Mps1 is associated with the BRAFV600E mutation and predicts poor outcome in patients with colorectal cancer.

Colorectal cancer (CRC) with the V600E mutation of B-Raf proto-oncogene serine/threonine kinase (BRAFV600E) mutation is insensitive to chemotherapy and is indicative of a poor patient prognosis. Although BRAF inhibitors have a marked effect on malignant melanoma harboring the BRAFV600E mutation, they have a limited effect on patients with CRC with the same BRAF mutation. A previous study identified a novel gene, monopolar spindle protein kinase 1 (Mps1), a downstream target of BRAFV600E only, rather than of wild-type BRAF as well, which contributes to tumorigenesis in melanoma. In the present study, the incidence of BRAFV600E in patients with CRC was identified and the correlation of Mps1, phospho-extracellular-signal-regulated kinase (p-ERK) and BRAFV600E was investigated. The results indicated that the mutation rate of BRAFV600E was 5.2% in CRC. Poorly differentiated tumors and mucinous tumors have a significantly higher incidence of BRAFV600E compared with well-differentiated tumors and non-mucinous tumors (P<0.05). Kaplan-Meier survival analysis indicated that the survival rate was markedly lower in patients with BRAFV600E compared with in patients with wild-type BRAF (BRAFWT). The expression of p-ERK and Mps1 in CRC with BRAFV600E was significantly higher compared with in CRC with BRAFWT (P<0.05), and their expression is associated with cancer classification, degree of differentiation and lymph node transfusion (P<0.05). In addition p-ERK expression was positively correlated with Mps1 expression, with a contingency coefficient of 0.679 (P=0.002). In conclusion, the results of the present study indicated that Mps1 was significantly associated with BRAFV600E/p-ERK and may serve a crucial function in the development of CRC. The results of the present study raise the possibility that targeting the oncogenic BRAF and Mps1, particularly when in conjunction, could provide promising therapeutic opportunities for the treatment of CRC.

Introduction

Colorectal cancer (CRC) is one of the most common malignant tumors and is the third highest cancer for incidence and has the second highest mortality (9.2%) of different types of cancer worldwide (1). The principal treatments used for CRC are surgery, radiation therapy, chemotherapy and molecular targeted therapy. As a novel treatment type, molecular targeted therapy has been used in a variety of tumors including CRC (2,3). The therapeutic strategy to target the selected epidermal growth factor receptor has been developed in clinical trials (4). However, it possesses drug resistance in the treatment of patients with CRC harboring the V600E mutation of B-Raf proto-oncogene serine/threonine kinase (BRAFV600E) mutation (5,6). BRAF is an important oncogene and mutant BRAF has been implicated in the pathogenesis of several types of cancer. The 1796T>A mutation results in an amino acid substitution at position 600 in BRAF, from valine to glutamic acid. This mutation occurs within the activation segment of the kinase domain and leads to the continuous activation of the MAPK/ERK signaling pathway (7,8). Although oral BRAF inhibitors have remarkable clinical activity in metastatic melanomas with BRAFV600E, resistance to therapy invariably develops in patients with CRC with the same BRAF mutation (9,10). Therefore, there is an urgent requirement to develop a novel and effective treatment for these patients.

Our previous study identified a novel gene monopolar spindle protein kinase 1 (Mps1), which is a downstream target of BRAFV600E, and continuously activated BRAFV600E signaling may be a potential mechanism for the deregulation of Mps1 stability and kinase activity in human malignancies (11,12). Persistent phosphorylation of Mps1 through BRAFV600E signaling is a key event in disrupting the control of centrosome duplication and chromosome stability that may contribute to tumorigenesis (13,14). Notably, phospho (p)-Ser281 Mps1 staining was demonstrated to be positively associated with p-mitogen-activated protein kinase (MAPK) [extracellular-signal-regulated kinase ERK)1/2] in human melanoma tissues (13). However, to the best of our knowledge, no previous study has investigated whether a correlation exists between BRAF and Mps1 in CRC.

