Underexpression of LATS1 TSG in colorectal cancer is associated with promoter hypermethylation

Abstract

Aim: To investigate large tumor suppressor 1 (LATS1) expression, promoter hypermethylation, and microsatellite instability in colorectal cancer (CRC).

Methods: RNA was isolated from tumor tissue of 142 CRC patients and 40 colon mucosal biopsies of healthy controls. After reverse transcription, quantitative polymerase chain reaction (PCR) was performed, and LATS1 expression was normalized to expression of the ACTB and RPL32 housekeeping genes. To analyze hypermethylation, genomic DNA was isolated from 44 tumor CRC biopsies, and methylation-specific PCR was performed. Microsatellite instability (MSI) status was checked with PCR using BAT26, BAT25, and BAT40 markers in the genomic DNA of 84 CRC patients, followed by denaturing gel electrophoresis.

Results: Decreased LATS1 expression was found in 127/142 (89.4%) CRC cases with the average ratio of the LATS1 level 10.33 ± 32.64 in CRC patients vs 32.85 ± 33.56 in healthy controls. The lowest expression was found in Dukes' B stage tumors and G1 (well-differentiated) cells. Hypermethylation of the LATS1 promoter was present in 25/44 (57%) CRC cases analyzed. LATS1 promoter hypermethylation was strongly associated with decreased gene expression; methylated cases showed 162× lower expression of LATS1 than unmethylated cases. Although high-grade MSI (mutation in all three markers) was found in 14/84 (17%) cases and low-grade MSI (mutation in 1-2 markers) was found in 30/84 (36%) cases, we found no association with LATS1 expression.

Conclusion: Decreased expression of LATS1 in CRC was associated with promoter hypermethylation, but not MSI status. Such reduced expression may promote progression of CRC.

Keywords: Colorectal cancer; Large tumor suppressor 1; Microsatellite instability; Promoter hypermethylation; Quantitative polymerase chain reaction; Reduced expression; Salvador-Warts-Hippo pathway.

Figure 1

Large tumor suppressor 1 mRNA levels in colorectal cancer and control colon biopsies. Quantitative polymerase chain reaction results of large tumor suppressor 1 (LATS1) expression in 142 colorectal cancer (CRC) samples compared with 40 colon biopsies of healthy patients. CRC cases were divided according to clinicopathological data: tumor stage - Dukes’ A (n = 27), B (n = 41), C (n = 54), D (n = 20); histological differentiation of tumor cells (G staging): G1 (n = 3), G2 (n = 48), G3 (n = 88), G4 (n = 3). Vertical bars represent LATS1 fold ratio calibrated to the average C_t of control (ΔΔC_t^LATS1 = ΔC_t^{LATS1, sample} - ΔC_t^{LATS1, control}), error bars: SE. ^aP < 0.05 vs control group; ^cP < 0.05 between subgroups (Mann-Whitney U test).

Figure 2

Microsatellite instability status and large tumor suppressor 1 expression. Comparison of large tumor suppressor 1 (LATS1) mRNA levels in cases divided by the mutations observed in the BAT26, BAT25, and BAT40 microsatellite markers. Samples were considered to have microsatellite stability (MSS; no mutation), microsatellite instability-low grade (MSI-L; 1-2 mutations, light grey box), and microsatellite instability-high grade (MSI-H; mutations in all three markers, dark grey box). MSS (n = 40), MSI-L (n = 30), MSI-H (n = 14). Vertical bars represent LATS1 fold ratio calibrated to the average C_t of MSS cases - (ΔΔC_t^LATS1 = ΔC_t^{LATS1, MSI} - ΔC_t^{LATS1, MSS}), error bars: SE.

Figure 3

Large tumor suppressor 1 expression in colorectal cancer in relation to promoter methylation status. Comparison between large tumor suppressor 1 (LATS1) mRNA expression and epigenetic hypermethylation (M) or absence of hypermethylation (UM) of CpG islands located within the LATS1 promoter region in a total of 44 colorectal cancer (CRC) cases (black vertical bars, n = 25 for M and n = 19 for UM). Vertical bars represent the LATS1 fold ratio calibrated to the average C_t of control (ΔΔC_t^LATS1 = ΔC_t^{LATS1, sample} - ΔC_t^{LATS1, control}), error bars: SE. CRC cases were further divided into two subgroups: absence or presence of metastasis in lymph nodes/distant organs: N0M0: Light grey bars (n = 20; M: n = 9; UM: n = 11); N1-2/M0-1: Dark bars (n = 24; M: n = 16; UM: n = 8), respectively. ^aP < 0.05 vs control group; ^cP < 0.05 between subgroups (Mann-Whitney U test).

Figure 4

Methylation status of large tumor suppressor 1 in relation to the expression ratio and histological staging of cells. Forty-four colorectal cancer cases were classified according to histological examination: G1: Well-differentiated cells, n = 2 (light grey bars), G2: Moderately differentiated cells, n = 11 (grey bars), G3: Poorly differentiated cells, n = 28 (dark grey bars), G4: Undifferentiated cells, n = 3 (black bars). G1 epigenetic hypermethylation (M) (n = 1), G1 absence of hypermethylation (UM) (n = 1), G2 M (n = 3), G2 UM (n = 8), G3 M (n = 20), G3 UM (n = 8), G4 M (n = 1), G4 UM (n = 2). Vertical bars represent the large tumor suppressor 1 (LATS1) fold ratio calibrated to the average C_t of control (ΔΔC_t^LATS1 = ΔC_t^{LATS1, sample} - ΔC_t^{LATS1, control}), error bars: SE. ^aP < 0.05 vs control group; ^cP < 0.05 between subgroups (Mann-Whitney U test).