Abstract
Aim: To investigate the clinical significance of ATP-binding cassette transporter 11 (ABCC11) protein expression in colon cancer. Materials and Methods: One hundred thirty nine patients with colon cancer resection between 2009 and 2011 were enrolled. The relationship with immunohistochemical ABCC11 staining and clinicopathological factors was retrospectively analyzed. Results: Median age was 70 years including 67 males and 72 females. The patients with Stage 0, 1, 2, 3a and 4 were 4, 20, 43, 35, 7 and 30, respectively. The patients with curability (Cur) A, B and C were 109, 11 and 19, respectively. Positive expression of ABCC11 was observed in 31 patients (22.3%). There were no significant differences regarding age, gender, location, serum tumor markers, T category, lymphatic invasion and stage in relation to ABCC11 protein expression. Cases with node metastasis and venous invasion as well as unresectable cases were significantly more often found negative for ABCC11 protein (p=0.0246, 0.0285 and 0.0422, respectively). Concerning the 3 year disease free survival (DFS) and the 5 year overall survival (OS) in Stage 2/3 and in Stage 3 with adjuvant chemotherapy, no significant differences were found. However, OS in ABCC11 negative cases was 81.1%, which was significantly lower compared to positive cases, where OS was 96.2%. Conclusion: There was significant correlation with ABCC11 expression and lymph node metastasis, venous invasion and curability. The prognosis in ABCC11 negative cases was poor because of increased cases without curative resection.
- Colon cancer
- ATP-binding cassette transporter 11
- 5-fluorouracil
- prognosis
Colorectal cancer is the third most common cancer worldwide and the fourth most common cause of cancer-related deaths (1). Globally, cancer detection and treatment, including endoscopic and laparoscopic resection, chemotherapy and surveillance are known to significantly decrease mortality (2-4).
ATP-binding cassette transporter 11 (ABCC11) was identified as a multidrug resistance-associated protein (MRP) family based upon analyses of its predicted protein (5-7). ABCC11 overexpressing LLC-PK1 cells have been reported to be 2.9 fold more resistant to 5-FU (8). ABCC11 directly diminishes the effect of 5-FU through the transport of the active metabolite FdUMP (9).
Concerning breast cancer, Japanese and European women with wet earwax–associated single-nucleotide polymorphisms (SNPs) have a higher relative risk of developing breast cancer than those with dry earwax (10, 11). Patients with HER2+ and triple-negative tumor subtypes that expressed high levels of ABCC11 had a significantly worse disease-free survival (12). ABCC11 is involved in eribulin resistance in breast cancer cells in vitro, regardless of the subtype (13).
ABCC11 has been reported to be involved in anticancer drug resistance, by releasing 5-FU outside the cell through this membrane protein, in ABCC11 overexpressing cells (14). However, for colon cancer, transcript levels of ABCC11 were significantly lower in non-responders of palliative chemotherapy compared to responders. The relapse interval in patients with adjuvant chemotherapy was significantly shorter in patients with low transcript levels of ABCC11 (15). Low level of ABCC11 expression increased the hazard ratio of disease relapse in patients almost four times. Analysis of patients treated with 5-FU based regimens revealed that patients with low ABCC11 expression in their tumors had shorter disease-free interval (DFS) than those with higher content (15).
In this report, we aimed to identify the clinical significance of ABCC11 expression in colorectal cancer, including the distribution of expression, the relation with clinicopathological factors and prognosis. We retrospectively analyzed a cohort of colon cancer patients that underwent resection to assess the expression of ABCC11 protein in cases with colon cancer.
Materials and Methods
Patients. One hundred thirty-nine patients with colon cancer who consecutively underwent resection of primary tumor with written informed consent between 2009 and 2011 were enrolled. The relationship with the expression of ABCC11 protein performed with immunohistochemical staining and the clinicopathological factors was described based on the Japanese classification of colorectal carcinoma (16).
Immunohistochemistry. Tissue-specific staining was achieved through immunohistochemistry using the biotin-streptavidin-peroxidase method with antibodies against MRP8 (ab92331; Abcam, Cambridge, MA, USA). A staining procedure was performed according to the manufacturer's instructions. In brief, the deparaffinized tissue samples by xylene were hydrated in a series of decreasing alcohols. Blocking of endogenous peroxidase was performed by incubating the sections in 3% hydrogen peroxide for 45 min. For antigen retrieval, the sections were incubated in citrate buffer (pH 6.0) at 90°C for 20 min. Non-specific proteins were blocked by incubation of the sections in 10% skim milk for 30 min. The slides were then incubated with the primary antibodies diluted in 1% bovine serum albumin for 14 h. They were then washed in 1× phosphate-buffered saline (PBS) and incubated with the secondary antibody [labeled streptavidin biotin (LSAB), Dako Cytomation, Glostrup, Denmark] for 30 min at 37°C. The slides were washed again in 1× PBS and incubated with streptavidin peroxidase (LSAB) for 30 min at 37°C. The color detection was performed with the addition of 0.03% diaminobenzidine plus 3% hydrogen peroxide as the chromogen solution. They were finally stained with Harris hematoxylin solution for 1 min, dehydrated in increasing alcohols and cleared in xylene (17, 18).