In the present study, the incidence of BRAFV600E was determined in CRC tissues, and the correlation of Mps1 and BRAFV600E and p-ERK in Chinese patients with CRC was determined. The results raise the possibility that targeting the oncogenic BRAF and Mps1, particularly when used in combination, may potentially provide effective therapeutic opportunities for the treatment of CRC.

Materials and methods

Patients and samples

The present study was approved by the Ethics Committee of Shanxi Medical University (Taiyuan, China), and patients provided written informed consent for their inclusion. A total of 288 (156 male and 132 female) paraffin-embedded tissue sections containing the carcinoma and its adjacent non-neoplastic colorectal tissue were obtained from The First Hospital of Shanxi Medical University and TaiYuan Municipal No. 2 People's Hospital collected between January 2009 and June 2015. Among them, there were 284 adenocarcinoma, 1 glandular squamous cell carcinoma, 1 signet-ring cell carcinoma and 2 neuroendocrine carcinoma tissues. The age of patients at the time of diagnosis with CRC ranged between 25 and 92 years, and the median age was 64 years. On the basis of Tumor-Node-Metastasis classification, there were 169 patients at stage I and II, 119 patients at stage IIIb and IV (15) 110 cases with lymph metastasis, and 178 cases without lymph node (LN) metastasis (Table I). All patients were diagnosed with CRC, with no previous history of other malignant tumor types, and had not received other treatments prior to surgery. In 183 cases [168 cases with wild-type BRAF (BRAFWT) and 15 cases with BRAFV600E], no cases of squamous cell carcinoma were identified. The complete clinical data and follow-up data were included in the survival analysis.

Table I.

Clinicopathological information of the 288 patients with colorectal cancer in the present study.

Clinicopathological feature	n
Sex
Male	156
Female	132
Age, years
>60	195
≤60	93
Smoking status
Yes	47
No	241
Drinking status
Yes	259
No	29
Differentiation
Well	51
Medium and poor	237
Pathological pattern
Mucinous carcinoma	24
Other	264
T[a] (infiltration depth)
>T3	79
≤T3	209
Lymph node metastasis
Positive	110
Negative	178
Clinical stage (15)
I–II	169
III–IV	119
Location
Rectum	125
Colon	163

>T3 includes T4a (tumor penetrates to the surface of the visceral peritoneum) and T4b (tumor directly invades or is adherent to other organs or structures). ≤T3 includes T1 (tumor invades submucosa), T2 (tumor invades muscularis propria) and T3 (tumor invades through the muscularis propria into the pericolorectal tissues) according to NCCN Guidelines version 2.2015 Staging Colon Cancer (15). LN, lymph node.

DNA extraction

Surgically removed tissue was fixed in 10% buffered formalin for 24 h at room temperature and embedded in paraffin. Hematoxylin and eosin-stained tumor tissues (stained for 5 min and 30 sec at room temperature, respectively) were independently reviewed by two pathologists. DNA was extracted using the FFPE DNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA), according to the manufacturer's protocol. The DNA concentration was determined using a NanoDrop 2000 instrument and the 260/280 nm ratio was calculated to evaluate the quality of DNA. The sample concentration was adjusted to 200–300 ng/µl, and DNA was stored at −80°C.

Polymerase chain reaction (PCR)

Using the DNA template above, and a 252-bp fragment of BRAF exon 15 was obtained using PCR. The primers of BRAF were 5′-CTTGCCACAGGTCTCCCC-3′ (forward) and 5′-TCTAGTAACTCAGCAGCATCTCAGG-3′ (reverse). The PCR was carried out in 10 µl PCR buffer, 4 µl dNTP, 2 µl DNA, 1.5 µl forward primer, 1.5 µl reverse primer, 1 µl DNA polymerase (PrimeSTAR GXL DNA Polymerase TAKARA Japan) and double-distilled water for a total reaction volume of 50 µl. The amplification procedure was pre-denaturation for 3 min at 98°C, followed by 30 cycles of 30 sec at 98°C, 30 sec at 58°C and 30 sec at 72°C. The PCR product was examined by 2% agarose electrophoresis and stained by ethidium bromide, then detected under UV. Sanger sequencing was performed by Beijing Liuhe Huada Gene Technology Company (Beijing, China).

