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. 2017 Dec 26;9(11):9672-9684.
doi: 10.18632/oncotarget.23822. eCollection 2018 Feb 9.

TMIGD1 acts as a tumor suppressor through regulation of p21Cip1/p27Kip1 in renal cancer

Rosana D Meyer  1 , Xueqing Zou  2 , Marwa Ali  1 , Esma Ersoy  1 , Philip Apraku Bondzie  1 , Mehrdad Lavaei  1 , Ilya Alexandrov  3 , Joel Henderson  1 , Nader Rahimi  1
Affiliations

TMIGD1 acts as a tumor suppressor through regulation of p21Cip1/p27Kip1 in renal cancer

Rosana D Meyer et al. Oncotarget. .

Abstract

Renal cell carcinoma (RCC) is a high-risk metastasizing tumor with a poor prognosis and poorly understood mechanism. In this study, we demonstrate that transmembrane and immunoglobulin domain-containing 1 (TMIGD1) is a novel tumor suppressor that is highly expressed in normal renal tubular epithelial cells, but it is downregulated in human renal cancer. We have identified CCAAT/enhancer-binding proteinβ (C/EBPβ, also called LAP) as a key transcriptional regulator of TMIGD1, whose loss of expression is responsible for downregulation of TMIGD1 in RCC. Transcriptionally active C/EBPβ/LAP physically interacted with and increased TMIGD1 promoter activity and expression of TMIGD1. Re-introduction of TMIGD1 into renal tumor cells significantly inhibited tumor growth and metastatic behaviors such as morphogenic branching and cell migration. Restoring TMIGD1 expression in renal tumor cells stimulated phosphorylation of p38MAK, induced expression of p21CIP1 (cyclin-dependent kinase inhibitor 1), and p27KIP1 (cyclin-dependent kinase inhibitor 1B) expression, key cell cycle inhibitor proteins involved in regulation of the cell cycle. The present study identifies TMIGD1 as a novel candidate tumor suppressor gene and provides important insight into pathobiology of RCC that could lead to a better diagnosis and possible novel therapy for RCC.

