The CXCR2 antagonist, SCH-527123, demonstrates antitumor ... · oxaliplatin-based therapy in CRC cell line models and genetic variants in IL-8 and CXCR2 both predict tumor recurrence

The CXCR2 antagonist, SCH-527123, demonstrates antitumor activity and sensitizes cells to oxaliplatin in

preclinical colon cancer models

Yan Ning1*, Melissa J. Labonte1*, Wu Zhang1, Pierre O. Bohanes1, Armin Gerger1, Dongyun Yang2, Leonor Benhaim1, David Paez1, David O. Rosenberg3, Kalyan C. Nagulapalli Venkata3, Stan G. Louie4, Nicos A. Petasis3,5, Robert D. Ladner6,, Heinz-Josef Lenz1,5,7

1Division of Medical Oncology, Sharon A. Carpenter Laboratory; 2Department of Preventive Medicine; 3Department of Chemistry; 4School of Pharmacy; 5Norris Comprehensive Cancer Center; 6Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089.

7To whom correspondence should be addressed: Heinz-Josef Lenz, M.D., Norris Comprehensive Cancer Center, Keck School of Medicine, 1441 Eastlake Ave, Suite3456, University of Southern California, Los Angeles, CA 90089; Tel:(323)865-3955; Fax (323)865-0061; E-mail: [email protected].

This study was funded by the NIH grant 5 P30CA14089-27S1, the. Kroha/Casner Family Foundation, Dhont Family and Wunderglo Foundation. *Both authors contributed equally to this work. Running title: SCH-527123 combined with oxaliplatin increases antitumor activity Key words: SCH-527123; oxaliplatin; colon cancer; CXCR2; Interleukin-8

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 5, 2012; DOI: 10.1158/1535-7163.MCT-11-0915

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Abstract Colorectal cancer (CRC) is the second most common cause of cancer-related death in the

United States. Recent studies showed that interleukin-8 (IL-8) and its receptors (CXCR1

and CXCR2) are significantly upregulated in both the tumor and its microenvironment,

and act as key regulators of proliferation, angiogenesis and metastasis. Our previous

study demonstrated that IL-8 overexpression in CRC cells triggers the upregulation of the

CXCR2-mediated proliferative pathway. The aim of this study was to investigate if the

CXCR2 antagonist, SCH-527123, inhibits CRC proliferation and if it can sensitize CRC

cells to oxaliplatin both in vitro and in vivo. SCH-527123 demonstrated concentration

dependent anti-proliferative effects in HCT116, Caco2 and their respective IL-8

overexpressing variants CRC cell lines. Moreover, SCH-527123 was able to suppress

CXCR2 mediated signal transduction as demonstrated through decreased phosphorylation

of the NF-κB/MAPK/AKT pathway. These findings corresponded with decreased cell

migration and invasion, while increased apoptosis in CRC cell lines. In vivo results

verifed that SCH-527123 treatment decreased tumor growth and microvessel density

when compared with vehicle treated tumors. Importantly, these preclinical studies

demonstrated that the combination of SCH-527123 and oxaliplatin resulted in a greater

decrease in cell proliferation, tumor growth, apoptosis and angiogenesis that was superior

to single agent treatment. Taken together, these findings suggest that targeting CXCR2

may block tumor proliferation, migration, invasion and angiogenesis. In addition,

CXCR2 blockade may further sensitize CRC to oxaliplatin treatment.

Introduction

Colorectal cancer (CRC) is the leading cause of death from gastrointestinal

malignancies, resulting in approximately 51,690 deaths in 2012 (1). Since 2005, no new




chemotherapeutic agents have been approved by the Food and Drug Administration for

treatment of patients with metastatic CRC, resulting in a significant need to develop more

effective targeted drugs aiming at both the tumor and its microenvironment. Recent

studies have identified that certain chemokines and their receptors act as key regulators of

CRC progression and may be important targets for novel drug development strategies (2).

Interleukin-8 (IL-8), a member of the neutrophil-specific C-X-C subfamily of

chemokines, acts on endothelial cells via binding onto either CXCR1 or CXCR2 to

promote invasion, proliferation and angiogenesis (2-5). IL-8 expression is upregulated by

hypoxia, cytokines and other environmental stresses, which are mediated by transcription

factors, including NF-κB and AP-1 (6, 7). The upregulation of CXCR2 has also been

correlated with promotion of tumorigenesis and angiogenesis in lung, melanoma and

ovarian cancers (8-10). Previous studies by our group and others have shown that IL-8

and its receptor CXCR2 are significantly upregulated in the tumor and its

microenvironment in CRC (11-13). These studies demonstrated that expression levels of

IL-8 and CXCR2 were associated with tumor proliferation, progression and sensitivity of

oxaliplatin-based therapy in CRC cell line models and genetic variants in IL-8 and

CXCR2 both predict tumor recurrence and oxaliplatin efficacy in patients (11, 14, 15). It

is well known that oxaliplatin-based chemotherapy is a standard-of-care agent used most

commonly in combination with 5-fluorouracil (5-FU) in patients with CRC (16).

Overexpression of IL-8 level in CRC cells decreased sensitivity to the cytotoxic effects of

oxaliplatin and contributed to oxaliplatin chemoresistance (11). Therefore, targeting IL-8

or CXCR2, in addition to having a direct antiangiogenic and antitumor effect, may also

increase chemosensitivity to oxaliplatin.




Recently, our group focused on inhibitors of the IL-8/CXCR2 pathway, which will

have the potential not only to have antitumor activity, but to increase the efficacy of

already available cytotoxic and targeted drugs, such as oxaliplatin, for patients with CRC

by targeting the tumor and its microenvironment. SCH-527123, as a novel and selective

antagonist of the CXCR2, has demonstrated efficacy in the treatment of inflammatory

diseases (17). Moreover, Singh et al showed that SCH-527123 treatment inhibited human

melanoma cancer growth and CRC liver metastases by decreasing tumor cell

proliferation, angiogenesis and enhancing the apoptosis of malignant cells (18, 19).

In this study, we demonstrate that treatment with SCH-527123 alone and in

combination with oxaliplatin is effective in synergistically inhibiting proliferation,

angiogenesis, and enhancing chemosensitivity in CRC cells and xenografts. Taken

together, these findings suggest that CXCR2 antagonists, such as SCH-527123, may be

important therapeutic candidates in treating CRC through attenuating the IL-8/CXCR2

signaling cascade, which influences disease progression and modulates the response to

oxaliplatin sensitivity.

