Metastasis-associated In Colon Cancer-1 mRNA Research Articles

Elevated MACC1 Expression in Colorectal Cancer Is Driven by Chromosomal Instability and Is Associated with Molecular Subtype and Worse Patient Survival.

Simple SummaryElevated expression of Metastasis-Associated in Colon Cancer 1 (MACC1) has been identified as a strong prognostic marker of adverse outcomes for human colorectal (CRC) and other solid cancers. The biological basis of high MACC1 expression and the context of its occurrence are still poorly understood. This study investigated whether chromosomal instability and somatic copy number alterations (SCNA) frequently occurring in CRC contribute to MACC1 dysregulation, with prognostic and predictive impact.Metastasis-Associated in Colon Cancer 1 (MACC1) is a strong prognostic biomarker inducing proliferation, migration, invasiveness, and metastasis of cancer cells. The context of MACC1 dysregulation in cancers is, however, still poorly understood. Here, we investigated whether chromosomal instability and somatic copy number alterations (SCNA) frequently occurring in CRC contribute to MACC1 dysregulation, with prognostic and predictive impacts. Using the Oncotrack and Charité CRC cohorts of CRC patients, we showed that elevated MACC1 mRNA expression was tightly dependent on increased MACC1 gene SCNA and was associated with metastasis and shorter metastasis free survival. Deep analysis of the COAD-READ TCGA cohort revealed elevated MACC1 expression due to SCNA for advanced tumors exhibiting high chromosomal instability (CIN), and predominantly classified as CMS2 and CMS4 transcriptomic subtypes. For that cohort, we validated that elevated MACC1 mRNA expression correlated with reduced disease-free and overall survival. In conclusion, this study gives insights into the context of MACC1 expression in CRC. Increased MACC1 expression is largely driven by CIN, SCNA gains, and molecular subtypes, potentially determining the molecular risk for metastasis that might serve as a basis for patient-tailored treatment decisions.

Pro-inflammatory TNF-α and IFN-γ Promote Tumor Growth and Metastasis via Induction of MACC1.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Early stage CRC patients have a good prognosis. If distant metastasis occurs, the 5-year survival drops below 10%. Despite treatment success over the last decades, treatment options for metastatic disease are still limited. Therefore, novel targets are needed to foster therapy of advanced stage CRC patients and hinder progression of early stage patients into metastasis. A novel target is the crucial oncogene Metastasis-Associated in Colon Cancer 1 (MACC1) involved in molecular pathogenesis of CRC metastasis. MACC1 induces cell proliferation and motility, supports cellular survival and rewires metabolism resulting in increased metastasis in vivo. MACC1 is a prognostic biomarker not only for CRC but for more than 20 solid cancer entities. Inflammation plays a pivotal role in tumorigenesis, tumor progression and metastasis. For CRC, inflammatory bowel disease and ulcerative colitis are important inflammation associated risk factors. Certain cytokines, such as TNF-α and IFN-γ, are key factors in determining the contribution of the inflammatory process to CRC. Knowledge of the connection between inflammation and MACC1 driven tumors remains unclear. Gene expression analysis of CRC cells after cytokine stimulation was analyzed by qRT-PCR and Western blot. Cellular motility was assessed by Boyden chamber assays. MACC1 promoter activity after stimulation with pro-inflammatory cytokines was measured using promoter-luciferase constructs. To investigate signal transduction from receptor to effector molecules, blocking experiments using neutralizing antibodies and knockdown experiments were performed. Following TNF-α stimulation, MACC1 and c-Jun expression were significantly increased at the mRNA and protein level. Knockdown of c-Jun reduced MACC1 inducibility following TNF-α stimulation. TNF-α promoted MACC1-induced cell migration that was reverted following MACC1 knockdown. Moreover, MACC1 and c-Jun expression were downregulated by blocking TNFR1, but not TNFR2. Knock down of the NF-κB subunit, p65, reduced basal MACC1 and c-Jun mRNA expression levels. Adalimumab, a clinically approved monoclonal anti-TNF-α antibody, hindered MACC1 induction. The present study highlights that TNF-α regulates the induction of MACC1 via the NF-κB subunit p65 and the transcription factor c-Jun in CRC cells. This finding unravels a novel signaling pathway upstream of MACC1 and provides a potential therapeutic target for the treatment of CRC patients with an associated inflammation.

Prognostic Characteristics of MACC1 Expression in Epithelial Ovarian Cancer.

Recent studies have shown that overexpression of metastasis-associated in colon cancer 1 (MACC1) is significantly associated with adverse prognoses of patients with different kinds of cancer. However, the exact survival effect of MACC1 on epithelial ovarian cancer (EOC) patients has not yet been established. Thus, the objective of this study was to explore the prognostic role of MACC1 mRNA in EOC by using Kaplan-Meier (KM) plotter and ONCOMINE database. Our results indicated that MACC1 mRNA high expression was significantly associated with unfavorable overall survival (hazard ratio (HR) = 1.51 (95% confidence interval (CI): 1.21 – 1.88), P = 0.00025) and progression-free survival (HR = 1.53 (95% CI: 1.24 – 1.89), P = 5.8e-05) in EOC patients. We also found that the expression of MACC1 mRNA in EOC was 2.5 times higher than that in normal surface ovarian epithelium, which was statistically significant (P = 2.86e-7). Our results suggest that MACC1 expression might be a biomarker for poor prognosis in individual EOC patients.

