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. 2019 Aug;26(8):1453-1466.
doi: 10.1038/s41418-018-0223-3. Epub 2018 Oct 25.

Deletion of TMEM268 inhibits growth of gastric cancer cells by downregulating the ITGB4 signaling pathway

Dubeiqi Hong  1   2   3 , Xuan Zhang  1   2   3 , Riyong Li  1   2   3 , Jiahong Yu  1   2   3 , Yaxin Lou  4 , Qihua He  4 , Xuanze Li  5 , Dong Xu  6 , Ping Lv  1   2   3 , Jian Lin  7 , Yingyu Chen  8   9   10
Affiliations

Deletion of TMEM268 inhibits growth of gastric cancer cells by downregulating the ITGB4 signaling pathway

Dubeiqi Hong et al. Cell Death Differ. 2019 Aug.

Abstract

Transmembrane protein 268 (TMEM268) encodes a novel human protein of previously unknown function. This study analyzed the biological activities and molecular mechanisms of TMEM268 in vivo and in vitro. We found that TMEM268 deletion decreases cell viability, proliferation, and cell adhesion as well as causing S-phase cell cycle arrest and disrupts cytoskeleton remolding. Xenograft tumor mouse model studies showed that TMEM268 deletion inhibits the tumorigenesis of BGC823 gastric cancer cells. In addition, TMEM268-deleted BGC823 cells failed to colonize the lungs after intravenous injection and to form metastatic engraftment in the peritoneum. Molecular mechanism studies showed a C-terminal interaction between TMEM268 and integrin subunit β4 (ITGB4). TMEM268 knockout promotes ITGB4 ubiquitin-mediated degradation, increasing the instability of ITGB4 and filamin A (FLNA). The reduced ITGB4 protein levels result in the disassociation of the ITGB4/PLEC complex and cytoskeleton remodeling. This study for the first time demonstrates that TMEM268 plays a positive role in the regulation of ITGB4 homeostasis. The above results may provide a new perspective that targeting the TMEM268/ITGB4 signaling axis for the treatment of gastric cancer, which deserves further investigation in the future.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
TMEM268 knockout inhibits cell growth and reduces tumorigenicity. a Western blot analysis of TMEM268 expression in control cells (TMEM268 WT) and Cas9-TMEM268/BGC823 cells. b Control and Cas9-TMEM268/BGC823 cells were seeded in six-well plates (1×105 cells/well). Seventy-two hours later, representative images were obtained by optical microscopy. c Control and Cas9-TMEM268/BGC823 cells were seeded in 96-well plates (3×103 cells/well; five replicates), serum-starved for 18 h and then pulsed with 10% FCS for different lengths of time. Cell viability was detected by MTS assay. Data represent the mean ± SD of three independent experiments. d Representative images of colony formation by indicated cells. e Number of clones counted in three independent experiments. Data are expressed as mean ± SD. *P < 0.05, ***P < 0.001. f The Control or Cas9-TMEM268/BGC823 cells were injected subcutaneously in BALB/c nude mice. Development of tumors (mean volume ± SD) was monitored using calipers. g, h Excised xenograft tumors were imaged and weighed on day 19. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 2
Fig. 2
TMEM268 knockout causes S-phase cell cycle arrest. a Control and CAS9-TMEM268/BGC823 cells were serum-starved for 24 h and then pulsed with 10% FCS for different amounts of time. Cell cycle distribution was assayed by flow cytometry. b Percentages of G0/G1, S, and G2/M phase cells. Each bar represents the mean ± SD of three independent experiments. c Cells were treated as described in (a), and protein levels detected by western blotting. d Quantification of the levels of the indicated proteins relative to GAPDH in cells treated as described in (a). Average value for control cells (24 h) was normalized to 1. Data shown represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3
