Myosin light chain kinase is a potential target for hypopharyngeal cancer treatment

Abstract

Hypopharyngeal cancer is squamous cell carcinoma (SCC) with the worst prognosis among the head and neck cancers. Overall, the 5-year survival rate remains poor although diagnostic imaging, radiation, chemotherapy, and surgical techniques have been improved. The mortality of patients with hypopharyngeal cancer is partly due to an increased likelihood of developing a second primary malignancy and metastasis. In this study, we found that MLCK expression, compared to healthy tissue, was up-regulated in hypopharyngeal tumor tissue. Of particular interest, a low 5-year survival rate was positively correlated with MLCK expression. We hypothesized that MLCK might be a target for hypopharyngeal cancer prognosis and treatment. In order to explore the function of MLCK in the development of cancer, we knockdown MLCK in hypopharyngeal cancer FaDu cells. The results showed that MLCK knockdown reduced the migration and invasion of FaDu cells. 4-amino-2-trifluoromethyl-phenyl retinate (ATPR) is the derivative of all-trans retinoic acid (ATRA), which was able to reduce both MLCK expression and activity in FaDu cells. ATPR induced FaDu cells apoptosis in a dose-dependent manner and also inhibited cell growth both in vivo and in vitro. Further experiments showed that overexpression of MLCK reduced ATPR induced-migration inhibition while increase of ATPR induced apoptosis, which suggested that MLCK was involved in ATPR's anti-cancer function. In conclusion, MLCK is a novel prognostic marker and therapeutic target for hypopharyngeal cancer. By targeting MLCK, ATPR exhibits its potential application in the treatment of this type of cancer.

Keywords: ATPR; ATRA; Hypopharyngeal cancer; MLCK.

Fig. 1.

MLCK is up-regulated in human hypopharyngeal carcinoma tissue and correlates with the 5-year survival rate. Expression profiles of MLCK and the markers of human hypopharyngeal cancer, including Cox-2 and E-cadherin, were analyzed. (A) Cox-2, (B) E-cadherin, and (C) MLCK expression in human hypopharyngeal carcinoma tissue were compared to adjacent non-lesion tissues. Cox-2 protein was up-regulated (A), whereas E-cadherin was down-regulated (B), demonstrating characteristics of hypopharyngeal cancer. Of interest, MLCK expression was obviously increased in hypopharyngeal carcinoma compared to adjacent healthy tissue. Bar = 50 μm (100X, upper panel), 10 μm (400X, lower panel), and arrows indicate the adjacent non-lesion tissues. (D) MLCK expression can be used as a prognostic marker for patients with hypopharyngeal cancer. The 5-year survival was negatively correlated with MLCK expression. The patients with low MLCK expression has higher survival rate. In contrast, the patients with high MLCK expression has low survival rate.

Fig. 2.

MLCK expression is associated with cell migration in hypopharyngeal cancer FaDu cells. (A) GFP expression in shMLCK transfected FaDu cells confirming the transfection efficiency of shMLCK. Bar = 50 μm. (B) MLCK transcript in MLCK knockdown cells compared to wide-type cells. The expression level of MLCK was decreased by ~70% in knockdown cells. (C) Wound healing assay showed that MLCK knockdown reduces FaDu cell migration. Bar = 50 μm (D) The quantitative migration distance ratio of wound healing assay is around 60-70% in MLCK knockdown FaDu cells compared to wide-type cells. (E) Representative images of transwell cell migration assay indicated that migration was attenuated in MLCK knockdown FaDu cells. Bar = 50 μm. (F) Quantitative analyses of transwell cell migration assay. Similar to the data from wound healing assay, MLCK suppression reduced cell migration ~60 - 70%. *p < 0.05 compared with wide-type cells.

Fig. 3.

