NT-RNAi) and CXCR4-targeted shRNA (CXCR4-RNAi). (C) Cells were exposed to SDF-1α for 60 minutes in the presence of the PI3-kinase inhibitor LY294002 at the designated concentrations. (D) Cells were exposed to SDF-1α for 60 minutes and temsirolimus. Lane 3 demonstrates exposure to temsirolimus for 30 minutes before SDF-1α stimulation. In lanes 4 to 7, HS766T cells were exposed to SDF-1α for a period of 60 minutes. Temsirolimus (20 nM) was added for incubation at specified times within the 60-minute incubation period.

SDF-1α serves as the ligand for two G protein-linked receptors, CXCR4 and CXCR7, both of which may have a role in pancreatic cancer tumorigenesis [28]. Cell surface expression analysis of CXCR4 and CXCR7 by FACS demonstrated that 0.29% of HS766T cells express CXCR4, representing a 10-fold increase over CXCR7 (0.02%). Virtually no cells possessed dual cell surface expression of CXCR4 and CXCR7 (Figure W2A). RNA interference was used to demonstrate that CXCR4 is the primary receptor responsible for the propagation of SDF-1α-mediated regulation of the mTOR pathway in pancreatic cancer cells. This was accomplished by incubating SDF-1α-stimulated HS766T cells with either nontarget small interfering RNA (NT-RNAi) or CXCR4 targeting shRNA (CXCR4-RNAi) for 60 minutes. Knockdown of CXCR4 by CXCR4-RNAi specifically inhibited SDF-1α-induced Akt phosphorylation at both Thr308 and Ser473 residues, as well as S6-RP phosphorylation when compared to nontargeting siRNA (Figure 2B). In contrast, CXCR7-targeted RNA interference assays failed to demonstrate alterations in SDF-1α-mediated mTOR pathway activation (Figure W2, B and C). These data define CXCR4 as the primary receptor used by SDF-1α to regulate mTOR pathway activation.

The role of PI3-kinase in SDF-1α-CXCR4-mediated mTOR pathway activation was demonstrated by exposing HS766T cells to SDF-1α in the presence of the PI3-kinase inhibitor LY294002. HS766T cells were stimulated with SDF-1α for 60 minutes with increasing concentrations of LY294002 ranging from 0.5 to 10 µM. LY294002 exposure inhibited SDF-1α-induced Akt and S6-RP phosphorylation in a concentration-dependent manner (Figure 2C). Micromolar LY294002 concentrations inhibited Akt phosphorylation at both Thr308 and Ser473, which was associated with near-total inhibition of S6-RP phosphorylation. In contrast, LY294002 concentrations of 1 µM or less inhibited Akt phosphorylation only at the Thr308 residue and were unable to prevent SDF-1α-induced S6-RP phosphorylation. These finding substantiate the importance of dual phosphorylation of Akt at residues Thr308 and Ser473 to result in maximal mTOR pathway activation by SDF-1α/CXCR4 (Figure 2A).

The ability of SDF-1α-CXCR4 to serve as a compensatory signal transduction pathway to promote temsirolimus resistance by maintaining fidelity of the mTOR pathway activation was investigated. First, SDF-1α (100 ng/ml) induced Akt and S6-RP phosphorylation in HS766T cells after stimulation for 60 minutes as predicted (lanes 1 and 2 of Figure 2D). Next, HS766T cells were treated with temsirolimus either before or after SDF-1α stimulation to define the ability of SDF-1α to maintain mTOR pathway activation relative to the timing of temsirolimus exposure in an attempt to mimic physiologic conditions associated with in vivo temsirolimus treatment. HS766T cells were pretreated with temsirolimus (20 nM) for 30 minutes before SDF-1α stimulation (lane 3 of Figure 2D). Temsirolimus pretreatment inhibited SDF-1α-induced S6-RP phosphorylation but promoted Akt Ser473 phosphorylation without altering Thr308 phosphorylation. Next, the ability of antecedent SDF-1α-CXCR4 to maintain mTOR pathway fidelity in association with temsirolimus exposure was analyzed. To accomplish this, HS766T cells were stimulated with SDF-1α (100 ng/ml) for 60 minutes and temsirolimus was added either simultaneously or at 15-minute intervals (after 0, 15, 30, and 45 minutes) throughout the SDF-1α incubation period (lanes 4 to 7 of Figure 2D). Simultaneous exposure to SDF-1α and temsirolimus inhibited S6-RP phosphorylation (0 minute; lane 4 of Figure 2D). In contrast, SDF-1α stimulation before temsirolimus exposure promoted S6-RP phosphorylation in a time-dependent manner (after 15, 30, and 45 minutes). The ability of SDF-1α to maintain S6-RP phosphorylation was associated with the incremental inhibition of Ser473 phosphorylation. Together, these data demonstrate that SDF-1α-induced CXCR4 activation provides a secondary signal to propagate PI3-kinase/Akt-dependent mTOR pathway activation that can overcome temsirolimus-induced mTOR pathway inhibition. Furthermore, these data provide evidence that a microenvironmental factor present within the tumor milieu can directly promote resistance to mTOR-targeted therapy despite adequate tumor cell exposure.

