Comparative Analysis of the Immunohistochemical Expression of the Parkin and APC proteins in Polyps and Colorectal Adenocarcinomas

A marked decline in the colorectal cancer (CRC) burden was observed in the twentieth century,1 probably due to the enhancement in colonoscopy screening programs and the improvement in treatment strategies.2 Despite this, CRC remained the third leading cancer and second leading cause of cancer-related deaths worldwide in 2020.3

Many hereditary cancer syndromes have been implicated in the pathogenesis of CRC; however, most cases are sporadic.4 It is believed that most CRC cases arise from precursor lesions, such as adenomas, and follow a predictive disease-progression model called the adenoma-carcinoma sequence. This model of carcinogenesis describes progressive phenotypic changes that reflect the acquisition of nonrandom somatic mutations.5 Specifically, mutations in the APC gene are found in most CRC cases,6 and the loss of its tumor-suppressive activity is considered a fundamental and early step in CRC carcinogenesis.7 Since the first description of the adenoma-carcinoma sequence in 1988,8 more than 20 genes involved in this pathway have been identified.9

Ubiquitin ligases (E3) are proteins involved in cellular responses to environmental stress and DNA damage, including the regulation of apoptotic pathways.10 Impairment of E3 favors key processes of cellular carcinogenesis, including genetic instability and deregulated cellular proliferation.10 One of the human E3 is parkin, a protein encoded by the PARK2 (Parkinson disease [autosomal recessive, juvenile] 2) or PRKN (parkin RBR E3 ubiquitin protein ligase) gene. Parkin is present in an autoinhibited form,11 and it has been postulated12 that its activation occurs via posttranslational reversible changes in response to cell stress. A growing body of evidence suggests that PARK2 is involved in diverse carcinogenic pathways, such as mitochondrial quality control, apoptosis, metabolism, and cell-cycle progression,13 and that impaired PARK2 expression promotes cancer progression.14 PARK2 is located in a chromosomal region prone to instability and breakages, called FRA6E, which has already been associated with cancer development.15 A preclinical study16 showed that parkin deficiency increases the susceptibility to cancer development in mice after exposure to an oncogenic insult. Deficiency in parkin expression has been found in various human cancers, including gliomas,17 18 leukemia,19 ovarian cancer,15 lung cancer,20 21 and hepatocarcinoma,22 23 and previous evidence13 has led to the belief that PARK2 acts as a tumor suppressor gene.

Recent studies24 25 26 27 have proposed a role for parkin in CRC development. Decreased parkin expression has been reported in colorectal precursor lesions in mice24 and humans,25 as well as in human CRC.24 26 27 Parkin expression has been found to correlate with APC expression24 26 and with a better prognosis in CRC.26 However, the exact role of parkin in the adenoma-carcinoma sequence remains unknown. No previous studies have compared parkin expression in CRC to that in its precursor lesions to determine whether parkin expression changes during CRC carcinogenesis.

The present study aimed to compare the immunohistochemical expression of parkin and APC in colorectal polyps and adenocarcinomas.

We included 295 individuals, 222 (75.2%) of whom underwent polypectomy for neoplastic polyps, and 73 (24.7%) underwent colorectal cancer surgery for adenocarcinoma. The clinical characteristics of the sample are summarized in Table 1. Among the 222 (75.2%) individuals who underwent polypectomy, 122 (55%) presented more than 1 polyp. In total, 284 polyps were included in the study. A descriptive analysis of the neoplastic lesions is shown in Table 2.

Parkin and APC expression in high- and low-grade polyps and adenocarcinomas is shown in Table 3. We found a statistically significant difference in parkin expression between the lesions: in low-grade lesions, Parkin expression was higher in adenocarcinomas than in polyps (p < 0.001), and, in high-grade lesions, parkin expression was also higher among adenocarcinomas, although the difference was not statistically significant. No significant difference was found between polyps and adenocarcinomas when comparing low- and high-grade lesions. There was also a statistically significant difference in APC expression between the groups; it was higher in adenocarcinomas than in polyps in low- (p <0.001) and high-grade (p = 0.001) lesions.

The results of the univariate multilevel linear regression for the comparison of protein expression showed that adenocarcinomas had a significantly higher expression of parkin (6.19; 95%CI: 4.43–9.95; p < 0.001) (Fig. 3A) and APC (13.5; 95%CI: 11–15.9; p < 0.001) (Fig. 3B) than polyps. Among the colorectal polyps, a positive correlation was found between parkin and APC expression (0.23; p <0.001).

