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. 2013 Mar;2(3):233-42.
doi: 10.5966/sctm.2012-0096. Epub 2013 Feb 13.

Synergistic effect of the γ-secretase inhibitor PF-03084014 and docetaxel in breast cancer models

Cathy C Zhang  1 , Zhengming Yan, Qing Zong, Douglas D Fang, Cory Painter, Qin Zhang, Enhong Chen, Maruja E Lira, Annette John-Baptiste, James G Christensen
Affiliations

Synergistic effect of the γ-secretase inhibitor PF-03084014 and docetaxel in breast cancer models

Cathy C Zhang et al. Stem Cells Transl Med. 2013 Mar.

Abstract

Notch signaling mediates breast cancer cell survival and chemoresistance. In this report, we aimed to evaluate the antitumor efficacy of PF-03084014 in combination with docetaxel in triple-negative breast cancer models. The mechanism of action was investigated. PF-03084014 significantly enhanced the antitumor activity of docetaxel in multiple xenograft models including HCC1599, MDA-MB-231Luc, and AA1077. Docetaxel activated the Notch pathway by increasing the cleaved Notch1 intracellular domain and suppressing the endogenous Notch inhibitor NUMB. PF-03084014 used in combination with docetaxel reversed these effects and demonstrated early-stage synergistic apoptosis. Docetaxel elicited chemoresistance by elevating cytokine release and expression of survivin and induced an endothelial mesenchymal transition (EMT) phenotype by increasing the expressions of Snail, Slug, and N-cadherin. When reimplanted, the docetaxel-residual cells not only became much more tumorigenic, as evidenced by a higher fraction of tumor-initiating cells (TICs), but also showed higher metastatic potential compared with nontreated cells, leading to significantly shortened survival. In contrast, PF-03084014 was able to suppress expression of survivin and MCL1, reduce ABCB1 and ABCC2, upregulate BIM, reverse the EMT phenotype, and diminish the TICs. Additionally, the changes to the ALDH(+) and CD133(+)/CD44(+) subpopulations following therapy corresponded with the TIC self-renewal assay outcome. In summary, PF-03084014 demonstrated synergistic effects with docetaxel through multiple mechanisms. This work provides a strong preclinical rationale for the clinical utility of PF-03084014 to improve taxane therapy.

