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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

The Mus81 gene encodes a critical endonuclease involved in DNA repair and tumor suppression. Our previous study has shown reduced expression of Mus81 in hepatocellular carcinoma and its association with the metastastic potential and prognosis of hepatocellular carcinoma. However, the role of Mus81 in colorectal carcinoma is currently unknown. We therefore carried out the present study to explore the correlation between Mus81 expression and the progression of colorectal carcinoma. Mus81 expression in 92 cases of colorectal carcinoma and matched normal tissues was determined by quantitative real-time polymerase chain reaction. Our results showed that Mus81 expression in colorectal carcinoma tissues was significantly reduced compared with the corresponding normal tissues (< 0.001) and the downregulation of Mus81 (decreased by more than 50%) was found in 60.9% (56/92) of colorectal carcinoma. Moreover, Mus81 downregulation correlated significantly to hepatic metastasis (= 0.019) and a high TNM stage (= 0.025) of colorectal carcinoma. In addition, the decrease of Mus81 was also detected in 10 cases of hepatic metastasis tissues compared with the corresponding primary colorectal carcinoma tissues (= 0.016). More importantly, colorectal carcinoma patients with apparent Mus81 downregulation have shown significantly poorer overall survival than those with little Mus81 downregulation (= 0.0374). Also, multivariable Cox regression analysis identified Mus81 downregulation as an independent prognostic factor for colorectal carcinoma (hazard ratio, 1.678; = 0.040). In conclusion, the reduced expression of Mus81 is closely related to hepatic metastasis and poor prognosis of colorectal carcinoma, indicating Mus81 as a novel prognostic marker for colorectal carcinoma. (Cancer Sci 2011; 102: 472–477)

Colorectal carcinoma (CRC) ranks as the third most common cancer in the world and the incidence of CRC is increasing rather rapidly in the Asia–Pacific region, especially in Japan.(1,2) Up until now, abundant evidence has shown that a variety of genetic and epigenetic alterations in both oncogenes and tumor suppressors are involved in the pathogenesis of CRC. Activation of oncogenes such as the ras gene and the inactivation of tumor suppressors such as APC and p53 genes have been documented in CRC.(3–5) In addition, we have identified some genetic as well as epigenetic changes related to this disease.(6–11) However, further investigations are still necessary to clarify the tumorigenic pathway of CRC.

The Mus81 gene encodes a structure-specific DNA endonuclease, which resolves holliday junctions through constituting a heterodimer with Eme1/Mms4 and plays a critical role in the repair of double-strand breaks of DNA and maintenance of chromosomal integrity.(12–15) McPherson et al.(16) have shown that 73%Mus81−/− mice and 50%Mus81+/− mice died of various spontaneous tumors such as lymphoma, breast cancer and prostate cancer, implicating Mus81 as a potent tumor suppressor in mice. Moreover, Mus81 was found to interact with p53 and checkpoint genes such as Cds1/chk2 and Rhp51/Rad51.(17–19) Recently, Mus81 has also been shown to be required in the survival of telomerase-negative cancer cells by alternative lengthening of the telomeres pathway.(20) Also, the single nucleotide polymorphisms of Mus81 were found to be associated with an increased risk of developing breast cancer.(21) Although these studies have indicated the potential role of Mus81 in human malignancies, the evidence for its expression pattern in human malignancies is still limited.(22,23)

In a previous study, we have documented the decrease of Mus81 expression in human hepatocellular carcinoma (HCC) tissues and its association with metastasis and poor prognosis, which provided the first evidence for the expression pattern and potential role of Mus81 in human malignancy.(23) The results suggest that Mus81 might play a similar role in other solid tumors. A recent in vitro study revealed that the haploinsufficiency of Mus81 in human colon cancer cell line HCT116 could activate the intra-S-phase and G2/M checkpoints and promote cellular replication, which implicates the involvement of Mus81 in the carcinogenesis of CRC.(24) Moreover, Tomoda et al.(25) have found that the expression of Mus81 decreases in another human colon cancer cell line LS180, which may correlate to cisplatin sensitivity of this cell line. However, the role of Mus81 in human CRC remains largely unknown. Thus, we carried out the present study to determine the expression patterns of Mus81 in human CRC tissues and to explore its correlation with the clinicopathological characteristics as well as the prognosis of CRC.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

