High expression of KIBRA in low atypical protein kinase C-expressing gastric cancer correlates with lymphatic invasion and poor prognosis

Abstract

Overexpression of atypical protein kinase Cλ/ι (aPKCλ/ι), a regulator of cell polarity, is frequently associated with the poor prognoses of several cancers, including gastric cancer. Recent studies revealed a molecular link between aPKC and KIBRA, an upstream regulator of tumor suppressor Hippo pathway that regulates cell proliferation and apoptosis. Further, KIBRA directly inhibits the kinase activity of aPKC to regulate epithelial cell polarity. These observations suggest that the KIBRA-aPKC connection plays a role in cancer progression; however, clinical significance of the correlation between these factors remains unclear. Here we examined the correlation between KIBRA/aPKCλ/ι expression, as detected by immunohistochemistry, and clinicopathological outcomes in 164 gastric cancer patients using Fisher's exact test and Kaplan-Meier log-rank test. We found an intimate correlation between the expression level of KIBRA and aPKCλ/ι (P = 0.012). Furthermore, high expression of KIBRA is correlated with lymphatic (P = 0.046) and venous invasion (P = 0.039). The expression level of KIBRA by itself did not correlate with the prognosis; however, high expression of KIBRA in low aPKCλ/ι-expressing gastric cancer correlated with disease-specific (P = 0.037) and relapse-free survival (P = 0.041) by Kaplan-Meier with log-rank test and higher lymphatic invasion cases by Fisher's exact test (P = 0.042). Furthermore, overexpression of the aPKC-binding region of KIBRA disrupted tight junctions in epithelial cells. These results suggest that high expression of KIBRA in low aPKC-expressing cells causes massive loss of aPKC activity, leading to loss of polarity and invasiveness of gastric cancer cells.

Figure 1

Expression and localization of KIBRA in non‐neoplastic and cancerous gastric tissues. Representative images of non‐neoplastic and cancerous gastric tissues immunostained for KIBRA are shown: non‐neoplastic epithelium (a), gastric epithelium with intestinal (b), signet ring cells (c), gastric cancer tissue diagnosed as 0 +  (d), 1 +  (e), 2 +  (f), and 3 +  (g). Bars: 20 μm.

Figure 2

No correlation between the expression of KIBRA and disease‐specific survival or relapse‐free survival. Kaplan–Meier plot comparing positive (line) and negative (dashed line) staining of KIBRA for disease‐specific survival (DSS) (a) and relapse‐free survival (RFS) (b) are shown. P‐values were calculated by log‐rank test.

Figure 3

Association between high expression of KIBRA in aPKCλ/ι‐low gastric cancer and poor prognosis. Kaplan–Meier plot comparing positive (line) and negative (dashed line) staining of KIBRA for disease‐specific survival (DSS) and relapse‐free survival (RFS) in a aPKCλ/ι‐high group (a & b) or in a aPKCλ/ι‐low group (c & d) are shown. P‐values were calculated by log‐rank test.

Figure 4

Overexpression of the atypical protein kinase C (aPKC) binding region of KIBRA affects tight junction formation. (a) Madin‐Darby canine kidney (MDCK) cells were transfected to express indicated proteins and then subjected to calcium switch assays. Cells were fixed at indicated times after calcium switch and stained for ZO‐1 (green), tRFP (red), and F‐actin (blue). Scale bars, 10 μm. (b) Cells that showed disrupted ZO‐1 staining in tRFP‐ or tRFP‐aBR‐expressing MDCK cells were counted, and percentages for total RFP positive cells were calculated. Values represent the mean ± standard deviation (SD) of three independent experiments. P‐values were calculated by Student's t‐test.