Colorectal carcinomas (CRC) is one of the most prevalent malignancy worldwide, and the third most deadly cancer (1). Despite the significant advancement in diagnosis and treatment of colorectal cancer over the past decade, the survival rate of advanced colorectal cancer remains poor owing to tumor recurrence, distant metastasis, and lack of diagnosis markers. Therefore, there is an urgent need for new prognostic and diagnostic markers and therapeutic targets, and a better understanding of molecular mechanisms underlying tumor development and progression.

TMEM16A, also known as ANO1, DOG1, ORAOV2, or TAOS2, was identified as a novel molecular component of calcium-activated chloride channel (CaCC) in 2008 (2–4). TMEM16A regulates many cellular functions, including epithelial secretion, cardiac, and neuronal excitation, smooth muscle contraction (5–8). Before the discovery of TMEM16A as a CaCC, TMEM16A has been described as a biomarker for gastrointestinal stromal tumor (GIST) (9, 10). Recently, growing evidences have shown that TMEM16A is overexpressed or amplified in many tumors, such as head and neck squamous cancer cells (HNSCC), esophageal squamous cell cancer (ESCC), and breast cancer (11–17), and overexpression of TMEM16A plays an important role in the development and progression of tumors (18–22). Duvvuri et al. found that TMEM16A overexpression correlated with decreased overall survival in patients with HNSCC (19). Ayoub et al. found ANO1 amplification and expression in human papilloma virus (HPV)-negative HNSCC accompanied with a high propensity for future distant metastasis (20). Shi et al. reported that TMEM16A mRNA expression and protein overexpression was associated with lymph node metastasis and advanced clinical stage in ESCC patients (21). Bae et al. found that TMEM16A expression is associated with shorter survival and progression of breast cancer (22). Thus, TMEM16A was thought as a new promising prognostic and diagnostic marker and potential therapeutic target for the treatment of some types of cancers. However, current available information is very limited regarding biological function and clinical significance of TMEM16A expression in CRC.

In our previous study, we found that TMEM16A expression in colorectal cancer SW620, HCT116, and LS174T cells, not in SW480 and HCT8 cells, and inhibition of TMEM16A expression decreased the growth, migration, and invasion ability of SW620 cells (23). These interesting findings prompted us to investigate clinical relevance of TMEM16A expression in CRC tissues. In the present study, we detected TMEM16A mRNA expression and protein expression in clinical CRC samples. Then, we analyzed the correlations of TMEM16A expression with clinicopathological parameters and patient prognosis. Finally, we investigated the relation between TMEM16A and tumor proliferation and metastasis related molecules, including KRAS/NRAS/BRAF mutations status, and expression of known proteins involved in CRC (Braf, CDX2, EGFR, p53, Ki67, CD34, MLH1, MSH2, MSH6, and PMS2).

In this study, we performed a detail analysis of TMEM16A mRNA and protein expression in human CRC tissue samples. Consistent with earlier studies (25–28), TMEM16A mRNA expression was found to be significantly up-regulated in CRC tissues compared with para-cancerous normal tissues, indicating that TMEM16A may participate in the process of carcinogenesis. Furthermore, TMEM16A mRNA expression was detected in 76% CRC cases, whilst TMEM16A protein expression occurred at a lower frequency (26%). There is a weak positive correlation between TMEM16A mRNA expression and protein expression. (Spearman's = 0.337, p = 0.019). TMEM16A mRNA expression didn't lead to TMEM16A protein expression in more than half of cases. Although TMEM16A mRNA expression may be responsible for TMEM16A protein expression, it is obviously not the only mechanism of TMEM16A expression in CRC. Similar discrepancys for TMEM16A mRNA and protein expression have been reported in previous studies (19, 25–28). For instance, TMEM16A gene amplification was frequently detected than protein expression in HNSCC samples (25, 27). In contrast, TMEM16A overexpression was more pervasive than gene amplification in human breast cancer and human gastric cancer (27, 29). We will discuss in detail the possible mechanisms leading to this interesting difference later.