Materials and Methods Compounds and Reagents

SCH-527123 was obtained from Schering Plough (Whitehouse Station, NJ) and

synthesized in Petasis group. Compound purity was verified by NMR and LC/MS.

Oxaliplatin was purchased from Sigma-Aldrich (St. Louis, MO). CellTiter96 Aqueous

One Solution was purchased from Promega (Madison, WI). Recombinant human IL-8

(rhIL-8) was purchased from R&D systems (Minneapolis, MN). Protease inhibitor

cocktail was purchased from Roche Molecular Biochemical (Indianapolis, IN).




Cell Lines

The human HCT116 and Caco2 CRC cells were purchased from American Type Culture

Connection (ATCC, Lockville, MD) in August 2008 (no authentication was done by the

authors). HCT116 and Caco2 IL-8 overexpressing isogenic cell lines (E2 and IIIe) were

generated as previously described (11). HCT116 and Caco2 cell lines were maintained in

McCoy’s5A and MEM media, respectively, and supplemented with 10% fetal bovine

serum (Lonza, East Rutherford, NJ), 5% penicillin/streptomycin, sodium pyruvate and L-

Glutamine (Mediatech, Manassas, VA). IL-8 overexpression cells were maintained as

described above with the addition of 5 μg/ml blasticidin (Invitrogen, Carlsbad, CA). Cells

were maintained in an incubator at 37oC with 5% CO2. The cell lines were routinely

tested to confirm that they were Mycoplasma free by using Mycoalert mycoplasma

detection Kit (Lonza).

ELISA

The quantification of IL-8 protein was determined using the Quantikine IL-8 ELISA kit

(R&D systems) according to manufactures instructions. More information is described in

Supplement methods and materials.

TUNEL Assay

Cells were plated in adherent conditions in 6-well plates at 50,000 cells per well. After

treatment, cells were fixed and stained for the presence of apoptotic cells by using the in

Situ apoptosis detection kit (Trevigen, Gaithersburg, MD). Nuclei were counterstained

with DAPI in red.

Growth Inhibition Assay and Drug Combination Analysis




Growth inhibition was measured as previously described (11, 20, 21). The combination

effect was determined using the combination index (CI) analysis methods of Chou and

Talalay (22) by utilizing Calcusyn software (Biosoft, Ferguson, MO) which quantifies the

degree of synergy between two agents that both induce a linear pharmacologic response.

Fraction Affected (FA) was calculated from the percent growth inhibition: FA=(100-%

growth inhibition)/100. CI values: <1=synergism; 1–1.2=additive and >1.2=antagonism.

Results are representative of three independent experiments conducted in duplicate.

Clonogenicity Assay

The clonogenicity assay were performed as previously described (20). Cells were

subsequently treated with SCH-527123 and oxaliplatin either alone or in combination for

a period of 72h. Cells were washed and incubated in drug-free media for 3 weeks to allow

colony formation. All experiments were performed in triplicate. Drug treated samples

were compared directly to untreated controls set at 100%.

siRNA Analysis

siRNA analysis was performed as previously described (11). siRNAs against CXCR2

were purchased from Ambion (Austin, TX). Two different siRNA oligonucleotides

[#14777/siRNA#1 and #4067/siRNA#2] were tested and siRNA#2 was used in the

described knockdown experiments. Knockdown was validated by mRNA analysis at 72h

post-transfection. siRNA-treated cells were normalized to negative control siRNA (NC

siRNA) for Q-PCR and growth inhibition analyses.

Quantitative reverse transcription-PCR




Total RNA was extracted with Qiagen Kit (Qiagen, Valencia, CA) according to the

manufacturer's instructions. IL-8 /CXCR2/ERCC1 primers were used for RT-PCR, and

target genes were normalized to β-actin and quantified using the 2–ΔΔCT method (23).

Western Blot

Cells were solubilized in cell lysis buffer containing a protease inhibitor cocktail. For

tissue samples, Tissue Protein Extraction Reagent was used for tumor tissue protein

extraction. Primary-rabbit-polyclonal antibodies were purchased from Cell Signaling

Technology (Beverly, MA) and rabbit-anti-β-tubulin antibodies were purchased from

Santa Cruz Biotechnology (Santa Cruz, CA). Quantitative analysis of Western blots was

performed by Image J.

In Vivo Studies

Xenograft experiments were conducted in male C57Bl/6 Balb/c mice (Taconic Labs,

Hudson, NY) that were 6-8-week old. Subcutaneous HCT116 and E2 xenografts were

established and allowed to grow until they reached ~100mm3 (day0). Animals were

randomized to treatment groups: vehicle, SCH-527123, oxaliplatin and combination of

SCH-527123 and oxaliplatin (n=6/group). SCH-527123 was administered at 50mg/kg by

oral gavage once daily. Oxaliplatin was administered at 7.5mg/kg by intraperitoneal

injection every 4 days.

Immunohistochemistry (IHC) and Quantitation of Microvascular Density (MVD)

IHC was performed as previously described (11). Formalin-fixed and snap-frozen

fragments of tumor specimens were paraffin embedded and sectioned to 4µm thicknesses.

Primary antibody Rat-anti-mouse-CD31 (BD Pharmingen) was added. MVD was




quantified from 4 different tumor samples by counting the total number of CD31-positive

vessels across the whole section of tumors for each experimental condition.

Statistical Analysis

For all analyses, the difference between each cell lines compared and/or treatment

groups were evaluated using a two-tailed t-test or ANOVA from GraphPad software.

P<0.05 was considered statistically significant.

Results

IL-8 overexpression modulated CXCR2 mRNA level in CRC xenografts.

To investigate the gene expression of IL-8 and CXCR2 from xenograft samples of

the HCT116 and HCT116-IL-8-overexpressing cells (E2), which were established from

subcutaneous injections in nude mice, qRT-PCR was used and data revealed that IL-8 and

CXCR2 mRNA expression were significantly increased at 26-fold (p<0.005) and 2.5-fold

(p<0.05), respectively, in E2 xenografts when compared with HCT116 xenografts (Fig.