Abstract 2778: Combination of DNA mismatch repair status and MACC1 expression in patients with stage II colon cancer: The BIOGRID studies

Abstract We have previously identified the gene Metastasis-Associated in Colon Cancer 1 (MACC1). MACC1 acts a prognostic biomarker for tumor progression, metastasis and patient survival for a broad variety of solid cancer types. Here we assessed if the MACC1 gene could separate stage II colon cancer patients with proficient mismatch repair (pMMR) into high- and low-risk groups who might benefit from or be spared adjuvant chemotherapy based on their prognosis. In the Charité 1 discovery cohort (n=61), MACC1 expression and MSI status were assayed by qRT-PCR in cryo-preserved tumors from CRC patients. MSS/MSI-low/MACC1-low tumors showed better survival vs. MSS/MSI-low/MACC1-high (P<0.0001). Patients with MSS/MSI-low/MACC1-low tumors had a similar prognosis as patients with MSI-H tumors. The Charité 2 comparison cohort (n=40) was used to translate MACC1 qRT-PCR analyses to FFPE samples. MACC1 expression was significantly higher in metachronously metastasizing tumors linked to shorter relapse-free survival (RFS), independent of the tissue type analyzed (cryo-preserved or FFPE). Next we translated MACC1 mRNA levels from qRT-PCR to MACC1 protein levels from immunohistochemistry (IHC) by comparing them in consecutive FFPE tumor sections in the BIOGRID 1 training cohort (n=189) enriched for disease recurrence. Chemotherapy-naïve patients with unfavorable pMMR status separated into MACC1-high and -low groups. Better RFS was seen in the pMMR/MACC1-low vs. pMMR/MACC1-high group using MACC1 mRNA and protein expression. pMMR/MACC1-low expression was seen in 12% and 8% of patients by qRT-PCR and IHC; interestingly, they had the same favorable prognosis as the deficient MMR (dMMR) group. Prognostic and predictive findings from BIOGRID 1 were confirmed in the independent BIOGRID 2 validation cohort (n=306) unenriched for recurrence. Better RFS was again seen in chemotherapy-naïve patients in the pMMR/MACC1-low (6%) vs. pMMR/MACC1-high group. No patients with pMMR/MACC1-low phenotype had disease recurrence. Remarkably, pooling BIOGRID 1 and 2, 5-year RFS was 100% and thus significantly longer in the pMMR/MACC1-low vs. pMMR/MACC1-high group (P=0.037). Taken together, MACC1 expression, measured by qRT-PCR or IHC, differentiates patients with unfavorable pMMR status. Patients with stage II colon cancer and pMMR/MACC1-low tumor status have a similar favorable prognosis as those patients with dMMR status who might not benefit from adjuvant therapy. Citation Format: Ulrich-Peter Rohr, Pia Herrmann, Katharina Ilm, Hai Zhang, Sabine Lohmann, Astrid Reiser, Andrea Muranyi, Janice Smith, Susen Burock, Marc Osterland, Katherine Leith, Shalini Singh, Patrick Brunhoeber, Rebecca Bowermaster, Jeanne Tie, Michael Christie, Hui-Li Wong, Paul Waring, Kandavel Shanmugam, Peter Gibbs, Ulrike S. Stein. Combination of DNA mismatch repair status and MACC1 expression in patients with stage II colon cancer: The BIOGRID studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2778. doi:10.1158/1538-7445.AM2017-2778

Effects of small interfering RNA silencing metastasis-associated in colon cancer-1 gene on the proliferation, migration and invasion of liver cancer cells