The roles of TMEM16A expression in multiple tumor samples has been extensively investigated. Most investigators reported that TMEM16A expression promotes tumor growth and metastasis, and is associated with poor patient prognosis (21, 25–28, 30–34). However, some researchers demonstrated different effects of TMEM16A on these hallmarks. For example, Shiwarski et al. demonstrated that primary tumors exhibit a high level of TMEM16A, whereas metastasis from lymph nodes have a low expression of TMEM16A (28). Wu et al. reported that TMEM16A overexpression is associated with good prognosis in PR-positive or HER2-negative breast cancers patients following Tamoxifen treatment, especially in those patients with the low expression of Ki67 (35). Dixit et al. found that TMEM16A/ANO1 was preferentially overexpressed in HPV negative HNSCC compared with HPV positive HNSCC, and that this overexpression was associated with decreased patient survival (36). Rodrigo et al. observed that there was no correlation between TMEM16A and clinical parameters in HNSSC and patients with TMEM16A-positive oropharyngeal tumors exhibited a significantly improved disease-specific survival, compared to hypopharyngeal, and laryngeal tumors (18). These results indicated the multifaceted role of TMEM16A in various cancers may be cell type-dependent.

The present study is the first conducted to explore the predictive and prognostic value of TMEM16A in patients with CRC by analyzing the correlation between TMEM16A mRNA expression or protein expression and clinical parameters. The results demonstrated that TMEM16A mRNA expression is correlated with tumor TNM stage and lymph node metastasis. The positive rate of TMEM16A mRNA expression in primary colorectal cancer showed a significant increase with lymph node metastasis and late TNM stage. Univariate and multivariate logistic regression analysis suggested that TMEM16A mRNA expression was an important independent predictive factor of lymph node metastasis in CRC. These results showed that TMEM16A mRNA expression may be a promising predictive biomarker just as previously reported (36–38). However, we found that higher levels of TMEM16A mRNA expression mainly occurred in N0 phase and TNM II stage (Supplementary Figure 2), suggesting that TMEM16A mRNA expression level may not be positively correlated with lymph node metastasis and late tumor stage, which was inconsistent with the results by Park et al. (26). Various factors could contribute to these disagreement results, such as differences in the treatment regiments of patient enrollment, particular tumor environment, sample size, and/or detection methods.

We next studied the relationship of TMEM16A expression with clinical prognosis of CRC patients. The results showed that TMEM16A mRNA expression tended to shorter disease-free survival and overall survival for CRC patients, although it is not statistically significant. This could because TMEM16A mRNA expression was closely related to TNM stage and lymph node metastasis, which contribute to clinical prognosis of CRC patients. In addition, other factors such as the patient's age, tumor location, venous invasion, and the treatment after operation, such as radiotherapy, chemotherapy, and targeted therapy, might affect the prognosis of patients. Another possibility is that this may be due to cell type-dependent, which needs to be further confirmed by increasing the sample size and prolonging the observation time of patients.

In order to verify our results, we made further bioinformatics analysis of TMEM16A mRNA expression in TCGA database (http://ualcan.path.uab.edu/). The results were showed in Supplementary Figure 3. The results demonstrated that TMEM16A was upregulated in colon adenocarcinoma (n = 286) comparing with normal tissues (n = 41), which had statistical significance (P = 1.62E−12). There was no significant difference (p = 0.36) in overall survival between patients with high TMEM16A mRNA expression (n = 70) and those with low TMEM16A mRNA expression (n = 209). These results are consistent with our results.

It has been reported that TMEM16A contributed to tumor progression by modulating other factors and their downstream signaling pathways (27, 29, 30, 38–42). However, the mechanisms underlying regulation of tumor tumorigenesis, growth, and metastasis by TMEM16A remained unclear. To explore the potential mechanism, we investigated the correlation between TMEM16A expression and mutation status of KRAS, NRAS, and BRAF, and the protein expression of most commonly used IHC Characteristics including BRAF (V600E), CDX2, EGFR, p53, Ki67, CD34, PMS2, MLH1, MSH6, and MSH2 in clinical CRC sample.

We found that there was a significant positive correlation between MSH2 and TMEM16A mRNA expression. Previous studies have shown that MLH1, MSH2, MSH6, and PMS2 as main proteins of mismatch repair proteins (MMR) are used to repair DNA replication errors. MMR deficient (dMMR) leads to microsatellite instability (MSI), which is an important cause of CRC (43). In the occurrence and development of CRC, 90% of MMR gene mutations are mainly caused by the inactivation of MLH1and MSH2 (44). It seems that MSH2 may be involved in the role of TMEM16A in the occurrence and development of CRC.