Pharmacodynamic Analysis of CXCR4-Mediated Temsirolimus Resistance

A pharmacodynamic approach was used to directly test the ability of the SDF-1α-CXCR4 ligand-receptor pair to mediate temsirolimus resistance. First, we reasoned that CXCR4 inhibition should overcome resistance to temsirolimus as defined by previous observations that tumor growth inhibition of 40% or greater in xenografts predicts clinical resistance. Combined treatment with temsirolimus and the CXCR4 inhibitor, AMD3465 of the temsirolimus-resistant human pancreatic cancer explant xenograft Panc159 resulted in synergistic growth inhibition in comparison to treatment with either drug alone (Figure 3A). CXCR4 inhibition by AMD3465 was not associated with significant intratumoral pS6-RP reduction compared to temsirolimus-treated tumors (data not shown). Decreased expressions of cyclin D1 and c-myc serve as measurements of 4E-BP1 function and have previously been demonstrated to promote the growth-inhibitory effects of rapalogs [29]-Dual CXCR4 and mTOR inhibition modulated 4E-BP1 function by synergistically inhibiting temsirolimus-induced intratumoral cyclin D1 and c-myc gene expression (Figure 3, B and C). Next, we confirmed that both the ligand and the receptor are necessary in vivo for SDF-1α.CXCR4 ligand-receptor pair-mediated mTOR-targeted therapy resistance. The CXCR4+/SDF-1α- temsirolimus-sensitive human pancreatic cancer explant xenograft Panc410 (Figure W3B) was exposed to exogenous human SDF-1α by tail vein injection to demonstrate that SDF-1α-mediated CXCR4 activation induces a temsirolimus-resistant phenotype. The injection of SDF-1α resulted in moderate increased tumor growth relative to treatment with temsirolimus alone (Figure 4A). Concomitant treatment with SDF-1α and AMD3465 nullified SDF-1α-augmented tumor growth in the presence of temsirolimus. Furthermore, SDF-1α exposure specifically induced both cyclin D1 and c-myc gene expression that was inhibited by AMD3465 treatment (Figure 4B). In combination, the Panc159 and Panc410 xenograft experiments provide evidence that the SDF-1α/CXCR4 ligand-receptor pair specifically mediate resistance to temsirolimus therapy in vivo.

Figure 3.

Figure 3

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Temsirolimus-resistant xenograft Panc159 treated with temsirolimus and AMD3465. (A) Growth curve of Panc159 xenograft treated with temsirolimus and AMD3465: control (◆), AMD3465 (Inline graphic), temsirolimus (Inline graphic), or the combination of temsirolimus and AMD3465 (Inline graphic) for 28 days. Growth curves represent percent growth of treated tumors throughout the study treatment. The experiments were conducted with 10 tumors per treatment group, and error bars represent ±SEM. (B) Intratumoral cyclin D1 mRNA expression. (C) Intratumoral c-myc mRNA expression. Presented data for B and C are from three individual tumors per group replicated three times, and error bars represent ±SEM. *P < .05, **P < .01.

Figure 4.

Figure 4

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SDF-1α exposure specifically induces resistance to temsirolimus. (A) Temsirolimus-sensitive patient-derived pancreas xenograft Panc410 (CXCR4+/SDF-1α-) was treated with either temsirolimus (◆), temsirolimus and SDF-1α (Inline graphic), or temsirolimus plus SDF-1α and AMD3465 (Inline graphic) for 28 days. Growth curves represent percent tumor growth of treated tumors relative to control (temsirolimus): temsirolimus plus SDF-1α, temsirolimus plus SDF-1α, and AMD3465. The experiments were conducted with eight tumors per treatment group, and error bars represent ±SEM. (B) Intratumoral gene expression for human cyclin D1 and c-myc mRNA expression. Presented data are from three individual tumors per group replicated three times, and error bars represent ±SEM. *P < .05, **P < .01.

