ER maleate is a novel anticancer agent in oral cancer: implications for cancer therapy

Abstract

ER maleate [10-(3-Aminopropyl)-3, 4-dimethyl-9(10H)-acridinone maleate] identified in a kinome screen was investigated as a novel anticancer agent for oral squamous cell carcinoma (OSCC). Our aim was to demonstrate its anticancer effects, identify putative molecular targets and determine their clinical relevance and investigate its chemosensitization potential for platinum drugs to aid in OSCC management. Biologic effects of ER maleate were determined using oral cancer cell lines in vitro and oral tumor xenografts in vivo. mRNA profiling, real time PCR and western blot revealed ER maleate modulated the expression of polo-like kinase 1 (PLK1) and spleen tyrosine kinase (Syk). Their clinical significance was determined in oral SCC patients by immunohistochemistry and correlated with prognosis by Kaplan-Meier survival and multivariate Cox regression analyses. ER maleate induced cell apoptosis, inhibited proliferation, colony formation, migration and invasion in oral cancer cells. Imagestream analysis revealed cell cycle arrest in G2/M phase and increased polyploidy, unravelling deregulation of cell division and cell death. Mechanistically, ER maleate decreased expression of PLK1 and Syk, induced cleavage of PARP, caspase9 and caspase3, and increased chemosensitivity to carboplatin; significantly suppressed tumor growth and increased antitumor activity of carboplatin in tumor xenografts. ER maleate treated tumor xenografts showed reduced PLK1 and Syk expression. Clinical investigations revealed overexpression of PLK1 and Syk in oral SCC patients that correlated with disease prognosis. Our in vitro and in vivo findings provide a strong rationale for pre-clinical efficacy of ER maleate as a novel anticancer agent and chemosensitizer of platinum drugs for OSCC.

Keywords: ER maleate; OSCC; Syk / PLK1; anticancer agent; tumor xenografts.

Figure 1. ER maleate inhibited cell proliferation, survival, spheroid formation and colony formation in OSCC cells

A. The chemical structure of ER maleate. B. ER maleate showed cytotoxic effect using three doses (4μM, 1.33μM and 0.44μM) in SCC4, Cal33, HSC2 and MDA1986 cells from second round validation of 48 inhibitors (15). C&D. ER maleate inhibited cell proliferation in a dose dependent manner (0-5 μM) in SCC4 cells (C) and Cal33 cells (D) by MTT assay. E. ER maleate (0-2 μM) enhanced carboplatin (0-50 μM) inhibited cell proliferation in SCC4 cells by MTT assay. F. Spheroid formation. ER maleate incubation with SCC4 cells decreased the cell density and size of spheroids in a dose dependent manner (0-2 μM) and failed to form the spheroid at 5μM (lower panel). Representative SCC4 cell spheroid is shown from each group (upper panel). Data are represented as mean ±SEM relative to the control from three independent experiments. G. Colony formation assay. SCC4 cells were treated with ER maleate (0.5 – 1 μM), carboplatin (CBP, 25μM) or in combination of ER maleate and carboplatin for 9 days. Colonies formed were stained and counted. Histogram analysis showed a significant reduction in colony forming ability in ER maleate treated cells with a further reduction in combination of ER maleate and carboplatin treatment (lower panel). Representative stained colonies were shown from each group (upper panel). Data are represented as mean ±SEM relative to the control from three independent experiments. Treatment groups denoted by different letters represent a significant difference at p<0.05 (ANOVA followed by Fisher's LSD test).

Figure 2. ER maleate inhibited cell invasion and migration potential, and modulated the expression of TIMP-MMPs in OSCC cells

A. ER maleate significantly inhibited invasive capability of SCC4 cells in a dose dependent manner (0 – 2 μM) after 24 h incubation by transwell invasion assay. Bar graphs show the decrease in invaded cell number with ER maleate treatment in a dose dependent manner. B. ER maleate significantly suppressed cell migration to the wound area in SCC4 cells in comparison with vehicle control cells in 24 h by wound healing assays. Histogram analysis showing significantly low number of cells in wound of ER maleate treated cells. C. ER maleate treatment decreased the expression of MMP-1, MMP-10, MMP-12 and MMP-13, while TIMP-2 expression increased with no significant change in TIMP-1 at the mRNA level in SCC4 cells analyzed by illumine mRNA profiles. The bar graph data presented as mean ± SEM; groups denoted by different letters represent a significant difference at p < 0.05(ANOVA followed by Fisher's LSD test).

