Cell-selective inhibition of NF-κB signaling improves therapeutic index in a melanoma chemotherapy model

Abstract

The transcription factor NF-κB promotes survival of cancer cells exposed to doxorubicin and other chemotherapeutic agents. IκB kinase is essential for chemotherapy-induced NF-κB activation and considered a prime target for anticancer treatment. An IκB kinase inhibitor sensitized human melanoma xenografts in mice to killing by doxorubicin, yet also exacerbated treatment toxicity in the host animals. Using mouse models that simulate cell-selective targeting, we found that impaired NF-κB activation in melanoma and host myeloid cells accounts for the therapeutic and the adverse effects, respectively. Ablation of tumor-intrinsic NF-κB activity resulted in apoptosis-driven tumor regression following doxorubicin treatment. By contrast, chemotherapy in mice with myeloid-specific loss of NF-κB activation led to a massive intratumoral recruitment of interleukin-1β-producing neutrophils and necrotic tumor lesions, a condition associated with increased host mortality but not accompanied by tumor regression. Therefore, a molecular target-based therapy may be steered toward different clinical outcomes depending on the drug's cell-specific effects.

Significance: Our findings show that the IκB kinase–NF-κB signaling pathway is important for both promoting treatment resistance and preventing host toxicity in cancer chemotherapy; however, the two functions are exerted by distinct cell type–specific mechanisms and can therefore be selectively targeted to achieve an improved therapeutic outcome.

Keywords: NF-κB; efficacy; melanoma; neutrophil; toxicity.

Figure 1. Doxorubicin-induced NF-κB activation and NF-κB-mediated chemoresistance in Hs944T and Roth human melanoma cells

A, Cytoplasmic (Cyto) and nuclear (Nuc) extracts from Hs944T and Roth cells treated with doxorubicin (Dxr; 2 μg/ml) were analyzed by immunoblotting with anti-RelA antibody. Values in the plot represent relative amounts of nuclear RelA. B, Hs944T and Roth cells were treated with doxorubicin and BMS (75 μM; added 1 h prior to doxorubicin treatment) as indicated. Cyto and Nuc extracts were prepared 2 h after doxorubicin treatment and analyzed as in A. C, Hs944T and Roth cells were treated with doxorubicin and BMS as in B. Cell viability was determined by MTT assay 24 h after doxorubicin treatment.

Figure 2. The efficacy and toxicity of doxorubicin and IKK inhibitor treatment in mice harboring Hs944T and Roth tumors

Hs944T and Roth cells were infected with lentivirus expressing luciferase (Luc) and injected s.c. into RAG-2^−/−γc^−/− mice (n=3). After tumors grew to 100 mm³ in volume, mice were administered i.p. with doxorubicin (Dxr; 2 mg/kg) and BMS (125 mg/kg) as indicated on day 0, 2, 4, 6, and 8. A, Tumors were visualized in pseudocolor by bioluminescence imaging on day 10. Representative images from each group are shown along with the reference scale of bioluminescence depicted on the right. B and C, Tumor sizes (B) and the severity of illness (C) were determined on day 10. Triangles and circles indicate values in individual mice. Severity of illness was expressed in scores representing decrease in liveliness (DIL) and grade of emaciation (GOE). D, Tumor sections from the indicated groups were analyzed by TUNEL staining (upper) and immunostaining with Ly6G-specific antibody (lower). The number within each image indicates relative fluorescence intensity (TUNEL and Ly6G signal, respectively) and represents mean ± standard deviation from three independent areas. Apoptotic nuclei (green) and neutrophils (red) are shown together with the counter staining of DNA (blue). Scale bar, 100 μm.

Figure 3. Doxorubicin-induced NF-κB activation and NF-κB-mediated chemoresistance in B16 mouse melanoma cells

A, Cytoplasmic (Cyto) and nuclear (Nuc) extracts from B16 cells treated with doxorubicin (Dxr; 2 μg/ml) were analyzed by immunoblotting with antibodies against the proteins indicated on the left. B, B16 cells stably expressing GFP (B16GFP) and an IκBα “super-repressor” (B16SR) were treated with doxorubicin (2 μg/ml). Whole cell lysates (upper), and Cyto and Nuc extracts (lower) were prepared and analyzed as in A. Anti-IκBα antibody detects both transfected (SR) and endogenous IκBα (endo). C, B16SR and BMS-preincubated B16 cells (SR and BMS, respectively) were treated with doxorubicin (2 μg/ml) for 4 h. Total RNA was isolated and analyzed by quantitative real-time polymerase chain reaction using primers specific to the genes indicated on the left. Values represent percent mRNA relative to that in B16GFP (SR) and DMSO-preincubated B16 (BMS) cells. Expression is relative to that of Ppia (encoding cyclophilin). D, B16GFP and B16SR cells were treated with DMSO and doxorubicin (0.5 μg/ml) as indicated. Shown are viable adherent cells stained with crystal violet 24 h after treatment.

Figure 4. Increased sensitivity to doxorubicin treatment and apoptotic tumor regression resulting from ablation of tumor-intrinsic NF-κB activity

B16GFP and B16SR cells were injected s.c. into C57BL/6 mice (n=8). After tumors grew to 100 mm³ in volume, mice were treated with DMSO (solid line) and doxorubicin (Dxr; dotted line) as in Figure 2. A, Tumor sizes were determined at the indicated time points. Red arrow, time of doxorubicin treatment. **, P < 0.01; *, P < 0.05. B and C, B16GFP and B16SR tumor sections were prepared on day 28 and analyzed by H&E staining (B), TUNEL staining (C, upper), and immunostaining with F4/80- and Ly6G-specific antibodies (C, middle and lower, respectively). The number within each image indicates relative fluorescence intensity (TUNEL, F4/80, and Ly6G signal, respectively) and represents mean ± standard deviation from three independent areas (C). Apoptotic nuclei (green), macrophages (red), and neutrophils (red) are shown together with the counter staining of DNA (blue). Scale bar, 100 μm.

Figure 5. Increased host mortality with necrotic tumor lesions and intratumoral neutrophil infiltration in doxorubicin-treated mice defective in myeloid NF-κB signaling

B16 cells were injected s.c. into WT and IKKβΔM mice (n=34 and 18, respectively). After tumors grew to 100 mm³ in volume, mice were treated with doxorubicin as in Figure 2. A, Tumor sizes (left) and host survival (middle) were determined at the indicated time points. Red arrow, time of doxorubicin treatment. **, P < 0.01. The incidence of necrotic tumor lesions in each group was determined and expressed as percent animals with varying scores (right). Necrotic lesions were scored according to the degree of maximum necrosis throughout the treatment period in each host animal. B and C, Tumor sections were prepared on day 20 and analyzed as in Figure 4 by H&E staining (B), TUNEL staining (C, upper), and immunostaining with F4/80- and Ly6G-specific antibodies (C, middle and lower, respectively). The number within each image indicates relative fluorescence intensity (TUNEL, F4/80, and Ly6G signal, respectively) and represents mean ± standard deviation from three independent areas (C). Scale bar, 100μm. D, Percentage of CD11b⁺Ly6G⁺ cells in tumors of the indicated host mice was determined by flow cytometry.

Figure 6. Inflammatory responses and necrotic tumor lesions driven by neutrophil-derived IL-1β

A–D, Tumors were prepared and analyzed as in Figure 5 by immunostaining (A, C), immunoblotting (B), and flow cytometry (D) with antibodies against IL-1β, F4/80, and Ly6G. E, Whole cell lysates from WT and IKKβΔM neutrophils treated with lipopolysaccharide (LPS; 100 ng/ml) were analyzed by immunoblotting with IL-1β- and actin-specific antibodies. F–H, IKKβΔM mice were administered with doxorubicin (2 mg/kg) alone on day 0, 2, 4, 6, and 8 or in conjunction with anakinra (25 mg/kg) on day 3, 5, 7, 9, 11, and 13. Tumor sections were analyzed as in Figure 5 by H&E staining (F), immunostaining with Ly6G-specific antibodies (G, upper), and TUNEL staining (G, lower). The number within each image indicates relative fluorescence intensity (Ly6G and TUNEL signal, respectively) and represents mean ± standard deviation from three independent areas (G). Scale bar, 100 μm. Serum amounts of IL-1β in the indicated mice were determined on day 14 by ELISA (H).

Figure 7. Clinical outcomes resulting from inhibition of tumor-intrinsic and myeloid NF-κB activation in melanoma chemotherapy

Dark and light gray areas denote tumor and tumor stromal tissues, respectively. The outer unshaded area indicates the circulation. Tumor stromal macrophages (M) and circulating neutrophils (N) are recruited to tumor tissues (T) depending on the cell type in which NF-κB activation is inhibited and the mode of tumor cell death.