EGFR Partners with CXCR4 to Induce mTOR-Targeted Therapy Resistance

Epidermal growth factor receptor (EGFR) is a primary receptor involved in microenvironmental regulation of pancreatic cancer cell function. As such, targeting EGFR with erlotinib is a clinically used strategy for the treatment of patients with pancreatic cancer [30]. c-Fos expression has previously been demonstrated to correlate with erlotinib growth-inhibitory effects in in vivo preclinical pancreatic cancer models [31]. Treatment of the temsirolimus-resistant Panc159 xenograft with AMD3465 resulted in the attenuation of both c-fos and EGFR gene expression (Figure 5, A and C). Induction of a temsirolimus-resistant phenotype by SDF-1α in the temsirolimus-sensitive explant Panc410 resulted in a two-fold induction of intratumoral c-fos gene expression (Figure 5B). Combined, these data provide evidence that SDF-1α-CXCR4-mediated signal transduction may directly regulate EGFR expression to promote mTOR-targeted therapy resistance. Based on these observations, we hypothesized that EGFR-targeted therapy would augment the ability of AMD3465 to overcome temsirolimus resistance. As hypothesized, treatment of the ultimate temsirolimus-resistant human pancreas explant Panc194 with the combination of AMD3465 and erlotinib resulted in significant tumor regression when combined with temsirolimus, thereby overcoming temsirolimus resistance (Figures W3C and 5D). The effect of combining AMD3465 and erlotinib with temsirolimus on tumor growth inhibition seems additive to the effects of combining AMD3465 or erlotinib with temsirolimus. The impact of the combination of AMD3465, erlotinib, and temsirolimus on tumor growth was also demonstrated by total growth inhibition in some tumors within the cohort, resulting in near-total growth inhibition in comparison to control by day 21, which was not obtained in the other treatment groups (Figure W4). Combined, these data demonstrate that secondary signals propagated by the interaction of physiologic stromal components with cancer cell surface receptors possess the ability to directly initiate temsirolimus resistance in part by maintaining the fidelity of the PI3-kinase/Akt/mTOR pathway. The hypoxic nature of pancreatic cancer makes these observations particularly important because hypoxia promotes the transcription of SDF-1α, CXCR4, and EGFR.

Figure 5.

Figure 5

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CXCR4 inhibition implicates EGFR as a potential partner with CXCR4 for the induction of temsirolimus resistance. (A) Intratumoral c-fos mRNA expression obtained fromthe Panc159 xenograft treated with AMD3465 and temsirolimus. (B) Panc410 intratumoral c-fos mRNA expression treated with temsirolimus and exposed to exogenous human SDF-1α and AMD3465. (C) Intratumoral EGFR mRNA expression obtained from the Panc159 xenograft treated with AMD3465 and temsirolimus. Presented data for A, B, and C are from three individual tumors per group replicated three times, and error bars represent ±SEM. *P < .05, **P < .01. (D) Growth curve of temsirolimus-resistant Panc194 xenograft treated with temsirolimus, AMD3465, and erlotinib: control (⛆), AMD3465 (Inline graphic), temsirolimus (Inline graphic), temsirolimus and AMD3465 (Inline graphic), temsirolimus and erlotinib (□), erlotinib and AMD3465 (○), or temsirolimus, AMD3465 and erlotinib (◊) for a period of 28 days. Growth curves represent percent growth throughout the study drug exposure period. The experiments were conducted with 10 tumors per treatment group, and error bars represent ±SEM. *P < .01.