Objective To investigate the effects of small interfering RNA (siRNA) silencing metastasis-associated in colon cancer-1 (MACC1) gene on the proliferation, migration and invasion of liver cancer cells, and the possible mechanism. Methods HepG2 cells were cultured. According to the different transfectants, the cells were divided into siRNA-MACC1 group, siRNA-NC group and blank control group. The cell proliferations in the different transfected groups were measured by methyl thiazol tetrazolium (MTT) assay. The cell apoptosis in the different transfected groups was examined by flow cytometry. The cell migration and invasion abilities in the different transfected groups were tested by Transwell methods. The expression levels of MACC1, hepatocyte growth factor (HGF) and c-Met genes in the different transfected groups were detected by real-time fluorescent quantitative polymerase chain reaction(FQ-PCR). The expression levels of MACC1, HGF, c-Met, MEK, phosphorylated MEK (p-MEK), extracellular signal-regulated kinase (ERK) and phosphorylated ERK (p-ERK) proteins in the different transfected groups were detected by Western blotting. Results (1) MTT results showed that absorptivity (A) values in the siRNA-MACC1 group at 24, 48, 72 and 96 h (0.27±0.05, 0.36±0.06, 0.46±0.05, 0.58±0.07) were lower than the siRNA-NC group (0.39±0.06, 0.47±0.05, 0.64±0.07, 0.75±0.08) and blank control group (0.41±0.05, 0.49±0.07, 0.62±0.06, 0.77±0.09), the differences were statistically significant (F=25.945, 32.286, 40.264, 38.165, P=0.000, 0.000, 0.000, 0.000). (2) Flow cytometry results showed that the apoptosis rate in the siRNA-MACC1 group was (13.52±2.61)%, which was significantly higher than the siRNA-NC group and blank control group [(4.94±1.84)%, (5.10±2.13)%, F=73.918, P=0.000]. (3) The number of migration cells and the number of invasive cells in the siRNA-MACC1 group (73.62±12.71, 59.23±11.53) were significantly lower than those in the siRNA-NC group and blank control group (98.53±14.22, 85.44±13.12 and 102.51±15.13, 87.62±14.24, F=11.149, 9.500, P=0.000, 0.000). (4) Compared with siRNA-NC group and blank control group, the relative expression levels of MACC1, HGF and c-Met mRNA in the siRNA-MACC1 group were significantly lower than the siRNA-NC group and blank control group (F=51.235, 137.507, 139.708, P=0.000, 0.000, 0.000). (5) Compared with the siRNA-NC group and blank control group, the relative expression levels of MACC1, HGF, c-Met, p-MEK and p-ERK proteins in the siRNA-MACC1 group were decreased (F=49.803, 33.564, 24.391, 228.400, 137.410, P=0.000, 0.000, 0.000, 0.000, 0.000), while the the relative expression levels of MEK and ERK proteins were increased (F=12.751, 23.829, P=0.000, 0.000). Conclusion Specific inhibition of MACC1 gene expression in HepG2 cells could effectively inhibit cell proliferation, decrease cell migration and invasion, and induce cell apoptosis. The mechanism might be related to HGF/Met and MEK/ERK signaling pathways. Key words: Liver cancer; Metastasis-associated in colon cancer-1; Small interfering; Biological characteristics

Abstract P6-01-20: A role of MACC1 expression and its regulation of the HGF/c-Met pathway in breast cancer

Abstract Background The newly identified gene, metastasis-associated in colon cancer 1 (MACC1), is suggested to be a transcriptional regulator of the receptor tyrosine kinase gene c-Met, leading to cancer progression and metastasis in colorectal cancer. Also in breast cancer, aberrant hepatocyte growth factor (HGF) / c-Met signaling has been shown to contribute to worse prognosis and confer resistance to endocrine therapy or trastuzumab treatment, however, little is known of the role of MACC1. Here, we report its impact on the survival for breast cancer patients and the biological function in the cell lines. Methods A total of 300 breast cancer patients who received both surgery and adjuvant treatment at Kumamoto University Hospital between 2001 and 2009 were selected. We analyzed expressions of MACC1 by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) to evaluate the associations of its expression with breast cancer survival. In an in vitro study, the expressions of MACC1 were examined by Western blotting in breast and colorectal cancer cell lines. After transfection with a MACC1-harboing plasmid, we evaluated the activities of cMet protein and cell motility and proliferation. Further, the binding ability of MACC1 to the cMet promoter was evaluated using chromatin immunoprecipitation (ChIP) assay. Results In survival analyses, reduced MACC1 expressions were associated with patient mortality. Cox proportional hazards model showed that MACC1 mRNA (HR = 0.25, P = 0.001), MACC1 protein (HR = 0.37, P = 0.016), as well as axillary nodal status and estrogen receptor status, were independent predictors of mortality. No significant correlations between MACC1 expression and other clinicopathological factors were found. We found no strong positive correlation between MACC1 protein and c-Met mRNA expression with a Spearman’s coefficient of 0.16 (P = 0.0067). In the cell lines tested, MACC1 expression was much higher in colorectal cancer cells (DLD-1) than breast cancer cells (MCF7 and MDA-MB-231). To investigate the impact of MACC1 on the biological function of the cells, we transfected with MACC1 in breast cancer and colorectal cancer cells (SW480). MACC1 overexpression did not induce cMet expression in MCF7, whereas the corresponding cMet expression was upregulated in SW480 cells. Further, SW480 cells transfected with MACC1 showed enhanced migratory ability, whereas in MDA-MB-231 cells, transfection of MACC1 had no impact on this ability. In ChIP assay, the binding of MACC1 to the cMet promoter region was suggested in SW480 cells, but not in MCF7 cells. Conclusions Our findings provide some novel insights into the role of MACC1 for breast cancer, indicating that it plays different roles in breast cancer and in several other cancers. The biological mechanism of MACC1 which underlies improvement of breast cancer prognosis remains unelucidated. There is possibility that MACC1 does not act as the exclusive master regulator of the HGF/c-Met signaling involved in disease progression in breast cancer. Further studies to validate our results are needed. Citation Format: Aiko Sueta, Yutaka Yamamoto, Mitsuhiro Hayashi, Takashi Takeshita, Mutsuko Ibusuki, Hirotaka Iwase. A role of MACC1 expression and its regulation of the HGF/c-Met pathway in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-20.