Strikingly, our statistical analysis showed that TMEM16A protein expression was positively correlated with KRAS mutation status, and negatively correlated with mutant p53 protein expression. Recent comprehensive genome analyses have identified frequently mutated genes in human CRC, including APC, KRAS, TGFBR2, and Tp53 (45). Among them, KRAS and p53 mutations have been found in ~40 and ~60% of CRC (45, 46). Gene mutations in KRAS and p53 are thought to be essential events for colorectal cancer development. Previous findings suggested that mutated KRAS continuously activates RAS-RAF-MAPK signaling pathway, which leads to uncontrolled cell proliferation and canceration (46, 47). Thus, we infer that TMEM16A protein may promote the occurrence and growth of colorectal cancer by activating mutated KRAS. In addition, it is generally believed that mutant p53 overexpression is related to tumor metastasis, recurrence, and poor prognosis (48, 49). More importantly, our logistic regression analysis further confirmed that mutant p53 protein was an independent predictive factor of lymph node metastasis in CRC samples. Therefore, we speculate that TMEM16A protein might suppress tumor metastasis indirectly by decreasing mutant p53 protein expression. Considering this, our data evoke an intriguing possibility that TMEM16A protein may play a dual role in tumor formation and metastasis by interacting with mutated KRAS and mutant p53 protein in CRC tissues. It should be noted that other proteins might be involved in the regulation of tumor growth and metastasis by TMEM16A, so further studies are required.

Although it has been recently reported that TMEM16A activated EGFR signaling pathway in HNSCC, breast cancer and pancreatic cancer (15, 30, 31, 50, 51). However, in this study, we did not find that TMEM16A was significantly associated with EGFR in human CRC tissues. One of the possible reasons is that TMEM16A regulates cancer cell function via its different protein networks in different cancer cells.

Previous studies demonstrated that TMEM16A expression could be regulated at transcriptional, translational, and post-translational level, and TMEM16A expression is able to modulate different molecules through multiple ways in various cancers (52). Based on our results, we speculated that MSH2 might be involve in regulating of TMEM16A mRNA expression at transcriptional level or act as a regulator in the translation process of TMEM16A, while KRAS and p53 might interact with TMEM16A protein at post-translational level. However, there is no direct evidence to demonstrate how they regulate each other. Here, we found this phenomenon and tried to explain it, but further investigation is needed to elucidate the underlying mechanism. In our study, we observed that with lymph node metastasis and higher TNM stage, the positive rate of TMEM16A mRNA expression increased significantly, but the positive rate of TMEM16A protein did not change significantly. We supposed that the difference might be related to MSH2, KRAS, and p53. In addition, we believe that the coordination of TMEM16A and various factors leads to this result. For example, TMEM16A mRNA expression might be regulated by other factors such as hypermethylation of the TMEM16A promoter, the signal transducer and activator of transcription (STAT) and some soluble factors in the tumor micro-environment at the transcriptional level during lymph node metastasis. TMEM16A expression might be controlled by a number of microRNAs such as miR-9, miR-144, and miR-132 at translational level, as previously reported (13, 26, 52). TMEM16A protein might interact with other molecular targets including ERK1/2, AKT, camodulin kinase II (CaMKII), EGFR, secreted calcium-activated chloride channel regulator 1 (CLCA1), and Coatomer protein complex subunit beta 1 (COPB1) at post-translational level. Due to the heterogeneity of cell subsets expressing different molecules in colorectal cancer, it is necessary to further study the molecular targets involved in the regulation of lymph node metastasis by TMEM16A and the interaction mechanism between TMEM16A and these molecular targets.

In summary, we newly described the prognostic role of TMEM16A expression and its correlation with clinical pathological parameters. We found that TMEM16A mRNA expression was more frequently detected than TMEM16A overexpression in human colorectal cancer tissue samples. TMEM16A mRNA expression can be used as an independent predictor for lymph node metastasis in CRC. TMEM16A mRNA expression was significantly associated with MSH2 protein. TMEM16A protein expression TMEM16A was positively correlated with KRAS mutation, and negatively correlated with mutant p53 protein. Our finding provides original evidence that TMEM16A mRNA expression may be a potential marker for predicting lymph node metastasis and TMEM16A protein may be a marker between tumor growth and metastasis in CRC.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Ethics Committee of the Second Hospital of Jilin University. The patients/participants provided their written informed consent to participate in this study.

YS, HL, and QY conducted experimental operations, sample processing, data analysis, and article writing. ZD, FW, HZ, LY, and SC performed the experiments. SH and ZM performed sample pretreatment. YS conceived and designed the experiments. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.