We found that loss of USP10 and p14ARF protein expression correlates with worse prognosis in colorectal cancer patients, and we confirmed a positive correlation between the molecules. Interestingly, USP10loss and USP10loss/p14ARFloss were significant markers of poor prognosis in patients without LN metastasis at the time of surgery, suggesting that USP10loss/p14ARFloss could predict disease progression in node-negative colorectal cancer. In addition, the methylation analysis revealed that the promoter region for USP10 is frequently hypermethylated in colorectal cancer cell lines and colorectal cancer primary tumors but not in normal colon tissues.
USP10 deubiquitinates p53 via MDM2, which regulates cellular p53 via reverse translocation and degradation of the protein . Also involved in the deubiquitination and stabilization of PTEN in lung cancer cells, USP10 has been reported to be a tumor suppressor . In addition, is an important mediator in the c-Myc-USP10-p14ARF axis because it deubiquitinates and stabilizes p14ARF . Lu et al. also suggested that USP10 behaves as a tumor suppressor in hepatocellular carcinoma . They demonstrated that USP10 inhibits the mTOR signaling pathway to deter cell growth in hepatocellular carcinoma. Further evidence that corroborates our results indicates that USP10 is a tumor suppressor in various cancer types, including gastric cancer , hepatocellular carcinoma , non-small cell lung cancer , small intestinal adenocarcinoma , and ovary cancer . These studies suggest that the loss of USP10 expression is significantly correlated with poor patient outcomes. On the contrary, Ouyang et al. proposed that USP10 plays an oncogenic role in colon cancer . They reported that USP10 promotes the expression of the oncogenic factor Musashi-2 (MSI2) by preventing its proteasome-dependent degradation. In addition, they demonstrated that USP10 promotes colon cell proliferation by deubiquitinating MSI2. Other evidence supports USP10 as an oncogenic factor in other cancers, including prostate cancer , glioblastoma multiforme , and breast cancer . Thus, the role of USP10 in cancer progression is controversial. One of the main reasons for this inconsistency in the data could be that cell conditions, such as the genetic mutation of tumor protein 53 (TP53), dictate the role of USP10 as a tumor suppressor or oncogenic factor. It is well-known that TP53 encodes a 53-kDa phosphoprotein and is commonly inactivated in a wide range of tumors, including colorectal cancer. Although we did not assess it in this study, the mutation rate of TP53 is reported to be about 50% in colorectal cancer. Notably, the prevalence of the TP53 mutation in colorectal patients depends on multiple factors, including tumor stage, location, and the status of hypermutation . Therefore, further studies are needed to clarify the association between the functional role of USP10 and the TP53 genotype in colorectal cancer.
Although several previous studies reported that USP10 is associated with tumor suppression in colon cancer, it remains unclear why USP10 expression is often downregulated in human cancers. In terms of the tumor suppressive role of USP10 in cancer, USP10 could contribute indirectly to regulating cell proliferation or tumor formation by interacting with other tumor suppressor proteins such as p53 or SIRT6 [28, 29]. Recently, overwhelming evidence suggests that promoter methylation is a key epigenetic mechanism that regulates gene expression, and that tumor-suppressor gene silencing in most cancers is caused by aberrant hypermethylated gene promoter regions. We had previously studied whether the CpG islands of the USP10 promoter region were hypermethylated in small intestinal adenocarcinoma , and our results imply that USP10 methylation occurs in an early stage of colorectal cancer development. Thus, the results of our previous and present studies strongly support that the downregulation of USP10 is epigenetically regulated in CRC.
The alternative reading frame (ARF) protein is frequently mutated in human cancer, and the CDKN2a locus encodes two different proteins (p14ARF and p16INK4a) . ARF is involved in regulating cell cycle arrest and apoptosis through p53-dependent and -independent pathways and is a potent tumor suppressor . The promoter region of p14ARF is known to be hypermethylated in a wide spectrum of human cancers, including colorectal cancer . According to one recent meta-analysis studying the prognostic value of p14ARF, the hypermethylation of p14ARF was more frequently observed in right side colon cancer and microsatellite instability (MSI)-associated cancer than in left side colon cancer and non-MSI associated cancer . In addition, methylation was not associated with tumor differentiation or colorectal cancer stage . Although a CpG island within the promoter region of p14ARF has been widely studied, p14ARF protein expression has not been thoroughly assessed via immunohistochemistry in colorectal cancers. A previous study reported that intact p14ARF expression was found in 36.9% (17/46) of colorectal cancers in Korean patients . On the other hand, we observed that p14ARF expression was not decreased in 71.4% (200/280) of colorectal cancer cases (Table 1). This discrepancy might be explained by the lack of well-defined p14ARF cut-off values for immunohistochemical scoring methods. Recently, Ko et al. demonstrated a positive correlation between USP10 and p14ARF expression in non–small cell lung cancer and found that the loss of both molecules correlated with poor prognosis. The role of USP10 and p14ARF as tumor suppressors has also been shown in small intestinal adenocarcinoma  and epithelial ovarian cancer . Corroborating previous studies, we we found that downregulation of both molecules was associated with poor patient prognosis. Interestingly, the prognostic significance of USP10loss/p14ARFloss was especially prominent in the group of patients that was LN-negative at the time of surgery (Supplementary Fig. S1). Therefore, to predict patients' prognoses, immunohistochemical staining for both USP10 and p14ARF is recommended if no LN metastasis is identified during surgery.
Our study has a few limitations. First, we used combined conventional immunohistochemistry and digital image analysis to quantitate the USP10 and p14ARF markers. Although the conventional manual scoring of immunohistochemical staining provides reasonable intra- and inter-observer reproducibility, this methodology has issues that make optimal scoring and deciding standard cut-off values for positivity difficult. We previously demonstrated that continuous immunohistochemical scoring via digital image analysis could improve the identification of optimal cut-off points compared with manual visual scoring , but that methodology needs to be standardized. For instance, a previously validated, commercially available antibody clone and a well-defined algorithm and cut-off value are essential for clinical utility. Second, although drug resistance in neoadjuvant therapy is a major factor contributing to patients’ negative outcomes, this single-institution study considered patients who did not undergo chemotherapy or radiation therapy prior to surgery. Interestingly, a previous study reported that the loss of USP10 correlated significantly with chemoresistance in epithelial ovarian cancer . Therefore, further multiple-institution studies considering a broad spectrum of diseases, including chemosensitivity response and disease recurrence cases, is warranted to validate optimal cut-offs and prognostic values. Third, our study could not include the results of various molecular tests, including MSI. The molecular characterization of patients with colorectal cancer has become a routine examination in clinical practice because genetic information about oncogenes and tumor suppressor genes provides insight into disease progression and response to therapy. However, we could not evaluate the association between molecular variation and USP10 or p14ARF protein expression. Further investigation with a larger number of cases and more molecular profiling data could be necessary to maximize the clinical value of USP10 or p14ARF information in colorectal cancer.