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Keywords:

  • breast cancer;
  • CXCR4;
  • primary tumor;
  • metastasis;
  • therapeutic target;
  • transgenic mouse

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

CXCR4 is a chemokine receptor implicated in the homing of cancer cells to target metastatic organs, which overexpress its ligand, stromal cell-derived factor (SDF)-1. To determine the efficacy of targeting CXCR4 on primary tumor growth and metastasis, we used a peptide inhibitor of CXCR4, CTCE-9908, that was administered in a clinically relevant approach using a transgenic breast cancer mouse model. We first performed a dosing experiment of CTCE-9908 in the PyMT mouse model, testing 25, 50 and 100 mg/kg versus the scrambled peptide in groups of 8–16 mice. We then combined CTCE-9908 with docetaxel or DC101 (an anti-VEGFR2 monoclonal antibody). We found that increasing doses of CTCE-9908 alone slowed the rate of tumor growth, with a 45% inhibition of primary tumor growth at 3.5 weeks of treatment with 50 mg/kg of CTCE-9908 (p = 0.005). Expression levels of VEGF were also found to be reduced by 42% with CTCE-9908 (p = 0.01). In combination with docetaxel, CTCE-9908 administration decreased tumor volume by 38% (p = 0.02), an effect that was greater than that observed with docetaxel alone. In combination with DC101, CTCE-9908 also demonstrated an enhanced effect compared to DC101 alone, with a 37% decrease in primary tumor volume (p = 0.01) and a 75% reduction in distant metastasis (p = 0.009). In combination with docetaxel or an anti-angiogenic agent, the anti-tumor and anti-metastatic effects of CTCE-9908 were markedly enhanced, suggesting potentially new effective combinatorial therapeutic strategies in the treatment of breast cancer, which include targeting the SDF-1/CXCR4 ligand/receptor pair.

Although the survival of patients with breast cancer has improved in recent years, further progress will require a better understanding of the metastatic process, which is the underlying cause of breast cancer mortality. The process of metastasis has been divided into several stages including the invasion of the primary tumor, intravasation into the bloodstream, circulation, extravasation and proliferation at the distant metastatic site.1 A recently proposed model that explains the process of extravasation of tumor cells from the peripheral circulation into target organs of metastasis is the chemokine-receptor model. Muller et al. showed that chemokines are overexpressed by those organs to which cancer metastasizes and serves to attract cancer cells which express their receptor,2 analogous to their function in recruiting inflammatory cells to sites of tissue injury. They identified the chemokine/receptor pair, stromal cell-derived factor (SDF)-1/CXCR4 as a candidate metastasis promoter in breast cancer.2 We have since shown that CXCR4 is overexpressed in 67% of breast cancer samples, that elevated expression of CXCR4 carries a poor prognosis, and that CXCR4 expression is strongly associated with human epidermal growth factor receptor 2 (HER2) expression in these tumors.3 Therefore, it appears plausible that inhibition of CXCR4 activity may hinder the metastatic process. Moreover, the SDF-1/CXCR4 ligand/receptor pair has also been implicated in regulating primary tumor growth. Overexpression of SDF-1 in cancer-associated fibroblasts present in the tumor microenvironment was reported to promote primary tumor growth by inducing tumor neo-angiogenesis via both paracrine (direct stimulation of tumor CXCR4), and endocrine (recruitment of endothelial progenitor cells from the bone marrow) mechanisms.4 Hence, targeting CXCR4 may be effective in inhibiting primary tumor growth as well as the formation of distant metastasis. Indeed, blocking CXCR4 activity has been found to inhibit primary tumor growth and metastasis in xenograft models, albeit using treatment strategies that may not translate well to the clinic.5–7

To target CXCR4, we selected a peptide antagonist that is in the advanced stages of clinical development for the treatment of solid tumors. CTCE-9908 (Chemokine Therapeutics Corp, Vancouver, BC), is an SDF-1 analog consisting of a dimer of the first 8 amino acids of SDF-1 (Supporting Information Fig. S1a), and serves as a competitive inhibitor to SDF-1. Radioligand binding assays have shown that CTCE-9908 competitively binds to CXCR4. CTCE-9908 has previously been reported to inhibit metastasis in osteosarcoma, melanoma, prostate and breast cancer mouse models.8–11 Unlike most other CXCR4 antagonists, evaluation of CTCE-9908 has already begun in patients. Safety of CTCE-9908 has been demonstrated in both a single dose Phase I trial in healthy adults and in a Phase I/II trial in cancer patients.12 To test the efficacy and toxicity of CTCE-9908 in a breast cancer model, as well as in combination with other treatments, we selected a well-known transgenic mouse model for breast cancer, mouse mammary tumor virus (MMTV)-driven Polyoma Middle T Antigen (PyMT) model. The PyMT mammary tumor has been shown to overexpress HER2/neu, and demonstrates progression from hyperplasia to pre-invasive, invasive and distant metastasis.13 Thus, this model is particularly appropriate to test both the growth inhibitory and the anti-metastatic potential of anti-CXCR4 therapy. We examined the anti-tumor effect of CTCE-9908 in the PyMT mouse model both alone, and in combination with other anti-cancer therapies such as an anti-angiogenic agent or a cytotoxic agent that is commonly used in breast cancer patients.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Transgenic mouse model