Patients and specimens.  Matched cancer and normal specimens were obtained from 92 cases of CRC patients who underwent operation at Gastroenterological Surgery, Showa University Fujikaoka Hospital from February 2007 to March 2008. All tissue specimens were collected and frozen in liquid nitrogen immediately after operation and then stored at −80°C until analysis. Hepatic metastases occurred in 10 patients within this cohort and hepatectomy was subsequently fulfilled in our hospital. Hepatic metastasis specimens from these 10 patients were therefore collected and conserved by the same method as mentioned above. All diagnoses of CRC and hepatic metastasis were confirmed by histopathological examination. Prior informed consent was obtained from all patients, as required by the Institutional Review Board. The characteristics of the colorectal tumors including tumor location, TNM stage, tumor category, lymph node category and histological type are shown in Table 1. The characteristics of all hepatic metastasis lesions and their primary colorectal tumors are listed in Table 2.

Table 1.   Characteristics of the colorectal tumors
CharacteristicsNumber
  1. †Proximal colon includes the cecum, ascending colon and transverse colon. ‡Distal colon includes the descending colon and sigmoid colon. §TNM classification is according to the International Union against Cancer(36). ¶Histological type is according to the Japanese Classification of Colorectal Carcinoma(37).

Tumor location
 Proximal colon†39
 Distal colon‡26
 Rectum27
TNM stage§
 I18
 II29
 III30
 IV15
T category§
 T114
 T210
 T364
 T44
N category§
 N059
 N122
 N211
Histological type¶
 Adenocarcinoma78
 Mucinous adenocarcinoma13
 Signet ring cell1
Table 2.   Characteristics of the hepatic metastasis lesions and their corresponding primary colorectal tumor
Case code†Hepatic metastasis lesionPrimary colorectal tumor
NumberLocation‡Size§ (mm)Tumor differentiation¶LocationTumor differentiation¶
  1. †The code of the cases begins with HM, which is the abbreviation for hepatic metastasis. ‡The location of the hepatic metastasis lesion is described according to Couinaud’s numbering system. §Maximal size of the hepatic metastasis lesion. ¶Tumor differentiation is according to the Japanese Classification of Colorectal Carcinoma(37). Mod, moderately differentiated adenocarcinoma; Well, well-differentiated adenocarcinoma.

HM0195Segment 630ModTransverse colonWell
Segment 715Mod
Segment 54Mod
Segment 83Mod
Segment 22Mod
HM0282Segment 77Well-ModAscending colonWell
Segment 54Well-Mod
HM0321Segment 850ModSigmoid colonWell
HM0421Segment 819ModTransverse colonWell
HM0511Segment 540ModSigmoid colonWell
HM0553Segment 723WellSigmoid colonWell
Segment 517Well
Segment 613Well
HM0561Segment 745Well-ModTransverse colonWell
HM0641Segment 620ModRectumWell
HM0721Segment 530ModDescending colonMod
HM0782Segment 520ModCecumWell
Segment 85Mod

RNA preparation and reverse transcription.  Total RNA was extracted from CRC tissues, corresponding normal tissues and hepatic metastasis tissues by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, as described previously.(26) The amount of total RNA was measured by absorbance at 260 nm with a U-2001 spectrophotometer (Hitachi Ltd, Tokyo, Japan). First-strand cDNA was generated from RNA as described previously.(27)

Quantitative real-time polymerase chain reaction (QRT-PCR).  QRT-PCR was performed in a Thermal Cycler Dice Real-time System TP800 (TaKaRa Bio Inc., Shiga, Japan) using SYBR Premix Ex Taq II kit (TaKaRa Bio Inc.). Thermocycling was done in a final volume of 25 μL containing 1.0 μL of cDNA sample, 0.5 μL of each primers (forward and reverse, 100 nM), 12.5 μL of SYBR Premix Ex Taq II (including Taq DNA polymerase, reacting buffer and deoxynucleotide triphosphate mixture; TaKaRa Bio Inc.). The PCR amplification consisted of 40 cycles (95°C for 5 s, 55°C for 30 s after an initial denaturation step [95°C for 10 s]). The PCR primers for Mus81 were described previously:(23) forward, 5′-TGTGGACATTGGCGAGAC-3′; and reverse, 5′-GCTGAGGTTGTGGACGGA-3′. To correct the differences in both quality and quantity of cDNA samples, the β-actin gene was used as an internal control and measured in the same samples. All PCR analyses were performed in duplicate.