Differential role of MACC1 expression and its regulation of the HGF/c‑Met pathway between breast and colorectal cancer.

The newly identified gene, metastasis‑associated in colon cancer 1 (MACC1), is suggested to be a transcriptional regulator of c‑Met, leading to cancer progression in colorectal cancer. To date however, little is known of the role of MACC1 in breast cancer. In a series of 300 breast cancer patients, we analyzed the association of MACC1 mRNA and protein expression with breast cancer survival using Cox proportional hazard models. In an in vitro study, we evaluated activities of c‑Met protein after transfection with a MACC1‑harboring plasmid as well as the binding ability of MACC1 to the c‑Met promoter using a chromatin immunoprecipitation (ChIP) assay. In survival analyses, reduced MACC1 expression was associated with patient mortality. MACC1 expression was an independent prognostic factor in multivariate analysis. In the cell lines tested, MACC1 expression was much higher in colorectal than in breast cancer cells. After cells were transfected with MACC1, c‑Met expression was not induced in MCF7 cells, whereas corresponding c‑Met expression was upregulated in SW480 cells. Further, SW480 cells transfected with MACC1 showed enhanced migratory ability, whereas in MDA‑MB‑231 cells, transfection of MACC1 had no impact on this ability. In ChIP assay, the binding of MACC1 to the c‑Met promoter was suggested in SW480 cells, but not in MCF7 cells. In conclusion, our findings provide some novel insights into the role of MACC1 in breast cancer, indicating that it plays different roles in breast and several other cancers. There is a possibility that MACC1 does not modulate the transcriptional role of c‑Met signaling in breast cancer.

Overexpression of MACC1 and the association with hepatocyte growth factor/c-Met in epithelial ovarian cancer.

Metastasis-associated in colon cancer-1 (MACC1) is a gene that has been newly identified by a genome-wide search for differentially expressed genes in human colon cancer tissues, metastases and normal tissues. MACC1 exerts an important role in colon cancer metastasis through upregulation of the c-Met proto-oncogene. The tyrosine kinase receptor encoded by the c-Met oncogene exhibits the unusual property of mediating the invasive growth of epithelial cells upon binding with the hepatocyte growth factor (HGF). MACC1 has been investigated with regard to colon carcinoma and MACC1 expression is associated with metastasis in various types of human cancer. However, the value of MACC1 as a potential biomarker for ovarian cancer remains unknown, although the c-Met/HGF receptor has been shown to be overexpressed in epithelial ovarian cancer tissues. To investigate the role of MACC1 in epithelial ovarian tumors, the expression levels of MACC1 mRNA in ovarian tumor specimens were analyzed together with the prognostic significance. MACC1 protein expression was also detected in the epithelial ovarian tissue specimens, and the effects of MACC1 overexpression on ovarian cancer migration, invasion and prognosis were evaluated. Due to the close association between MACC1 and c-Met expression levels in colon cancer, the expression levels of HGF/c-Met in the ovarian specimens were also examined to determine whether such a correlation is also present in epithelial ovarian cancer. A total of 92 epithelial ovarian tissue samples were used to assess the expression levels of MACC1 mRNA and protein using reverse transcription-polymerase chain reaction and immunohistochemical methods, respectively. The serum levels of MACC1 protein expression in patients with epithelial ovarian cancer were detected by enzyme-linked immunosorbent assay. The results indicated that MACC1 may be important in the malignant progression of epithelial ovarian tumors, in particular for early stage patients. Thus, MACC1 may become a predictor of prognosis and a therapeutic target in the treatment of ovarian tumors. The combined detection of MACC1 and HGF/c-Met is therefore important in assessing the prognosis of patients with malignant epithelial ovarian tumors.

Circulating MACC1 as a novel diagnostic and prognostic biomarker for nonsmall cell lung cancer.

Metastasis-associated in colon cancer-1 (MACC1) is a newly identified gene that plays an important role in cancer progression and metastasis. MACC1 has important functions in the differentiation, invasion, and metastasis of nonsmall cell lung cancer (NSCLC). However, the value of circulating MACC1 as a potential diagnostic and prognostic biomarker for NSCLC remains unknown. Plasma MACC1 mRNA levels were examined in 272 patients with NSCLC, 61 with benign lung disease, and 80 healthy volunteers using reverse transcription quantitative real-time polymerase chain reaction. MACC1 was more highly expressed in NSCLC patients than in patients with benign disease (P < 0.001) or in healthy volunteers (P < 0.001). High MACC1 expression was significantly associated with NSCLC stage (P = 0.013) and lymph node metastasis (P = 0.016). The area under the receiver operating characteristic curve was 0.766, and the optimal cutoff value was 0.105, providing a sensitivity of 71.4 % and a specificity of 89.1 %. The diagnostic capability of circulating MACC1 mRNA was higher than that of carcinoembryonic antigen (P = 0.025) or cytokeratin-19 (P = 0.010). Furthermore, high MACC1 expression was associated with poor overall survival (OS) and disease-free survival (DFS) and predicted poor survival in NSCLC patients. Consequently, MACC1 mRNA was an independent prognostic factor of OS and DFS. We concluded that circulating MACC1 mRNA represents a potential noninvasive, diagnostic and prognostic marker for NSCLC.