FVB/N TgN (MMTV-PyMT)634 male mice were obtained from Dr. William Muller (McGill University, Québec, Canada), and were mated with FVB/N from Taconic Farms (Albany, New York). Female mice heterozygous for the PyMT oncogene were identified by extracting tail DNA as per Ref. 14 using PCR to amplify a 540 base pair fragment with the following primers: forward, 5′-GGA AGC AAG TAC TTC ACA AGG G-3′; reverse, 5′-GGA AAG TCA CTA GGA GCA GGG - 3′.

Tumor monitoring

All experimental procedures were conducted according to McGill University Animal Care Committee. In the MMTV-PolyMT model, all tumors from each of the mammary fat pads (total number 10) were measured twice a week using calipers. Length and width of each tumor were measured in mm and tumor volume was estimated using the following formula: (x2×y)/2, where x refers to the shortest diameter, and y refers to the larger diameter.15 Tumors from all 10 fat pads were summed up together. After the final dose of treatment was administered, mice were sacrificed within 24 hr. All 10 tumors were harvested and fragments were either snap-frozen or fixed in 10% buffered formalin for paraffin embedding. Lungs were examined, macroscopic nodules counted, weighed and fixed in formalin. Kidneys, liver, spleen and spine were also grossly examined.

Pharmacological agents

CTCE-9908 was obtained from Chemokine Therapeutics Corp. and reconstituted in sterile water for injection. The control used for CTCE-9908 was either the scrambled peptide, SC-9908 (Supporting Information Fig. S1b), reconstituted in sterile water for injection at a dose of 25 mg/kg, or its vehicle, sterile water for injection. Docetaxel (Sanofi-Aventis; Jewish General Hospital Oncology Pharmacy) was diluted in normal saline; normal saline was used as the vehicle control. To target the vascular endothelial growth factor (VEGF) pathway in this transgenic mouse model, we used DC101, an anti-VEGF receptor 2 (VEGFR2) rat IgG1 monoclonal antibody (courtesy of Imclone Systems Incorporated, New York, NY), with its control purified rat IgG (Chrompure rat IgG, Jackson Immunoresearch Laboratories).

Three major experiments were conducted in the PyMT transgenic mouse model in total. First, a CTCE-9908 dosing experiment was performed to identify the most effective dose in the PyMT model. CTCE-9908 was administered at 25, 50 and 100 mg/kg subcutaneously (s.c.), at four alternating sites of injection, starting from 7 weeks of age, 5 days per week for a total of 4.5 weeks (Supporting Information Fig. S2a). Second, CTCE-9908 was given at 25 mg/kg in combination with docetaxel (Supporting Information Fig. S2b). One dose of docetaxel was administered first, and in the subsequent week, CTCE-9908 was started 5 days per week, for a total of 4.5 weeks.16 A total of three doses of docetaxel were administered, one dose per week at 35 mg/kg intraperitoneally (I.P.), a dose that was previously reported as non-toxic.17 Third, CTCE-9908 was given at 25 mg/kg, concomitantly with DC101 at 1000 μg per dose I.P. twice per week for a total duration of 4.5 weeks (Supporting Information Fig. S2c). We also tested CTCE-9908 at 50 mg/kg administered simultaneously with DC101 at 400 μg per dose, twice per week, for a total duration of 4.5 weeks. Because of the transgenic nature of our mouse model, all experiments were designed such that the mean age at sacrifice of the mice from each treatment or control group was standardized at 82 days, thereby allowing every mouse the same lifespan for tumor growth and metastasis formation.18 An average of 8–16 mice were dosed in each treatment group in each of the three major experiments.

Protein extraction, Western blot analysis

Proteins from five-six representative MMTV-PyMT mice from each group of the dosing experiment (total 20 mice) were extracted from frozen tumor samples with Cell Lysis Buffer (Cat. No. 9803, Cell Signalling Technology, Danvers, MA) supplemented with phenylmethylsulphonyl fluoride (PMSF) (Cat No. P7626; Sigma). Protein concentrations were measured via spectrophotometry using the bicinchoninic acid (BCA) kit for protein determination (Cat. No. BCA-1, Sigma). For western blot analysis, Hybond ECL nitrocellulose membranes (Cat No. RPN203D, Amersham Biosciences) were used with the enhanced chemiluminescence method (Cat No. RPN2132, Amersham ECL Plus Western Blotting Detection Reagent; GE Healthcare, Buckinghamshire, UK). The following primary antibodies were used: Phospho-Akt (Ser473) (D9E) (Cat No. 4060, Cell Signalling Technology); Akt (pan) (C67E7) (Cat No. 4691, Cell Signalling Technology); VEGF (Cat No. ab46154, Abcam), alpha-tubulin for loading control (Cat No. ab7291, Abcam). Protein detection was carried out as per manufacturer's protocol. Images were acquired on GeneSnap from SynGene software (Version 6.08, Cambridge, England), and quantification of western blot bands were performed on GeneTools from SynGene software (Version 3.06, Cambridge, England).

