Sorafenib and Quinacrine Target Anti-Apoptotic Protein MCL1: A Poor Prognostic Marker in Anaplastic Thyroid Cancer (ATC)

Abstract

Purpose and experimental design: Anaplastic thyroid cancer (ATC) comprises approximately 2% of all thyroid cancers, and its median survival rate remains poor. It is responsible for more than one third of thyroid cancer-related deaths. ATC is frequently resistant to conventional therapy, and NFκB signaling has been proposed to be a feature of the disease. We aimed to assess the activity of the antimalaria drug quinacrine known to target NFκB signaling in combination with the clinically relevant kinase inhibitor sorafenib in ATC cells. The presence of NFκB-p65/RELA and its target MCL1 was demonstrated in ATC by meta-data gene set enrichment analysis and IHC. We assessed the responses of a panel of human ATC cell lines to quinacrine and sorafenib in vitro and in vivo RESULTS: We detected increased expression of NFκB-p65/RELA and MCL1 in the nucleus of a subset of ATC compared with non-neoplastic thyroid. ATC cells were found to respond with additive/synergistic tumor cell killing to the combination of sorafenib plus quinacrine in vitro, and the drug combination improves survival of immunodeficient mice injected orthotopically with ATC cells as compared with mice administered either compound alone or doxorubicin. We also demonstrate that the combination of sorafenib and quinacrine is well tolerated in mice. At the molecular level, quinacrine and sorafenib inhibited expression of prosurvival MCL1, pSTAT3, and dampened NFκB signaling.

Conclusions: The combination of quinacrine and sorafenib targets emerging molecular hallmarks of ATC and shows promising results in clinically relevant models for the disease. Further testing of sorafenib plus quinacrine can be conducted in ATC patients. Clin Cancer Res; 22(24); 6192-203. ©2016 AACR.

Figure 1:. Nuclear NFκB-p65/RelA and Mcl-1 is overexpressed in anaplastic thyroid cancer and may be associated with markers of poor prognosis.

(A) IHC for NFκB-p65/RelA on clinical FFPE non-neoplastic thyroid (NT) and anaplastic thyroid cancer (ATC) specimens. Each panel shows a representative image of a unique individual patient sample with cytoplasmic (Cyto.) and nuclear (Nuc.) expression of NFκB-p65/RelA in ATC or the expression of NFκB-p65/RelA in non-neoplastic thyroid (NT) as detected by IHC. A 400x magnification is shown. (B) Mcl-1 expression in clinical FFPE specimens of NT and ATC. Representative images are shown. (C) Overall survival (OS) of ATC patients with nuclear (Nuc.) and cytoplasmic (Cyto.) expression of NFκB-p65/RelA. The P-value was determined using the Log-rank test. (D) Quantitation of FFPE immunohistochemistry for Mcl-1 in NT (N=11) and ATC tissues (N=11). Medians are shown by black horizontal lines and each data point represents a stained specimen from one patient. Tumors expressing Mcl-1 that had nuclear NFκB-p65/RelA are indicated. The P-value was determined using the Mann-Whitney test. (E) OS of ATC patients in relation to the expression levels of Mcl-1 in their tumors. A “low” and “high” expression level indicates expression (% positive cells in the specimen) below or above the median respectively (as shown in ‘B’). The P-value was determined using the Log-rank test.

Figure 2:. Meta analysis of expression profiles from normal thyroid (NT) tissue and ATC.

(A) Gene set enrichment analysis of clinical meta-data from Giordano et al (Giordano et al 2005). The GSEA analysis was performed for ATC (N=3) and NT (N=3). Gene set enrichment was assessed for the MSigDB gene sets HALLMARK_INFLAMMATORY_RESPONSES (A), HALLMARK_TNF_SIGNALING_VIA_NFKB (B) and N$YKAPPA (genes with RelA binding sites within 2kb of their core promoter) (C).

Figure 3:. Quinacrine and sorafenib synergize in the killing of ATC cells.