1A). CXCR1 mRNA expression was not significantly different between the HCT116 and

E2 xenografts.

Knockdown of CXCR2 increased sensitivity to oxaliplatin in CRC cells.

To examine the role of CXCR2 in CRC cell proliferation, CXCR2 siRNA was

used to suppress CXCR2 expression in CRC cell lines. Two different CXCR2 siRNA

oligonucleotides were tested. After 72h post-transfection, CXCR2 knockdown was

validated by RT-PCR and qRT-PCR and siRNA#2 was used in subsequent CXCR2

siRNA knockdown experiments (Fig.1B). qRT-PCR analysis revealed that transfection of

CXCR2 siRNA#2 reduced the mRNA expression of CXCR2 by 45%±0.039 and

59%±0.049 (siRNA#1 and siRNA#2) in HCT116 cells compared to mock-transfected




cells (p<0.005, Fig.1B). E2 cells transfected with control mock and negative control

siRNA had no evidence of CXCR2 knockdown (Fig.1B). Our previously study

demonstrated that IL-8 overexpression in HCT116 cells decreased sensitivity to the

cytotoxic effects of oxaliplatin (11). Having confirmed CXCR2 mRNA knockdown, we

examined if decreased CXCR2 mRNA expression in CRC cell lines can reduce the IL-8-

induced chemoresistance to oxaliplatin. The growth profiles of HCT116, E2, Caco2 and

Caco2-IIIe (IIIe) were analyzed after 24h treatment with CXCR2 siRNA, and then

subsequently treated for 72h with increasing concentrations of oxaliplatin by growth

inhibition assay (Fig.1C,D). There was a decrease in the IC50(72h) of the CXCR2

siRNA/oxaliplatin-treated cells when compared with mock and β-actin control

siRNA/oxaliplatin-treated cells. Importantly, IC50(72h) of CXCR2 siRNA/oxaliplatin-

treated cells that overexpressed IL-8 (E2 and IIIe) had significant differences than

parental siRNA/oxaliplatin-treated cells. These results suggest that the knockdown of

CXCR2 increases sensitivity to oxaliplatin in CRC cells, especially in IL-8-

overexpressing CRC cells.

SCH-527123 inhibited CRC proliferation, migration, and invasion.

To investigate the impact of CXCR2 inhibitors, the effect of SCH-527123 was

evaluated in CRC cells. Single agent efficacy of SCH-527123 was evaluated by the

growth inhibition assays in the HCT116, E2, Caco2 and IIIe cells. Cells were treated

with increasing concentrations of SCH-527123 for 72h and showed dose-dependent

growth inhibitory activity with IC50(72h) values ranging from 18-40µM (Fig.2A).

Importantly the IL-8 overexpressing cells demonstrated a higher IC50(72h) concentration of

SCH-527123 when compared to parental cells [HCT116 and E2 (p<0.005): 28.9±0.02μM




and 39.5±0.01μM, respectively; Caco2 and IIIe(p<0.005): 18.8±0.03μM and

25.5±0.02μM, respectively] (Fig.2A). Therefore, we concluded that SCH-527123,

decreased growth inhibitory activity in CRC cell lines. Next, the effect of SCH-527123

on inducing apoptosis was evaluated using a TUNEL assay in HCT116 and E2 cells.

Data showed a significant increase in apoptosis in cells treated with SCH-527123, with a

10%±0.5 (p<0.005) (HCT116) and 25%±0.8 (p<0.005) (E2) increase in apoptotic cell

after 4-day treatment compared to untreated control cells (Fig.2B). This result suggests

that SCH-527123 resulted in a decrease of cell growth followed by induction of

significant apoptosis in CRC cells. Moreover, we found that 25µM SCH-527123 was

sufficient to block IL-8-mediated CXCR2 activation in all cell lines analyzed, where

phosphorylation of downstream kinases (MAPK/NF-κB) of CXCR2 was reduced in a

concentration-dependent manner (Supplement fig.1).

Since IL-8 functions to promote cellular migration and invasion, we evaluated the

effect of SCH-527123 on migration and invasion of CRC cells. Treatment with SCH-

527123 (24h) significantly inhibited migration and invasion of HCT116 (24%±0.084%,

p<0.05; 60%±0.003, p<0.005) and E2 (39%±0.01, p<0.005; 49%±0.004, p<0.005) cells

when compared to control treated cells (Fig.2C). Similar results were observed after the

addition of 10ng/ml RhIL-8 to HCT116 cells, where following treatment with 25μM

SCH-527123 there was a significant inhibition of migration (59%±0.015, p<0.005) and

invasion (57%±0.004, p<0.005) (Fig.2C, supplement fig.2).

SCH-527123 in combination with oxaliplatin synergistically suppressed cell growth

and survival.




We next investigated the antiproliferative effects of combining SCH-527123 and

oxaliplatin. All four cell lines were treated with increasing concentrations of SCH-

527123 and oxaliplatin alone and in combination for 72h, then growth inhibition was

measure by a MTS assay. The median-effect analysis method (22) was used in the

evaluation of the combination drug effect. The effects of simultaneous treatment with

both agents in HCT116 and Caco2 cell lines produced synergistic growth inhibition

resulting in synergistic combination index (CI) value<1 for the majority of concentrations

tested at 0.5 fraction affected (FA) (Fig.3A,3C. left). The combination of SCH-527123

(10-18µM) and oxaliplatin (0.25-1.5µM) in E2 and IIIe cell lines also produced

synergistic growth inhibition resulting in FA range of 0.6-0.8 and synergistic CI value<1

(Fig.3B,3D. left). These results displayed that SCH-527123 synergistically increased

sensitivity to oxaliplatin in HCT116 and Caco2 and IL-8 overexpressing cells, which was

consistent with CXCR2 siRNA results described above.

To determine whether alterations in sensitivity to SCH-527123 and oxaliplatin

observed by growth inhibition assay translated to changes in the ability of cells to recover

from drug treatment, a clonogenicity assay was performed. All four cell lines were

treated with SCH-527123 and oxaliplatin alone and selected combinations for 72h

followed by outgrow in drug-free medium for 21 days. Combined drug analysis was

performed using increasing concentrations of both agents. As shown in Fig. 3A-D (right)

and supplement fig.2, in all cell lines, SCH-527123 and oxaliplatin alone resulted in a

dose-dependent suppression of colony formation. Importantly, the combinations of SCH-

527123 and oxaliplatin synergistically suppressed colony formation at all combinations

tested in all cell lines (HCT116: 60%, E2: 64%, Caco2: 72%, IIIe: 60%). Therefore, these




findings suggest that targeting CXCR2 increases drug sensitivity of CRC cells to the one

of the current CRC chemotherapies, oxaliplatin.