Immunohistochemistry

Expression of CXCR4 in the PyMT model was verified on a section of a tissue microarray constructed from a total of 33 tumors and in-situ lesions obtained from 11 different mice, using immunohistochemistry via the labeled-streptavidin method. CXCR4 expression was measured using an anti-CXCR4 antibody (Cat No. 2074, Abcam, Cambridge, MA) at 1/50 dilution overnight at 4°C. The secondary antibody used was a biotin-labeled, goat anti-rabbit (Cat. No. 111-065-003, Jackson Immunoresearch Laboratories), at 2.75 μg/mL. Images were visualized on a UNICO H602 compound microscope. Images were acquired on a 10x objective lens using a Nikon digital camera.

Statistical analysis

In order to determine if the comparison of two particular treatment groups demonstrated statistical significance, a Mann–Whitney test was performed. p-values < 0.05 were considered statistically significant. All statistical analysis was performed using Graph Pad Prism 4.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

CXCR4 is expressed in PyMT mouse tumors

We first verified the degree of CXCR4 protein expression in mammary tumors from the PyMT mouse model. We determined the expression of CXCR4 in 18 tumors from 7 untreated mice. All 18 tumors demonstrated ≥50% cells for positive nuclear staining, and 17/18 tumors demonstrated ≥50% cells positive for cytoplasmic staining. Thus, the expression of CXCR4 is relatively uniform amongst different mice and within different tumors from the same mouse (Supporting Information Figs. S3a and 3b).

CXCR4 antagonist delays primary tumor growth rate

Since patients with breast tumors are diagnosed and treated only when they become clinically evident (often palpable), we first performed a dosing trial in the PyMT mouse with the CXCR4 antagonist administered only when the first tumors became palpable. We tested three doses of CTCE-9908: 25 (CTCE-9908-25), 50 (CTCE-9908-50) and 100 (CTCE-9908-100) mg/kg and compared each to the scrambled peptide. Treatment with CTCE-9908 for 2.5 weeks already resulted in a delay in the growth of the primary tumor, with a 45% inhibition in tumor growth with CTCE-9908-50, and a 68% inhibition in tumor growth with CTCE-9908-100 (p = 0.05; p = 0.03, respectively) (Fig. 1a). A maximal effect upon primary tumor growth inhibition was observed at 3.5 weeks, with a 45% reduction in tumor volume with 50 mg/kg (p = 0.005), and a 56% reduction with 100 mg/kg (p = 0.001). At necropsy, a less pronounced inhibitory effect was observed with CTCE-9908-50, which reduced primary tumor volume by 23% with near statistical significance (p = 0.07), while CTCE-9908-100 resulted in a 36% reduction (p = 0.05) (Fig. 1b). To summarize, CTCE-9908 administered at 50 and 100 mg/kg slowed tumor growth with a maximal inhibitory effect obtained at 3.5 weeks. Thereafter, the rate of tumor growth subsequently increased, such that the reduction of tumor volume was less evident at necropsy.

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Figure 1. CTCE-9908 inhibits primary tumor growth in MMTV-PyMT transgenic mice. (a) Growth curves of primary mammary tumors, representing the sum of the volume of 10 tumors from the different fat pads of each mouse, measured twice weekly with calipers. A total of 8–16 mice were treated with three different doses of CTCE-9908 (25 or 50 or 100 mg/kg s.c. daily for 5 days per week) or with control. The p-values refer to the differences between the control and the 50 mg/kg dose of CTCE-9908 at different time points. (b) Bar graphs of sums of the volumes of 10 primary tumors measured at necropsy of each mouse treated with different doses of CTCE-9908; p-values for comparisons between CTCE-9908-25 versus control, p = 0.75; CTCE-9908-50 versus control, p = 0.07; CTCE-9908-100 versus control, p = 0.05. Error bars refer to standard error of the mean.

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Effect of CXCR4 antagonist upon VEGF expression in the primary tumor

In order to determine if the expression of VEGF was modulated by CTCE-9908, we identified the expression of VEGF in the primary tumor. Due to the positive feedback loop between CXCR4 and VEGF expression in endothelial cells, whereby VEGF upregulates CXCR4 expression, and CXCR4 stimulation promotes VEGF expression,19, 20 we determined if blocking CXCR4 activity would affect tumor expression of VEGF. The administration of CTCE-9908 alone resulted in a significant decrease in tumor VEGF levels by Western blot (p = 0.01), with a 42% decrease in VEGF expression observed at the 50 mg/kg dose (Figs. 2a and 2b).