TMEM268 knockout decreases cell adhesion. a Control and Cas9-TMEM268/BGC823 cells were allowed to attach to 96-well plates precoated with fibronectin or laminin. The unadhered cells were removed at 30 min, and the number of adhered cells was determined using an MTS assay (*P<0.05, **P<0.01). b−f Control and Cas9-TMEM268/BGC823 cells were plated on glass slides for 24 h, incubated with the indicated FITC-conjugated antibodies. Nuclei were stained with Hoechst 33342. Representative images obtained from confocal microscopy are shown. Scale bars, 10 μm
Fig. 4
Fig. 4
The TMEM268/ITGB4 interaction occurs at their C-terminals. a GST or GST-TMEM268 fusion protein were immobilized on glutathione-Sepharose beads and incubated with BGC823 cell lysates at 4 °C for 4 h. ITGB4 and GST were detected in the washed beads by western blotting. b Total BGC823 cell extracts were subjected to IP using either an anti-TMEM268 antibody or an IgG isotype control. ITGB4 was detected in the immunoprecipitates by western blotting. c, d Construction of truncated TMEM268 and ITGB4 plasmids. e BGC823 cells were transfected with FLAG-TMEM268, FLAG-TMEM268-N, or FLAG-TMEM268-C for 24 h. Total cell extracts were subjected to IP using either an anti-FLAG antibody or IgG isotype control. ITGB4 and FLAG were detected in the immunoprecipitates by western blotting. f GST and GST-TMEM268, GST-TMEM268-N or GST-TMEM268-C fusion protein immobilized on glutathione-Sepharose beads were incubated with BGC823 cell lysates. ITGB4 and GST were detected in the washed beads by western blotting. g 293T cells were cotransfected with FLAG-TMEM268 and either HA-ITGB4-C1, HA-ITGB4-C2 or HA-ITGB4-C3 for 24 h. Total cell extracts were subjected to IP using an anti-HA antibody. ITGB4 mutants and FLAG-TMEM268 were detected in the immunoprecipitates by western blotting
Fig. 5
Fig. 5
TMEM268 knockout promotes ITGB4 ubiquitination and degradation. a Control and Cas9-TMEM268/BGC823 cells were treated with different small molecules for different time (10 nM of Baf.A1 or 25 μM of CQ for 4 h; 10 mM MG-132 for 6 h). The cell lysates were then probed with anti-ITGB4 and anti-FLNA antibodies. GAPDH was used as the loading control. b Control and Cas9-TMEM268/BGC823 cells were treated with cycloheximide (CHX, 10 nM) for the indicated times. The levels of endogenous ITGB4 and FLNA were measured by western blotting. c, d RT-PCR and quantitative real-time RT-PCR were performed to assess mRNA levels of ITGB4 and FLNA in Cas9-TMEM268/BGC823 cells. e Control and Cas9-TMEM268/BGC823 cells were transfected with the indicated plasmids for 24 h. The cell lysates were then immunoprecipitated with an anti-FLAG antibody and probed with an anti-ITGB4 antibody
Fig. 6
Fig. 6
TMEM268 deficiency causes cytoskeleton remodeling and cytoskeletal network damage. a, b Control and Cas9-TMEM268/BGC823 cells were plated on glass slides for 24 h, then an immunofluorescence assay performed with the indicated antibodies. Nuclei were stained with Hoechst 33342. Representative images obtained from confocal microscopy are shown. Scale bars, 10 μm. c Changes in the microfilament network structure in control and Cas9-TMEM268/BGC823 cells were observed by transmission electron microscopy. Representative images obtained are shown
Fig. 7
Fig. 7
TMEM268 deletion prevents the metastasis of BGC823 cells. a Control or Cas9-TMEM268/BGC823 cells (1×106 cells/mouse) were injected into BALB/c nude mice (n = 6) via the tail vein. On day 54, the mice from each group were sacrificed, and the lungs filled with India ink via the upper trachea and fixed. Metastatic lesions (red arrows) on the black lung surface were observed and photographed. b H&E staining of lung tissues. c Control or Cas9-TMEM268/BGC823 cells (8×106 cells/mouse) were injected into the abdominal cavity of mouse (n = 6). The changes in body weight were monitored every 4 days after injection (*P < 0.05; **P < 0.01). d Images of the peritoneal metastatic nodules were obtained at day 32. Arrows indicate the disseminated tumor nodules.
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