Score of Mus81 expression and grouping of CRC patients.  The relative expressions of Mus81 in tissue samples were normalized to the internal control β-actin and calculated by the inline image method. The downregulation of Mus81 was scored to positive when the relative expression of Mus81 in the CRC tissue was < 0.5 of that in the corresponding normal tissue, as described elsewhere.(28) Based on the score of Mus81 downregulation, all CRC patients could be divided into two groups: (i) the low Mus81 expression group (patients with positive Mus81 downregulation); and (ii) the high Mus81 expression group (patients with negative Mus81 downregulation).

Follow up and candidate prognostic factors.  Follow-up data were obtained from all 92 patients. The follow-up period was defined as the interval between the date of operation and the date of the patient’s death or the last follow up. Deaths from other causes were treated as censored cases. The prognosis of CRC patients was compared between the low Mus81 expression group and the high Mus81 expression group. To determine factors influencing postoperative survival, 10 conventional variables along with the downregulation of Mus81 were tested in all 92 patients: age (years); gender (male versus female); maximal tumor size (mm); depth of tumor invasion (< muscular tunic invasion versus ≥ muscular tunic invasion); pathological type (well-moderately differentiated CRC versus poorly differentiated CRC); lymph node metastasis (presence versus absence); hepatic metastasis (presence versus absence); peritoneal dissemination (presence versus absence); distant metastasis (presence versus absence); TNM stage (I, II, III stage versus IV stage); and downregulation of Mus81 (positive versus negative).

Statistical analysis.  The non-parametric Mann–Whitney U-test was applied to analyze the Mus81 expression levels in CRC tissues, normal tissues and hepatic metastasis tissues. The associations between Mus81 downregulation and clinicopathological characteristics were analyzed by the Chi-squared test (categorical data) and Student’s t-test (continuous data). Survival curves were constructed using the Kaplan–Meier method and the difference of overall survival between the low Mus81 expression group and the high Mus81 expression group was evaluated by the Log-Rank test. The Cox proportional hazards regression model was constructed to identify factors that were independently associated with overall survival. All statistical analyses were performed by using the SPSS software package (version 10.0; Chicago, IL, USA). All tests were two-sided and < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

Downregulation of Mus81 in CRC. Mus81 was detectable in all specimens of CRC tissues and corresponding normal tissues. However, the relative expression level of Mus81 in CRC tissues (mean ± SE: 0.00175 ± 0.00049; median: 0.00057) was significantly lower than that in the corresponding normal tissues (mean ± SE: 0.00627 ± 0.00148; median: 0.00184; < 0.001) (Fig. 1). According to the score and grouping criterion, the downregulation of Mus81 was scored positive in 60.9% (56/92) of CRC patients and therefore all CRC patients could be divided into the high Mus81 expression group (= 56) and the low Mus81 expression group (= 36).

image

Figure 1.  Downregulation of Mus81 in colorectal cancer (CRC) tissues. Mus81 expression levels in 92 cases of CRC tissues and the corresponding normal tissues were determined by quantitative real-time PCR and the results showed that the expression levels of Mus81 in the CRC tissues were significant lower than those in the corresponding normal tissues (< 0.001). The bars in the figure indicate the mean of the relative expression of Mus81.

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Correlations between Mus81 expression and clinicopathological characteristics.  To explore the clinical association of Mus81 downregulation in CRC, the clinicopathological data were correlated with the Mus81 expression scores. Our results showed that the downregulation of Mus81 significantly correlated to hepatic metastasis (= 0.019) and high TNM stage (= 0.025) of CRC. There was no significant association between downregulation of Mus81 and the other clinicopathological features such as age, gender, maximal tumor size, depth of tumor invasion, pathological type, lymph node metastasis, peritoneal dissemination and distant metastasis (Table 3).