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Figure 2. The effects of CTCE-9908 treatment on VEGF expression. (a) Representative western blot of mammary tumor lysates comparing VEGF expression in scrambled peptide control, CTCE-9908-25 and CTCE-9908-50. Decreased expression of VEGF is observed in those mice treated with CTCE-9908-50. Alpha-tubulin is used as loading control and is shown in the row below. (b) Bar graphs of relative VEGF expression (ratio of VEGF to loading control) obtained from western blot band densitometry for five representative mice from each treatment group. p-values for comparisons between CTCE-9908-25 versus control, p = 0.18; CTCE-9908-50 versus control, p = 0.01. Error bars refer to standard error of the mean. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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CXCR4 antagonist may inhibit AKT activity in primary tumor

We next examined the activity of AKT, a critical mediator of cell survival and migration downstream of the CXCR4 receptor. To determine if blocking CXCR4 with CTCE-9908 could affect AKT activity in the mouse tumors, we measured the expression of phosphorylated (p)-AKT in relation to total AKT in five representative tumors per dosing group. Although not statistically significant, a 14% reduction in expression of p-AKT/AKT was observed with CTCE-9908-50 (p = 0.69), while a 30% reduction in expression was seen with CTCE-9908-100 in comparison to the scrambled control peptide (p = 0.84). Thus, the p-AKT/AKT expression ratio may reflect the effectiveness of CTCE-9908 blockade of the CXCR4 receptor on the primary mammary tumor in our mouse model (Supporting Information Figs. S4a and 4b).

Effect of CXCR4 antagonist on mammary tumor metastasis

We tested the effect of each dose of CTCE-9908 on the formation of pulmonary metastasis. The number of visible lung nodules were counted at necropsy, and a very strong correlation was identified between the number of macroscopic nodules and microscopic metastatic lesions (ρ = 1.0; p = 0.02) in five representative mice, confirming that the nodules identified grossly could be used as surrogate markers for total metastatic tumor burden in the lungs. We found that mice treated with 25 mg/kg of CTCE-9908 showed a 32% decrease in the number of macroscopic lung nodules in comparison with control (p = 0.38). Administration of 50 and 100 mg/kg of CTCE-9908 resulted in similar effects of approximately 40% inhibition of lung metastasis (p = 0.07; p = 0.23, respectively) (Fig. 3).

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Figure 3. CTCE-9908 inhibits metastatic tumor development in MMTV-PyMT transgenic mice. (a) Average number of visible tumor nodules in lungs obtained at necropsy from 8 to 16 mice in each treatment group. p-values for comparisons between CTCE-9908-25 versus control, p = 0.38; CTCE-9908-50 versus control, p = 0.07; CTCE-9908-100 versus control, p = 0.23. Error bars refer to standard error of the mean. (b) Whole lung from mouse treated with vehicle control. (c) Whole lung from mouse treated with 25 mg/kg of CTCE-9908. (d) Lung tissue section from control treated mouse stained with hematoxylin and eosin, showing microscopic tumor deposits within the hatched area. (e) Lung tissue section from mouse treated with CTCE-9908, stained with hematoxylin and eosin, without tumor deposits.

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CTCE-9908 combines with docetaxel to further delay primary tumor growth

Biologic agents do not show clinical efficacy when administered alone. Moreover, the administration of chemotherapeutic agents, and especially, taxanes, has shown to result in a compensatory mobilization of endothelial precursor cells mediated by SDF-1 increase in plasma.21 Therefore, we combined CTCE-9908 with a known cytotoxic chemotherapeutic agent of the taxane family, docetaxel, which is one of the most active agents currently used against breast cancer. Both docetaxel and the combination of docetaxel and CTCE-9908 resulted in similar marked delays in primary tumor growth (Fig. 4a). However at necropsy, we found a 19% decrease in tumor volume with docetaxel alone (p = 0.28), while the combination of CTCE-9908 and docetaxel resulted in an enhanced effect, with a 38% decrease in tumor volume (p = 0.02) (Fig. 4b). Thus, CTCE-9908 can further enhance and sustain the inhibitory effect of docetaxel upon primary mammary tumor growth. Interestingly, the two treatment groups also had a remarkable impact upon metastasis. Docetaxel alone had a marked effect upon distant metastasis, with a 91% decrease in the number of visible nodules (p = 0.0003). This suggests that the dose of docetaxel used was a therapeutic dose for this model. The combination of docetaxel and CTCE-9908 resulted in a similar 83% reduction in the number of metastasis when compared to control (p = 0.002).

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Figure 4. CTCE-9908 combines with docetaxel to further inhibit primary tumor growth in MMTV-PyMT transgenic mice. (a) Growth curves of primary mammary tumors, representing the sum of the volume of 10 tumors from the different fat pads of each mouse, measured twice weekly with calipers. A total of 8–16 mice were treated with control scrambled peptide, docetaxel or a combination of docetaxel and CTCE-9908. The p-values refer to the significance of the differences between the control and the combination of docetaxel and CTCE-9908 at various time points (from 2.5 to 5 weeks). (b) Sums of the volume of 10 primary tumors per mouse measured at necropsy in mice treated with different doses of control scrambled peptide, docetaxel or a combination of docetaxel and CTCE-9908. p-values for comparisons between docetaxel versus control, p = 0.28; combination of docetaxel and CTCE-9908-25 versus control, p = 0.02; combination of docetaxel and CTCE-9908-25 versus docetaxel, p = 0.33. Error bars refer to standard error of the mean.