SCH-527123 alone and in combination with oxaliplatin suppressed downstream

signaling and modulated apoptotic markers in CRC cell lines.

To evaluate the expression of CXCR2 and IL-8 in our CRC cell lines treated with

SCH-527123 and oxaliplatin alone and combination, qRT-PCR analysis and ELISA

assay were performed. Although there were no differences in the level of IL-8 mRNA

expression in all cell lines with treatment (data not shown), IL-8 protein expression was

significantly downregulated in HCT116, E2 and IIIe cells with both agents alone and

combination (Fig.4A). These data suggest that SCH-527123-mediated antagonism may

decrease IL-8 protein expression at the post-translational level.

To evaluate the effect of combination treatment on IL-8/CXCR2 downstream

signaling, the activation of NF-κB/AKT/MAPK pathway was tested by Western blot. All

four cell lines were treated with SCH-527123 and oxaliplatin alone and combination for

24h. Phospho-NF-κB/phospho-MAPK/phospho-AKT levels were significantly

suppressed in all cell lines with combination treatment compared to untreated and single

agents alone (Fig.4B). These results suggest that the effects of SCH-527123 alone and in

combination with oxaliplatin may be mediated by NF-κB/AKT/MAPK signaling cascade.

Furthermore, using immunoblotting assay, we observed NF-κB targeted anti-apoptotic

gene BCL-2 expression, pro-apoptotic protein BAX expression, which acts as an

antagonist of BCL-2 and promotes apoptosis by forming a heterodimer (BCL-2/BAX)

and therefore losing the pro-apoptotic effect of BCL-2, and apoptotic protein Poly (ADP-

ribose) polymerase (PARP) activation, which is involved in DNA repair (Fig.4C). After




exposure of all cells to SCH-527123 and oxaliplatin alone and combination, levels of

BCL-2 protein expression decreased and BAX protein expression increased. HCT116 and

E2 cells showed enhanced cleavage of PARP into 116-/89-kDa fragments by combination

of both agents compared to oxaliplatin alone (Fig.4C). These findings suggest that SCH-

527123 alone and in combination with oxaliplatin increased apoptosis in CRC cells.

SCH-527123 alone or in combination with oxaliplatin enhanced antiproliferative

activity in tumor xenografts.

To explore the enhanced anti-proliferative effects of SCH-527123 alone or in

combination with oxaliplatin on tumor growth in vivo, and to assess the potential for

SCH-527123 to compromise oxaliplatin activity, nude mice implanted with HCT116 or

E2 cells were exposed to both single agents and their combination. SCH-527123 was

administered at 50mg/kg/day, where the dose was determined by the in vitro IC50(72h), and

previously published studies. Oxaliplatin was administered at 7.5mg/kg oxaliplatin every

4 days. Following 21 days of treatment, average tumor volume (TV) for the vehicle

treated control, oxaliplatin and SCH-527123 single agent treatment groups were

831±133mm3, 382±69mm3 and 386±64mm3 respectively in the HCT116 xenografts. .

Interestingly, in the E2 xenografts, SCH-527123 alone decreased the TV with an average

of 229±56mm3 and was more potent than oxaliplatin alone with a TV of 295±61mm3

where vehicle-treated control tumors had TV of 391±121mm3. Importantly, the

combination displayed a significant decrease in TV [HCT116: 340±63mm3(p<0.05) and

E2: 123±26mm3(p<0.05)], compared to either single agent alone from day13 through the

end of the study (Fig.5A). Moreover, combination treatment did not cause any significant

difference in body weight compared to vehicle-treated control (p=0.67, Fig.5B).




On day21, the tumors were excised and evaluated for the mRNA expression of

CXCR2 and IL-8 following treatment with either single agent alone or in combination by

qRT-PCR. qRT-PCR analysis showed that the combination of SCH-527123 and

oxaliplatin resulted in a significant reduction of CXCR2 and IL-8 mRNA in HCT116

(p<0.05) and E2 (p<0.005) xenografts when compared to vehicle-treated controls

(Fig.5C). Measurement of circulating IL-8 levels in serum which were collected at the

time of necropsy showed a dramatic decrease in both HCT116 and E2 tumor-bearing

mice with agent alone and combination compared with vehicle-treated controls (Fig.5D).

Excision repair cross-complement group1 (ERCC1) is a key element in the

nucleotide excision repair pathway which has previously been associated with oxaliplatin

resistance (24-26). Therefore, the effect of treatment on ERCC1 mRNA expression in the

xenografts following SCH-527123, oxaliplatin alone and their combination was measured

by qRT-PCR. The analysis confirmed that both the HCT116 and E2 xenografts displayed

a significant decrease in ERCC1 mRNA expression following treatment with single

agents alone and their combination in comparison with vehicle controls (Fig.5C).

The combination of SCH-527123 and oxaliplatin synergistically suppresses

downstream signaling and angiogenic activity in xenograft model.

We examined the activity of NF-κB/AKT/MAPK by Western blot in each of the

xenografts after SCH-527123 and oxaliplatin treatments at the end of the study. In

HCT116 and E2 xenografts, SCH-527123 alone and in combination with oxaliplatin

resulted in a significant decrease in the protein activation of phospho-NF-κB/phospho-

AKT/phospho-MAPK (Fig.6A). These in vivo data are consistent with the in vitro data




and confirm that SCH-527123 inhibits the NF-κB/AKT/MAPK signaling cascades in

both models.