Abstract 45: MACC1 expression and KRas13 mutation for improved survival prognosis of colorectal cancer patients

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide. The metastatic dissemination of the primary tumor is directly linked to patient survival. We previously discovered the gene metastasis-associated in colon cancer 1 (MACC1). MACC1 has been identified as a new remarkable biomarker for disease prognosis and prediction of therapy response for CRC and for a variety of solid cancers. MACC1 expression in tumor tissues and patient blood correlates with tumor formation and progression, development of metastases and patient survival representing a decisive driver for tumorigenesis and metastasis. Here we aim at the refinement of risk assessment in a retrospective study of 100 CRC patients with tumors staged I, II and III by combinatorial analyses of MACC1 with the KRas and BRaf mutation status. First, we correlated the single parameters, KRas12, KRas13, BRaf600 mutation and MACC1 expression with clinical outcome data, such as metastasis-free survival (MFS). We found correlations of high MACC1 expression with shorter MFS (P&amp;lt;0.001). Likewise, patients with a somatic KRas13 mutation showed decreased MFS (P=0.003). Interestingly, KRas12 or BRaf600 mutations as well as their wild type counterparts had no impact on survival prognosis. Multivariate analyses combining MACC1 expression level with the single parameters KRas12, KRas13 and BRaf600 mutation revealed an improved prognosis exclusively for KRas13 mutated tumors vs. MACC1 expression alone. High MACC1 expressers with KRas13 mutation had the poorest median MFS (17.2 months) compared to MACC1 high/KRas wild type patients (37.2 months; P=0.039). Further, we addressed the impact of KRas13 on regulation of MACC1 expression in vitro. We used RNAi to analyze the impact of KRas signaling on MACC1 in several CRC cell lines with different KRas mutation status. In addition, we employed HCT116 cells harboring the activating G13D KRAS mutation together with the HCT116-derived sublines Hkh-2 and Hke-3 lacking the mutated KRas13 allele by homologous recombination. Disrupting the constitutive active KRas signaling led to increased MACC1 mRNA and protein expression levels in HCT116 cells, either by knock-down of the endogenous G13D mutated KRas expression by RNAi or through gene targeting. In summary, MACC1 expression levels together with KRas13 mutation status was the only combination that improved the identification of CRC patients at high risk for metastasis formation and the prediction of MFS when compared to MACC1 alone Citation Format: Katharina Ilm, Wolfgang Kemmner, Gudrun Koch, Pia Herrmann, Marc Osterland, Senji Shirasawa, Takehiko Sasazuki, Ulrike S. Stein. MACC1 expression and KRas13 mutation for improved survival prognosis of colorectal cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 45. doi:10.1158/1538-7445.AM2014-45

MACC1 induces metastasis in ovarian carcinoma by upregulating hepatocyte growth factor receptor c-MET.

Metastasis-associated in colon cancer 1 (MACC1) is a newly identified gene that has been shown to promote tumor cell invasion and metastasis. The present study investigated the effect of MACC1 downregulation on the biological characteristics of the ovarian cancer OVCAR3 cell line. In this study, MACC1 expression was blocked using the RNA interference technique. The downregulation of MACC1 mRNA and protein expression was confirmed using quantitative polymerase chain reaction and western blot analysis. The proliferative activity and adhesion rate of the cells were detected using cell counting kit-8 and a cell adhesion assay, while cell invasion was determined using a Matrigel invasion assay and migration capacity was observed using migration and wound-healing assays. A tube formation assay was also used to examine the angiogenic capacity of cells, and a luciferase assay was performed to assess whether MACC1 binds to the c-MET gene. The MACC1 mRNA and protein expression levels were significantly downregulated using sequence-specific small interfering RNA (siRNA). The inhibition of MACC1 expression markedly decreased the invasive, metastatic and angiogenic capacities of the cells, but only slightly inhibited growth and adhesion. In addition, a putative MACC1-binding site was identified in the 3′-untranslated region of c-MET. MACC1-siRNA was also found to significantly reduce the expression of the c-MET protein and a luciferase reporter assay confirmed that c-MET was the target gene of MACC1. These results demonstrated that the attenuation of MACC1 suppresses cell invasion and migration and that MACC1 may regulate cell metastasis through targeting the expression of c-MET. Inhibition of the function of MACC1 may represent a new strategy for treating ovarian cancer.