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CTCE-9908 combines with DC101 to inhibit primary tumor volume and distant metastasis

Due to the role of SDF-1 in promoting endothelial cell proliferation and tumor angiogenesis,4, 22, 23 and our results of decreased VEGF expression with anti-CXCR4 treatment, we combined CTCE-9908 with an anti-angiogenic agent, the anti-VEGFR2 antibody DC101 (Imclone), in our transgenic mouse model. We first tested DC101 at 1000 μg/dose, the average dose previously used in xenograft breast cancer mouse models,24, 25 in conjunction with CTCE-9908 at 25 mg/kg. At necropsy, DC101 treatment alone inhibited tumor growth by 39% (p = 0.05), whereas the combination of DC101 and CTCE-9908-25 resulted in a 48% reduction in primary tumor volume (p = 0.01) compared to treatment with scrambled peptide, representing the greatest anti-tumor effect we observed in this mouse model. No statistically significant reduction in pulmonary metastasis was noted with the combination of DC101 and CTCE-9908-25 compared to DC101 alone (data not shown). To better uncover an effect of CTCE-9908 on the activity of DC101, we increased the dose of CTCE-9908 to 50 mg/kg (maximum efficacy as shown above), and decreased DC101 to 400 ug/dose, a dose that has also been used in mouse models of different cancer types.26, 27 Our necropsy measurements showed that DC101 treatment alone was much less effective in inhibiting tumor growth (13% decrease in primary tumor volume compared to scrambled peptide, p = 0.24). However, the combination of DC101 and CTCE-9908-50 resulted in a 37% decrease in primary tumor volume (p = 0.01) compared to the scrambled peptide (Fig. 5a). This combination also resulted in an impact upon distant metastasis. In particular, there was a remarkable 75% inhibition in the number of visible lung metastasis (p = 0.009), compared to 58% with DC101 alone (p = 0.09) (Fig. 5b). Thus, we identified an enhancing effect of CTCE-9908 when combined with DC101 in inhibiting primary tumor growth and lung metastasis.

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Figure 5. CTCE-9908 combines with the anti-VEGFR2 antibody, DC101, to inhibit primary tumor growth in MMTV-PyMT mice with 7–9 mice per group. (a) Bar graph showing the average primary tumor volumes at necropsy in mice treated with control scrambled peptide, DC101 alone, and DC101 with CTCE-9908. p-values for the comparison of DC101 and control, p = 0.24; comparison of combination and DC101 and CTCE-9908 versus control, p = 0.01; comparison of DC101 versus combination of DC101 and CTCE-9908, p = 0.14. (b) Bar graph showing the average number of macroscopic lung metastases at necropsy in mice treated with control scrambled peptide, DC101 alone and DC101 with CTCE-9908. p-values for the comparison of DC101 and control, p = 0.09; comparison of combination of DC101 and CTCE-9908 versus control, p = 0.009; comparison of DC101 versus combination of DC101 and CTCE-9908, p = 0.47. Error bars refer to standard error of the mean.

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Toxicity of CTCE-9908 alone and in combination

The toxicity of CTCE-9908 alone and in combination was documented by the animals' weight prior to and post administration of the drug. No change in percentage of weight gain was noted amongst the three different doses of CTCE-9908 given alone or in combination with docetaxel or DC101, in comparison to control. Furthermore, no macroscopic changes were observed in other organs such as liver, spleen, kidney and spine.

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The SDF-1/CXCR4 ligand/receptor pair has been shown to play a critical role in many aspects of breast tumorigenesis. Although initially implicated as a key regulator of metastasis, and specifically, of the extravasation of circulating tumor cells into target metastatic organs, this ligand/receptor pair also plays a role in primary tumor growth. This may be due to the role of SDF-1/CXCR4 in recruiting endothelial precursor cells for neo-angiogenesis4 and to its transactivation of the HER2 signaling pathway.28 Various preclinical approaches have been used to inhibit SDF-1/CXCR4 activity in primary breast tumor growth and metastasis. These modalities, including siRNA knockdown, a neutralizing anti-CXCR4 mouse monoclonal antibody, a polypeptide inhibitor of CXCR4, TN14003, or a bicyclam inhibitor, AMD3100, demonstrated either inhibition of primary tumor or metastasis, or prolonged survival in animal models.2, 5, 6 The shortcoming of these studies is that they either used agents that may not be administered in the clinic (such as TN14003) or that these agents were administered in ways that are not analogous to clinical treatment. We selected CTCE-9908, not only due to its efficacy previously demonstrated in the inhibition of metastasis in pre-clinical mouse models, but also due to the excellent safety profile observed in patients.8, 12 CTCE-9908 is the only CXCR4 antagonist that has been tested in a Phase I/II clinical trial in cancer patients and has demonstrated no major adverse effects in these patients.