We demonstrated a significant correlation between parkin and APC expressions in polyps and adenocarcinomas. Previous research24 found that the deletion or alteration in the PARK2 gene is intrinsically related to a deficiency in the expression of the APC gene, both of which cooperate to accelerate tumor progression. Accelerated adenoma progression and multiplicity in APC were found in mutant mice when PARK2 knockout was present compared with those with an isolated APC mutation.24 However, isolated parkin loss did not lead to adenoma development.24 This suggests that PARK2 loss plays an additive role in CRC development but is insufficient if not accompanied by other key genetic events. Genomic analyses24 revealed a deficiency in PARK2 expression in approximately one-third of sporadic CRC cases, and PARK2 deficiency was found to be associated with APC deficiency.

This positive correlation was also found in another study26 that observed higher expressions of the APC and parkin proteins in tumor and normal tissues respectively. Both studies24 26 suggested that initial APC suppression can influence subsequent changes in parkin expression, which may be another mechanism by which parkin could be involved in cancer pathophysiology. Therefore, the tissue expression of parkin in colorectal polyps and adenocarcinomas is consistent with the expression of APC, a protein that is better established as a tumor suppressor. Regarding its role in the Vogelstein8 tumor progression model, the APC mutation is admitted as an initial event in tumors of the “classic” pathway, that is, in the left colon. Thereafter, a sequence of events occurs at different stages of the evolution of a polyp until it accumulates enough mutations to present dysplasia, which will invariably progress to carcinoma. We suggest that, in the initial stages of the Volgestein model, parkin has a tumor suppressor function, acting in conjunction with APC.

In addition, we observed a higher immunoexpression of parkin and APC in (high- and low-grade) adenocarcinomas than in polyps (with high- and low-grade dysplasia). There are no previous studies showing overexpression of such proteins in adenocarcinomas compared with neoplastic polyps. This suggests that, as a neoplastic polyp dedifferentiates toward the formation of adenocarcinoma, the expressions of parkin and APC increase, in contrast to what we initially expected. As both are products of tumor suppressor genes, when mutated, we expected that they would favor neoplastic evolution, thus being expressed in progressively smaller amounts. However, this assumption was not confirmed by the results observed. In fact, after the multilevel analysis with four subgroups of polyps and carcinomas, we confirmed that there was a greater expression of parkin and APC in the group of adenocarcinomas than in the polyp groups. Likewise, when analyzing the multilevel hierarchical structure for APC immunoexpression, a higher expression of this protein was identified in polyps, which is expected in the classic pathway of the carcinogenesis model. Thus, corroborating previous findings,29 the results observed with these markers may be associated with the progression of precursor lesions to colorectal cancer. A recent study25 reported high parkin expression in colonic polyps. On the other hand, other studies26 27 found lower PARK2 expression in adenocarcinomas than in normal tissue, and lower parkin protein expression levels in adenocarcinomas than in adjacent normal tissue.26 However, as in the current study we did not compare the results with normal tissue and polyps may eventually show alterations, the results may suggest that the eventual mutation of these genes, which leads to an expected reduction in their expression in the first events of the classic pathway, paradoxically results in a subsequent increase in expression when the cell becomes malignant and has a high potential for tumor invasion and aggressiveness (poorly differentiated neoplasms). Thus, analyzing the expression of parkin transversally, it could be increased in normal tissue, decreased in polyps, and again increased in adenocarcinomas, trying to contain tumor progression, but with expression still below the levels found in normal tissue.

Another alternative is that parkin mediates mitochondrial mitophagy. Mitochondrial damage caused by changes in tumor cells can lead to increased generation of reactive oxygen species and alterations in membrane potential. Parkin ensures mitochondrial homeostasis by inducing mitophagy in damaged mitochondria through an increase in the BAX/BCL-2 ratio. Thus, it protects cells from apoptosis and contributes to cell viability and consequent tumor progression.33 In an antagonistic manner, high levels of parkin in adenocarcinomas could contribute to tumor progression, resulting in increased expression accompanied by tumor progression.

However, it is important to note that the immunohistochemical analysis performed did not enable us to investigate whether the expressed protein was functional. Additional studies are needed to identify the functionality of this protein.

Therefore, we conclude that parkin and APC have a similar biological behavior in tumor suppression, but with a tendency towards a “de novo” increase in their expression as the neoplastic cell advances in the adenoma-carcinoma sequence. These findings indicate that parkin and APC may be involved in the late mechanisms of tumor progression control in the carcinogenesis pathway.