Dose-response curves for the 72-hr survival of ATC cell lines THJ-16T, THJ-21T and THJ-29T following sorafenib (A) and quinacrine (B). The X-axis are shown as log scale. (C) Flow cytometric sub-G1 programmed cell death (apoptosis) assay of the ATC cell line 8505C subjected to treatment with sorafenib (S) and quinacrine (Q) and the combination (S/Q). Averages (N=3) +/− SEM are shown. Statistical analysis was performed using Students t-test where P<0.05 was considered statistically significant. Dose-response modulation of the combinatorial response of Q and S. (D) Cell survival assay (CellTiter-Glo®) of ATC cells treated with different combinatorial concentrations of Q and S. A representative figure of the result of a 72-hr Cell Titer-Glo® assay for the ATC cell line THJ-29T is shown. (C) Combinatorial indices generated using the CalcuSyn 2.0 software employing the method by Chou Talalay for S and Q in the sorafenib-refractory ATC cell line THJ-16T. Red indicates ‘strong drug synergism’, blue – ‘drug synergism’, green – ‘moderate drug synergism’.

Figure 4:. Sorafenib (S) and quinacrine (Q) target the anti-apoptotic protein Mcl-1 in ATC cells.

(A) Combination treatment with S and Q triggers inhibition of Stat3 phosphorylation (Stat3PY) in 8505C cells. (B) The effect of S and Q on PARP cleavage, Mcl-1 and Stat3PY expression in 8505C cells. (C) Western blot to analyze impact of S/Q on expression of NFκB-p65/RelA in 8505C and THJ-16T cells. The doses of S and Q used were 10 and 20 µM respectively. (D) Western blot analysis show efficient targeting of p65/RelA in 8505C ATC cells by specific siRNA (p65) (left panel). No effect on p65/RelA is observed by siRNA-mediated targeting of Stat3 (S3 [1+2]) (right panel). Scrambled (Scr) siRNA was used as a control for specific targeting of p65/RelA and Actin was used as a loading control. (E) Western blot analysis show efficient targeting of total Stat3 by two different siRNA (S3[1] and S3[2]) in 8505C cells. A combination of both siRNA (S3[1+2]) was used to achieve efficient knock down of total Stat3. Actin was used as a loading control. Sorafenib (F) and quinacrine (G) dose-response curves following depletion of Stat3 and p65/RelA in 8505C cells using the CellTiter-Glo® assay. The 8505C cells were treated for 72 hrs with the drugs.

Figure 5:. Mcl-1 expression is required in ATC cells for combinatorial efficacy of the sorafenib (S) and quinacrine (Q).

(A) Western blot assessment of the efficacy of small interfering RNA (siRNA)-dependent targeting of Mcl-1 expression in 8505C cells. (B) CellTiter-Glo® cell survival assay. Sorafenib (C) and quinacrine (D) dose-response curves in 8505C cells following siRNA mediated targeting of Mcl-1. The X-axis are shown as log scale. (E) The observed IC₅₀-values following S and Q treatment with and without Mcl-1-targeting. Isobolograms for 8505C cells treated with various effective S and Q concentrations (IC_S80/Q30, IC_S80/Q50 and IC_S80/Q60) in the presence (F) and absence (G) of Mcl-1 knock-down.

Figure 6:. The sorafenib (S) and quinacrine (Q) combination improves the survival of mice subjected to a thyroid orthotopic injection of ATC cells.

(A) Live whole-body near-infrared imaging (NIR) of whole mice (left panel) and tumor engraftment of 8505C cells in the thyroid of a Nu/Nu mouse (right panel). (B) NIR-imaging of a Nu/Nu mouse subjected to thyroid orthotopic injection of CellVue® NIR815-labeled 8505C cells after the removal of the salivary gland. (C) NIR-imaging of a dissected and isolated tumor ATC-xeno-engrafted mouse (Nu/Nu) thyroid. The injected 8505C cells were labeled with CellVue® NIR815-labeled Survival curves of mice subjected to thyroid orthotopic injection of 8505C cells and subjected to no treatment (Control) and sorafenib (D), quinacrine (E), doxorubicin (F) and the combination of S and Q (G). (H) Immunohistochemistry for TUNEL (apoptosis) and Ki-67 (proliferating cells) on mouse thyroid xenograft tumors of the ATC cell line 8505C. (I) Growth signals trigger constitutively active NFκB signaling and Mcl-1 expression in ATC cells. Mcl-1 inhibits the activity of the pro-apoptotic proteins Bak, Bid, Noxa and Bim. In addition, Mcl-1 may also influence cell cycle progression and proliferation (not depicted). (J) Both S and Q inhibit NFκB activity and ameliorate Mcl-1 expression. Selectivity of the compounds is achieved through increased activity of NFκB in ATC’s. Reduced expression of Mcl-1 allows the pro-apoptotic proteins Bak, Bid, Noxa and Bim to trigger cell death (apoptosis) and a therapeutically relevant anti-tumor response in ATC.