We selected the PyMT model as it allowed us to simultaneously test the efficacy of anti-CXCR4 therapy on primary tumor growth and metastatic tumor formation and growth, both of which are likely dependent on CXCR4 activity. First, we found that CTCE-9908 slowed the rate of growth of the primary tumor in the PyMT model. Administration of CTCE-9908 at 50 or 100 mg/kg resulted in a delay in tumor growth first observed after 2.5 weeks of treatment, with the maximal effect at 3.5 weeks, whereby both 50 mg/kg and 100 mg/kg of CTCE-9908 inhibited tumor growth by about 50%. Although the effect of these two doses also demonstrated a similar trend at necropsy, it was less pronounced, suggesting that the tumors were beginning to escape the inhibitory effect of the drug. The inhibitory effect on tumor growth was associated with a decrease in VEGF expression in the primary tumors at the time of necropsy. This is in agreement with previous studies, which showed that tumor secretion of VEGF was reduced with anti-CXCR4 therapy.9, 20

The administration of CTCE-9908 resulted in a 30–40% decrease in lung metastasis at the time of necropsy of transgenic mice. This effect was not statistically significant in our study, and indeed, is a less dramatic effect than reported previously for this CXCR4 antagonist in pre-clinical models of breast and other cancers.8, 10 In our opinion, the use of the PyMT model, which expresses a strong genetic drive for tumor progression, makes therapeutic intervention even more challenging. However, one of the drawbacks of this model is the variability in tumor size and metastasis development within each treatment group, which may explain the weak statistical significance. Moreover, unlike all previous studies with CXCR4 antagonists in breast cancer,2, 5, 6, 10, 11 we administered the CXCR4 antagonist only when the tumor was palpable, in an attempt to determine its potential use in a clinical setting similar to that of breast cancer patients. We believe that the stringent conditions due to the selection of a transgenic mouse model as well as a more clinically relevant dosing schedule may more effectively screen novel therapeutics before human clinical trials are initiated.7