Immunohistochemistry (IHC)

According to the sequencing results, 15 cases harboring BRAFV600E and the same number of patients harboring BRAFWT were randomly selected for IHC, with rabbit anti-human Mps1 antibody (cat. no. ab135819; Abcam, Cambridge, UK) and rabbit anti-human p-ERK antibody (cat. no. 4376; Cell Signaling Technology, Inc., Danvers, MA, USA) as primary antibodies, and horseradish peroxide (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG; cat. no. A20120A0704; BioTNT, Shanghai, China) as a secondary antibody. A known positive tissue section served as a positive control. A negative control was established using PBS instead of the primary antibody.

The IHC analysis was performed as follows: 4 micron thick sections were moved to anhydrous ethanol for 2 min at room temperature, then placed in 95% ethanol liquid cylinder for 2 min, then moved to 85% ethanol liquid cylinder for 2 min, and finally placed in 75% ethanol liquid cylinder for 2 min, and then incubated in 3% hydrogen peroxide to block endogenous peroxidase for 15 min at room temperature, followed by washing with PBS three times and soaking in distilled water for 5 min. Antigen retrieval was performed with citrate liquid in a microwave at 92–98°C for 15 min. Sections were blocked with 10% goat serum (1201A Shanghai Biyun day Biotechnology Co., Ltd China) at 37°C for 30 min and rabbit anti-human Mps1 monoclonal antibody (1:100) or anti-p-ERK antibody (1:400) was added, and incubated at 4°C overnight. The slides were then washed with PBS three times and incubated with HRP-labeled goat anti-rabbit IgG at 37°C for 20 min, followed by washing with PBS three times and using 3,3′-diaminobenzidine as a chromogen. Sections were counterstained with hematoxylin for 1 min at room temperature, dehydrated in graded alcohol and sealed with resin sealing agent.

Fully automatic digital pathological scanning apparatus (Leica Microsystems, Inc., Buffalo Grove, IL, USA) were used to obtain high-resolution digital images. A total of five high-power fields of vision (×400 magnification) were randomly selected and analyzed using Image Scope software (12.0; Leica Microsystems, Inc.). The average value of the positive rate of five randomly selected fields of view was obtained using standardized cell nuclear analysis parameters. Nuclear p-ERK expression was classified as negative for 0–35 and positive for >35. Mps1 expression in the cytoplasm was classified as negative for 0–70 and positive for >70.

Statistical analysis

Continuous variables are presented as the mean (SD), and categorical variables are presented as count values (percentages). The p-ERK and Mps1 positive expression levels were divided into four groups based on inter-quartile range respectively, which were 35–45.71, 45.71–51.14 51.14–65.54, and ≥65.54 for p-ERK, as well as 70–88.73, 88.73–117.33, 117.33–157.12 and ≥157.12 for Mps1. The differences were analyzed by χ2 test. χ2 test or Fisher's exact test was also used to assess the association between BRAF mutation status and clinical parameters. Univariate and multivariate survival analyses were performed using a Cox proportional hazards regression model. The contingency coefficient was used to evaluate the correlation between p-ERK and Mps1 expression. A sensitivity analysis was conducted using Spearman's rank analysis was performed to determine the correlation between p-ERK and Mps1. Kaplan-Meier survival analysis and the log-rank test were used to analyze the association between BRAFV600E and prognosis. Data were analyzed using SPSS software (version 20.0; IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