Figure 3. ER maleate induced apoptosis in OSCC cells by Annexin-V and 7-ADD double staining assay

A. A significant increase in cell apoptosis/death was observed in SCC4 cells on treatment with ER maleate (2μM), or CBP (25μM) alone, or their combination for 24h, 48h and 72h, respectively. CBP treatment induced apoptotic cell population and this induction was further enhanced by combining with ER maleate. B. Histogram showed the change in apoptotic cell percentage of SCC4 cells on treatment with ER maleate (2μM), or CBP (25μM) alone or their combination. C. An increase in apoptosis was also observed in Cal33 cells on treatment with ER maleate, or CBP (25μM) alone or their combination for 24h, 48h and 72h, respectively. CBP treatment induced apoptotic cell population and this induction was further enhanced by combining with ER maleate. D. Histogram showed the change in apoptotic cell percentage of Cal33 cells on treatment with ER maleate (2μM), or CBP (25μM) alone or their combination. The bar graph data were presented as mean ± SEM; groups denoted by different letters represent a significant difference at p < 0.05 (ANOVA followed by Fisher's LSD test).

Figure 4. ER maleate induced cleavage of PARP, caspase9 and caspase3 in OSCC cells

A, B. ER maleate treatment for 24 h induced the expression of cleaved PARP, and caspase3 and reduced levels of full length caspase 9 and caspase 3 in SCC4 (A) and Cal33 cells (B) by western blot analysis. C. ER maleate induced Bad mRNA expression in both SCC4 and Cal33 cells at 24 h. The bar graph data were presented as mean ± SEM; groups denoted by different letters represent a significant difference at p < 0.05 (ANOVA followed by Fisher's LSD test).

Figure 5. ER maleate arrested cell in G2 phase and induced polyploid population

A. FACS analysis of SCC4 cells. ER maleate treatment decreased the diploid fraction from 95.42% to 70.82%, whereas it increased polyploid population from 4.58% to 29.18% in a dose dependent manner (0 – 2 μM, Supplementary Table S1). For the diploid cells, cell population in G₂ phase was increased from 15.37% to 43.44% and in G₁ phase decreased from 46.11% to 16.56% in a dose dependent manner (0 – 2 μM) with ER maleate treatment of SCC4 cells for 48 h through Modfit analysis. For the polyploid cell population, most cells (99.68%) accumulate in S phase on treatment with ER maleate at 2μM. B. FACS analysis of Cal33 cells. ER maleate decreased the diploid population from 100% to 43.86% but increased the polyploid population from 0 to 56.14% in Cal33 cells with ER maleate treatment (0-2 μM) for 48 h (Supplementary Table S2).

Figure 6. ImageStream FACS of OSCC cells

A. Imagestream and Ideas program analysis showed ER maleate induced the tetraploid/anueploid (polyploid) cell population in SCC4 cell. Two dimensional plot shows the cell size and DNA content (right panel) and histogram shows cell DNA content (left panel) in SCC4 cells at 24 h (Upper panel) and 48 h (Bottom panel); B. Imagestream nuclear morphology of SCC4. Cell nuclei stained with PI and run on Amnis Imagestream MKII reveal a significant increase in tetraploid/anueploid (polyploid) cell population in SCC4 cells; C. Imagestream and Ideas program analysis showed ER maleate induced the tetraploid/anueploid (polyploid) cell population in Cal33 cell. Two dimensional plot shows the cell size and DNA content (Right panel) and histogram shows cell DNA content (Left panel) in Cal33 cells at 24 h (Upper panel) and 48 h (Bottom panel). D. Imagestream nuclear morphology of Cal33. Cell nuclei stained with PI and run on Amnis Imagestream MKII reveal a significant increase in tetraploid/anueploid (polyploid) cell population in Cal33 cells.

Figure 7. ER maleate inhibits the expression of Syk, PLK1, and CHEK2 and modulates PI3K/Akt signaling in OSCC cells

A–D. Illumin mRNA profiling revealed ER maleate down-regulated gene expression of Syk (A), PLK1 (B), and CHEK2 (C) at mRNA level, but not PLK4 expression (D) in both SCC4 and Cal33 cells. E–J. Real-time PCR quantification showed the expression of Syk and PLK1was decreased in a dose dependent manner in SCC4 (E, F) and Cal33 cells (H, I) without significant change in PLK4 (G, J) in both SCC4 and Cal33 cells. The bar graph data were presented as mean ± SEM; groups denoted by different letters represent a significant difference at p < 0.05 (ANOVA followed by Fisher's LSD test). K, L. The expression of PLK1, Syk and Cyclin D1 was decreased in a dose dependent manner in SCC4 (K) and Cal33 cells (L) after treatment with ER maleate (0-2 μM) with or without CBP (25μM) for 48 h. Both phosphorylation level of Akt and the expression of total Akt were suppressed with ER maleate treatment in SCC4 (K) and Cal33 cells (L). EGFR kinase was also decreased at protein level by ER maleate in SCC4 (K) and Cal33 cells (L). GAPDH served as a loading control.

Figure 8. PLK1 siRNA mimics ER maleate effect on inhibition of cell viability, and PLK1 and Syk partially rescue ER maleate-reduced cell viability

A, B. PLK1 siRNA transfection decreased cell viability in SCC4 (A) and Cal33 cells (B). Inset: siRNA mediated knockdown of PLK1 expression was shown in both cells by western blot (A, B). β-actin served as a loading control. C–F. Overexpression of PLK1 and Syk increased cell viability in a dose dependent manner in SCC4 (C, E) and Cal33 cells (D-F) and partially rescued ER maleate (1 μM) reduced cell viability both cells (C-F). The bar graph data were presented as mean ± SEM; groups denoted by different letters represent a significant difference at p < 0.05 (ANOVA followed by Fisher's LSD test).

