Metastasis-associated In Colon Cancer-1 Protein Research Articles

Core-Clock Genes Regulate Proliferation and Invasion via a Reciprocal Interplay with MACC1 in Colorectal Cancer Cells.

Simple SummaryColorectal cancer (CRC) belongs to the top three most common malignancies and is one of the deadliest cancers worldwide. Advancements in the understanding of CRC pathophysiology can lead to the development of novel treatments preventing cancer progression while prolonging overall survival. Numerous studies have shown a role for the biological clock in the regulation of cancer hallmarks and in CRC. However, the mechanistic link between the circadian clock and CRC progression is not fully understood. In the current study, we aimed to investigate the effects of a genetically disrupted clock on cancer properties using different CRC cell lines, with a focus on metastasis-related components. Our results demonstrate a reciprocal interplay between the circadian clock and the metastasis associated gene MACC1 (metastasis-associated in colon cancer 1), pointing to the circadian clock-regulation of CRC invasiveness. A circadian MACC1 expression, as shown by our data, may be considered to optimize MACC1-targeted CRC treatment.The circadian clock coordinates the timing of several cellular processes including transcription, the cell cycle, and metabolism. Disruptions in the clock machinery trigger the abnormal regulation of cancer hallmarks, impair cellular homeostasis, and stimulate tumourigenesis. Here we investigated the role of a disrupted clock by knocking out or knocking down the core-clock (CC) genes ARNTL, PER2 or NR1D1 in cancer progression (e.g., cell proliferation and invasion) using colorectal cancer (CRC) cell lines HCT116, SW480 and SW620, from different progression stages with distinct clock phenotypes, and identified mechanistic links from the clock to altered cancer-promoting cellular properties. We identified MACC1 (metastasis-associated in colon cancer 1), a known driver for metastasis and an EMT (epithelial-to-mesenchymal transition)-related gene, to be significantly differentially expressed in CC manipulated cells and analysed the effect of MACC1 manipulation (knockout or overexpression) in terms of circadian clock phenotype as well as cancer progression. Our data points to a bi-directional MACC1-circadian clock interplay in CRC, via CC genes. In particular, knocking out MACC1 reduced the period of oscillations, while its overexpression increased it. Interestingly, we found the MACC1 protein to be circadian expressed in HCT116 WT cells, which was disrupted after the knockout of CC genes, and identified a MACC1-NR1D1 protein–protein interaction. In addition, MACC1 manipulation and CC knockout altered cell invasion properties of HCT116 cells, pointing to a regulation of clock and cancer progression in CRC, possibly via the interaction of MACC1 with core-clock genes.

Circ_0006174 Accelerates Colorectal Cancer Progression Through Regulating miR-138-5p/MACC1 Axis.

BackgroundCircular RNAs (circRNAs) were reported to be involved in the progression of a variety of cancers, including colorectal cancer (CRC). However, the precise functions and mechanism of circRNAs in CRC have not been elucidated. This study aimed to investigate the effect and mechanism underlying circ_0006174 in CRC.MethodsThe expression of circ_0006174, microRNA (miR-138-5p) and metastasis associated in colon cancer 1 (MACC1) mRNA was detected by quantitative real-time polymerase chain reaction (RT-qPCR) assay. Western blot was employed to measure MACC1 protein expression. The effects of circ_0006174 knockdown, MACC1 overexpression or miR-138-5p inhibition on cell proliferation, migration, invasion, and apoptosis were assessed by cell counting kit 8 (CCK-8) assay, clone formation assay, transwell assay and flow cytometry assay, respectively. The interaction between miR-138-5p and circ_0006174 or MACC1 was confirmed by RNA pull down assay or dual-luciferase reporter assay. Xenograft tumor model in nude mice was used to verify the function of circ_0006174 in vivo.ResultsCirc_0006174 and MACC1 expression was highly expressed, while miR-138-5p expression was downregulated in CRC cells and tissues. Meanwhile, circ_0006174 functioned as a sponge of miR-138-5p to upregulate MACC1 expression. Furthermore, circ_0006174 knock down-mediated suppression on cell proliferation, migration and invasion, and promotion on cell apoptosis could be alleviated by MACC1 overexpression or miR-138-5p inhibition in CRC cells. Besides, circ_0006174 knockdown also inhibited CRC procession in vivo.ConclusionCirc_0006174 advanced CRC progression via sponging miR-138-5p to upregulate MACC1 expression, which may provide a promising molecular target for CRC treatment.

MiR-338-3p regulates the proliferation, apoptosis and migration of SW480 cells by targeting MACC1.

The mortality and incidence rates of colorectal cancer (CRC) vary widely worldwide. miR-338-3p inhibits tumor cell proliferation in several types of cancer, however, the role of miR-338-3p on CRC remains unknown. The aim of the current study was to investigate the cellular function of miRNA-338-3p (miR-338-3p) in CRC, the malignant behavior of CRC cells and the interaction between miR-338-3p and metastasis-associated in colon cancer-1 (MACC1). miR-338-3p expression was significantly decreased in CRC tissue compared with adjacent normal tissue. In the CRC cell line SW480, miR-338-3p overexpression suppressed cell proliferation and migration and induced G1/S cell cycle arrest and apoptosis. By contrast, miR-338-3p knockdown significantly enhanced cell proliferation and migration, and suppressed G1/S cell cycle arrest and apoptosis. Furthermore, the dual-luciferase reporter assay confirmed MACC1 as a direct target of miR-338-3p. In addition, miR-338-3p overexpression reduced the level of MACC1 protein expression and MACC1 expression was significantly upregulated in CRC tissue samples. MACC1 siRNA significantly reduced CRC cell proliferation and migration, whilst cell apoptosis was significantly increased. In conclusion, miR-338-3p expression was decreased in CRC. miR-338-3p regulated the proliferation, apoptosis and migration of CRC cells by targeting MACC1.

