Autophagy-related Proteins as a Prognostic Factor of Patients With Colorectal Cancer

Abstract

Objectives: Autophagy plays a dual role in tumorigenesis. In the initial stages, it promotes cell survival and suppresses carcinogenesis, whereas in cancer development, it induces cancer cell survival. In this study, we investigate the role of autophagy as a protective or tumor suppressor mechanism in colorectal cancer (CRC) cell lines and evaluate its role as a potential biomarker in human tumor samples.

Materials and methods: The data of 68 patients with CRC treated at our Department from January 1 to December 31, 2016 were analyzed. Immunohistochemistry evaluation of p62, LC3B, Beclin-1, and Rab-7 in formalin-fixed paraffin-embedded tissue samples was performed and their expression was correlated with clinicopathologic characteristics, mutation status, and therapeutic approach. The χ was used to test an association among categorical variables. Survival curves were estimated using the Kaplan-Meier method and differences were assessed using the log-rank test. Colo-205, HT29, SW-480, and Caco-2 cell lines were also used so as to test the autophagy markers with oxaliplatin, irinotecan, hydroxychloroquine, and 3-methyladenine.

Results: Overexpression of Beclin-1 is associated with poor survival (P=0.001) in patients with CRC treated with chemotherapy, irrespective of the stage and mutational status. Rab-7 is also correlated with progression-free survival (PFS) (P=0.088). Oxaliplatin (10 and 20 μΜ) and irinotecan (10 and 20 μΜ) inhibit autophagy in microsatellite stable (MSS) CRC cell lines. The inhibition of autophagy in MSS CRC cell lines after treatment with oxaliplatin and irinotecan is further identified through monodancylcadaverine staining. Moreover, inhibition of autophagy with molecules such as hydroxychloroquine (20 μΜ) and 3-methyladenine (5 mM) was identified by the accumulation of p62 and LC3B.

Conclusions: Beclin-1 is an independent prognostic factor of overall survival and PFS. Also, Rab-7 is identified as an independent prognostic factor of PFS. Besides, several chemotherapeutic drugs such as oxaliplatin and irinotecan inhibit autophagy in MSS CRC cell lines in a similar way like hydroxychloroquine and 3-methyladenine. Thus, in MSS patients who develop chemoresistance, a combination of other therapies that include an autophagy inhibitor could be more beneficial. Further clinical trials are needed to investigate these therapeutic strategies.

FIGURE 1

Immunohistochemical staining of p62, LC3B, Beclin-1, and Rab-7 in human colorectal carcinomas (×40).

FIGURE 2

A, Kaplan-Meier estimates of overall survival of patients with CRC with low and high expression of autophagy markers p62, LC3B, Beclin-1, and Rab-7. High expression of Beclin-1 reduces life span. B, Kaplan-Meier estimates of PFS of patients with CRC with low and high expression of autophagy markers p62, LC3B, Beclin-1, and Rab-7. In low expression of Beclin-1 and Rab-7, we observed better PFS. CRC indicates colorectal cancer; PFS, progression-free survival.

FIGURE 3

Oxaliplatin and irinotecan inhibit autophagy in microsatellite stable colorectal cancer cell lines. Western blot analysis after 24-hour exposure of cells in 10 and 20 μM of oxaliplatin or irinotecan. The protein levels of Beclin-1, p62, LC3, and Rab-7 were identified by specific antibody. The quantification of LC3 reflects the whole protein levels as compared with the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). Protein levels were normalized against actin (A). The formation of autophagic vacuoles in Caco-2, Colo-205, HT29, and SW-480 cells because of treatments was determined with 0.1 mM of MDC (light blue) through confocal microscopy, whereas phalloidin staining (red) was used for cytoskeleton detection. The yellow arrows show the autophagic vacuoles. The graph represents the quantification of MDC in each cell line (B). MDC indicates monodansylcadaverine.

FIGURE 4

The protein levels of autophagy markers after treatment with 5 mM of 3-MA and 20 μM of HCQ in colorectal cancer cell lines. Western blot analysis after 24-hour exposure of cells in 5 mM of 3-MA and 20 μM of HCQ. The protein levels of Beclin-1, p62, LC3, and Rab-7 are presented. The quantification of LC3 reflects the whole protein levels as compared with the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). Protein levels were normalized against actin. HCQ indicates hydroxychloroquine; 3-MA, 3-methyladenine.