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Figures

Figure 1.
Figure 1.
The residual tumor cells display higher tumorigenic and metastatic potential after docetaxel therapy. MDA-MB-231Luc tumor-bearing mice were treated with an efficacious dose of docetaxel (15 mg/kg, i.p.) once per week for three cycles prior to harvesting tumors. Tumor cells were dissociated from the vehicle- and docetaxel-residual tumors (n = 10 each group) for fluorescence-activated cell sorting (FACS) analyses and secondary reimplant. The values are the means ± SEM. (A): Western blot analysis of NUMB and the endothelial mesenchymal transition markers. (B): Representative images of ALDH+ cells via FACS. (C): The percentages of CD133+/CD44+, ALDH+, and CD44+/CD24− subpopulations. For FACS analysis, the isotype control was used for each individual sample. (D, E): The secondary reimplant experiment was performed by implanting cells subcutaneously (D) or intravenously (E) in SCID Beige mice (n = 10 mice per group). A Kaplan-Meier survival plot showed disease progression of the mice with intravenous tumor implant (E). The representative bioluminescence images were collected on day 30 after the reimplant. Abbreviations: ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; Doce, docetaxel; E-Cad, E-cadherin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SSC, side scatter.
Figure 2.
Figure 2.
PF-03084014 exhibits synergistic effects with docetaxel in breast xenograft models. Size-matched tumor-bearing mice were treated as described in Materials and Methods. The docetaxel dose was 15 mg/kg (MDA-MB-231Luc and AA1077) or 25 mg/kg (HCC1599). (A): The dosing schedule for the efficacy and pharmacodynamic assessments. (B–D): PF-03084014 significantly improved the efficacies of docetaxel in the MBA-MB-231Luc (B), HCC1599 (C), and AA1077 (D) tumor models (n = 10 mice per group). The values are the means ± SEM. *, p < .05 versus the treatment group of PF-03084014 or docetaxel alone. Abbreviations: d, days; PD, pharmacodynamic endpoint.
Figure 3.
Figure 3.
Pharmacodynamic assessment of PF-03084014 alone and in combination with docetaxel in breast xenograft models (n = 5 mice per group). The values are the means ± SEM. (A–C): The data were generated using HCC1599 tumors. (A, C): Western blot analysis of biomarkers on day 2 (A) and day 19 (C) after dosing commencement. (B): Notch target gene modulations on day 19 after treatment. (D): Serum levels of IL-6 or IL-8 on day 15 after dosing commencement in multiple models. (E): Changes in the endothelial mesenchymal transition markers and ABC transporters were observed in MDA-MB-231Luc tumor on day 19. Abbreviations: Doce, docetaxel; E-Cad, E-cadherin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PF, PF-03084014; Veh, vehicle.
Figure 4.
Figure 4.
PF-03084014 reduces cancer stem cell frequency in the Notch1mut HCC1599 model (n = 5 mice per group). The values are the means ± SEM. To perform the analyses, tumor cells were dissociated on day 19 after dosing commencement. (A): The representative images in the MMFE test. The tumor-derived cells were seeded at a density of 3,000 cells per well in a 24-well plate for 6 days before analysis. (B): The percentage of ALDH-positive cells. ALDH activity was analyzed by flow cytometry. An aliquot from each sample was tested under identical conditions in the presence of the ALDH inhibitor diethylaminobenzaldehyde. All gates in each sample were created using the DEAB-treated corresponding sample. (C): Quantitative measurement of MMFE, tumor size by serial in vivo transplant, and ALDH+ subpopulation. In vivo self-renewal analysis was performed by implanting 5 × 106 tumor-derived cells per SCID Beige mouse. Abbreviations: ALDH, aldehyde dehydrogenase; Doce, docetaxel; MMFE, mammosphere forming efficiency; PF, PF-03084014; SSC, side scatter.
Figure 5.
Figure 5.
PF-03084014 alone and in combination with docetaxel diminishes the cancer stem cells in MBA-MB-231Luc xenografts. Size-matched tumor-bearing mice were treated as described in Materials and Methods. Tumors were harvested and dissociated on day 19 after dosing commencement to perform fluorescence-activated cell sorting (FACS) and functional analysis (n = 5 mice per group). The values are the means ± SEM. (A): The representative images in the MMFE test. Tumor-derived cells were seeded at a density of 1,500 cells per well in a 24-well plate for 6 days before analysis. (B): The representative bioluminescence imaging images of tumor growth after the secondary implant on day 45. In vivo self-renewal analysis was done by implanting 10 × 104 tumor-derived cells per SCID Beige mouse. (C): Quantitative assessment of the stem cell self-renewal ability. (D): Representative FACS analysis of CD133+/CD44+ cells from each group. Each sample was stained with an isotype control to ensure that nonspecific staining was excluded. (E): Percentage of ALDH+ and CD133+/CD44+ cells in each treatment group. Abbreviations: Doce, docetaxel; Max, maximum; Min, minimum; MMFE, mammosphere forming efficiency.
Figure 6.
Figure 6.
The tumorigenicity of CD133+/CD44+ cells derived from MDA-MB-231Luc tumors. (A): FACS analysis demonstrated that the CD133+/CD44+ subpopulation was enriched after a 3-week treatment of docetaxel (15 mg/kg). (B): Study design for the isolation and reimplant of CD133+/CD44+ and CD133−/CD44− cells. The cells were isolated and sorted after docetaxel treatment. The cell doses for reimplant were 10, 100, 1,000, and 10,000 cells for each of the experimental groups (10 mice per group). (C): The tumorigenic frequency of CD133+/CD44+ or CD133−/CD44− subpopulations. (D): The representative bioluminescence imaging images of the tumor-bearing mice transplanted with 10,000 cells. Abbreviations: FACS, fluorescence-activated cell sorting; Tx, treatment; SOC, standard of care agent.
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References

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