Recent experience has shown that the use of biological agents or targeted therapy alone has not been met with great clinical success in the treatment of both primary and metastatic cancers. The clinical utility of these agents is often dependent on their combination with a cytotoxic agent. Moreover, it may be more effective to combine biological agents so as to target more than one molecular factor involved in the growth and propagation of tumors. Indeed, a new rationale was recently proposed for combining anti-angiogenic therapy with anti-metastatic agents, since anti-VEGF treatment was shown to enhance the metastatic potential of tumors.29–31 Also, Shaked et al. reported that the administration of paclitaxel resulted in the mobilization of endothelial precursor cells from the bone marrow, mediated by SDF-1, the ligand for CXCR4.21 We found that the administration of CTCE-9908 further enhanced the inhibitory effect of docetaxel, a cytotoxic agent. Interestingly, CTCE-9908 was also recently shown to induce mitotic catastrophe by dysregulating the G2/M cell cycle checkpoint in ovarian cancer cells in-vitro. In these cells, the combination of CTCE-9908 and paclitaxel led to additive cytotoxicity, similar to our results.32 The combination of CTCE-9908 at 50 mg/kg and DC101 resulted in a remarkable 75% decrease in lung metastases, underlining the significance of targeting angiogenesis in order to inhibit the metastatic process. Moreover, we were able to demonstrate a maximal inhibitory effect upon metastasis using a lower dose of DC101. This is particularly important since anti-VEGF therapies, such as bevacizumab, are associated with significant toxicity, including mortality, in patients.33, 34 Therefore, our combinatorial drug approach may allow for a decrease in the dosing of an anti-VEGF therapy without significantly compromising overall efficacy. We also observed the greatest sustained tumor inhibition in our transgenic model when CTCE-9908 was combined with DC101 treatment at 1000 μg/dose (48% tumor inhibition at necropsy), suggesting that combining two “biologic” therapies may result in similar efficacy to combining a single biologic with a cytotoxic chemotherapeutic agent. Such a finding is important, because of the increased toxicities resulting from presently used combinations of cytotoxic chemotherapies with targeted therapies.35 In summary, we have demonstrated that treatment with CTCE-9908 delays tumor growth and impacts metastasis in the PyMT model using a clinically relevant treatment strategy, and can be combined with either docetaxel or DC101 to reduce tumor burden and inhibit metastasis, showing potential for future clinical development in breast cancer. Our findings provide a roadmap for the design of clinical trials for anti-CXCR4 agents in breast cancer.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank the Canadian Surgery Research Fund Operating Grant 2006 and Chemokine Therapeutics Corp for funding support of experiments. CTCE-9908 was graciously provided by Chemokine Therapeutics Corp (Vancouver, British Columbia), and the animal experiments were partially funded by Chemokine Therapeutics. They also thank Véronique Michaud and Rob Schamborski, Animal Quarters, Lady Davis Institute, for their technical assistance.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • 1
    Weinberg RA. The biology of cancer. New York: Garland Science, 2007. 591 p.
  • 2
    Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN, Barrera JL, Mohar A, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 2001; 410: 506.
  • 3
    Salvucci O, Bouchard A, Baccarelli A, Deschenes J, Sauter G, Simon R, Bianchi R, Basik M. The role of CXCR4 receptor expression in breast cancer: a large tissue microarray study. Breast Cancer Res Treat 2006; 97: 27583.
  • 4
    Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem R, Carey VJ, Richardson AL, Weinberg RA. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 2005; 121: 33548.
  • 5
    Liang Z, Wu T, Lou H, Yu X, Taichman RS, Lau SK, Nie S, Umbreit J, Shim H. Inhibition of breast cancer metastasis by selective synthetic polypeptide against CXCR4. Cancer Res 2004; 64: 43028.
  • 6
    Smith MC, Luker KE, Garbow JR, Prior JL, Jackson E, Piwnica-Worms D, Luker GD. CXCR4 regulates growth of both primary and metastatic breast cancer. Cancer Res 2004; 64: 860412.
  • 7
    Sharpless NE, Depinho RA. The mighty mouse: genetically engineered mouse models in cancer drug development. Nat Rev Drug Discov 2006; 5: 74154.
  • 8
    Kim SY, Lee CH, Midura BV, Yeung C, Mendoza A, Hong SH, Ren L, Wong D, Korz W, Merzouk A, Salari H, Zhang H, et al. Inhibition of the CXCR4/CXCL12 chemokine pathway reduces the development of murine pulmonary metastases. Clin Exp Metastasis 2008; 25: 20111.
  • 9
    Porvasnik S, Sakamoto N, Kusmartsev S, Eruslanov E, Kim WJ, Cao W, Urbanek C, Wong D, Goodison S, Rosser CJ. Effects of CXCR4 antagonist CTCE-9908 on prostate tumor growth. Prostate 2009; 69: 14609.
  • 10
    Huang EH, Singh B, Cristofanilli M, Gelovani J, Wei C, Vincent L, Cook KR, Lucci A. A CXCR4 antagonist CTCE-9908 inhibits primary tumor growth and metastasis of breast cancer. J Surg Res 2009; 155: 2316.
  • 11
    Richert MM, Vaidya KS, Mills CN, Wong D, Korz W, Hurst DR, Welch DR. Inhibition of CXCR4 by CTCE-9908 inhibits breast cancer metastasis to lung and bone. Oncol Rep 2009; 21: 7617.
  • 12
    Wong D, Korz W. Translating an antagonist of chemokine receptor CXCR4: from bench to bedside. Clin Cancer Res 2008; 14: 797580.
  • 13
    Lin EY, Jones JG, Li P, Zhu L, Whitney KD, Muller WJ, Pollard JW. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. Am J Pathol 2003; 163: 211326.
  • 14
    Wang Z, Storm DR. Extraction of DNA from mouse tails. Biotechniques 2006; 41: 410, 12.
  • 15
    Kim LS, Price JE. Clinically relevant metastatic breast cancer models to study chemosensitivity. Methods Mol Med 2005; 111: 28595.
  • 16
    Wong D, Korz W, Salari H. Anticancer effect of a combination of CXCR4 antagonist CTCE-9908 and Docetaxel in a murine model of human prostate cancer. In: Proceedings of the AACR/NCI/EORTC Molecular Targets and Cancer Therapeutics: Discovery, Biology, and Clinical Applications, San Francisco, California. October 22–26, 2007. (Abstract C20).
  • 17
    Dykes DJ, Bissery MC, Harrison SD,Jr., Waud WR. Response of human tumor xenografts in athymic nude mice to docetaxel (RP 56976, Taxotere). Invest New Drugs 1995; 13: 111.
  • 18
    Lifsted T, Le Voyer T, Williams M, Muller W, Klein-Szanto A, Buetow KH, Hunter KW. Identification of inbred mouse strains harboring genetic modifiers of mammary tumor age of onset and metastatic progression. Int J Cancer 1998; 77: 6404.
  • 19
    Bachelder RE, Wendt MA, Mercurio AM. Vascular endothelial growth factor promotes breast carcinoma invasion in an autocrine manner by regulating the chemokine receptor CXCR4. Cancer Res 2002; 62: 72036.
  • 20
    Liang Z, Brooks J, Willard M, Liang K, Yoon Y, Kang S, Shim H. CXCR4/CXCL12 axis promotes VEGF-mediated tumor angiogenesis through Akt signaling pathway. Biochem Biophys Res Commun 2007; 359: 71622.
  • 21
    Shaked Y, Henke E, Roodhart JM, Mancuso P, Langenberg MH, Colleoni M, Daenen LG, Man S, Xu P, Emmenegger U, Tang T, Zhu Z, et al. Rapid chemotherapy-induced acute endothelial progenitor cell mobilization: implications for antiangiogenic drugs as chemosensitizing agents. Cancer Cell 2008; 14: 26373.
  • 22
    Salcedo R, Wasserman K, Young HA, Grimm MC, Howard OM, Anver MR, Kleinman HK, Murphy WJ, Oppenheim JJ. Vascular endothelial growth factor and basic fibroblast growth factor induce expression of CXCR4 on human endothelial cells: In vivo neovascularization induced by stromal-derived factor-1alpha. Am J Pathol 1999; 154: 112535.
  • 23
    Feil C, Augustin HG. Endothelial cells differentially express functional CXC-chemokine receptor-4 (CXCR-4/fusin) under the control of autocrine activity and exogenous cytokines. Biochem Biophys Res Commun 1998; 247: 3845.
  • 24
    Fenton BM, Paoni SF, Ding I. Pathophysiological effects of vascular endothelial growth factor receptor-2-blocking antibody plus fractionated radiotherapy on murine mammary tumors. Cancer Res 2004; 64: 57129.
  • 25
    Rakhmilevich AL, Hooper AT, Hicklin DJ, Sondel PM. Treatment of experimental breast cancer using interleukin-12 gene therapy combined with anti-vascular endothelial growth factor receptor-2 antibody. Mol Cancer Ther 2004; 3: 96976.
  • 26
    Wang ES, Teruya-Feldstein J, Wu Y, Zhu Z, Hicklin DJ, Moore MA. Targeting autocrine and paracrine VEGF receptor pathways inhibits human lymphoma xenografts in vivo. Blood 2004; 104: 2893902.
  • 27
    Zhang L, Hannay JA, Liu J, Das P, Zhan M, Nguyen T, Hicklin DJ, Yu D, Pollock RE, Lev D. Vascular endothelial growth factor overexpression by soft tissue sarcoma cells: implications for tumor growth, metastasis, and chemoresistance. Cancer Res 2006; 66: 87708.
  • 28
    Cabioglu N, Summy J, Miller C, Parikh NU, Sahin AA, Tuzlali S, Pumiglia K, Gallick GE, Price JE. CXCL-12/stromal cell-derived factor-1alpha transactivates HER2-neu in breast cancer cells by a novel pathway involving Src kinase activation. Cancer Res 2005; 65: 64937.
  • 29
    Loges S, Mazzone M, Hohensinner P, Carmeliet P. Silencing or fueling metastasis with VEGF inhibitors: antiangiogenesis revisited. Cancer Cell 2009; 15: 16770.
  • 30
    Ebos JM, Lee CR, Cruz-Munoz W, Bjarnason GA, Christensen JG, Kerbel RS. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell 2009; 15: 2329.
  • 31
    Paez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, Vinals F, Inoue M, Bergers G, Hanahan D, Casanovas O. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell 2009; 15: 22031.
  • 32
    Kwong J, Kulbe H, Wong D, Chakravarty P, Balkwill F. An antagonist of the chemokine receptor CXCR4 induces mitotic catastrophe in ovarian cancer cells. Mol Cancer Ther 2009; 8: 1893905.
  • 33
    Jain RK, Duda DG, Clark JW, Loeffler JS. Lessons from phase III clinical trials on anti-VEGF therapy for cancer. Nat Clin Pract Oncol 2006; 3: 2440.
  • 34
    Cannistra SA. Challenges and pitfalls of combining targeted agents in phase I studies. J Clin Oncol 2008; 26: 36657.
  • 35
    Popat S, Smith IE. Therapy Insight: anthracyclines and trastuzumab--the optimal management of cardiotoxic side effects. Nat Clin Pract Oncol 2008; 5: 32435.