Expression of metastasis-associated in colon cancer-1 in different stages of epithelial ovarian cancer

To investigate the expression of metastasis-associated in colon cancer-1 (MACC1) in different International Federation of Gynecology and Obstetrics(FIGO)stages of epithelial ovarian cancer and its relationship with prognosis. Between May 2008 and August 2010, 52 epithelial ovarian cancer patients were selected from the Department of Obstetrics and Gynecology of the First Affiliated Hospital of Zhengzhou University. Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression of MACC1 mRNA and protein in the primary lesions of epithelial ovarian cancer patients, the levels of MACC1 in different stage patients were compared, and the relationship between expression of MACC1 and prognosis of ovarian cancer patients was analyzed by Kaplan-Meier analysis. The relative expression levels of MACC1 mRNA in epithelial ovarian cancer from 1 stage to 4 stage were 0.72±0.01, 0.75±0.01, 0.78±0.01, and 0.81±0.02, respectively (F=51.305, P=0.000). The expression levels of MACC1 protein from 1 stage to 4 stage were 0.71±0.04, 0.73±0.02, 0.76±0.01, and 0.84±0.05, respectively (F=65.142, P=0.000). At the end of the follow-up, the expression of MACC1 protein in recurrence and dead patients of 3-4 stages was obviously higher than that in the patients with stable disease (0.85±0.03 vs.0.74±0.05, F=72.324, P=0.000). Compared to 1-2 stage patients with lower MACC1 expression, the survival time of 3-4 stage patients with higher MACCC1 expression was significantly shorter (χ(2)=29.804, P=0.000). Increased expression of MACC1 may indicate poor prognosis of ovarian cancer patients. Therefore, MACC1 may be a potential biomarker for advanced ovarian cancer.

MACC1 overexpression predicts a poor prognosis for non-small cell lung cancer

The expression of metastasis-associated in colon cancer-1 (MACC1) in non-small cell lung cancer (NSCLC) and its association with pathological characteristics and prognosis for NSCLC patients were investigated retrospectively. The expression of MACC1 was evaluated through immunohistochemical staining of tissue microarrays from 180 samples of resected lung cancer tissues and adjacent normal lung tissues. MACC1 protein and mRNA expression were also examined from lung cancer cell lines with different metastatic potentials, 28 pairs of samples of resected fresh non-small cell lung cancer tissues, and adjacent normal lung tissues. Immunohistochemical staining of tissue microarrays showed that MACC1 was located in the cytoplasm. In addition, the expression of MACC1 protein in NSCLC was significantly higher compared to adjacent normal tissues (P < 0.001). The expression of MACC1 was positively associated with differentiation grade (P = 0.020), postoperative pathological TNM stage (P = 0.033), and lymph node metastasis (P = 0.028). Disease-free survival (DFS) and overall survival (OS) for the high MACC1 expression group were lower than the low expression group; univariate and multivariate regression analyses showed that MACC1 was an independent prognostic indicator for DFS (HR 3.124, P = 0.01) and OS (HR 2.905, P = 0.01) in NSCLC patients. The expression of MACC1 protein and mRNA was also upregulated in highly metastatic human lung cancer. In conclusion, the overexpression of MACC1 protein and mRNA may represent a potentially useful biomarker for the prognosis of NSCLC patients and might be involved in progression of NSCLC.

Abstract C9: MACC1 enhances intestinal tumorigenesis in ApcMin mice.

Abstract Colorectal cancer is one of the most frequently occurring malignancies worldwide. Although about 50% of the patients with colorectal cancer can be cured by surgery and multimodal treatment, the successful outcome of CRC patients is seriously affected by the metastatic dissemination of primary tumors. We have previously identified a novel gene termed MACC1 (metastasis-associated in colon cancer 1), which has a strong prognostic value for colon cancer metastasis, enhancing the metastatic potential of colon cancer cells both in vitro and in vivo. To further understand the role of MACC1 on colorectal carcinogenesis, we generated a transgenic mouse model with intestine-specific overexpression of MACC1 (vil-MACC1). Additionally, we crossed these animals with ApcMin mice to create vil-MACC1/ApcMin mice. Four vil-MACC1 founder lines showed MACC1 transgene mRNA and protein expression in the small intestine and colon, but not in other organs including liver, lung, and kidney. Although the villin promoter drove MACC1 expression through the whole vertical (crypt-villus) and horizontal (duodenum-colon) intestinal axes, higher levels of transgenic MACC1 were detected in the small intestine as compared with the colon, and in the villi as compared with the crypts. Histopathological analysis of the intestine of 3-months old vil-MACC1 mice showed no abnormalities as compared with age-matched wild-type littermate controls. Similar analysis is currently being performed in older animals (≥ 1 year old). In line with what we observed in the vil-MACC1 animals, the vil-MACC1/ApcMin mice displayed a similar pattern of MACC1 mRNA and protein expression throughout the whole small intestine and colon. Remarkably, transgenic overexpression of MACC1 in the intestine of vil-MACC1/ApcMin mice significantly increased the total number of adenomas, as compared with ApcMin littermate controls (P = 0.0146). Moreover, the small intestine of vil-MACC1/ApcMin mice displayed an increased number of large-sized adenomas (diameter ≥ 3 mm; P = 0.0033). Additionally, we also observed a slight increase in spleen size in vil-MACC1/ApcMin mice, as compared with ApcMin controls. Despite the increased tumor burden on vil-MACC1/ApcMin mice, we didn't observe a significant difference on survival between the two animal groups. We report here for the first time the generation of a mouse model with genetically engineered MACC1 expression. More importantly, our current findings demonstrate that MACC1 is instrumental for intestinal adenoma formation and development in vivo. These results further strengthen the importance of MACC1 for intestinal tumorigenesis and metastasis, underlying its potential as a new target for colorectal cancer treatment. Funded by the Alexander von Humboldt Foundation, the Wilhelm Sander Foundation, the Preclinical Comprehensive Cancer Center and the German Cancer Consortium Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C9. Citation Format: Clara Lemos, Cynthia Voss, Boris Jerchow, Wolfram Haider, Ulrike Stein. MACC1 enhances intestinal tumorigenesis in ApcMin mice. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C9.