CXCR4 Regulates VEGF Expression

Pancreatic cancer cells use hypoxia-inducible proteins to adapt to the hypoxic microenvironment in which they grow. CXCR4, SDF-1α, and VEGF gene expression are all regulated by hypoxia. In this context, SDF-1α-CXCR4 signaling directly regulates VEGF transcription [32]. Initial evaluation of baseline VEGF gene expression did not demonstrate a relationship between baseline VEGF gene expression and temsirolimus growth inhibition (data not shown). However, baseline SDF-1α- gene expression was directly associated with that of VEGF. Tumors from temsirolimus-resistant xenografts demonstrated in an increased frequency of VEGF expressing tumors in six (86%) of seven in contrast to four (44%) of nine temsirolimus-sensitive xenografts (Figure 6A). In addition, VEGF-containing temsirolimus-resistant xenografts possessed increased levels of VEGF in comparison to their counterpart temsirolimus-sensitive xenografts. Temsirolimus-resistant tumors also tended to maintain VEGF expression after temsirolimus exposure (Figure W5A). The importance of this observation is that VEGF protein expression has been associated with mTORC1 inhibitor resistance and may serve as a marker of persistent PI3-kinase/Akt/mTOR pathway activation. Consistent with the hypothesis of interplay between SDF-1α-CXCR4 signaling and VEGF, both VEGF and CXCR4 were expressed in pancreas tumor cells at the leading edge of the explanted tumor (Figure W5, C and D). The direct relationship between SDF-1α-CXCR4 signaling and VEGF expression was evaluated in subsequent experiments.

Figure 6.

Figure 6

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Intratumoral VEGF expression is inhibited by pharmacologic inhibition of CXCR4. (A) Western blot of basal protein expression in explant probed for CXCR4 and VEGF. (B) Intratumoral VEGF mRNA expression obtained from the Panc159 xenograft treated with AMD3465 and temsirolimus. (C) Panc410 intratumoral VEGF mRNA expression in xenografts treated with temsirolimus and exposed to exogenous human SDF-1α and AMD3465. Presented data for B and C are from three individual tumors per group replicated three times, and error bars represent ±SEM. **P < .01. (D) VEGF protein expression from Panc159-xenografted tumors treated with temsirolimus and AMD3465. (E) Panc410 VEGF protein expression in xenografts treated with temsirolimus and exposed to exogenous human SDF-1α and AMD3465. Protein lysates were immunoblotted with antibodies recognizing VEGF protein. β-Actin serves as the protein loading control. Presented data are from a mean of three individual replicate experiments, and error bars represent ±SEM. ***P < .001.

The regulation of angiogenesis by mTOR through its effects on VEGF production is an important adaptive process used by cancer cells to survive hypoxic conditions. SDF-1α-CXCR4 ligand-receptor pair signaling promotes angiogenesis by a number of mechanisms including the direct promotion of VEGF transcription [32]. The relationship between SDF-1α-CXCR4 signaling, and VEGF expression was evaluated using the pharmacodynamic approach. As such, intratumoral VEGF mRNA and protein expression in tumors obtained from the aforementioned pharmacodynamic experiments were evaluated. AMD3465 treatment of the temsirolimus-resistant xenograft Panc159 inhibited intratumoral VEGF gene and protein expression (Figure 6, B and D). In contrast, SDF-1α exposure of the temsirolimus-sensitive xenograft Panc410 increased intratumoral VEGF gene and protein expression, which was specifically inhibited by AMD3465 (Figure 6, C and E). The pancreatic cancer cell line HS766T was used to demonstrate the direct ability of SDF-1α to regulate VEGF transcription in pancreatic cancer cells. SDF-1α (100 ng/ml) stimulation of HS766T cells resulted in an eight-fold increase in VEGF expression occurring at 4 hours, thus demonstrating the direct effects of SDF-1α-CXCR4 on VEGF transcription (Figure W5B). These data provide evidence that CXCR4 regulates VEGF intratumoral production by pancreatic cancer cells in an mTOR-dependent manner.

Discussion

Pancreatic cancer remains a devastating disease with limited therapeutic options. Gemcitabine combined with either capecitabine or erlotinib remains the standard of care and provides marginal impact on patient survival [30,33–35]. The early acquisition of activating mutations of K-ras in pancreatic adenocarcinoma oncogenesis places mTOR as a central node in the initiation and maintenance of pancreatic adenocarcinoma oncogenesis [3]. A fundamental set of 12 signaling pathways has been demonstrated to undergo genetic alteration during pancreatic cancer oncogenesis [36]. Interestingly, many of these pathways interact with mTOR. These observations, combined with preclinical testing of mTOR inhibitors, support the development of this class of compound for pancreatic cancer therapy. However, recent clinical trials have failed to demonstrate the degree of clinical efficacy as would be predicted by the available preclinical models [13–15]. The inability to translate preclinical observations of tumor growth inhibition by mTOR inhibitors into clinical benefit for patients with pancreatic cancer may be explained by the utilization of inappropriate thresholds to define sensitivity in preclinical models [15]. Alternatively, undetermined secondary signals provided by the tumor microenvironment or intratumoral genetic alterations may be able to directly overcome mTOR inhibition. In this study, we demonstrate that activation of the G-protein-linked receptor CXCR4 by the chemokine SDF-1α (CXCL-12) mediates resistance to mTOR-targeted therapy in pancreatic cancer.