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_25665_sm_suppfig-S1.eps681KSupplementary Figure S1. Chemical structures of CTCE-9908 and control scrambled peptide. (a) Structure of CTCE-9908. (b) Structure of control scrambled peptide, SC-9908.
IJC_25665_sm_suppfig-S2.eps395KSupplementary Figure S2. Dosing schedules for different treatment cohorts in MMTV-PyMT mice. (a) Dosing schedule for CTCE-9908 alone trial. (b) Dosing schedule for the combination therapy of CTCE-9908 and docetaxel. Note that docetaxel is started prior to initiation of CTCE-9908 therapy. (c) Dosing schedule for the combination therapy of CTCE-9908 and DC101.
IJC_25665_sm_suppfig-S3.eps6380KSupplementary Figure S3. CXCR4 immunohistochemical staining of mammary tumors from MMTV-PyMT mice. (a), (b) CXCR4 staining of primary mammary tumor.
IJC_25665_sm_suppfig-S4.eps1627KSupplementary Figure S4. Administration of CTCE-9908 to MMTV-PyMT transgenic mice decreases phosphorylated-AKT expression in mammary tumors. (a) Western blot of lysates from mammary tumors from mice treated with the 3 doses of CTCE-9908 as well as with scrambled peptide, showing increasing inhibition of phosphorylated-AKT expression with increasing dose. Total AKT protein expression is used as control in lower row. (b) Bar graph of relative levels of expression of phosphorylated-AKT to total AKT protein in mammary tumors obtained at necropsy from 5 mice from each treatment group. P-values for comparisons between CTCE-9908-25 versus control, P = 0.69; CTCE-9908-50 versus control, P = 0.69; CTCE-9908-100 versus control P = 0.84. Error bars refer to standard error of the mean.

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