MACC1 is involved in the regulation of proliferation, colony formation, invasion ability, cell cycle distribution, apoptosis and tumorigenicity by altering Akt signaling pathway in human osteosarcoma

There is mounting evidence that metastasis-associated in colon cancer-1 (MACC1) plays pivotal roles in development and progression of many tumors, particularly in osteosarcoma (OS). However, its precise roles and molecular mechanisms remain to be delineated in OS. In the current study, we found that the levels of MACC1 mRNA and protein in four OS cell lines (MG-63, HOS, SaOS-2 and U2OS) were significantly higher than that in hFOB1.19 osteoblast (P < 0.05). The vector pcDNA-MACC1 contributed to the increase of MACC1 level in MG-63 cells, whereas MACC1 siRNA evoked the decrease of MACC1 level in U2OS cells. In addition, MACC1 downregualtion caused the inhibition of cell proliferation in vitro, colony formation, invasion and tumor growth in vivo, arrested cell cycle in G0/G1 phase and induced cell apoptosis in U2OS cells, and reversed effects were observed in MG-63 cells by MACC1 upregulation. Most notably, MACC1 depletion markedly inactivated Akt signaling pathway in U2OS cells, conversely, MACC1 upregulation evidently activated Akt signaling pathway in MG-63 cells. Collectively, our data presented herein suggest that biological implications triggered by MACC1 may be tightly associated with the status of Akt signaling pathway in OS.

High expression of MACC1 predicts poor prognosis in patients with osteosarcoma

Increasing evidence has demonstrated that high metastasis-associated in colon cancer-1 (MACC1) level is tightly associated with the development, progression, and poor prognosis of a variety of tumors. However, the relationship between MACC1 and the occurrence, development, and progression of osteosarcoma (OS) remains to be clarified. To facilitate and deepen the understanding of the associations of MACC1 with the development and progression of OS, in the current study, we detected the expressions of MACC1 mRNA and protein, and investigated the relationship between MACC1 expression and prognosis of the patients with OS. Our findings demonstrated that expressions of MACC1 mRNA and protein in OS tissues were significantly higher than those in paired normal bone tissues (P < 0.05). Additionally, the level of MACC1 mRNA in the patients with higher clinical stage and distant metastasis was markedly higher than those with lower clinical stage and without metastasis (P < 0.05). Furthermore, high MACC1 level was closely correlated with clinical stage and distant metastasis (P < 0.05), but not related to the patients' age, gender, tumor size, and anatomical location (P > 0.05). Stepwise investigation revealed that survival time of the patients with high MACC1 level was obviously lower than that with low MACC1 level (P < 0.05). Collectively, our data suggest that MACC1 may play important roles in the development and progression of OS, and thus may be considered as a novel molecular target for therapy of the patients with OS.

Overexpression of MACC1 protein and its clinical implications in patients with glioma

Metastasis associated in colon cancer 1 (MACC1) has been regarded as a novel potential therapeutic target for multiple cancers. However, the impact of MACC1 in glioma remains unclear. The aim of this study was to analyze the correlation of MACC1 expression with the clinicopathological features of glioma. MACC1 mRNA and protein expression levels in human glioma tissues were detected by quantitative real-time polymerase chain reaction and immunohistochemistry assays, respectively. MACC1 mRNA and protein expression were both significantly higher in glioma tissues than in corresponding noncancerous brain tissues (both P < 0.001). In addition, statistical analysis suggested that high MACC1 expression was significantly correlated with advanced pathological grade (P = 0.004) and that patients with high expression of MACC1 protein exhibited a poorer prognosis than those with low MACC1 expression. Furthermore, Cox multivariate analysis showed that MACC1 overexpression was an independent prognostic factor for predicting the overall survival of glioma patients. In conclusion, expression of MACC1 in glioma could be adopted as a candidate biomarker for the diagnosis of clinical stage and for assessing prognosis, indicating for the first time that MACC1 may play an important role in the tumor development and progression in glioma. MACC1 might be considered as a novel therapeutic target against this cancer.