BRAFV600E mutation and its association with clinical parameters in CRC

Sanger DNA sequencing was used to detect the 1796T>A (V600E) mutation, which was the most frequently observed mutation site in BRAF (Fig. 1A and B). In 288 cases of colorectal cancer, 15 cases of BRAF mutation were identified. The rate of BRAFV600E was 5.2% in CRC. A statistical analysis of BRAF mutations and clinical parameters revealed that BRAFV600E was further associated with the age, infiltrating depth, pathological pattern of CRC, were more prevalent in older patients (>60 years), infiltrating depth>T3 stage patients and the mucinous tumors (χ2 test or Fisher's exact test P<0.05; Table II). However, no association of the BRAF mutation with location, clinical stage, LN metastasis, differentiation and sex were identified (χ2 test or Fisher's exact test P>0.05; Table II). Despite previous studies attempting to identify specific risk factors, no dietary and lifestyle factors have been clearly associated with the development of BRAF mutated CRC (16–18). The results of the present study suggest that the BRAF mutation was not associated with smoking, alcohol intake (χ2 test or Fisher's exact test P>0.05; Table II).

Figure 1.

Sanger sequencing peak map of BRAF. (A) Sanger sequencing peak map of BRAFV600E. The BRAFV600E mutation shows additional peaks compared with the wild-type, which is indicated by the grey box. (B) Sanger Sequencing peak map of BRAFWT. The site at position 1796 was not mutated. (C) Kaplan-Meier survival analysis indicated that the survival rate was significantly lower in patients with BRAFV600E mutation compared with those with BRAF. BRAF, B-Raf proto-oncogene serine/threonine kinase; WT, wild-type.

Table II.

Association between BRAFV600E mutation and clinicopathological parameters in colorectal cancer.

	BRAFV600E mutation
Clinicopathological feature	+ (n=15; 5.2%)	- (n=273; 94.8%)	P-value
Sex			0.739
Male	7	149
Female	8	124
Age, years			0.043
>60	14	181
≤60	1	92
Smoking status			0.142
Yes	0	47
No	15	226
Drinking status			0.378
Yes	0	29
No	15	244
Differentiation			0.082
Well	0	51
Medium and poor	15	222
Pathological pattern			0.001
Mucinous carcinoma	6	18
Others	9	255
T[a] (infiltration depth)			<0.001
>T3	15	64
≤T3	0	209
Lymph node metastasis			0.882
Positive	6	104
Negative	9	169
Clinical stage (15)			0.871
I–II	8	161
III–IV	7	112
Location			0.107
Rectum	3	122
Colon	12	151

>T3 includes T4a (tumor penetrates to the surface of the visceral peritoneum) and T4b (tumor directly invades or is adherent to other organs or structures). ≤T3 includes T1 (tumor invades submucosa), T2 (tumor invades muscularis propria) and T3 (tumor invades through the muscularis propria into the pericolorectal tissues) according to NCCN Guidelines version 2.2015 Staging Colon Cancer (15). BRAF, B-Raf proto-oncogene serine/threonine kinase.

BRAFV600E mutation is associated with a poor prognosis of patients with CRC

Kaplan-Meier survival analysis indicated that the survival rate was significantly lower in patients with BRAFV600E mutation compared with those with BRAFWT (Fig. 1C). The median survival time of patients with BRAFV600E and BRAFWT were 300 and 429.5 days respectively.

Cox regression analysis was used to assess the impact of BRAF mutations and clinical parameters on OS. Notably, the results revealed that the association of BRAF mutations with OS was statistically significant (Cox regression multivariate analysis HR=0.32, P=0.051; Fig. 2A; Cox regression univariate analyses, HR=0.36, P=0.03, Fig. 2B). Although the P-value of BRAFV600E in the multivariate analysis was 0.051These results suggested that BRAFV600E may serve an important function in specific pathological CRC, and may function as an independent prognostic factor and a novel oncological therapeutic strategy.

Figure 2.