Keywords: TMIGD1; cell cycle inhibitors; renal cancer; signal transduction; tumor suppressor.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. TMIGD1 expression is downregulated in human renal cancer
(A) Total mRNA extracted from various human organs was subjected to qPCR analysis using primers specifically designed to amplify exons 2 and 3 of TMIGD1. The relative levels of IGPR-1 are shown. (B) TMIGD1 expression of in mouse organs/tissues is shown. The data is extracted from the online publically available mouse genome data (http://biogps.org). (C) Whole cell lysates from human tissues was subjected to western blot analysis using anti-TMIGD1 antibody or anti-tubulin antibody as a loading control. The lower band of tubulin is likely due to protein degerdation. (D) Immunohistochemistry staining of human kidney tissue using anti-TMIGD1 antibody is shown, which shows renal tubular epithelial cells are highly positive (arrow), where podocytes of the glomerulus are negative for TMIGD1. Scale bars, 50 μm.
Figure 2
Figure 2. TMIGD1 expression is downregulated in human renal cancer
(A) The mRNA of human kidney tissue or human kidney tumor cell lines including, TK10, 786-0, A498, and 769P were subjected to qPCR analysis and the relative expression of TMIGD1 is presented. (B) Whole cell lysates from normal human kidney cell line, HK2, or kidney tumor cell lines were blotted for TMIGD1 or for a loading control b-actin. The blot for TMIGD1 was quantified using the Image J software. AU (arbitrary unit). (C) Human kidney cancer tissues were subjected to immunohistochemistry staining using anti-TMIGD1 antibody and three representative images were shown. Scale bars, 50 μm.
Figure 3
Figure 3. Re-expression of TMIGD1 in 786-0 cells inhibits tumor growth, cell invasion and branching morphogenesis
(A) Morphology of 786-0 cells expressing empty vector (EV) or TMIGD1 seeded on the collagen-coated plates for 24 hours. The same cells were seeded on the collagen-coated cover slips for 40 minutes, cells were fixed and stained with Rhodamine Phalloidin and pictures (40X) were taken under an immunofluorescence microscope. (B) Equal number of 786-0 cells expressing empty vector (EV) or TMIGD1 were plated in 24-well plates in quadruple in the presence of 10%FBS and proliferation of cells were measured at day 1, 2 and 3. *p < 0.05. (B) Similarly, equal number of 786-0 cells expressing empty vector or TMIGD1 were plated in 24-well plates in quadruple and subjected to BrdU staining and cell proliferation was measured at day 0, 24 h and 48 h. Experiments are presentative of three intendent experiments. *p < 0.05. (C) Equal number of 786-0 cells expressing empty vector (pMSCV) or TMIGD1 were mixed with matrigel and xenografted into athymic nude mice (4/group) and after 27 days tumors were excised, weighed and pictures were taken. P = 0.033. (D) 786-0 cells expressing empty vector or TMIGD1 were subjected to branching morphogenesis and pictures were taken after 9 days. Branching morphogenesis (four images/group) was quantified via Image J software. *p < 0.05. The data is representative of three independent experiments. (E) 786-0 cells expressing empty vector or TMIGD1 were seeded on the collagen-coated transwells in triplicates for about 12 hours. Cells were fixed and number of cells that were migrated to the other side of the transwells were counted under microscope. The data is representative of three independent experiments (quadruple wells/group). *p < 0.05.
Figure 4
Figure 4. Re-expression of TMIGD1 in 786-0 cells induces multiple pathways involved in the inhibition of cell cycle and cell proliferation
(A) 786-0 cells expressing empty vector (EV) or TMIGD1 were plated in 96-well plates in triplicates and subjected to ActiveSignal assay Assay analysis, which measures expression or activation of 70 proteins . The graph shows the major proteins involved in the regulation of cell cycle and are affected by TMIGD1. AU (arbitrary unit). (B) Whole cell lysates from 786-0 cells expressing empty vector (EV) or TMIGD1 were subjected to western blot analysis and western blots of a panel of selected proteins involved in the regulation of cell cycle and proliferation are shown. (C) 788-0 cells expressing empty vector or TMIGD1 were treated with p38-MAPK inhibitor (SB203580) for 30 minutes. Cells were lysed and whole cell lysates were blotted for total p38, phospho-p38, phospho-Rb, p21CIP1 and p27KIP1. (D) Schematic of pathways affected by TMIGD1 in 786-0 cells and the possible role of p38MAPK in the regulation of phospho-Rb, and induction of p21CIP1 and p27KIP1.
Figure 5
Figure 5. C/EBPβ transcription factor regulates expression of TMIGD1
(A) Shown is the schematic of full-length TMIGD1 promoter cloned into pHAGE-GFP reporter vector. (B) HEK-293T cells were transfected with TMIGD1- pHAGE-GFP or EFα1-pHAGE-GFP. Images were taken under a fluorescent microscope (20X) after 48 hours of transfection. (C) TMIGD1 and control EFα1 promoter activities was quantified by FACS analysis via measuring expression of GFP. (D) 5’ biotinylated TMIGD1 promoter mixed either with the cell lysates of HEK-293 cells over-expressing C/EBPβ (LAP) or empty vector and subjected to an electrophoretic mobility shift (EMSA) assay. (E) HEK-293T cells were transfected with full-length (FL) TMIGD1 promoter alone or with C/EBPβ (LAP). After 4 days transfection, cells were prepared and subjected to FACS analysis. The data presented in this figure were independently repeated at least three times.
Figure 6
Figure 6. C/EBPβ regulates expression of TMIGD1 in renal tumor
(A) Human kidney cancer tissues (5 cases) were subjected to IHC staining using anti-TMIGD1 antibody or anti-C/EBPβ antibody and representative images are shown. (B) 786-0 cells were transfected with either an empty vector or C/EBPβ (LAP) and the mRNA derived from cells subjected to qPCR analysis. GAPDH was used for internal control. P = 0.0058. (C) Similarly, 786-0 cells expressing empty vector or C/EBPβ (LAP) were lysed, subjected to western blot analysis and blotted for TMIGD1, C/EBPβ (LAP) using anti-FLAG antibody and GAPDH for loading control. (D) 786-0 cells 786-0 cells expressing empty vector or C/EBPβ (LAP) were prepared and subjected to branching morphogenesis assay. Pictures were taken under a microscope after 10days.
Figure 7
Figure 7. Proposed model for expression and signaling of TMIGD1
Based on the data presented in this study, we propose that downregulation of C/EBPβ or its activity in renal cancer cells is associated with suppression of TMIGD1 expression in RCC. Restoring TMIGD1 expression in renal tumor cells activated p38-MAPK that lead to induction of p21CIP1/p27KIP1 that inhibits cell proliferation and migration.
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