Loss of monopolar spindle-binding protein 3B expression promotes colorectal cancer invasiveness by activation of target of rapamycin kinase/autophagy signaling

Figure 1 Monopolar spindle-binding protein 3B downregulation is associated with a poor colorectal cancer prognosis. A: Immunohistochemistry images. Monopolar spindle-binding protein 3B (MOB3B) protein was detected in colorectal cancer and distant normal tissues (magnification, 400 ×); B: Kaplan-Meier curves stratified by MOB3B expression and the log-rank test.

Figure 2 Establishment of RKO and DLD1 cell subline with stable monopolar spindle-binding protein 3B overexpression or knockdown. A-D: Quantitative reverse transcription-polymerase chain reaction; E-H: Western blot; I: Quantified data of E; J: Quantified data of F; K: Quantified data of G; L: Quantified data of H. The data are summarized as the mean ± SD of three independent experiments. ^aP < 0.0001 vs RKO-NC+, ^bP < 0.0001 vs RKO-NC-, ^cP < 0.001 vs DLD1-NC+, ^dP < 0.0001 vs DLD1-NC-, ^eP < 0.001 vs RKO-NC+, ^fP < 0.001 vs RKO-NC-, ^gP < 0.001 vs DLD1-NC+, ^hP < 0.01 vs DLD1-NC-.

Figure 3 Effect of monopolar spindle-binding protein 3B on proliferation, migration, and invasion of RKO and DLD1 cells. A-D: Cell counting kit-8 assay; E-H: Transwell assay. The data are presented as the mean ± SD of three independent experiments. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001.

Figure 4 Antitumor activity of monopolar spindle-binding protein 3B in nude mice. A: Tumor cell xenograft assay using RKO-OE and RKO-NC+ cells; B: Weight of xenografts from RKO-OE vs RKO-NC+ cells; C: Tumor cell xenograft growth curves of RKO-OE and RKO-NC+ cells; D: Tumor cell xenograft assay using RKO-KD and RKO-NC- cells; E: Weight of xenografts from RKO-KD vs RKO-NC- cells; F: Tumor cell xenograft growth curves of RKO-KD vs RKO-NC- cells. n = 5, ^bP < 0.01.

Figure 5 Effects of monopolar spindle-binding protein 3B on expression of matrix metalloproteinase 2, matrix metalloproteinase 9, phosphorylated mechanistic target of rapamycin kinase, and autophagy related proteins in RKO cells. A: Representative Western blot images. Stable monopolar spindle-binding protein 3B (MOB3B)-overexpressing RKO cells were grown and subjected to Western blot analysis; B: Quantified data of A; C: Representative Western blot images. RKO cells with stable MOB3B knockdown were grown and subjected to Western blot analysis; D: Quantified data of C. The data are summarized as the mean ± SD of three replicates. ^aP < 0.05, ^bP < 0.01. mTOR: Mechanistic target of rapamycin; MMP: Matrix metalloproteinase.

Figure 6 Effect of monopolar spindle-binding protein 3B on number of autophagosomes/autophagy lysosomes in RKO cells. A: Transmission electron microscopy (TEM) images. Stable monopolar spindle-binding protein 3B (MOB3B)-overexpressing RKO cells were grown and treated with MHY 1485 (50 μM), a mechanistic target of rapamycin kinase (mTOR) agonist, for 48 h, and then subjected to TEM analysis of cell morphology; B: Quantified data of A; C: TEM images. RKO cells with stable MOB3B knockdown were grown and treated with rapamycin (100 nM), an mTOR inhibitor, for 48 h, and then subjected to TEM analysis of cell morphology; D: Quantified data of C. The arrows show autophagosomes/autophagy lysosomes. The data are shown as the mean ± SD of five replicates. ^aP < 0.05 vs RKO-NC+, ^bP < 0.05 vs RKO-OE, ^cP < 0.05 vs RKO-NC-, ^dP < 0.05 vs RKO-KD.

Figure 7 Monopolar spindle-binding protein 3B inhibition of colorectal cancer cell malignant behaviors through inactivation of mechanistic target of rapamycin kinase/autophagy signaling. A: Western blot images. Stable monopolar spindle-binding protein 3B (MOB3B)-overexpressing RKO cells were grown, treated with MHY 1485 (50 μM) for 48 h, and then subjected to Western blot analysis; B: Quantified data of A; C-H: RKO cells with with stable MOB3B knockdown were grown, treated with rapamycin (100 nM) for 48 h, and then subjected to wound healing assay (C and D), Transwell assay (E and F), and Western blot analysis (G and H). The data are presented as the mean ± SD of three independent experiments. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. mTOR: Mechanistic target of rapamycin; MMP: Matrix metalloproteinase; LC: Light chain.