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. 2021 Jan 13;9(1):68.
doi: 10.3390/biomedicines9010068.

High Monopolar Spindle 1 Is Associated with Short Survival of Cholangiocarcinoma Patients and Enhances the Progression Via AKT and STAT3 Signaling Pathways

Piya Prajumwongs  1   2 , Ratthaphong Phumphu  1   2 , Orawan Waenphimai  1   2 , Worachart Lert-Itthiporn  1   2 , Kulthida Vaeteewoottacharn  1   2 , Sopit Wongkham  1   2 , Yaovalux Chamgramol  2   3 , Chawalit Pairojkul  2   3 , Kanlayanee Sawanyawisuth  1   2
Affiliations

High Monopolar Spindle 1 Is Associated with Short Survival of Cholangiocarcinoma Patients and Enhances the Progression Via AKT and STAT3 Signaling Pathways

Piya Prajumwongs et al. Biomedicines. .

Abstract

Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium. The major problems of this cancer are late diagnosis and a high rate of metastasis. CCA patients in advanced stages have poor survival and cannot be cured with surgery. Therefore, targeting molecules involved in the metastatic process may be an effective CCA treatment. Monopolar spindle 1 (MPS1) is a kinase protein that controls the spindle assemble checkpoint in mitosis. It is overexpressed in proliferating cells and various cancers. The functional roles of MPS1 in CCA progression have not been investigated. The aims of this study were to examine the roles and molecular mechanisms of MPS1 in CCA progression. Immunohistochemistry results showed that MPS1 was up-regulated in carcinogenesis of CCA in a hamster model, and positive expression of MPS1 in human CCA tissues was correlated to short survival of CCA patients (n = 185). Small interfering RNA (siRNA)-induced knockdown of MPS1 expression reduced cell proliferation via G2/M arrest, colony formation, migration, and invasion. Moreover, MPS1 controlled epithelial to mesenchymal transition (EMT)-mediated migration via AKT and STAT3 signaling transductions. MPS1 was also involved in MMPs-dependent invasion of CCA cell lines. The current research highlights for the first time that MPS1 has an essential role in promoting the progression of CCA via AKT and STAT3 signaling pathways and could be an attractive target for metastatic CCA treatment.

Keywords: AKT; Cholangiocarcinoma; EMT; MMPs; MPS1; STAT3; invasion; migration.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
MPS1 is associated with cholangiocarcinogenesis in a hamster model. (A) Immunohistochemistry (IHC) staining of MPS1 in liver tissues from four groups of hamsters including control, carcinogen N-nitrosodimethylamine (NDMA)-treated, Ov-infected, and combination of NDMA + Ov at one, three, and six months after treatment. (B) Comparison of the percent of MPS1 positive cases between NBD, hyperplasia/dysplasia (HP/DP), and Cholangiocarcinoma (CCA). Median H-score of MPS1 in hamster CCA tissues was 70, which was used as a cut off value to categorize the samples into two groups; MPS1 negative and MPS1 positive.
Figure 2
Figure 2
Overexpression of MPS1 or TTK in human CCA tissues. (A) A box plot of MPS1 transcript level in 36 human CCA tissues compared to 9 normal bile ducts (NBD). Comparison of overall survival curves between negative and positive MPS1 expressing patients. Data were retrieved from GEPIA database. TPM is transcript per million. * p < 0.05. (B) A dot plot of MPS1 transcript level in 91 human CCA patients categorized in two groups: MPS1 negative and MPS1 positive, using the median value of MPS1 expression (7.8). Cumulative survival times of MPS1 positive patients were significantly shorter than those of MPS1 negative patients (p < 0.01). Data were retrieved from GEO database. (C) Representative IHC staining of MPS1 protein in 185 CCA patient tissues. MPS1 expression was not detected in NBD (magnification 400×) and MPS1 negative CCA tissues (magnification 200×). MPS1 positive CCA showed cytoplasmic staining of MPS1 (magnification 200×). (D) Distribution of MPS1 expression in 25 NBD and 185 CCA. Median H-score was 0, which was used as a cut-off value for dividing the MPS1 positive and negative cases. (E) Cumulative survival times of MPS1 positive patients (n = 74) were significantly shorter than those of MPS1 negative patients (n = 111) p < 0.001.
Figure 3
Figure 3
MPS1 contributed to cell proliferation, colony formation, and altered cell cycle of CCA cell lines. (A) Knockdown efficiency of MPS1 specific siRNA at 24 to 72 h in KKU-055 and KKU-213A cell lines. Quantification of each band was normalized to GAPDH and is shown as the numbers above the corresponding panels. (B) Cell proliferation rates of siMPS1 treated KKU-055 and KKU-213A were determined at 24 to 96 h. (C) Colony formation assay shows decreasing numbers of colonies after MPS1 knockdown in CCA cell lines. (D) Flow cytometry histograms demonstrate decreasing G1 phase and increasing G2/M arrest in siMPS1 treated cells at 72 and 96 h. (E) Sub-G1 populations were analyzed by flow cytometry. A time-dependent increase in the percentage of sub-G1 populations was observed upon MPS1 knockdown. Data are presented as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001.
Figure 4
Figure 4
MPS1 promoted EMT-mediated migration via AKT and STAT3 signaling pathways. (A) Boyden chamber assay was performed to evaluate the migration of siMPS1 treated KKU-055 and KKU-213A cells. (B) Cells were pre-treated with siMPS1 and an AKT inhibitor (MK2206) or an AKT activator (SC79). The expression of EMT-related proteins was analyzed using Western blot assay. Representative results of claudin-1, slug, vimentin, pAKT (s473), and pSTAT3 (Y705) are shown. GAPDH was used as an internal control. Quantification of protein expression was normalized with GAPDH and by assigning the scramble (sc) control as 1. Bar graph shows the mean ± SD band intensities of proteins from three independent experiments. (C) Combination of siMPS1 and a STAT3. inhibitor (Stattic) greatly altered the expression of EMT-related markers in KKU-055 and KKU-213A cell lines. Protein loading and protein quantification were performed as described for Figure 4B. Significant difference (p < 0.05) when compared with sc (*) or si (#).
Figure 5
Figure 5
Suppression of MPS1 attenuated MMPs-dependent invasion in CCA cell lines. (A) The percentage of invaded cells after knocking down MPS1 in KKU-055 and KKU-213A cell lines. Results are mean ± SD of three independent experiments. (B) Gelatin zymogram of MMP-2 and MMP-9 activities in siMPS1-treated cells. The enzyme activity was relatively quantified to scramble (sc) control. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001.
Figure 6
Figure 6
The proposed molecular mechanism of how MPS1 promotes the metastasis of CCA. MPS1 contributes to cell proliferation, colony formation, and alters cell cycle progression. MPS1 activates AKT and STAT3 signal transduction. These actions consequently enhance the expression of EMT markers slug, vimentin, and MMP-2/9, while modulating expression of claudin-1. These result in an increase in migration and invasion, which lead to metastasis in CCA.
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