To establish whether the decreased tumorigenicity and growth of tumors was

associated with decreased angiogenesis, IHC was utilized to measure the expression of

CD31 (mouse endothelial cell-specific) to evaluate microvessel density (MVD) in the

tumor specimens. MVD in the HCT116 xenografts was decreased following treatment

with SCH-527123 [28%±0.04 (p=0.02)] and oxaliplatin [10%±0.05 (p=0.06)] when

compared to vehicle-treated xenografts. Importantly, when used in combination, MVD

were significantly decreased by 41%±0.05 (p=0.006) in HCT116 xenografts. Similarly, in

the E2 xenografts, MVD was decreased by SCH-527123 by 44%±1.08 (p<0.05),

oxaliplatin by 32%±0.58 (p<0.005) and their combination by 67%±0.6 (p<0.005) when

compared to vehicle-treated xenografts (Fig.6B,C). These results demonstrate that SCH-

527123 in combination with oxaliplatin resulted in a significant inhibition of angiogenic

activity as determined by CD31 IHC expression.

Discussion The role of IL-8 and CXCR2 in tumor development and progression has been

well documented in a wide range of cancer types (8, 27-31). Our previous data and others

have demonstrated that the IL-8/CXCR2 pathway plays a key role in mediating CRC

development (11, 14, 15, 32). In follow-up to our previously published data in CRC, this

study focused on the impact of inhibition of CXCR2 on the proliferation, survival,

invasion and migration in CRC in vitro and in vivo models. To the best of our knowledge,

this study demonstrates for the first time that the CXCR2 antagonist, SCH-527123, has

significant antitumor activity in CRC pre-clinical models and can further sensitize colon




cancer cells to oxaliplatin based treatment. Furthermore, our key findings showed that

the antitumor activity of SCH-527123 resulted from inhibition of cancer cell growth,

motility and angiogenesis through the NF-κB/AKT/MAPK signaling pathways, and this

signaling could be further attenuated when SCH-527123 was co-administered with

oxaliplatin.

Previous data provides evidence that IL-8 is constitutively expressed in mCRC

and primarily associated with the proliferation, metastasis and the induction of

angiogenesis (11, 33). Important roles of its receptors, CXCR1 and CXCR2, have also

been defined in CRC progression (34, 35). Our previous study demonstrated that knock-

down IL-8 expression can inhibit CRC cell growth and metastasis. Conversely, IL-8

overexpression can increase CRC cell metastatic and angiogenic potential, as well as

increasing chemoresistance to oxaliplatin (11). However, neutralizing antibodies against

other chemokines [such as IL-6 (36), IL-5 (37)] also demonstrate similar results.

Importantly, CXCR2 has been shown to interact and bind diverse ligands in addition to

CXCR1, therefore, resulting in the targeting of IL-8/CXCR2 signaling demonstrating a

more effective and broader inhibition in CRC development and progression.

Several small molecule inhibitors targeting IL8/CXCR2 signaling have been

developed to suppress inflammatory diseases (38, 39). A recent study has demonstrated

the potential of SCH-527123 in inhibiting human colon liver metastases using in vivo

models (18). In our in vitro study, we demonstrated that SCH-527123 treatment inhibited

cell proliferation and induced apoptosis in both HCT116 and Caco2 parental and IL-8

overexpressing CRC cells. The in vitro findings were validated in an in vivo study where

mice were given SCH-527123 orally and exhibited a reduction in tumor volume when




compared with the vehicle-treated control group, which further supported the

antiproliferative effect of SCH-527123. SCH-527123 also demonstrated a decrease in

tumor vascular density when compared with the vehicle-treated control group. These

findings indicated the targeting CXCR2 with antagonists such as SCH-527123 may be a

promising therapeutic for CRC.

In prostate cancer cells it was reported that IL-8 signaling contributes to the

intrinsic resistance of the cancer cells to undergo apoptosis in response to either

environmental or chemical stress (40). One of the key investigations in this study was to

evaluate the antiproliferative effects of combining the CXCR2 antagonist with cytotoxic

chemotherapy. Oxaliplatin is a platinum-based DNA damaging chemotherapy that

demonstrates clinical benefit for patients with high-risk stage II and stage III CRC, as

well as patients with advanced disease (41, 42). However, cancer cells are frequently

resistant to oxaliplatin (43). Our present study demonstrated CXCR2 to be a key mediator

of IL-8-mediated chemoresistance to oxaliplatin and utilizing CXCR2siRNA to

knockdown its expression in CRC cells sensitized them to oxaliplatin. These results led to

the hypothesis that targeting IL-8/CXCR2 in combination with oxaliplatin may increase

sensitivity to oxaliplatin, providing enhanced efficacy and eventually benefit in CRC

treatment. Our novel observation showed that the combination of SCH-527123 and

oxaliplatin resulted in synergistic suppression of CRC cell proliferation and survival with

additive-to-synergistic effects. These synergistic antitumor properties were also observed

in the IL-8-overexpressing cell lines, which were more insensitive to single agent SCH-

527123 treatment. Our in vivo data confirmed the in vitro findings and showed that the

combination of SCH-527123 and oxaliplatin significantly inhibits tumor growth when




compared to single agents alone. ERCC1 mRNA levels have been shown to be predictive

of oxaliplatin cytotoxicity in the HCT116 cell line (25) and a useful marker in predicting

response to oxaliplatin-based treatment for CRC patients (44). High ERCC1 mRNA

expression has been demonstrated to be associated with resistance to oxaliplatin (26).

Supplement fig.3 showed, in an oxaliplatin-resistant cell line (HCT116-OR, where

resistance was induced by long-term culture in the presence of low dose oxaliplatin), or

in E2 cell line, that both cell lines demonstrated an increased level of ERCC1 mRNA

expression. Our in vivo data showed that treatment with both agents in combination

dramatically lowered ERCC1 mRNA expression, which suggests that SCH-527123 may

be associated with increased sensitivity to oxaliplatin through modulation of the DNA

nucleotide excision repair pathway.

To elucidate the mechanism of action of SCH-527123, we analyzed the activation

of the downstream pathway of IL-8/CXCR2. IL-8/CXCR2 has previously been shown to

signal through AKT and MAPK pathways (45, 46). Wilson group demonstrated that IL-

8/CXCR2 signaling confers resistance to oxaliplatin through NF-κB activity, which is an

important determinant of cancer cell sensitivity to oxaliplatin (40). In this study, our

findings suggest that SCH-527123 decreases NF-κB activity through IL-8/CXCR2

signaling, which is in turn responsible for enhancing sensitivity to oxaliplatin. Moreover,

our study demonstrated that SCH-527123 in combination with oxaliplatin significantly

increased apoptotic signaling in CRC cells. Therefore, we propose that inhibition of

CXCR2/IL-8 signaling increases oxaliplatin sensitivity that is mediated partially by

attenuating NF-κB activity and also inducing apoptosis. However, further investigations




are warranted to elucidate the mechanism of oxaliplatin sensitivity mediated through NF-

κB activity and apoptosis.