Table 3.   Correlations between downregulation of Mus81 and clinicopathological characteristics of colorectal cancer
Clinicopathological characteristicsVariablenDownregulation of Mus81
PositiveNegativeP-value†
  1. *< 0.05. †P-values were obtained from the Chi-squared test (categorical data) or Student’s t-test (continuous data) and all tests were two-sided. ‡Depth of tumor invasion and tumor differentiation are according to the Japanese Classification of Colorectal Carcinoma(37). §TNM classification is according to the International Union against Cancer(36). Mod, moderately differentiated adenocarcinoma; Mt, muscular tunic; Poor, poorly differentiated, mucinous or signet ring cell adenocarcinoma; Well, well-differentiated adenocarcinoma.

GenderMale5432220.706
Female382414
Age (years; mean ± SE) 9266.3 ± 1.668.2 ± 1.70.442
Maximal tumor size (mm; mean ± SE) 9247.9 ± 3.151.3 ± 6.00.578
Depth of tumor invasion‡<Mt14860.756
≥Mt784830
Tumor differentiation‡Well–Mod7644320.203
Poor16124
Lymph node metastasisPresence3320130.969
Absence593623
Hepatic metastasisPresence101000.019*
Absence824636
Peritoneal disseminationPresence8620.633
Absence845034
Distant metastasisPresence8620.633
Absence845034
TNM stage§I, II, III7743340.025*
IV15132

Mus81 expression was significantly decreased in hepatic metastasis tissues.  Because the downregulation of Mus81 correlated significantly with hepatic metastasis of CRC, we compared the Mus81 expression levels in the hepatic metastasis tissues with their corresponding primary CRC tissues. Mus81 was also detectable in all hepatic metastasis and the level of expression (mean ± SE: 0.00062 ± 0.00026; median: 0.00029) was significantly lower than the corresponding primary CRC tissues (mean ± SE: 0.00173 ± 0.00052; median: 0.00130; = 0.016) (Fig. 2).

image

Figure 2.  Decrease of Mus81 expression in hepatic metastasis tissues of colorectal cancer (CRC). Ten cases of hepatic metastasis tissues and the corresponding primary CRC tissues were detected by quantitative real-time PCR for Mus81 expression. The results showed that Mus81 expression was also decreased significantly in the hepatic metastasis tissues compared with the corresponding primary CRC tissues (= 0.016). The bars in the figure indicate the mean of the relative expression of Mus81.

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Downregulation of Mus81 correlated with poor prognosis of CRC.  To study the association of Mus81 downregulation with the prognosis of CRC patients, we compared the prognosis of CRC patients in the high Mus81 expression group with the low Mus81 expression group. The results showed that patients with low Mus81 expression had significantly poorer overall survival than those with high Mus81 expression (2-year survival rate, 84.4% versus 100.0%; = 0.0374) (Fig. 3). Furthermore, by univariable Cox regression analysis, Mus81 downregulation (hazard ratio [HR], 1.111; = 0.036), high TNM stage (HR, 0.123; = 0.006), hepatic metastasis (HR, 0.152; = 0.014) and poor pathological type (HR, 0.197; = 0.047) were correlated significantly with the overall survival of CRC patients. Interestingly, in the multivariable Cox regression model, only Mus81 downregulation (HR, 1.678; = 0.040) and high TNM stage (HR, 1.161; = 0.045) were found to be independent prognostic factors for the overall survival of CRC patients (Table 4).

image

Figure 3.  Estimated overall survival according to the expression of Mus81 in 92 cases of colorectal cancer (CRC). All 92 cases of CRC patients were divided into two groups according to the score of Mus81 downregulation: low Mus81 expression group (patients with positive downregulation of Mus81, = 56) and high Mus81 expression group (patients with negative downregulation of Mus81, = 36). The survival curves of the two groups were described using the Kaplan–Meier method. The Log-Rank test shows that the CRC patients in the low Mus81 expression group have significant poorer overall survival than those in the high Mus81 expression group (2-year survival rate, 84.4% versus 100.0%; = 0.0374).

