LIMD1 is a survival prognostic marker of gastric cancer and hinders tumor progression by suppressing activation of YAP1

Abstract

Purpose: The purpose of this study was to investigate the clinical significance of LIMD1 and its biological roles in gastric cancer (GC).

Materials and methods: The prognostic value of LIMD1 in GC patients was determined by the online tool Kaplan-Meier Plotter. The biological functions of LIMD1 in GC were examined by in vitro assays, including proliferation, anchorage-independent growth, migration, invasion, and epithelial to mesenchymal transition (EMT) assays. The levels of downstream YAP1 regulated by LIMD1 were measured by Western blot analysis, and the sub-localization of YAP1 in GC cells was visualized by immunofluorescence staining. Differential expression levels and copy number levels of LIMD1 between GC and normal tissues were compared using the Oncomine database. A correlation of LIMD1 mRNA level and the copy number level was depicted by cBioPortal. We also evaluated the methylation status around the LIMD1 genes by Wanderer.

Results: The expression level of LIMD1 positively correlated with the prognosis of GC patients regardless of tumor stage, size, lymph node, metastasis, Lauren's classification, differentiation, gender, treatment, and ERBB2 amplification status. Overexpression of LIMD1 impeded the tumor growth, cell motility, invasiveness, and metastasis, and knockdown of LIMD1 promoted these phenotypes in GC cells. Mechanistically, YAP1 was one of the downstream effectors of LIMD1; LIMD1 suppressed the expression of YAP1 as well as its intracellular translocation. Furthermore, we found that LIMD1 expression was reduced in some of the GC profiling datasets. Gene deletion, instead of DNA methylation, contributed to the reduced expression of LIMD1 in GC.

Conclusion: Our results identified LIMD1 as a convincing prognostic marker as well as a potentially therapeutic target for GC.

Keywords: LIMD1; YAP1; gastric cancer; oncosuppressor; prognostic marker.

Figure 1

The mRNA level of LIMD1 correlates with prognosis in patients with GC. Notes: (A) KM plots of PFS and LIMD1 expression are shown. (B) KM plots of OS and LIMD1 expression are shown. The expression data were interrogated from the GEO datasets including GSE14210, GSE15459, GSE22377, GSE29272, GSE38749, GSE51105, and GSE62254, and the results were downloaded from KM Plotter (http://kmplot.com/). (C) The HRs for OS and PFS in LIMD1 high-expressing and low-expressing patients are compared in the forest plot. The mOS, mPFS, HRs, and 95% CI are listed for each subgroup. (D–F) KM plots of OS and LIMD1 expression for patients in the subgroups of different stages (D), Lauren’s classification (E), and HER2 amplifications (F) are shown. ^*P<0.05; ^**P<0.01; ^****P<0.0001. Abbreviations: GC, gastric cancer; KM, Kaplan–Meier; mOS, median OS; mPFS, median PFS; PFS, progression-free survival; GEO, gene expression omnibus.

Figure 2

LIMD1 impedes tumor growth in GC cell lines. Notes: (A) Western blot shows the expression level of LIMD1 in four GC cell lines, including BGC823, SGC7901, MKN45, and MKN28, (B, C) Western blot shows the LIMD1 expression levels in BGC823 and SGC7901 cells transfected with LIMD1 overexpression vector and empty vector. (D) LIMD1 expression levels in BGC823 cells transfected with LIMD1si and NCsi. (E–G). Proliferation curves of BGC823 and SGC7901 cells transfected with LIMD1 overexpression vector (E and F) and LIMD1-siRNA (G). (H–J). Colony formation assay of BGC823 and SGC7901 cells transfected with LIMD1 overexpression vector (H and I) and LIMD1-siRNA (J). The representing plates are shown on the left, and the quantification bars are shown on the right. Each panel represents at least three independent experiments. ^*P<0.05; ^**P<0.01. LIMD1, LIMD1 overexpression vector; pc3.1, empty vector. Abbreviations: GC, gastric cancer; LIMD1si, LIMD1-siRNA; NCsi, negative control siRNA; NS, no significance.

Figure 3

LIMD1 inhibits cell migration and invasion in GC cells. Notes: (A–C). Cell migration assays of BGC823 and SGC7901 cells transfected with LIMD1 overexpression vector (A and B) and LIMD1si (C) are shown (40×). (D–F) Cell invasion assays of BGC823 and SGC7901 cells transfected with LIMD1 overexpression vector (D and E) and LIMD1si (F) are shown (40×). (G–I) LIMD1 alters EMT markers. Western blot analysis with the indicated antibodies in LIMD1-overexpressing (G and H) and LIMD1-downexpressing (I) GC cells is depicted. Cell morphology of BGC823 cells with altered LIMD1 is depicted in (J) (10×). Immunofluorescence assay of EMT markers, including E-cadherin, N-cadherin, and Vimentin, is shown in (K) (40×). ^*P<0.05; ^**P<0.01; ^****P<0.0001. LIMD1, LIMD1 overexpression vector; pc3.1, empty vector. Abbreviations: EMT, epithelial to mesenchymal transition; GC, gastric cancer; LIMD1si, LIMD1-siRNA; NCsi, negative control siRNA.

Figure 4

LIMD1 suppresses YAP1 expression and translocation from the cytoplasm into the nucleus. Notes: (A–C) YAP1 expression in LIMD1-overexpressing (A and B) and LIMD1-downexpressing (C) GC cells is shown. The names of the cell lines are indicated below the panels. (D) Localization of YAP1 in BGC823 cells transfected with scramble siRNA and LIMD1 siRNA is shown by immunostaining of YAP1. Each panel represents at least three independent experiments. LIMD1, LIMD1 overexpression vector; pc3.1, empty vector. Abbreviations: GC, gastric cancer; LIMD1si, LIMD1-siRNA; mAb, monoclonal antibody; NCsi, negative control siRNA.

Figure 5

Gene deletion contributes to the reduced expression of LIMD1. Notes: (A) LIMD1 expressions in mRNA profile datasets are shown. The names of the datasets are on the top of the figures. All the datasets were extracted from the Oncomine database. (B) Copy number variations in normal tissues and GC tissues with different pathological types are shown. The data were taken from TCGA database and extracted from Oncomine database. (C) LIMD1 levels between GC patients with and without LIMD1 deletions in tow cohorts are compared. The data were taken from TCGA database and extracted from cBioPortal database. ^*P<0.05; ^**P<0.01; ^****P<0.0001. (D) Methylation levels throughout the region around LIMD1 gene between normal tissues and GC tumors are compared. Green probes indicate GpC islands. Abbreviations: AC, adenocarcinoma; GC, gastric cancer; NS, no significance; TCGA, The Cancer Genome Atlas.