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

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.

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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

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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.

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Supplementary Fig. 1
figure6

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)

Supplementary Fig. 2
figure7

(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)

Supplementary Fig. 3
figure8

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)

Supplementary Fig. 4
figure9

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)

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Supplementary Table 1

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Supplementary Table 2

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Supplementary Table 3

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Supplementary Table 4

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Subbannayya, T., Leal-Rojas, P., Zhavoronkov, A. et al. PIM1 kinase promotes gallbladder cancer cell proliferation via inhibition of proline-rich Akt substrate of 40 kDa (PRAS40). J. Cell Commun. Signal. 13, 163–177 (2019). https://doi.org/10.1007/s12079-018-00503-5

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Keywords

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