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Table 4.   Analysis by Cox proportional hazards regression model
VariablesUnivariable analysisMultivariable analysis
HR (95% CI)P-valueHR (95% CI)P-value
  1. *< 0.05; **< 0.01. CI, confidence interval; HR, hazard ratio.

Gender4.576 (0.550–38.075)0.1599.802 (0.076–57.815)0.357
Age (years)3.890 (0.078–22.680)0.2416.200 (0.010–66.791)0.909
Maximal tumor size (mm)1.008 (0.987–1.030)0.4460.977 (0.913–1.045)0.496
Depth of tumor0.037 (0.000–3.039)0.4500.006 (0.001–0.072)0.662
Pathological type0.197 (0.040–0.979)0.047*0.207 (0.011–3.775)0.288
Lymph node metastasis0.787 (0.176–3.517)0.7546.413 (0.299–37.477)0.235
Hepatic metastasis0.152 (0.034–0.680)0.014*0.054 (0.013–5.319)0.213
Peritoneal dissemination0.226 (0.044–1.171)0.0760.593 (0.025–3.846)0.745
Distant metastasis0.436 (0.052–3.657)0.4440.061 (0.001–4.435)0.201
TNM stage0.123 (0.027–0.549)0.006**1.161 (1.007–1.388)0.045*
Downregulation of Mus811.111 (1.007–1.226)0.036*1.678 (1.127–3.920)0.040*

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

Although recent studies have implicated the potential role of Mus81 in the replication of colon cancer cell line HCT116 and have documented the downregulation of Mus81 in another colon cancer cell line LS180,(24,25) the expression pattern of Mus81 in human CRC is currently unknown. The present study has provided the first evidence showing a significant decrease of Mus81 expression in more than half of the patients with CRC. The downregulation of Mus81 expression in CRC is not only in agreement with the expression pattern of Mus81 in HCC but also consistent with the notion that Mus81 might be a potent tumor suppressor,(16,23) suggesting Mus81 downregulation is an important event during the carcinogenesis of CRC.

When correlating Mus81 downregulation with the clinicopathological characteristics of CRC, we found that Mus81 downregulation associated significantly with hepatic metastasis, which was further verified by the significantly decreased Mus81 expression in hepatic metastasis tissues compared with primary colorectal tumors. It is well known that hepatic metastasis is most common in CRC and has enormous implications on the prognosis of CRC patients.(29,30) Although the exact mechanisms underlying hepatic metastasis of CRC remain unknown, recent studies have shown that chromosomal aberration occurs more frequently in hepatic metastasis than the corresponding primary CRC,(31,32) implicating the important role of genomic instability such as chromosomal instability during the development of hepatic metastasis. In light of Mus81, as an important DNA repair gene, being critical in maintaining chromosomal stability,(16,17) we speculate that downregulation of Mus81 might lead to hepatic metastasis through increased chromosomal instability in CRC.

Our results also showed that the downregulation of Mus81 was significantly associated with high TNM stage (stage IV) of CRC, which might result from the correlation between Mus81 downregulation and hepatic metastasis of CRC. Considering the fact that the TNM stage is the most important prognostic factor for the survival of CRC patients,(33,34) we used the Kaplan–Meier method to assess the prediction power of Mus81 expression in the prognosis of CRC patients. Not surprisingly, the CRC patients with low Mus81 expression had poorer prognosis than those with high Mus81 expression. Moreover, multivariable Cox regression analysis also indicated that the downregulation of Mus81 was an independent risk factor for the prognosis of CRC patients, suggesting Mus81 downregulation as a novel prognostic marker for CRC. The latest study has shown that chromosomal aberration is significantly associated with poor prognosis of CRC patients,(35) implicating that Mus81 downregulation could also influence the prognosis of CRC patients through enhancing chromosomal instability.

Collectively, the current study has provided the first evidence indicating the significant decrease of Mus81 expression in human CRC tissues and its close association with hepatic metastasis and poor prognosis of CRC. Our study suggests that downregulation of Mus81 might serve as a novel prognostic marker for CRC. However, further studies are required to elucidate how and why Mus81 expression is downregulated in CRC.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

The authors would like to thank M. Ogada for her technical assistance in RNA preparation and reverse transcription.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References