The results of this study demonstrate two disparate but related mechanisms by which CXCR4 activation can mediate resistance to mTOR-targeted therapy. The role of SDF-1α-CXCR4 signaling in pancreatic cancer biology is becoming increasingly apparent, particularly because it promotes the metastasis of pancreatic cancer stem cells. As such, SDF-1α binding to its receptor CXCR4 may represent a mechanism by which the tumor cell microenvironment may regulate intracellular events to promote resistance to signal transduction pathway inhibitors. As such, CXCR4 possesses the ability to directly regulate the activation state of the mTOR pathway in a PI3-kinase-dependent manner. Specifically, stimulation of SDF-1α-CXCR4 signaling before temsirolimus exposure maintained the fidelity of mTOR signal transduction despite pharmacologic inhibition by temsirolimus. In contrast, SDF-1α-CXCR4 stimulation failed to mediate these effects in the context of prior temsirolimus exposure. This is a significant finding in that the hypoxic nature of pancreatic cancer would likely result in a basal level of intratumoral SDF-1α production, thereby initiating SDF-1α-CXCR4 signaling to promote mTOR pathway activation. As demonstrated by the aforementioned experiments, basal intratumoral SDF-1α production would be predicted to result in inherent resistance to mTOR-targeted therapy in patients. If true, this observation may provide insight into the lack of clinical efficacy of mTOR inhibitor in the treatment of patients with pancreas adenocarcinoma.

The CXCR4 receptor likely represents a minority of receptors expressed on pancreatic cancer cells. One might hypothesize that receptors such as EGFR, which possess receptor tyrosine kinase function, may be a more significant regulator of the mTOR pathway by extracellular signals. In this case, SDF-1α-CXCR4 may serve to amplify EGFR-mediated signal transduction. Interactions between SDF-1α and EGFR have previously been demonstrated in that SDF-1α promotes EGFR phosphorylation and gene expression in ovarian cancer cells [37]. In addition, EGFR expression combined with hypoxia-induced CXCR4 expression in primary non.small cell lung cancer cells has been demonstrated to promote metastasis [38]. Here we demonstrate that SDF-1α promotes both c-Fos and EGFR transcription in pancreatic cancer. c-Fos expression has previously been demonstrated to correlate with the antiproliferative effects of EGFR pathway inhibition in pancreatic cancer [31]. The functional relevance of SDF-1α-mediated EGFR regulation in pancreas was demonstrated by the ability of dual EGFR and CXCR4 inhibition combined with temsirolimus to overcome temsirolimus resistance in the human explant xenograft model. This observation is akin to the interplay of insulin-like growth factor receptor 1 and c-Met signal transduction and resistance to EGFR-targeted therapy in non-small cell lung cancer [39,40]. These data support the hypothesis that strategies incorporating mTOR-based therapy in pancreatic cancer may be effective but will require the use of vertical signal transduction inhibitory strategies. Such strategies should use proximal receptor kinase inhibition in conjunction with the nodal mTOR inhibition. This strategy may have the capacity to overcome the lack of oncogenic addition displayed in pancreatic cancer resulting from the multiple genetic pathways involved in pancreatic oncogenesis [36]. In addition, these results may be of significant clinical relevance because it provides evidence for a combinatorial molecular strategy to augment EGFR-targeted therapy in pancreatic cancer [35]. Taken together, these data identify SDF-1α-CXCR4-mediated signal transduction as a tumor microenvironment-regulated process that possesses the capacity to directly mediate mTOR-targeted therapy resistance.

