MACC1 expression levels as a novel prognostic marker for colorectal cancer

Metastasis-associated in colon cancer-1 (MACC1) is key in promoting tumor proliferation and invasion, and is mediated by the hepatocyte growth factor (HGF) and mesenchymal-epithelial transition factor. Previous reports have revealed that MACC1 is a novel oncogene that is expressed in various types of gastrointestinal cancer. The present study comprised of 174 patients who underwent curative surgery for colorectal cancer (CRC). The correlation between gene expression and clinical parameters of the patients was assessed. It was identified that patients exhibiting high MACC1 expression levels were statistically more susceptible to distant metastases and a poor prognosis, and those exhibiting low MACC1 expression showed improved disease-free and overall survival than those with high expression. Therefore, the present data indicates that MACC1 expression levels may present as a prognostic factor in CRC patients.


Introduction
In developed countries where the aging population is increasing, cancer is one of the most prominent diseases with regard to public welfare and health measures. One in four mortalities in the USA, for example, is due to cancer (1). In the USA, the incidence of colorectal cancer (CRC) has increased significantly in recent years due to the changing lifestyle of the population and is currently one of the most frequently exhibited malignancies and leading causes of cancer-related mortality. The metastatic dissemination of primary tumors is directly associated with patient survival, and distant metastases, such as of the liver or lungs, are the major cause of mortality in CRC patients. Furthermore, metastatic disease is the most frequent reason for treatment failure (2). Therefore, the identification of genes that are responsible for the development and progression of CRC, and comprehension of the clinical significance of these genes is critical for the diagnosis and adequate treatment of CRC. The characterization of these key molecules is promising for the development of novel treatment strategies for CRC.
T he hepatocy te g row t h factor (H GF )/mesenchymal-epithelial transition factor (MET) signaling pathway was reported to have a key role in cellular growth, invasiveness and metastasis (3)(4)(5). The metastasis-associated in colon cancer-1 (MACC1) induces MET expression and promotes HGF-induced scattering, which the mitogen-activated protein kinase (MAPK) signaling pathway prevents (6). This indicates that MACC1 expression in primary tumors is associated with metastasis and results in a poor prognosis.
The aim of the present study was to analyze the correlation between MACC1 expression levels, in tissue obtained from CRC patients, with their clinicopathological factors, and to investigate the possible functions of MACC1 in the metastasis of CRC. RNA preparation and expression analysis. Total RNA was prepared using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) or using DNase and a modified acid guanidinium-phenol-chloroform procedure (9). Reverse transcription (RT) was performed with SuperScript™ II (Invitrogen Life Technologies) or by the methods reported previously (10) and an MACC1 fragment was amplified by polymerase chain reaction (PCR). Two human MACC1 oligonucleotide primers were designed as follows: Forward, 5'-T TCT T T TGAT TCCTCCGGTGA-3' and reverse, 5'-ACTCTGATGGGCATGTGCTG-3' . A PCR kit (Takara Ex Taq; Takara Bio Inc., Shiga, Japan) on a GeneAMP ® PCR System 9600 (PE Applied Biosystems, Foster City, CA, USA) was used to perform 35 cycles of PCR with the following parameters: 95˚C for 40 sec, 45˚C for 40 sec and 72˚C for 60 sec. An 8-µl aliquot of each reaction mixture was size-fractionated in a 1.5% agarose gel and visualized with ethidium bromide staining. To ensure the RNA was not degraded, a PCR assay with primers specific for the glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene was performed for 1 min at 95˚C, 1 min at 56˚C, and 1 min at 72˚C for 30 cycles. The GAPDH primers were as follows: Forward, 5'-TTGGTATCGTGGAAGGACTCA-3' and reverse, 5'-TGTCATCATATTGGCAGGTT-3' and produced a 270-bp amplicon. Complementary DNA (cDNA) from Human Reference Total RNA (Clontech Laboratories, Mountain View, CA, USA) was analyzed concurrently and served as a positive control. For quantitative assessment, RT-qPCR was performed using a LightCycler ® FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Tokyo, Japan) for cDNA amplification of MACC1 and GAPDH. The amplification protocol consisted of 35 cycles of denaturation at 95˚C for 10 sec, annealing at 60˚C for 10 sec, and elongation at 72˚C for 10 sec. The products were then subjected to a temperature gradient from 55˚C to 95˚C with continuous fluorescence monitoring to produce a melting curve of the products. The expression ratios of the MACC1 mRNA copies in the tumor and normal tissues were calculated following normalization against the GAPDH mRNA expression.

