Significant correlation between LKB1 and LGR5 gene expression and the association with poor recurrence-free survival in rectal cancer after preoperative chemoradiotherapy

Abstract

Purpose: The aim of the present study was to investigate whether the gene expression levels of LKB1 and LGR5 correlated with clinical outcome in patients with locally advanced rectal cancer treated with preoperative chemoradiotherapy (CRT).

Methods: Residual cancer cells were obtained from 52 patients with locally advanced rectal cancer treated with preoperative CRT. Total RNA was then isolated from formalin-fixed, paraffin-embedded specimens using microdissection. The expression levels of LKB1 and LGR5 genes were measured using real-time reverse-transcription polymerase chain reaction and by immunohistochemistry. In addition, in vitro studies were performed using colon cancer cell lines to study the serial changes of LKB1, LGR5 and PRKAA1 (AMPK) gene expression levels after irradiation.

Results: Our data demonstrate that specimens obtained from patients with poor pathological response and tumor recurrence had significantly higher gene expression levels of LKB1 and LGR5 than those without them (P < 0.05), and there was a significant positive correlation between LKB1 and LGR5 gene expression after CRT (Spearman's ρ: 0.429, P = 0.0023). The patients with high expression levels of both LKB1 and LGR5 had a significantly lower recurrence-free survival compared with the other group (P = 0.0055, 95 % confidence interval: 1.39-11.08). Lastly, in vitro studies demonstrated a similar pattern of serial gene expression among LKB1, LGR5 and PRKAA1 after irradiation.

Conclusions: Our results suggest that LKB1 and LGR5 expression may be implicated in resistance to CRT, therefore contributing to tumor relapse in patients with locally advanced rectal cancer treated with preoperative CRT.

Fig. 1

Recurrence-free survival curves according to LKB1 (a), LGR5 (b) gene expression levels and LKB1/LGR5 combination (c)

Fig. 2

Positive correlation between LKB1 and LGR5 gene expression in rectal cancer after CRT

Fig. 3

Immunohistochemical findings of LKB1, LGR5 and AMPK. LKB1 expression is observed in the cytoplasm and nucleus of residual cancer cells (a, d). LGR5 expression is observed in the membrane and cytoplasm of residual cancer cells (b, e). AMPK expression was observed in the cytoplasm of residual cancer cells (c, f). Original magnification: ×100 (a–c), ×400 (d–f)

Fig. 4

Expression patterns of LKB1, LGR5 and PRKAA1 (AMPK) in HT29 and DLD1 after irradiation. The serial change of these expressions after irradiation showed similar trends, especially at a dose of 5 Gy. Solid line 2.5 Gy; dashed line 5 Gy