Modulation of VEGF expression represents the second mechanism by which SDF-1α-CXCR4 may promote mTOR-targeted therapy resistance. The linkage of VEGF production with CXCR4-regulated resistance to mTOR-targeted therapy implies that CXCR4 may be used by the pancreatic cancer cells to promote angiogenesis. In this context, CXCR4 regulation of Yin Yang 1 promotes VEGF transcription to promote angiogenesis during malignancy [32]. The finding that SDF-1α stimulation promotes VEGF transcription supports the ability of pancreatic cancer cells to directly regulate angiogenesis. The importance of angiogenesis in pancreatic cancer has recently been reevaluated in the context of sonic hedgehog pathway inhibition leading to altered angiogenesis in association with pronounced growth-inhibitory effects when combined with gemcitabine [41]. In this context, sonic hedgehog pathway inhibition results in the modulation of the pancreatic cancer stromal architecture allowing for increased vascular patency. Vascular patency is compromised as a function of the increased interstitial pressure exerted by the tumor-stromal components. The increased vascular patency in turn allows for increased transit of chemotherapeutic agents of a variety of sizes resulting in an increase in intratumoral pharmacokinetic parameters of the chemotherapeutic agent. This observation may explain some perplexing clinical findings such as the lack of clinical efficacy associated with EGFR-targeted antibodies, in contrast to that observed with the small-molecule inhibitor erlotinib [35,42]. Similarly, this observation may be key to the lack of bevacizumab efficacy in clinical evaluation when compared to preclinical analysis [43]. Furthermore, targeting angiogenesis by either antibodies or small-molecule inhibitors may have no therapeutic effect without altering the stroma architecture. Interestingly, preclinical models of sonic hedgehog and mTOR pathway inhibition demonstrate additive growth-inhibitory effects [44].

In terms of the angiogenesis and the tumor-stromal compartment, SDF-1α-CXCR4-mediated signal transduction may have multiple roles. Both bone marrow-derived endothelial cell precursors and cancer-associated fibroblasts have been demonstrated to migrate to cancer foci in a SDF-1α-CXCR4-dependent manner. Both of these events may occur in response to hypoxia. The regulation of endothelial cell precursors along with cancer-associated fibroblast may be not only important for conditioning of the nascent pancreatic cancer microenvironment but also critical for conditioning of the metastatic niche [45,46]. Conditioning of the metastatic microenvironmental niche to promote angiogenesis may facilitate the survival of the metastatic cell by allowing it to overcome the relative hypoxia associated with a new microenvironment [45]. The cancer-associated fibroblast cell population may ultimately give rise to liver stellate cells. Liver stellate cells have been demonstrated to function as nurse cells to support metastatic cancer cells within the liver in an SDF-1/CXCR4-dependent manner [47]. In addition to its role in the regulation of homing of potential stromal components, CXCR4 has been demonstrated to mediate the metastasis of pancreatic cancer stem cells to the liver [48]. Thereby, CXCR4 may have multiple functions in pancreatic cancer biology including direct effects on various subtypes of pancreatic cancer cells in addition to its effects on various stromal components, all of which may be regulated in part by VEGF in response to tumor-associated hypoxia.

In conclusion, we demonstrate that basal SDF-1α gene expression does not simply serve as a biomarker mTOR-targeted therapy resistance but functionally regulates the mTOR pathway to promote mTOR-targeted therapy resistance. Furthermore, SDF-1α-CXCR4 recruits EGFR to presumably further amplify mTOR pathway activation to promote mTOR-targeted therapy resistance. The combination of mTOR-targeted therapy with that of EGFR and CXCR4 may represent a novel therapeutic strategy to augment EGFR-targeted therapy in pancreatic cancer. Functional analysis of mTOR pathway activation demonstrated that VEGF signaling is regulated by SDF-1α-CXCR4, thereby providing implications for a role of angiogenesis in this process.

Supplementary Material

Supplementary Figures and Tables
neo1408_0690SD1.pdf (1.7MB, pdf)

Footnotes

1

C.D.W. is a recipient of a UNCF-Merck Postdoctoral Fellowship Award and Amgen Oncology Institute Hematology/Oncology Fellowship Award additional support from the Goldman Family, the Viragh Foundation for Cancer Research, National Institutes of Health grant CA113669, and Specialized Program of Research Excellence in Gastrointestinal Cancer grant CA62924.

2

This article refers to supplementary materials, which are designated by Figures W1 to W5 and are available online at www.neoplasia.com.

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Supplementary Materials

Supplementary Figures and Tables
neo1408_0690SD1.pdf (1.7MB, pdf)

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