In conclusion, our studies provide significant evidence that the CXCR2 antagonist,

SCH-527123 demonstrates antitumor effects and increases sensitivity to oxaliplatin

therapy in both in vitro and in vivo CRC models. The antitumor activity of SCH-527123

observed in CRC cells lines was shown to be due to decreased cell proliferation,

migration, invasion and increased apoptosis. In addition, it was demonstrated that

combination of SCH-527123 and oxaliplatin increased sensitivity to oxaliplatin in CRC

cells in vitro and in vivo. Moreover, the combination of SCH-527123 and oxaliplatin

synergistically inhibited in vivo tumor growth and vascularity. Based on these preclinical

results, CXCR2 may represent a novel therapeutic target in CRC, that when targeted in

combination with the DNA damaging agent, oxaliplatin will increase chemosensitivity.




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Figure Legends

Figure 1: CXCR2 mRNA expression in CRC HCT116 and E2 xenografts and the

influence of CXCR2 knockdown in CRC on growth of cells treated with oxaliplatin.

A. qRT-PCR determination of IL-8/CXCR2 mRNA expression in HCT116 and E2

xenografts. B. HCT116 cells were transfected with mock, β-actin siRNA and CXCR2

siRNA. Top, Q-PCR results are representative at 72h post-transfection. Bottom, qRT-PCR

results are representative of mean±SD from triplicate samples and presented as mRNA

fold change relative to mock transfected control. C,D. All cell lines were transfected with

mock, β-actin siRNA and CXCR2 siRNA, and then treated with increasing

concentrations of oxaliplatin for 72h.

Figure 2: SCH-527123 decreases cell proliferation, migration and invasion and

increases apoptosis in CRC cells.

A. Growth inhibition assay. IC50 (72h)

of SCH-527123 were determined using GraphPad

Prism software. B. After 72h treatment, the number of apoptotic cells was evaluated, and

apoptotic cells (blue) in HCT116 and E2 were detected in SCH-527123-treated cells

compared with controls, in which mostly viable cells (red) were present. C. Quantitation

of migration and invasion after SCH-527123 treatment. Histogram shows fold change

over the number of HCT116 and HCT116-rhIL-8 and E2 cells that migrated or invaded

compared to no treatment.

Figure 3: SCH-527123 combined with oxaliplatin synergistically suppresses CRC

cell proliferation and survival.

A-D (Left panel). Growth inhibition assay. A-D (Right panel). Clonogenicity assay. All

cell lines were treated with increasing concentrations of SCH-527123 and oxaliplatin




alone and in combination for 72h. Data is presented as histograms of the mean percentage

of colony formation compared with untreated controls (100%)±SD. The combined drug

effects were analyzed using the combination index (CI) with fraction affected (FA) values

for combinations.

Figure 4: SCH-527123 in combination with oxaliplatin modulates protein expression

of IL-8, PARP, BCL-2/BAX and decreased NF-κB/Akt/MAPK signaling activity in

CRC cells.

A. An ELISA assay was used to measure IL-8 production from all cell lines. The results

were normalized to total cell numbers. Histograms represent the mean fold change ±SD

of IL-8 overexpressing cells compared to parental cells. B and C. Western blot analysis.

All cells were treated with 25μM SCH-527123 and 0.5μM oxaliplatin alone and in

combination for 24h. β-tubulin was used to as a loading control and normalization protein

for quantitation. .

Figure 5: Antitumor activity of SCH-527123 combined with oxaliplatin in HCT116

and E2 xenografts.

A, Examination of tumor growth in xenografts of CRC cells. The graphs indicate the

mean tumor growth rates ±SEM of each group. Statistical significance was determined by

ANOVA using Graphpad Software, P<0.05. B, Mouse bodyweight represented as the

percent initial body weight at day21 compared to day1. C. qRT-PCR was used to

determine the level of IL-8, CXCR2 and ERCC1 mRNA in HCT116 and E2 xenografts

with treatment. D. Serum IL-8 production was measured by ELISA, and the results were

normalized to whole blood volume and presented as the mean±SD. The results are

representative of a minimum of three independent experiments.




Figure 6: SCH-527123 in combination with oxaliplatin significantly suppressed NF-

κB/Akt/MAPK downstream signaling and angiogenic activity in HCT116 and E2

xenografts.

A. Cell lysates of each tissue were immunoblotted with anti-phospho-NF-κB-

p65/Akt/p44/42MAPK or anti-total NF-κB-p65/Akt/p44/42MAPK antibodies, β-tubulin

as a loading control and normalization protein for quantitation. B,C.

Immunohistochemistry and Quantitation of MVD (CD31) from tumor tissue slides. In

HCT116 and E2 tumor specimens, rat-anti-mouse- CD31 antibodies were added to tissue

sections. Data shown represent the mean±SD (*, P<0.05; **, P<0.005).




Figure 1.

A. B.

C.

- 1 . 0 - 0 . 5 0 . 0 0 . 5 1 . 0

4 0

6 0

8 0

1 0 0

H C T 1 1 6 Mock Transfected I C 5 0 = 0 . 9 9 8 + / - 0 . 0 4 H C T 1 1 6 Negative control I C 5 0 = 1 . 1 3 2 + / - 0 . 0 2 H C T 1 1 6 C X C R 2 s i R N A I C 5 0 = 0 . 6 3 2 1 + / - 0 . 0 8

H C T 1 1 6 - Parental

L o g [ μM ] O x a l i p l a t i n

% C

o n t r

o l

- 1 . 0 - 0 . 5 0 . 0 0 . 5 1 . 0

4 0

6 0

8 0

1 0 0

H C T 1 1 6 - E 2 Mock Transfected I C 5 0 = 1 . 5 5 7 + / - 0 . 0 5 H C T 1 1 6 - E 2 Negative control I C 5 0 = 1 . 2 6 7 + / - 0 . 0 3 H C T 1 1 6 I C 5 0 = 0 . 6 7 + / - 0 . 1 1