Multivariate and univariate analyses of survival prognosis in CRC. (A) Multivariate analyses of survival prognosis in CRC (B) Univariate analysis of survival prognosis in CRC. BRAF, WT vs. V600E; pathological stage, I–II vs. III–IV; M stage, M0 vs. M1; N stage, N0 vs. N1; T stage, T1-2 vs. T3-4; differentiation, poor vs. medium + well; histological, mucous adenocarcinoma vs. others; family history, yes vs. no; drinking, yes vs. no; smoking, yes vs. no; age at diagnosis, ≥60 years vs. <60 years; sex, male vs. female. CRC, colorectal cancer; BRAF, B-Raf proto-oncogene serine/threonine kinase; WT, wild-type; HR, hazard ratio; T, tumor; N, node; M, metastasis. The bracketed values represent 95% confidence intervals.

IHC and evaluation of p-ERK and Mps1 in CRC

According to the sequencing results, 15 cases harboring BRAFV600E and the same number of patients harboring BRAFWT were randomly selected for IHC with anti-Mps1 and anti-p-ERK antibodies. The positive expression of p-ERK protein was brown and localized in the nucleus (Fig. 3). And the positive rate of p-ERK expression was 93.3% in colorectal cancer tissue with BRAFV600E, while the positive rate of p-ERK expression was 6.7% in paired normal tissues, which the difference was statistically significant in BRAFV600E mutation cases (Fig. 4A, χ2 test, P<0.05). However, In BRAFWT cases, there was no significant difference in the expression of p-ERK between colorectal cancer and paired normal tissues (Fig. 4A, χ2 test, P>0.05).

Figure 3.

Representative immunohistochemistry images revealed p-ERK or Mps1 staining in colorectal cancer and corresponding normal tissues with BRAFWT or BRAFV600E mutations, at ×100 magnifcation. In the BRAFV600E mutation of colorectal cancer, the positive expression of p-ERK and Mps1 protein were brown and localized in the nucleus and the cytoplasm respectively, while in the normal tissues with BRAFV600E mutation, the expression of pERK and Mps1 was negtive. In the colorectal cancer with BRAFWT, there was no significant difference in the expression of p-ERK or Mps1 between colorectal cancer and paired normal tissues. p-ERK, phospho-extracellular-signal-regulated kinase; Mps1, monopolar spindle protein kinase 1. (×100 magnification).

Figure 4.

Mps1 is significantly associated with BRAFV600E/p-ERK. (A) The expression of p-ERK between BRAFV600E and BRAFWT. (B) The expression of Mps1 between BRAFV600E and BRAFWT. (C) A sensitivity analysis was conducted using Spearman's rank correlation analysis in order to evaluate the correlation between p-ERK and Mps1. The results demonstrated that p-ERK expression was positively correlated with Mps1 expression (contingency coefficient, 0.623; P<0.001). BRAF, B-Raf proto-oncogene serine/threonine kinase; Mps1, monopolar spindle protein kinase 1; p-ERK, phospho-extracellular-signal-regulated kinase; WT, wild-type *P<0.05. The p-ERK and Mps1 positive expression levels between colorectal cancer and paired normal tissues in BRAFV600E group is statistically significant (P<0.05); NS. The p-ERK and Mps1 positive expression levels between colorectal cancer and paired normal tissues in BRAFWT group is not statistically significant (P>0.05).

Further evaluations were made concerning the expression of Mps1 in colorectal cancer. the positive expression of Mps1 protein was brown and localized in the cytoplasm (Fig. 3). The positive expression of Mps1 in CRC with BRAFV600E was significantly higher compared with that in paired normal tissues (Fig. 4B, χ2 test, P<0.05). While there was no significant difference in the expression of Mps1 between colorectal cancer and paired normal tissues in BRAFWT cases. (Fig. 4B, χ2 test, P>0.05).