MACC1 mRNA Levels Predict Cancer Recurrence After Resection of Colorectal Cancer Liver Metastases

Upon colon cancer metastasis resection in liver, disease outcome is heterogeneous, ranging from indolent to very aggressive, with early recurrence. The aim of this study is to investigate the capability of metastasis associated in colon cancer 1 (MACC1) levels measured in liver metastasis specimens to predict further recurrence of the disease. Gene expression and gene dosage of MACC1, hepatocyte growth factor (HGF), and hepatocyte growth factor receptor (MET) were assessed using quantitative realtime polymerase chain reaction on a cohort of 64 liver metastasis samples from patients with complete follow-up of 36 months and detailed clinical annotation. The most relevant mutations associated to prognosis in colorectal cancer, KRAS, and PIK3CA were assessed on the same specimens with Sanger sequencing. Receiver operating characteristic (ROC) analysis revealed that MACC1 mRNA abundance is a good indicator of metastatic recurrence (AUC = 0.65, P < 0.05), whereas no such results were obtained with MET and HGF, nor with gene dosage. Generation of MACC1-based risk classes was capable of successfully separating patients into poor and good prognosis subgroups [hazard ratio (HR) = 5.236, 95% confidence interval (CI) = 1.2068-22.715, P < 0.05]. Also KRAS mutation was significantly associated with higher risk of recurrence (HR = 2.07, 95% CI = 1.048-4.09, P < 0.05). Cox regression multivariate analysis supported the independence of MACC1, but not KRAS, from known prognostic clinical information (Node Size HR = 3.155, 95% CI = 1.4418-6.905, P < 0.001, Preoperative carcinoembryonic antigen HR = 2.359, 95% CI = 1.0203-5.452, P < 0.05, MACC1 HR = 7.2739, 95% CI = 1.6584-31.905, P < 0.01). MACC1, a new easily detectable biomarker in cancer, is an independent prognostic factor of recurrence after liver resection of colorectal cancer metastasis.

Metastasis-associated in colon cancer-1 upregulation predicts a poor prognosis of gastric cancer, and promotes tumor cell proliferation and invasion

Metastasis-associated in colon cancer-1 (MACC1) is a newly identified oncogene, and little is known about its role in gastric cancer (GC). Our study was performed to investigate whether MACC1 influences the prognosis of GC patients and to explore the potential mechanisms involved. MACC1 expression was verified to be higher in GC tissues than in adjacent nontumorous tissues by Western blotting. A retrospective analysis of 361 GC patients (Stages I-IV) revealed that higher MACC1 expression was associated with more advanced disease, more frequent postoperative recurrence, more metastases and a higher mortality rate. The disease-free survival of Stage I-III patients and overall survival of Stage-IV patients were significantly worse when their tumors showed high MACC1 expression. To investigate the underlying mechanisms, MACC1 overexpression and downregulation were established in two GC cell lines (BGC-823 and MKN-28 cells). MACC1 overexpression significantly accelerated tumor growth and facilitated metastasis in athymic mice. MACC1 also promoted the proliferation, migration and invasion of both GC cell lines. Moreover, gastric MACC1 mRNA expression levels were significantly correlated with markers of the epithelial-to-mesenchymal transition (EMT) in patients with GC. MACC1 overexpression upregulated mesenchymal-epithelial transition factor and induced changes to markers of EMT, whereas silencing of MACC1 reversed all these changes. These findings provide some novel insights into the role of MACC1, a gene that contributes to a poor prognosis of GC by promoting tumor cell proliferation and invasion as well as the EMT.

High intratumoral metastasis-associated in colon cancer-1 expression predicts poor outcomes of cryoablation therapy for advanced hepatocellular carcinoma

BackgroundCryoablation is one of the local therapies for hepatocellular carcinoma (HCC), but its safety and effect has not been studied in patients with Child class A or B and Barcelona Clinic Liver Cancer (BCLC) stage C HCC. Metastasis-associated in colon cancer-1 (MACC1) overexpression has been associated with poor prognosis of HCC, but its predictive value to post-cryoablation outcomes remains unknown in patients with BCLC stage C HCC.MethodsThis study assessed the safety and outcomes of cryoablation measured by time to progression (TTP) and overall survival (OS), and predictive value of MACC1 mRNA and protein overexpression in tumorous tissue to post-cryoablation outcomes in 120 advanced HCC patients with child-pugh class A or B by quantitative polymerase chain reaction and immunohistochemical staining. The potenial correlation of MACC1 and c-Met expression to tumor cell proliferation and apoptosis was also analyzed.ResultsThe cryoablation in patients with advanced unresectable HCC resulted in a median TTP and OS of 5.5 (4.2- 6.7) months and 10.5 (9.0-12.0) months, respectively and no significant complications, comparable to the historical report for RFA therapy. The MACC1 mRNA and nuclear protein expression was significantly increased in tumorous tissues in these patients than that in normal liver tissue controls. Higher expression of MACC1 mRNA and nuclear protein in tumorous tissues in these patients was associated with shorter post cryoablation median TTP and OS than that with lower MACC1 expression.ConclusionsCryoablation is a safe and effective therapeutic option for patients with advanced HCC and Child-pugh class A or B cirrhosis; and a higher intratumoral expression of MACC1 or nuclear translocation predicts poor outcomes of cryotherapy in these patients.