H C T 1 1 6 - E 2


% C

o n t r

o l

- 1 . 0 - 0 . 5 0 . 0 0 . 5 1 . 0 2 0

4 0

6 0

8 0

1 0 0

C a c o 2 Mock Transfected I C 5 0 = 0 . 6 7 + / - 0 . 0 9 C a c o 2 Negative control I C 5 0 = 0 . 6 0 + / - 0 . 0 3 C a c o 2 C X C R 2 s i R N A I C 5 0 = 0 . 4 1 + / - 0 . 0 5

C a c o 2 - Parental


% C

o n t r

o l

- 1 . 0 - 0 . 5 0 . 0 0 . 5 1 . 0

4 0

6 0

8 0

1 0 0

C a c o 2 - I I I e Mock Transfected I C 5 0 = 0 . 8 8 + / - 0 . 0 6 C a c o 2 - I I I e Negative control I C 5 0 = 0 . 8 2 + / - 0 . 0 8 C a c o 2 - I I I e C X C R 2 s i R N A I C 5 0 = 0 . 3 8 4 + / - 0 . 1 4

C a c o 2 - I I I e


% C

o n t r

o l

D.

Mock Negative control siRNA

CXCR2 siRNA #1 CXCR2 siRNA #2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

** **

CXCR

2 mRN

A fo

ld ch

ange

HCT116-parental 48 h HCT116-parental 72 h

Mock Negative control siRNA

CXCR2 siRNA #1 CXCR2 siRNA #2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

** **

CXCR

2 mRN

A fo

ld ch

ange

HCT116-parental 48 h HCT116-parental 72 h

actin

CXCR2

β-

SiRNA#2

HCT 116 72h Mock NC SiRNA#1

p < 0.05 p < 0.0001

p < 0.0001 p = 0.08

HCT116 (Parental xenograft) E2 (IL-8 transfected xenograft)

0

5

25

30 P<0.005

P<0.05 *

**

mRN

A Fo

ld C

hang

e

IL-8 mRNA CXCR2 mRNA CXCR1 mRNA

-E2 CXCR2 siRNA




A.

B.

C.

Figure 2.

HCT116 HCT116 +SCH 25 µM

HCT116 - E2 HCT116 - E2+SCH 25 µM

HCT116 HCT116 +SCH 25 µM

HCT116 - E2 HCT116 - E2+SCH 25 µM HCT116 HCT116-E2

0

5

10

15

20

25

30

* *

* *

Apop

totic

cel

ls (%

) Vehicle SCH-527123 (25 µM)

HCT116 HCT 116 + IL-8 HCT116-E2 0.0

0.2

0.4

0.6

0.8

1.0

* * *

* *

Fold

Cha

nge

in M

igra

tion

Vechicle SCH-527123 (25 µM)

HCT116 HCT 116 + IL-8 HCT116-E2 0.0

0.2

0.4

0.6

0.8

1.0

* * * * * *

Fold

Cha

nge

in In

vasi

on

Vechicle SCH-527123 (25 µM)

1.0 1.2 1.4 1.6 1.8 0

50

100

Caco2 IC 50 18.78+/- 0.02

Caco2-IIIe IC 50 25.45+/-0.02

HCT116 IC 50 28.95+/-0.02

HCT116-E2 IC 50 39.45+/-0.01

Log [ µM] SCH-527123

% C

ontr

ol




Figure 3.

B.

D.

A.

C.

20

40

60

80

100 Oxaliplatin SCH-527123 Combo

Caco2-parental

0.125 0.25 0.5 1 1.5 2 3.25 6.5 12.5 µM Oxaliplatin 5 10 15 18 22.5 25 30 40 50 µM SCH-527123

0.92 0.85 0.59 0.45 0.36 0.32 0.3 0.3 0.26 FA 1.33 0.39 0.83 0.78 0.82 0.85 1.1 1.77 2.43 CI

% C

ontr

ol

20

40

60

80


0.125 0.25 0.5 1 1.5 2 3.25 6.5 12.5 µM Oxaliplatin 5 10 15 18 22.5 25 30 40 50 µM SCh-527123