Clinical
Statistical analysis. The association between MACC1 expression and patient clinicopathological factors was analyzed using the χ 2 test. Kaplan-Meier survival curves were plotted and compared with the generalized log-rank test. Univariate and multivariate analyses were performed to identify prognostic factors using a Cox proportional hazards regression model. The in vitro assay values were analyzed using the Wilcoxon signed-rank test. All tests were analyzed with JMP software (SAS Institute Inc., Cary, NC, USA) and P<0.05 was considered to indicate a statistically significant difference.

Expression levels of MACC1 in clinical tissue specimens.
RT-qPCR analysis was performed on tissues from primary CRC and adjacent normal colorectal regions. MACC1 expression was calculated by normalising it to GAPDH expression for each tumor or normal tissue sample (Fig. 1). A significant difference was identified between the tissue types, with the average expression in tumor tissues larger than that of the corresponding normal tissues. In the following analyses, MACC1 expression, normalized to GAPDH expression, in tumor tissue was calculated following division by MACC1/ GAPDH expression in the normal tissue.
Expression levels of MACC1 and patient clinicopathological characteristics. For the clinicopathological evaluation, experimental samples were divided into two groups according to the  Table I. The data indicates that the level of MACC1 expression was not significantly correlated with the clinicopathological factors.
Association between MACC1 expression and prognosis. The data showed that the postoperative disease-free survival rate was significantly lower in patients in the high-expression group than that of the low-expression group (P=0.019; Fig. 2). The postoperative overall survival rate was lower in patients in the high-expression group than in the patients in the low-expression group (P=0.054; Fig. 3). The median follow-up was 4.1 years.

Discussion
HGF activates the HGF/MET signaling pathway, which is involved in metastasis of CRC. The HGF receptor, the gene for the receptor tyrosine kinase, MET was identified as the first transcriptional target of MACC1 (11). MACC1 is located Table II. Univariate and multivariate analyses for disease-free survival (Cox proportional hazards regression model).   on chromosome 7 and was identified through genome-wide expression analyses conducted on primary and metastatic colon cancer (12). MACC1 binds to, and promotes, MET gene expression by translocating from the cytoplasm to the nucleus, leading to migration, invasion and metastasis. The present study demonstrated that MACC1 expression levels are an independent factor of disease-free survival for CRC. Tumor malignancy was identified to correlate with MACC1 expression levels and MACC1 expression may affect the values of other prognostic factors in multivariate analysis, such as distant metastasis, which was found to be significant in univariate analysis. MACC1 expression levels were a significant prognostic factor, reflecting disease-free survival as well as the occurrence of distant metastasis. The present study is considered to be important as it has provided analyses of a large number of samples, which demonstrate that MACC1 expression levels may be used as a statistically significant marker for CRC metastasis following curative resection, along with other reported predictors (13). The present results support recent reports that a MACC1-dependent signaling pathway is involved in the progression of CRC (12,14,15).
In CRC, various adjuvant chemotherapeutic strategies are facilitative during certain disease stages and indicate the usefulness of less invasive surgery for CRC (13,(16)(17)(18)(20)(21)(22)(23)(24)(25)(26). For these cases, an informative prognostic marker, which is independent from the traditional TNM classification and contributes to diagnosis and treatment, is important. In conclusion, the present data indicates MACC1 expression levels as a potential prognostic marker for CRC. Whilst improved preoperative and postoperative treatment strategies for CRC, such as chemo-and radiotherapy combined with surgery, have contributed to the reduction of recurrences, eventually half of the cases metastasize despite the systemic chemotherapy and surgery (27). Adjuvant chemotherapy for CRC is advantageous in cases where recurrence is considered to be likely. In these cases, MACC1 expression level analysis may present as a tool to predict poor prognosis and provide adequate treatment for patients.