Evaluation of staining. Staining results were assessed independently by two investigators without any information on the patients. Cases were defined as positive for ABCC11 protein when tumor cells were diffusely stained or at least 5% were positive for ABCC11 in focally stained cases (Figure 1).
Statistical analyses. All statistical analyses were performed using JMP Pro version 13 (SAS institute Inc., Cary, NC, USA). Differences in groups based on ABCC protein expression were assessed by Chi square test and Student's t-test. The disease free survival and overall survival were calculated using the Kaplan Meier method. Significant differences were identified by the log rank test. A statistically significant difference was considered when p<0.05. The protocol of this study was approved by the Institutional Review Board of Tokyo Women's Medical University (approval no. 4563-R).
Results
Patient characteristics. The characteristics of the patients enrolled are summarized in Table I. Median age was 70 years, ranging from 43 to 89, including 67 males and 72 females. The tumor location was divided into proximal colon with 86 cases in the cecum, ascending, transverse and distal colon with 53 cases in descending, sigmoid and rectosigmoid colon. The patients with stage 0, 1, 2, 3a and 4 were 4, 20, 43, 35, 7 and 30, respectively. The patients with curability (Cur) A, B and C (16) were 109, 11 and 19, respectively. Positive expression of ABCC11 was observed in 31 patients (22.3%).
Correlation of ABCC11 protein expression with clinicopathological findings. There were no significant differences regarding age, gender, location of primary lesion and serum tumor markers in relation to ABCC11 protein expression. Additionally, there was no significant difference regarding T-category, lymphatic invasion and stage. Cases with node metastasis and venous invasion were significantly more often observed in the group without ABCC11 protein expression (p=0.0246 and 0.0285, respectively). There was significantly more cases with Cur C cases (unresectable cases) in the negative expression group (p=0.0422) (Table II).
Prognosis according to ABCC11 protein expression. The 3-year disease free survival (DFS) in Stage 2/3 was 79.3% in negative and 85.4% in positive ABCC11 cases. The 5 year overall survival (OS) was 90% in negative and 95% in positive ABCC11 cases. In Stage 3 cases performed with adjuvant chemotherapy, DFS and OS were both 80.0% in ABCC 11 positive cases, whereas in ABCC11 negative cases, DFS and OS were 76.4% and 94.7%, respectively. There were no significant differences of prognosis in both groups. However, in all cases, OS in negative cases was 81.1%. It was significantly poorer than in positive cases with 96.2% in OS (Table III).
Discussion
In the present study, the expression of ABCC11 protein was firstly assessed by immunohistochemical staining using an anti-ABCC11 antibody. The distribution of ABCC11 protein expression was broadly found from the early stages, i.e. without the ability of metastasis such as in situ to the advanced stage. There was no apparent relation between tumor growth and ABCC protein expression. However, lymph node metastasis was significantly more often observed in ABCC11 negative cases. Also, cases with venous invasion were more often found in ABCC negative patients. Although ABCC11 is not involved in tumor cell proliferation, it may be involved in metastasis and malignant transformation such as migration and invasion.
The function of ABCC11 protein was previously reported as an efflux pump of drugs, including anticancer drugs (6). ABCC11 is known to perform outward transport of nucleoside analogs such as 5-fluorouracil used for the treatment of colorectal cancer; nevertheless, there was no association of ABCC11 with relapse and survival in Stage III patients treated with adjuvant chemotherapy. Moreover, the OS in ABCC11 negative cases was found to be significantly worse compared to the OS in positive cases, by analysis of all patients in the present study. When the function of ABCC11 was maintained, anticancer drugs could not work against tumor cells expressing ABCC11 due to the efflux pump effect of the drug. Krizkova et al. also reported that analysis of patients with 5-FU based adjuvant chemotherapy revealed that patients with low ABCC11 expression in their tumors had shorter DFS than those with higher expression (13). These results suggest that the interaction between ABCC11 expression and 5-FU efflux should be found as the opposite effect in terms of 5-FU treatment. Additionally, the present study shows that the ABCC11 protein expression cannot predict the prognosis in cases with colon cancer.
Although this study obtained a certain result, there were several limitations to this report. First, this study was conducted as a retrospective study which may include selection bias, although consecutive cases were used. Second, the number of cases in this study was limited because it was performed in a single institution. Data from previous clinical trials or prospective studies with much more cases will be required. Finally, we could not offer a precise mechanistic explanation for the observed role of ABCC11 in colon cancer tissue.
In conclusion, there was a significant relation of ABCC11 expression levels and lymph node metastasis, venous invasion and curability. Prognosis in ABCC11 negative cases was poor because of more cases without curative resection. To elucidate the role of ABCC11 in colon cancer, further validation and functional analysis in colon cancer tissue must be performed.
Acknowledgements
The present study was supported by the Tokyo Association for Clinical Surgery. This agency has no effect on the study design and execution.
Footnotes
Authors' Contributions
YY and YK contributed to planning of the study, assessed the immunohistochemical staining, analyzed data and prepared the manuscript. YK contributed to the immunoshitochemical procedure. YH and OS supported the collection of the patient's data. SS supervised all of this work.
Conflicts of Interest
The Authors declare no competing interests.
- Received June 20, 2020.
- Revision received July 22, 2020.
- Accepted July 27, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved