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Journal of Cell Communication and Signaling

, Volume 13, Issue 2, pp 163–177 | Cite as

PIM1 kinase promotes gallbladder cancer cell proliferation via inhibition of proline-rich Akt substrate of 40 kDa (PRAS40)

  • Tejaswini Subbannayya
  • Pamela Leal-Rojas
  • Alex Zhavoronkov
  • Ivan V. Ozerov
  • Mikhail Korzinkin
  • Niraj Babu
  • Aneesha Radhakrishnan
  • Sandip Chavan
  • Remya Raja
  • Sneha M. Pinto
  • Arun H. Patil
  • Mustafa A. Barbhuiya
  • Prashant Kumar
  • Rafael Guerrero-Preston
  • Sanjay Navani
  • Pramod K. Tiwari
  • Rekha Vijay Kumar
  • T. S. Keshava Prasad
  • Juan Carlos Roa
  • Akhilesh Pandey
  • David Sidransky
  • Harsha Gowda
  • Evgeny IzumchenkoEmail author
  • Aditi ChatterjeeEmail author
Research Article
  • 220 Downloads

Abstract

Gallbladder cancer (GBC) is a rare malignancy, associated with poor disease prognosis with a 5-year survival of only 20%. This has been attributed to late presentation of the disease, lack of early diagnostic markers and limited efficacy of therapeutic interventions. Elucidation of molecular events in GBC can contribute to better management of the disease by aiding in the identification of therapeutic targets. To identify aberrantly activated signaling events in GBC, tandem mass tag-based quantitative phosphoproteomic analysis of five GBC cell lines was carried out. Proline-rich Akt substrate 40 kDa (PRAS40) was one of the proteins found to be hyperphosphorylated in all the invasive GBC cell lines. Tissue microarray-based immunohistochemical labeling of phospho-PRAS40 (T246) revealed moderate to strong staining in 77% of the primary gallbladder adenocarcinoma cases. Regulation of PRAS40 activity by inhibiting its upstream kinase PIM1 resulted in a significant decrease in cell proliferation, colony forming and invasive ability of GBC cells. Our results support the role of PRAS40 phosphorylation in GBC cell survival and aggressiveness. This study also elucidates phospho-PRAS40 as a clinical marker in GBC and the role of PIM1 as a therapeutic target in GBC.

Keywords

Cell survival Gastrointestinal cancer mTOR signaling Phosphoproteomics SGI-1776 Targeted therapy 

Abbreviations

GBC

Gallbladder cancer

PRAS40

Proline-rich Akt substrate 40 kDa

TEABC

Triethyl ammonium bicarbonate

TMT

Tandem mass tag

IHC

Immunohistochemistry

TMA

Tissue microarray

bRPLC

Basic reverse phase liquid chromatography

PI3K

Phosphatidylinositol 3 kinase

Notes

Acknowledgements

We thank the Department of Biotechnology (DBT), Government of India for research support to the Institute of Bioinformatics. IOB is supported by DBT Program Support on Neuroproteomics and infrastructure for proteomic data analysis (BT/01/COE/08/05). We thank the “Infosys Foundation” for the research support to the Institute of Bioinformatics. This work was supported by the Science and Engineering Research Board, Department of Science and Technology, Government of India grant “miRNAs in chronic tobacco-induced oral cancer (SR/S0/HS-02081/2012)”; NCI’s Clinical Proteomic Tumor Analysis Consortium initiative (U24CA160036) and FAMRI-funded 072017_YCSA. P.K. Tiwari acknowledges research support from the Indian Council of Medical Research (ICMR), MP Council of Science & Technology (MPCST), Bhopal and Department of Science and Technology, Government of India. Harsha Gowda is a Wellcome Trust/DBT India Alliance Early Career Fellow. Juan Carlos Roa acknowledges research support from the National Fund for Scientific and Technological Development (FONDECYT 1170893), and Millennium Institute on immunology and immunotherapy (IMII P09/016-F), Government of Chile. Pamela Leal acknowledges research support from the National Fund for Scientific and Technological Development (FONDECYT 1151008), Government of Chile. Niraj Babu is a recipient of Senior Research Fellowship from the Council for Scientific and Industrial Research (CSIR), Government of India. Remya Raja is a recipient of Research Associateship from Department of Biotechnology, Government of India. Sneha M. Pinto is a recipient of DST INSPIRE Faculty award from Department of Science and Technology, Government of India. We thank Dr. S. K. Shankar of National Institute of Mental Health and Neuro Sciences for providing the use microscope facility.

Compliance with ethical standards

Disclosure of interest

The authors report no conflict of interests.

Supplementary material

12079_2018_503_Fig6_ESM.png (168 kb)
Supplementary Fig. 1

The plots depict overall survival and disease-free status of 36 patients with cholangiocarcinoma as a function of PRAS40 mRNA expression. The plots were visualizing by cBioPortal. (PNG 167 kb)

12079_2018_503_MOESM1_ESM.eps (1.1 mb)
High Resolution Image (EPS 1086 kb)
12079_2018_503_Fig7_ESM.png (180 kb)
Supplementary Fig. 2

(a) Scatter plot representing phospho-PRAS40 (T2546) expression versus PIM1 expression in GBC cell lines. (b) Scatter plot representing phospho-PRAS40 (T2546) expression versus AKT expression in GBC cell lines. (c) Scatter plot representing colony forming ability versus phospho-PRAS40 (T246) expression in GBC cell lines. (d) Scatter plot representing invasive ability versus phospho-PRAS40 (T246) expression in GBC cell lines. (PNG 179 kb)

12079_2018_503_MOESM2_ESM.eps (1.2 mb)
High Resolution Image (EPS 1208 kb)
12079_2018_503_Fig8_ESM.png (1.1 mb)
Supplementary Fig. 3

Inhibition of PRAS40 phosphorylation at T246 using inhibitors of its upstream kinases. GBC cell lines TGBC24TKB, TGBC2TKB, OCUG-1, GB-d1 and G-415 were treated with PIM1 inhibitor SGI-1776 (5 μM) or PI3K inhibitor LY294002 (10 μM) or both. Western blot analysis of phospho-PRAS40 (T246), PRAS40, PIM1, phospho-AKT1 (S473) and AKT1 in GBC cell lines. β-actin was used as loading control. (PNG 1091 kb)

12079_2018_503_MOESM3_ESM.eps (11.8 mb)
High Resolution Image (EPS 12109 kb)
12079_2018_503_Fig9_ESM.png (294 kb)
Supplementary Fig. 4

Graphical representation of the western blot densitometry analysis for phospho-PRAS40 (T246), total PRAS40, PIM1, phospho-AKT (S473), total AKT, phospho-RPS6 (S240), total RPS6, pan 14–3-3, phospho-FOXO3A (S253) and total FOXO3A in GBC cell lines TGBC2TKB (a) and G-415 (b) treated with PIM1 inhibitor SGI-1776 (5 μM), PI3K inhibitor LY294002 (10 μM) or both. Phospho-PRAS40, phospho-AKT, phospho-RPS6 and phospho-FOXO3A normalized to their total expression levels, PIM1 and 14–3-3 normalized to β-actin (*p < 0.05; **p < 0.01); AU-Relative abundance. (PNG 294 kb)

12079_2018_503_MOESM4_ESM.eps (1.4 mb)
High Resolution Image (EPS 1479 kb)
12079_2018_503_MOESM5_ESM.pdf (11 kb)
Supplementary Table 1 (PDF 11 kb)
12079_2018_503_MOESM6_ESM.pdf (932 kb)
Supplementary Table 2 (PDF 932 kb)
12079_2018_503_MOESM7_ESM.pdf (960 kb)
Supplementary Table 3 (PDF 960 kb)
12079_2018_503_MOESM8_ESM.pdf (4 mb)
Supplementary Table 4 (PDF 4086 kb)

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Copyright information

© The International CCN Society 2019

Authors and Affiliations

  • Tejaswini Subbannayya
    • 1
  • Pamela Leal-Rojas
    • 2
    • 3
  • Alex Zhavoronkov
    • 4
  • Ivan V. Ozerov
    • 4
  • Mikhail Korzinkin
    • 4
  • Niraj Babu
    • 1
    • 5
  • Aneesha Radhakrishnan
    • 1
  • Sandip Chavan
    • 1
  • Remya Raja
    • 1
  • Sneha M. Pinto
    • 6
  • Arun H. Patil
    • 1
    • 7
  • Mustafa A. Barbhuiya
    • 8
  • Prashant Kumar
    • 1
  • Rafael Guerrero-Preston
    • 9
  • Sanjay Navani
    • 10
  • Pramod K. Tiwari
    • 11
    • 12
  • Rekha Vijay Kumar
    • 13
  • T. S. Keshava Prasad
    • 1
    • 6
    • 14
  • Juan Carlos Roa
    • 15
  • Akhilesh Pandey
    • 3
    • 16
    • 17
    • 18
  • David Sidransky
    • 9
  • Harsha Gowda
    • 1
    • 6
  • Evgeny Izumchenko
    • 9
    Email author
  • Aditi Chatterjee
    • 1
    • 6
    Email author
  1. 1.Institute of Bioinformatics, International Technology ParkBangaloreIndia
  2. 2.Center of Excellence in Translational Medicine (CEMT) &Scientific and Technological Bioresource Nucleus (BIOREN)Universidad de La FronteraTemucoChile
  3. 3.McKusick-Nathans Institute of Genetic MedicineJohns Hopkins University School of MedicineBaltimoreUSA
  4. 4.Insilico Medicine, Inc., Emerging Technology CentersJohns Hopkins University at EasternBaltimoreUSA
  5. 5.Manipal Academy of Higher EducationManipalIndia
  6. 6.Center for Systems Biology and Molecular MedicineYenepoya (Deemed to be University)MangaloreIndia
  7. 7.School of BiotechnologyKIIT (Deemed to be University)BhubaneswarIndia
  8. 8.Department of Pathology and Laboratory MedicinePennsylvania State University College of MedicineHersheyUSA
  9. 9.Department of Otolaryngology, Head and Neck SurgeryThe Johns Hopkins University School of MedicineBaltimoreUSA
  10. 10.Lab SurgpathMumbaiIndia
  11. 11.Centre for Genomics, Molecular and Human GeneticsJiwaji UniversityGwaliorIndia
  12. 12.School of Studies in ZoologyJiwaji UniversityGwaliorIndia
  13. 13.Department of PathologyKidwai Memorial Institute of OncologyBangaloreIndia
  14. 14.NIMHANS-IOB Proteomics and Bioinformatics Laboratory, Neurobiology Research CentreNational Institute of Mental Health and NeurosciencesBangaloreIndia
  15. 15.Department of Pathology, Millenium Institute on Immunology and Immunotherapy (IMII)Pontificia Universidad Católica de ChileSantiagoChile
  16. 16.Department of Biological ChemistryJohns Hopkins University School of MedicineBaltimoreUSA
  17. 17.Department of OncologyJohns Hopkins University School of MedicineBaltimoreUSA
  18. 18.Department of PathologyJohns Hopkins University School of MedicineBaltimoreUSA

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