Prognostic relevance of autophagy-related markers LC3, p62/sequestosome 1, Beclin-1 and ULK1 in colorectal cancer patients with respect to KRAS mutational status

Abstract

Background: Autophagy is a cellular pathway that regulates transportation of cytoplasmic macromolecules and organelles to lysosomes for degradation. Autophagy is involved in both tumorigenesis and tumour suppression. Here we investigated the potential prognostic value of the autophagy-related proteins Beclin-1, p62, LC3 and uncoordinated (UNC) 51-like kinase 1 (ULK1) in a cohort of colorectal cancer (CRC) specimens.

Methods: In this study, we analysed the immunoexpression of the autophagy-related proteins p62, LC3, Beclin-1 and ULK1 in 127 CRC patients with known KRAS mutational status and detailed clinical follow-up.

Results: Survival analysis of p62 staining showed a significant correlation of cytoplasmic (not nuclear) p62 expression with a favourable tumour-specific overall survival (OS). The prognostic power of cytoplasmic p62 was found in the KRAS-mutated subgroup but was lost in the KRAS wildtype subgroup. Survival analysis of Beclin-1 staining did not show an association with OS in the complete cohort. LC3 overexpression demonstrated a slight, though not significant, association with decreased OS. Upon stratifying cases by KRAS mutational status, nuclear (not cytoplasmic) Beclin-1 staining was associated with a significantly decreased OS in the KRAS-mutated subgroup but not in the KRAS wildtype CRCs. In addition, LC3 overexpression was significantly associated with decreased OS in the KRAS-mutated CRC subgroup. ULK1 expression was not correlated to survival.

Conclusions: Immunohistochemical analyses of LC3, p62 and Beclin-1 may constitute promising novel prognostic markers in CRC, especially in KRAS-mutated CRCs. This strategy might help in identifying high-risk patients who would benefit from autophagy-related anticancer drugs.

Keywords: Autophagy; Beclin-1; Colorectal cancer; LC3; ULK1; p62.

Fig. 1

p62 immunohistochemistry. Examples of p62 immunostaining in colorectal cancers. The upper row shows three tumours with strong cytoplasmic immunostaining and the lower row demonstrates three tumours with negative p62 staining (×400 magnification). Note the positively stained of stromal cells/macrophages, which can be used as internal controls

Fig. 2

Beclin-1 immunhistochemistry. Examples of Beclin-1 immunostaining in colorectal cancers. On the left, there is a cancer without any Beclin-1 immunostaining. In the middle, there is a cancer with prominent cytoplasmic Beclin-1 immunostaining whereas on the right a colorectal cancer exhibits a mainly nuclear Beclin-1 expression (×400 magnification)

Fig. 3

LC3 immunohistochemistry. On the left side, there is a cancer without any dot-like LC3 immunostaining. Notice the positively stained nerve, which can be considered as a positive internal control, since nerve fibers are constantly LC3 positive. On the right side, there is a CRC with a strong and characteristic dot-like LC3 immunostaining. A diffuse cytoplasmic staining was not noticed (×400 magnification)

Fig. 4

ULK1 immunhistochemistry. On the left, there is a picture of a cancer with strong cytoplasmic immunostaining. In the middle, there is a cancer with barely seen faint ULK1 staining, classified as negative. On the right side, missing staining in normal mucosa is demonstrated. Note the positivity in small blood vessels as an internal control (×400 magnification)

Fig. 5

Autophagy-related proteins in normal colonic epithelial tissue. In the upper left, the picture shows normal crypts lacking Beclin-1 immunoexpression. In the upper right, negative p62 staining can be seen in normal colonic crypts. Notice the positive staining in macrophages. The lower left picture demonstrates lack of LC3B expression in crypts. Notice the moderate LC3B positivity in nerve cells. The lower right light micrograph shows a lack of ULK1 staining in normal crypts (×400 magnification)

Fig. 6

Kaplan-Meier survival plot of 126 colorectal cancers (KRAS wildtype and KRAS mutated) in relation to p62 immunostaining intensity. p62-negative CRC revealed a significantly decreased OS (log-rank test: p = 0.043)

Fig. 7

Kaplan-Meier survival plot of 34 KRAS-mutated colorectal cancers in relation to nuclear Beclin-1 immunoexpression. Strong nuclear Beclin-1 expression was significantly associated with decreased OS survival (log-rank test: p = 0.010)

Fig. 8

Kaplan-Meier survival plot of 123 colorectal cancers (KRAS wildtype and KRAS mutated) in relation to dot-like LC3 immunostaining intensity. LC3 expression lacked a significant association with OS; however, there was a statistical trend of LC3 expression being associated with decreased OS (log-rank test: p = 0.063)

Fig. 9

Kaplan-Meier survival plot of 32 KRAS-mutated colorectal cancers in relation to dot-like LC3 immunoexpression. LC3 expression was significantly associated with decreased OS survival (log-rank test: p = 0.023)

Fig. 10

Kaplan-Meier survival plot of 62 colorectal cancers with advanced stage UICC III/IV (all treated with 5-FU-based chemotherapy) in relation to nuclear Beclin-1 immunoexpression. In this subgroup, strong Beclin-1 expression was significantly associated with decreased OS survival (log-rank test: p = 0.011)