ATPR reduces MLCK expression and activity in hypopharyngeal cancer cells and inhibits FaDu cell migration. (A) ATPR reduced MLCK expression in FaDu cells in a dose-dependent manner. (B) Quantification of MLCK protein level expression by Quantity one software. (C) ATPR inhibited MLC phosphorylation in FaDu cells. Since MLCK directly induced MLC phosphorylation, this data suggested that ATPR also suppressed MLCK activity. (D) Quantification of MLC phosphorylation by Quantity one software. (E) Effects of ATPR on ZIPK and ROCK expression in FaDu cells. ZIPK and ROCK have been considered as the other regulators of MLC phosphorylation. We therefore analyzed the expression of these two proteins. Although ATPR did not decrease ROCK expression, it dose-dependently increases ZIPK expression. (F) Quantification of ZIPK protein level expression in FaDu cells by Quantity one software. (G-J) Transwell cell migration and wound healing assays showed that ATPR significantly inhibited cell migration in a dose-dependent manner. Bar = 50 μm. *p < 0.05; **p < 0.01 compared with vehicle control.

Fig. 4.

ATPR is non-toxic and effective for hypophaiyngeal cancer treatment. (A) H&E staining of mouse tissues, including heart, liver, spleen, lung, and kidney, compared to control mice, ATPR treatment did not induce morphologic abnormalities in these vital organs. This result suggested that ATPR is non-toxic at this dose in vivo. Bar = 50 μm. (B) To generate a xenograft mouse model of hypophaiyngeal cancer, mice were subcutaneously injected with 10⁷ FaDu cells. Tumor size was measured after 6 weeks post-injection, ATPR reduced tumor growth in nude mice in a dose-dependent manner. Bar =1 mm. (C) Quantification of the effect of ATPR on tumor size in mice, five mice per group. (D) Flow cytometry was performed to detect the effects of ATPR in FaDu cell proliferation. (E) Quantitative analysis of flow cytometry. ATPR induced cell cycle arrest at the G0/G1 phase in FaDu cells. *** p < 0.001 compared with vehicle control.

Fig. 5.

ATPR induces hypopharyngeal cancer cell apoptosis. (A) Representative TUNEL staining in FaDu cells treated with or without ATPR. Bar = 20 μm. (B) Quantification of TUNEL staining. (C) ISOL staining was performed in FaDu cells treated with or without ATPR treatment. Bar = 20 μm. (D) Quantification of apoptotic cells in ISOL staining. (E) Flow cytometry analysis of cell apoptosis in ATPR-treated group compared to vehicle control FaDu cells. (F) Quantification of apoptotic cell ratio in flow cytometry assay. Based on TUNEL, ISOL, and flow cytometry assays, ATPR promoted apoptosis in FaDu cells in a dose-dependent manner. (G) Immunoblots of apoptotic associated protein signals in FaDu cells treated with or without ATPR. Importantly, ATPR increases expression of pro-apoptotic protein, including cleaved caspase-3 and Bax, in parallel with suppressing anti-apoptotic protein Bcl-xl. *p < 0.05; **p < 0.01 compared with vehicle control.

Fig. 6.

Anti-cancer activities of ATPR on MLCK-overexpressing FaDu cells. (A) EGFP-MLCK expression induced by doxycycline (dox) treatment in FaDu cells transfected with pPBH-TREtight-EGFP plasmid. Bar = 20 μm. (B, C–,) Wound healing assay revealed that, in inducible EGFP-MLCK overexpressing FaDu cells, the migration rate was increased. ***p < 0.001 compared with w/o dox. (D–,E) Effect of ATPR on cancer cell migration inhibition was attenuated in EGFP-MLCK overexpressing FaDu cells. Bar = 50 μm. ***p < 0.001 compared with w/o dox. (F,–) ATPR induced cell cycle arrest at G0/G1 phase in dox-treated and untreated EGFP-MLCK overexpressing FaDu cells. ***p < 0.001 compared with vehicle control. There is no difference between dox treatment and without dox group. (H, –I) Effect of ATPR-induced cancer cell apoptosis was potentiated in EGFP-MLCK overexpressing FaDu cells. ***p < 0.001 compared with w/o dox or vehicle.