0.93 0.85 0.74 0.63 0.55 0.47 0.39 0.30 0.23 FA 1.03 0.99 0.97 0.96 1.03 0.94 1 1.22 1.47 CI

HCT116-E2

% C

ontr

ol

20

40

60

80



0.89 0.81 0.66 0.52 0.42 0.31 0.26 0.24 0.20 FA 0.79 0.97 0.89 0.85 0.85 0.72 0.84 1.33 1.86 CI

HCT116-parental %

Con

trol

20

40

60

80


Caco2-IIIe


0.89 0.77 0.62 0.56 0.48 0.44 0.37 0.30 0.25 FA 0.76 0.85 0.88 1.05 1.13 1.19 1.13 1.67 2.17 CI

% C

ontr

ol

0

10

20

30

40

50

60

70

80

90

100 Caco2-IIIe

% C

ontro

l

Oxaliplatin Sch-527123 Combo

FA CI

µM Oxal µM SCH

.. .. 0.3

.. .. 0.835

0 1 1 25 0 25

.. .. 0.4

.. .. 0.625

0 0.5 0.5 15 0 15

.. .. 0.7

.. .. 0.913

0 0.25 0.25 10 0 10

FA CI

µM Oxal µM SCH

.. .. 0.3

.. .. 0.835

0 1 1 25 0 25

.. .. 0.4

.. .. 0.625

0 0.5 0.5 15 0 15

.. .. 0.7

.. .. 0.913

0 0.25 0.25 10 0 10

0

10

20

30

40

50

60

70

80

90 Caco2-parental

% C

ontro

l

Oxaliplatin SCH-527123 Combo

FA CI

µM Oxal µM SCH

.. .. 0.194

.. ..0.684

0 1 1 25 0 25

.. .. 0.28

.. .. 0.54

0 0.5 0.5 15 0 15

.. .. 0.626

.. .. 1.042

0 0.25 0.25 10 0 10

FA CI

µM Oxal µM SCH

.. .. 0.194

.. ..0.684

0 1 1 25 0 25

.. .. 0.28

.. .. 0.54

0 0.5 0.5 15 0 15

.. .. 0.626

.. .. 1.042

0 0.25 0.25 10 0 10

FA CI

µM Oxal µM SCH

.. .. 0.15

.. ..0.922

0 1 1 25 0 25

.. .. 0.4

.. .. 1.101

0 0.5 0.5 15 0 15

.. .. 0.73

.. .. 1.63

0 0.25 0.25 10 0 10

FA CI

µM Oxal µM SCH

.. .. 0.15

.. ..0.922

0 1 1 25 0 25

.. .. 0.4

.. .. 1.101

0 0.5 0.5 15 0 15

.. .. 0.73

.. .. 1.63

0 0.25 0.25 10 0 10

0

10

20

30

40

50

60

70

80

90

HCT116-parental

% Co

ntrol


0 10 20 30 40 50 60 70 80 90

100 110

HCT116-E2

% Co

ntrol


FA CI

µM Oxal µM SCH

.. .. 0.15

.. ..0.922

0 1 1 25 0 25

.. .. 0.4

.. .. 1.101

0 0.5 0.5 15 0 15

.. .. 0.73

.. .. 1.63

0 0.25 0.25 10 0 10

FA CI

µM Oxal µM SCH

.. .. 0.15

.. ..0.922

0 1 1 25 0 25

.. .. 0.4

.. .. 1.101

0 0.5 0.5 15 0 15

.. .. 0.73

.. .. 1.63

0 0.25 0.25 10 0 10




HCT116 HCT116-E2 Caco2 Caco2-IIIe

0

1000

2000

3000

IL-8

conc

entra

tion

(pg/

ml/1

x10 6 /48

h )

Vehicle SCH-527123 Oxaliplatin Combo

A.

B.

C.

Figure 4.

P - NF - κB

P - p42/44

β- Tubulin

SCH - 527123 Oxaliplatin

P - Akt

NF - κB

P42/44

Akt

HCT116 xenograft E2 xenograft - + - + - - + +

- + - + - - + +

1 1.25 1.25 1.75 1 1.2 1.2 1

1 0.85 0.28 0.1 1 0.6 0.01 0.1

1 1.5 2 2.5 1 1.1 1.25 1.25

1 0.2 0.6 0.2 0.7 0.2 0.2 0.02

1 1.25 1.5 1.4 1 0.9 0.85 0.85

1 1.6 0.4 0.1 1 2 0.2 0.1

PARP

BAX

BCL - 2

β- Tubulin


- + - + - - + +

HCT116 HCT116 - E2 - + - + - - + +

- + - + - - + +

Caco2 Caco2 - IIIe - + - + - - + +

1 5 2 5.5 1 1 1.5 2.3

1 1.16 1.3 2.3 1 1.4 1.6 2

1 1.4 0.2 0.04 1 1.1 0.7 0.6

1 1.3 4.2 2.3 1 1.25 4.25 4.75

1 1.25 3.6 8 1 1.3 5 5.3

1.0 0.6 0.4 0.4 1 0.4 0.3 0.4




A.

C.

B.

D.

Figure 5.

Vehicle

SCH527123 only Oxaliplatin only

Combo 0

200

800

1200

1600

**

** ** **

** **

IL-8 c

once

ntra

tion

(pg/m

l) HCT116 Xenograft HCT116-E2 Xenograft

CXCR2 IL-8 ERCC1 0.0

0.5

1.0

1.5

* * * * * *

** * *

mRN

A le

vel f

old

chan

ge

HCT116-E2 Vehicle HCT116-E2 SCH-527123 50 mg/kg HCT116-E2 Oxaliplatin 7.5 mg/kg HCT116-E2 Combo

CXCR2 IL-8 ERCC1 0.0

0.5

1.0

* *

*

mRN

A le

vel f

old

chan

ge

HCT116 Vehicle HCT116 SCH-527123 50 mg/kg HCT116 Oxaliplatin 7.5 mg/kg HCT116 Combo

0

25

50

75

100

Vehicle 50 mg/kg SCH-527123

7.5 mg/kg Oxaliplatin

Combo

HCT116-Parental

% In

itial

Bod

ywei

ght

0

25

50

75

100

Vehicle 50 mg/kg SCH-527123

7.5 mg/kg Oxaliplatin

Combo

HCT116-E2

% In

itial

Bod

ywei

ght

1 2 3 4 5 6 7 8 0

100

200

300

400

500

600

1 4 7 10 13 16 19 21

HCT116-E2

Day of Treatment

Tum

or V

olum

e (m

m 3 )

HCT116-Parental

1 2 3 4 5 6 7 8 0

250

500

750

1000

Vehicle 50 mg/kg SCH-527123 7.5 mg/kg Oxaliplatin Combo

1 4 7 10 13 16 19 21

Day of Treatment

Tum

or V

olum

e (m

m 3 )




A.

B.

C.

Vehicle SCH - 527123 Oxaliplatin Combo

E2 E2 E2 E2

HCT116 HCT116 HCT116 HCT116 Vehicle SCH - 527123 Oxaliplatin Combo

E2 E2 E2 E2

HCT116 HCT116 HCT116 HCT116

E2 E2 E2 E2 E2 E2 E2 E2

HCT116 HCT116 HCT116 HCT116

Vehicle SCH-527123

Oxaliplatin Combo

0

5

10

15

20

**

** *

* *

Mea

n m

icro

vess

el d

ensi

ty

HCT116 xenograft HCT116-E2 xenograft

Figure 6

P - NF - κB

P - p42/44

β- Tubulin


P - Akt

NF - κB

P42/44

Akt

HCT116 xenograft E2 xenograft - + - + - - + +

- + - + - - + +

1 1.25 1.25 1.75 1 1.2 1.2 1

1 0.85 0.28 0.1 1 0.6 0.01 0.1

1 1.5 2 2.5 1 1.1 1.25 1.25

1 0.2 0.6 0.2 0.7 0.2 0.2 0.02

1 1.25 1.5 1.4 1 0.9 0.85 0.85

1 1.6 0.4 0.1 1 2 0.2 0.1




Published OnlineFirst March 5, 2012.Mol Cancer Ther Yan Ning, Melissa J LaBonte, Wu Zhang, et al. cancer modelsactivity and sensitizes cells to oxaliplatin in preclinical colon The CXCR2 antagonist, SCH-527123, demonstrates antitumor

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The CXCR2 antagonist, SCH-527123, demonstrates antitumor ... · oxaliplatin-based therapy in CRC cell line models and genetic variants in IL-8 and CXCR2 both predict tumor recurrence