Association between p-ERK or Mps1 expression and clinical parameters

Subsequently, the association of expression of p-ERK and Mps1 with clinical pathological features was analyzed (Table III). p-ERK expression was associated with LN metastasis, pathology type and degree of differentiation (χ2 test, P<0.05; Table III). The expression of p-ERK was significantly higher in poorly differentiated adenocarcinoma and mucinous adenocarcinoma compared with that in highly differentiated adenocarcinoma and non-mucinous adenocarcinoma, as well as in the group with LN metastasis compared with without LN metastasis. (χ2 test, P<0.05; Table III); however, p-ERK expression was not associated with age, sex, smoking status, drinking status, location or T stage in CRC (χ2 test, P>0.05; Table III).

Table III.

Association between p-ERK/Mps1 and clinicopathological parameters in colorectal cancer.

	p-ERK in tumor			Mps1 in tumor
Clinicopathological feature	+ (n=17; 56.7%)	- (n=13; 13.3%)	P-value	+ (n=18; 60%)	- (n=12; 40%)	P-value
Sex
Male	10	9		9	10
Female	7	4	0.558	9	2	0.121
Age, years
>60	15	11		17	9
≤60	2	2	0.773	1	3	0.274
Smoking status
Yes	1	1		0	2
No	16	12	0.844	18	10	0.152
Drinking status
Yes	1	0		0	1
No	16	13	1.000	18	11	0.400
Differentiation
Well	1	6		1	6
Medium and poor	16	7	0.01	17	6	0.009
Pathological pattern
Mucinous carcinoma	6	0		6	0
Others	11	13	0.024	12	12	0.057
T[a] (infiltration depth)
>T3	4	1		5	0
≤T3	13	12	0.355	13	12	0.066
Lymph node metastasis
Positive	8	1		6	3
Negative	9	12	0.02	12	9	0.626
Clinical stage (15)
I–II	8	9		10	7
III–IV	9	4	0.225	8	5	0.88
Location
Rectum	13	9		14	8
Colon	4	4	0.698	4	4	0.679

>T3 includes T4a (tumor penetrates to the surface of the visceral peritoneum) and T4b (tumor directly invades or is adherent to other organs or structures). ≤T3 includes T1 (tumor invades submucosa), T2 (tumor invades muscularis propria) and T3 (tumor invades through the muscularis propria into the pericolorectal tissues) according to NCCN Guidelines version 2.2015 Staging Colon Cancer (15). p-ERK, phospho-extracellular-signal-regulated kinase; Mps1, monopolar spindle protein kinase 1.

Positive Mps1 expression was significantly greater in poorly differentiated carcinoma compared with in well-differentiated adenocarcinoma in CRC (χ2 test, P<0.05, Table III). There were no significant associations between positive expression of Mps1 and age, sex, smoking status, drinking status, location, pathological type, LN metastasis or T stage in CRC (χ2 test, P>0.05; Table III).

In addition, the expression of p-ERK and Mps1 between the BRAFV600E and BRAFWT groups were then compared, and the expression of p-ERK and Mps1 in the BRAFV600E group was higher compared with those in the BRAFWT group (Fisher's exact test, P<0.05). Expression of p-ERK was correlated positively with the Mps1 expression, with a contingency coefficient of 0.679 (P=0.002; Table IV). In the sensitivity analysis, it was also identified that p-ERK expression was positively correlated with the expression of Mps1 (Spearman's rank correlation analysis correlation coefficient 0.623; P<0.001; Fig. 4C).

Table IV.

Association between the p-ERK or Mps1 expression and BRAF mutation in colorectal cancer.

	p-ERK			Mps1
CRC	+	−	P-value	+	−	P-value
BRAFV600E	14 (93.3%)	1 (6.7%)		15 (100%)	0 (0%)
BRAFWT	3 (20%)	12 (80%)	<0.001	3 (20%)	12 (80%)	0.002

CRC, colorectal cancer; BRAF, B-Raf proto-oncogene serine/threonine kinase; p-ERK, phospho-extracellular-signal-regulated kinase; Mps1, monopolar spindle protein kinase 1; WT, wild-type.

