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Mol Cancer
2018 Aug 01;171:111. doi: 10.1186/s12943-018-0848-3.
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MASTL induces Colon Cancer progression and Chemoresistance by promoting Wnt/β-catenin signaling.
Uppada SB
,
Gowrikumar S
,
Ahmad R
,
Kumar B
,
Szeglin B
,
Chen X
,
Smith JJ
,
Batra SK
,
Singh AB
,
Dhawan P
.
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BACKGROUND: Chemotherapeutic agents that modulate cell cycle checkpoints and/or tumor-specific pathways have shown immense promise in preclinical and clinical studies aimed at anti-cancer therapy. MASTL (Greatwall in Xenopus and Drosophila), a serine/threonine kinase controls the final G2/M checkpoint and prevents premature entry of cells into mitosis. Recent studies suggest that MASTL expression is highly upregulated in cancer and confers resistance against chemotherapy. However, the role and mechanism/s of MASTL mediated regulation of tumorigenesis remains poorly understood.
METHODS: We utilized a large patient cohort and mouse models of colon cancer as well as colon cancer cells to determine the role of Mastl and associated mechanism in colon cancer.
RESULTS: Here, we show that MASTL expression increases in colon cancer across all cancer stages compared with normal colontissue (P < 0.001). Also, increased levels of MASTL associated with high-risk of the disease and poor prognosis. Further, the shRNA silencing of MASTL expression in colon cancer cells induced cell cycle arrest and apoptosis in vitro and inhibited xenograft-tumor growth in vivo. Mechanistic analysis revealed that MASTL expression facilitates colon cancer progression by promoting the β-catenin/Wnt signaling, the key signaling pathway implicated in colon carcinogenesis, and up-regulating anti-apoptotic proteins, Bcl-xL and Survivin. Further studies where colorectal cancer (CRC) cells were subjected to 5-fluorouracil (5FU) treatment revealed a sharp increase in MASTL expression upon chemotherapy, along with increases in Bcl-xL and Survivin expression. Most notably, inhibition of MASTL in these cells induced chemosensitivity to 5FU with downregulation of Survivin and Bcl-xL expression.
CONCLUSION: Overall, our data shed light on the heretofore-undescribed mechanistic role of MASTL in key oncogenic signaling pathway/s to regulate colon cancer progression and chemo-resistance that would tremendously help to overcome drug resistance in colon cancer treatment.
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30068336
???displayArticle.pmcLink???PMC6090950 ???displayArticle.link???Mol Cancer ???displayArticle.grants???[+]
BXBX002086 U.S. Department of Veterans Affairs, BX002761 U.S. Department of Veterans Affairs, DK088902 National Institutes of Health, R21CA216746 National Cancer Institute, R21CA216746 NCI NIH HHS , P30 CA036727 NCI NIH HHS , I01 BX002761 BLRD VA, P30 CA008748 NCI NIH HHS
Fig. 2. MASTL knockdown results in altered functional characteristics in colon cancer cell lines in vitro. a (i) MASTL knockdown in SW620 cells was confirmed by Western blot and qRT-PCR analysis. (ii) MASTL knockdown in HCT116 cells was confirmed by Western blot and qRT-PCR analysis. b Tumorigenic and invasive potential was determined by ability to form colonies in soft agar assay and invasion in (i) SW620C and SW620MKD cells (ii) HCT116C and HCT116MKD cells. For graphs, data represent mean ± SD; **, P < 0.001
Fig. 3. Cell are arrested in G2M phase as a result of MASTL knockdown in colon cancer. Control and MASTL knockdown cells were synchronized in serum free conditions for 72 h after which are treated with RO3306 for 16 h and then grown for one hour in fresh media. Cells were fixed immediately and cell cycle analysis was carried out via FACS. RO3306 is a selective ATP-competitive inhibitor of CDK1 that reversibly arrests proliferating human cells at the G2/M phase border, and the arrested cells enter mitosis rapidly after release from the G2 block [8] (a) HCT116 MASTL knockdown cells were unable to overcome the G2M block as compared to control cells (b) Similarly, the SW620 MASTL knockdown cells were also significantly arrested in G2M phase. For graphs, data represent mean ± SD; *, P < 0.01
Fig. 6. Effect of modulation of MASTL expression on tumor xenograft in vivo. (a-c) Flank tumor xenograft tumor development, after subcutaneous injection (n = 6 mice per group), was monitored for HCT116C, or HCT116MKD cells. Tumor volume and tumor weight of HCT116C and HCT116MKD cells after 4 weeks of inoculation in nude mice. (d) Tumors were evaluated for MASTL and β-catenin expression as well as markers of proliferation (Ki67) by immunohistochemistry; (e) Immunoblotting on tumors from 3 mice from each group (M1, M2 and M3) for Survivin, Bcl-xL and Cleaved PARP and normalized with tubulin as control. For graphs, data represent mean ± SD; **, P < 0.001; ***, P < 0.0001 versus control
Fig. 7. Overexpression of β-catenin-S33Y mutant rescues MASTL knockdown cells from apoptosis. β-catenin-S33Y mutant was transiently overexpressed (48 h) in MASTL knockdown cells and overexpression of activated β-catenin was confirmed by immunoblotting and topflash reporter assay in HCT116 (a) and SW620 (b) cells. c Caspase-3/7 activity as measured by luminescence in HCT116MKD and SW620MKD cells as compared to control cells and modulation due to overexpression of activated form of β-catenin in these cells
Fig. 8. MASTL imparts chemoresistance to 5-FU in colon cancer cell lines. (a) HCT116C and HCT116MKD cells were treated with 10 and 20 μM of 5-FU. Western blot analysis demonstrated induction of Survivin and Bcl-xL in control cells. However, inhibition of MASTL inhibited these protein expression even in presence of 5-FU. (b) MTT assay in HCT116C and HCT116MKD cells showed significant reduction in viable cells as compared to control treated cells. For graphs, data represent mean ± SD; **, P < 0.001; ***, P < 0.0001 versus control. (c) Model depicting the role of MASTL in regulation of colon cancer progression. When MASTL expression is increased it phosphorylates GSK-3β and inactivates it, thereby β-catenin is not phosphorylated and degraded. The β-catenin then is translocated into nucleus leading to active transcription of its target genes c-Myc, Survivin and Bcl-xL. These cells escape G2M arrest and apoptosis leading to cancer progression. Conversely, in MASTL inhibited cells, GSK-3β is active which then phosphorylates and degrades β-catenin thus preventing its translocation to nucleus and activation of target genes
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