Figure 9. ER maleate anticancer potential in tumor xenograft mice model and IHC analysis of Syk and PLK1 in human patient OSCC

A. ER maleate inhibits growth of tumor xenografts in mice. Cal33 cells were injected in the right flank of 6 weeks old immunocompromised mice (NOD/SCID/crl). ER maleate treatment was started after 3 weeks when tumor xenografts volume was about 250 mm³ with doses ranging from 0.1-3.0 mg/kg bwt for 10 weeks. Analysis of xenograft tumors from mice treated with ER maleate showed a dose dependent suppression of tumor growth within initial 6 weeks with an efficacious pharmacodynamic effect of complete inhibition of tumor growth at 1mg/kg bwt and 3mg/kg bwt by the 10^th week. From the 7^th week, the group of mice pre-treated with 0.1mg/kg bwt ER maleate was treated with CBP at 75mg/kg bwt and the group with ER maleate at 0.3mg/kg bwt within first 6 weeks received a combination treatment of ER maleate (1mg/kg bwt) and CBP (75mg/kg bwt) shown in the grey box. The combination treatment with ER maleate and CBP inhibited tumor growth in vivo from the 8^th week; in comparison, inhibition of tumor growth by CBP alone was lesser than in combination with ER maleate. B. Effect of ER maleate treatment on body weight of mice. Weekly measurements of mice body weight after Cal33 cell injection among different groups. C–H. Histogram of apoptotic markers in the frozen tumor tissues by western blot analysis showed the levels of PARP (C), cleaved PARP (D), caspase3 (E) and its two cleaved forms at 19 kDa (F) and 17 kDa (G) in xenograft tumor from groups receiving ER maleate of 1 mg/kg bwt or 3 mg/kg bwt compared to vechile control group. (H) Representative western blot showed the expression of PARP, cleaved PARP, caspase3 and its two cleaved forms at 19 kDa and 17 kDa. b-actin served as a loading control. I. Immunohistochemical analysis of Cyclin D1, Syk and PLK1 in tumor xenografts in immunocompromised mice. Panel I shows H&E stained tumor tissue sections in untreated control mice (a) and the treatment groups (b-e). Panels II, III and IV show nuclear Cyclin D1, Syk and PLK1 expression in untreated control mice (a); reduced Cyclin D1, Syk and PLK1 expression in CBP (75mg/kg bwt) treated tumors (b); combination of CBP (75mg/kg bwt) and ER maleate (1mg/kg bwt) shows further reduction in Cyclin D1, Syk and PLK1 (c); ER maleate treatment at 1mg/kg bwt and 3mg/kg bwt show reduced Cyclin D1, Syk and PLK1 expression in comparison with untreated controls (d and e), respectively. Original magnification is x400.

Figure 10. IHC analysis of Syk and PLK1 in tumor tissues and ER maleate effect on human OSCC derived cells

A. IHC studies showed no detectable expression of Syk in normal oral mucosa. In OSCC, both nuclear (N) and cytoplasmic (C) expression of Syk were increased. Positive control OSCC tissue showing Syk overexpression was included in each batch of immunostaining. Original magnification is 400x. B. IHC analysis showed no detectable expression of PLK1 in normal oral mucosa. In OSCC, both nuclear (N) and cytoplasmic (C) expression of PLK1 were increased. Positive control OSCC tissue showing PLK1 overexpression was included in each batch of immunostaining. Original magnification is 400x. C. Kaplan-Meier survival analysis of Syk. OSCC patients showing nuclear Syk overexpression (Syk positive, Nuc_score≥3) followed up over a period of up to 140 months showed a significant increase in mean disease free survival (DFS) (DFS = 41.03 months) as compared to patients who didn't show nuclear Syk positivity (Syk negative, Nuc_score<3) (DFS = 10.58 months; p = 0.017). D. Kaplan-Meier survival analysis of PLK1. Survival analysis over a period of up to 100 months showed a significant reduction in mean DFS in OSCC patients overexpressing nuclear PLK1 (PLK1 positive, Nuc_score≥3.5) (DFS =58.7 months) as compared to patients who didn't show nuclear PLK1 positivity (PLK1 negative, Nuc_score<3.5) (DFS = 89.8 months; p = 0.004). E. Human OSCC derived cells showed a dose-dependent reduction in cell survival with ER maleate treatment for 48 h. F. PLK1 mRNA level was decreased by ER maleate in a dose dependent manner (0-2 μM) and this decreasing effect was further enhanced in OSCC derived cells with treatment of CBP at 25μM by qPCR assay. The bar graph data were presented as mean ± SEM; groups denoted by different letters represent a significant difference at p < 0.05 (ANOVA followed by Fisher's LSD test).