Discussion

The results of the present study demonstrated that the incidence of BRAFV600E was 5.2% in CRC, which was consistent with previously published rates, between 5 and 15% (19–24). The difference mentioned above may be due to the complicated genetic background of different ethnicities. In the study by Yoon et al (25), BRAF mutation frequency in CRC from Caucasians (13.9%) was twice that of tumors from Asians (5.6%) or individuals of African (6.4%) descent.

Malignant tumors with the BRAFV600E mutation have been demonstrated to be associated with mortality in patients with colorectal cancer (26). Numerous studies have demonstrated that the malignant tumor with BRAFV600E is insensitive to the traditional treatments and patients have a poor prognosis (27–29). With the success of BRAF inhibitors in malignant melanoma, there is concern about the efficacy of BRAF inhibitors in other tumors with BRAFV600E mutations (9,30). However, BRAF inhibitors exhibited severe adverse effects in the treatment of patients with CRC harboring the BRAFV600E mutation (31). A previous clinical study compared the expression of p-ERK between pre- and post-treatment with BRAF inhibitors, but the results showed that the downregulation of p-ERK only occurred in 47% of patients with CRC harboring the BRAFV600E mutation (32). This indicates that the inhibition of the MAPK signaling pathway by this BRAF inhibitor is insufficient, which may be a principal reason for the low response rate of BRAF inhibitors. Thus, identifying novel strategies for the full and sustained inhibition of the MAPK pathway in patients with CRC with the BRAF mutant is of marked clinical importance.

Mps1, a member of the spindle-monitoring complex, is involved in centrosome duplication and spindle checkpoint (33) and cell cycle regulation, and has maximum kinase activity in the M phase of the cell cycle (34). Typically, Mps1 is an unstable protein, which is degraded by the ubiquitin-proteasome pathway when centrosome duplication is completed, and cells enter anaphase (35). It has been reported that either too high or too low Mps1 kinase activity results in aberrations in centrosome duplication (36), lead to aneuploidy formation and result in malignant tumor formation (37). Currently, Mps1 has been reported in the breast, colon and other malignant tumors with high expression, and may facilitate tumor cell evasion from apoptosis, culminating in carcinogenesis (38–40). Our previous study identified that Mps1 is a downstream target of BRAFV600E (12). Persistent phosphorylation of Mps1 through BRAFV600E signaling is a key event in disrupting the control of centrosome duplication and chromosome stability that may contribute to tumorigenesis (13). Thus, Mps1 may serve as a novel therapeutic target for patients with CRC harboring the BRAFV600E mutation.

The effect of Mps1 kinase inhibitors have been investigated in a variety of malignant tumors, with promising results (41,42). The present study initially identified that Mps1 was significantly associated with BRAFV600E/p-ERK in CRC. It was indicated that Mps1, the downstream target of the BRAFV600E/MAPK/ERK kinase/ERK signaling pathway, may serve a significant function in the development of CRC. The use of a BRAF inhibitor combined with an Mps1 inhibitor may provide a novel therapeutic approach for treating patients with CRC harboring the BRAFV600E mutation, who were previously resistant or insensitive to the BRAF inhibitor.

However, there were some limitations to the present study. For example, the data pertaining to the 5-year survival rate are still being collected, and the sample size of BRAFV600E is not large enough, which leads to the lack of representativeness. However, even if the sample size of BRAFV600E is small, the data still conform to the normal distribution, ensuring the accuracy and integrity of the results. We will analyze the 5-year survival data in further study. More samples of BRAFV600E will be selected in statistical analysis in further research.

In conclusion, the present study demonstrated that Mps1 was significantly associated with BRAFV600E/p-ERK and may serve a crucial function in the development of CRC. Targeting the oncogenic BRAFV600E and Mps1, particularly when used in combination, could potentially provide therapeutic opportunities for the treatment of cancer.