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Glycogen synthase kinase-3β mediated regulation of matrix metalloproteinase-9 and its involvement in oral squamous cell carcinoma progression and invasion

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Abstract

Purpose

Oral squamous cell carcinoma (OSCC)-related deaths mainly result from invasion of the tumor cells into local cervical lymph nodes. It has been reported that progressive basement membrane loss promotes the metastatic and invasive capacities of OSCCs. Matrix metalloproteinase-9 (MMP-9) is known to play a central role in tumor progression and invasion. However, the role of MMP-9 in OSCC invasion has so far remained paradoxical and little is known about its regulation. Here, we aimed to assess MMP-9 expression regulation and its activation by glycogen synthase kinase-3β during human OSCC progression and invasion.

Methods

In the present study, 178 human OSCC samples, including 118 fresh samples (18 adjacent normal, 42 noninvasive and 58 invasive tumor samples) and 60 archival human tissue microarray (TMA) tongue cancer samples, were included. mRNA expression, protein expression, MMP-9/-2 activity, protein-protein interaction and Snail, c-Myc, β-catenin and TIMP1 expression were assessed using RT-PCR, immunohistochemistry, Western blotting, co-immunoprecipitation and gelatin zymography analyses, respectively. Wnt5a and LPA mediated MMP-9 regulation was assessed in OCSCC-derived SCC-9 cells exogenously expressing GSK3β (WT) or non phosphoryable GSK3β (S9A).

Results

We observed a progressive up-regulation/activation of MMP-9 at various stages of oral tumor progression/invasion. Positive correlations were observed between MMP-9 and c-Myc expression, MMP-9 and MMP-2 activity, MMP-9 and TIMP1 expression and MMP-9 activity and TIMP1-MMP-9 interaction. In contrast, a negative correlation between phosphorylated β-catenin and MMP-9 expression was observed. Conversely, we found that in oral tongue SCC MMP-9 expression was positively correlated with inactivation of GSK3 signaling. Finally, we found that Wnt5a and LPA mediated increased MMP-9 and decreased GSK3β activities in tongue SCC-derived SCC-9 cells. MMP-9 regulation by GSK3β was confirmed by using phosphoryable/regulatory GSK3β (WT construct) and not by non-phosphoryable GSK3β (S9A construct).

Conclusions

Collectively, our results show that MMP-9 overexpression and activation are important events occurring during OSCC progression/invasion and that this overexpression/activation is regulated by c-Myc, active MMP-2 and inactive GSK3β mediated pathways.

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Acknowledgements

The authors wish to acknowledge Prof. M.K. Rai (Pathologist), Director, RIMS, Ranchi and Prof. N.K. Jha, Head Dept. of Surgery (and his colleagues) RIMS, Ranchi and the Director of CARA, Cancer Hospital, Ranchi and his colleagues Dr. M. Akhouri, Dr. Aftab A. Ansari and Dr. K. Saurav and Dr. Raghav Sharan (Clinic), Ranchi for their cooperation. The fellowship of KKP (CSIR), PM (CUJ), AKS (DBT-RA) and the financial support of DBT, New Delhi (ProjectNo.BT/PR4624/MED/30/701/2012; Departmental DBT Builder Programme No. BT/PR9028/INF/22/193/ 2013) are acknowledged.

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Authors and Affiliations

  1. Centre for Life Sciences, School of Natural Sciences, Central University of Jharkhand, Ratu-Lohardaga Road, Brambe, Ranchi, Jharkhand, -835205, India

    Kamdeo K. Pramanik, Abhay K. Singh, Tanushree Kashyap, Nidhi Nath, Manzar Alam & Rajakishore Mishra

  2. Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu, -608 002, India

    Siddavaram Nagini

  3. Centre for Applied Chemistry, School of Natural Sciences, Central University of Jharkhand, Ratu-Lohardaga Road, Brambe, Ranchi, Jharkhand, -835205, India

    Prajna Mishra

  4. Division of Surgical Oncology, Department of Surgery, College of Medicine, The University of Illinois at Chicago, 840 S. Wood Street, Suite 601 Clinical Sciences Building, MC 958, Chicago, IL, 60612, USA

    Ajay Rana

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  1. Kamdeo K. Pramanik
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Contributions

KKP performed the IHC, WB, RT-PCR and zymography experiments; KKP, PM, AKS, MA, TK and NN performed the cell culture experiments; KKP and RM analyzed the results. RM, SN and AR provided their knowledge of biochemistry and molecular biology. RM has written the MS and the final version of the MS has been approved by all the authors.

Corresponding author

Correspondence to Rajakishore Mishra.

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Electronic supplementary material

13402_2017_358_MOESM1_ESM.jpg

The quantitative expression of associated molecules that comes under GSK3β pathway and may promotes the activation of MMP-9 in SCC-9 cells. Quantitative expression (mean and SD) of representative immunoblots (WB) of (A1) β-catenin, (A2) c-myc (A3) Twist, (A4) MMP9 protein expression; and gel picture (RT-PCR) of (A5) MMP-9, (A6) MMP-2 and (A7) TIMP-2, showing the differential expression, at various treated conditions, as indicated in the figure. (* p < 0.05, **p < 0.01, ***p < 0.001). (JPEG 200 kb)

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Pramanik, K.K., Nagini, S., Singh, A.K. et al. Glycogen synthase kinase-3β mediated regulation of matrix metalloproteinase-9 and its involvement in oral squamous cell carcinoma progression and invasion. Cell Oncol. 41, 47–60 (2018). https://doi.org/10.1007/s13402-017-0358-0

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