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

The expression of metastasis-associated in colon cancer-1 and KAI1 in gastric adenocarcinoma and their clinical significance

BackgroundThe most common reason for malignant tumor treatment failure is recurrence and metastasis. Metastasis-associated in colon cancer-1 (MACC1) was originally identified as a metastatic and prognostic biomarker for colon cancer and later other solid tumors. Kangai 1 (KAI1), a marker of suppressor of metastasis, is also associated with metastasis and poor prognosis in many tumors. However, the prognostic value of either MACC1 or KAI1 in gastric adenocarcinoma (GAC) is unclear. In this study, we explored the relationship between MACC1 and KAI1 expression, as well as their respective correlation with clinicopathological features, to determine if either could be helpful for improvement of survival prognosis in GAC patients.MethodsThe expression levels of both MACC1 and KAI1 in 325 whole-tissue sections of GAC were examined by immunohistochemistry. Clinical data was also collected.ResultsMACC1 was significantly overexpressed in GAC tissues when compared to levels in normal gastric tissues; KAI1 was significantly down-expressed in GAC tissues when compared to levels in normal gastric tissues. Investigation of association between MACC1 and KAI1 protein levels with clinicopathological parameters of GAC indicated association between the expression of each with tumor grade, lymph node metastasis, invasive depth, and TNM stages. The overall survival time of patients with MACC1- or KAI1-positive GAC tumors was significantly shorter or longer than that of those who were negative. Importantly, multivariate analysis suggested that positive expression of either MACC1 or KAI1, as well as TNM stage, could be independent prognostic factors for overall survival in patients with GAC.ConclusionsMACC1 and KAI1 may represent promising metastatic and prognostic biomarkers, as well as potential therapeutic targets, for GAC.

Targeting H19 by lentivirus-mediated RNA interference increases A549 cell migration and invasion

ABSTRACTBackground: Lung cancer is one of the most common and a lethal malignancy in the world and non-small cell lung cancer (NSCLC) is the most usual type. H19 long non-coding RNA (lncRNA) plays essential roles in tumor development. But its role in tumor metastasis is still unclear. Materials and Methods: MACC1 RNAi and Lentivirus-mediated H19-specific shRNA was used to establish H19 stable knocking-down A549 cells. Transwell assays were performed to examine the effect of H19 knocking-down on A549 cells migration and invasion. The downstream signaling proteins targeted by H19 were also examined by western blot. AG1478 and U0126 were used as the inhibitor of EGFR and ERK1/2, respectively. Results: The knockdown of H19 increased the migration and invasion of A549 cells, and knockdown of metastasis-associated in colon cancer 1 (MACC1) decreased the migration and invasion of A549 cells. Furthermore, MACC1 protein targeted by H19 was upregulated as well as the downstream signaling proteins including epidermal growth factor receptor (EGFR), β-catenin, extracellular-signal-regulated kinase 1/2 (ERK1/2). Inhibited the expression of EGFR or ERK1/2 significantly decreased the migration and invasion of tumor cells. Conclusion: Our findings showed that H19 functions as a suppressor of NSCLC and plays an important role in the migration and invasion of NSCLC. More importantly, H19 may regulate NSCLC metastasis through modulating cellular signaling pathway proteins related to cell proliferation and cell adhesion, including MACC1, EGFR, β-catenin and ERK1/2. These results put forward our understanding of the detailed mechanism of H19 lncRNA regulating the process of NSCLC metastasis.

Increased MACC1 levels in tissues and blood identify colon adenoma patients at high risk.

BackgroundColorectal cancer is a preventable disease if caught at early stages. This disease is highly aggressive and has a higher incidence in African Americans. Several biomarkers and mutations of aggressive tumor behavior have been defined such as metastasis-associated in colon cancer 1 (MACC1) that was associated with metastasis in colorectal cancer patients. Here, we aim to assess colon tissue MACC1 protein and circulating MACC1 transcripts in colon preneoplastic and neoplastic African American patients.MethodsPatients’ tissue samples (n = 143) have been arranged on three tissue microarrays for normal (n = 26), adenoma (n = 68) and cancer (n = 49) samples. Immunohistochemistry was used to detect MACC1 expression. Blood samples (n = 93) from normal (n = 45), hyperplastic (n = 15) and tubular adenoma (n = 33) patients were used to assess MACC1 transcripts using qRT-PCR. Distribution of continuous variables was tested between different diagnoses with Kruskal–Wallis test. Categorical variables were tested by Chi square test. We assessed the prognostic ability of IHC staining by calculating area under receiver operating characteristics curve (ROC) for adenoma and cancer separately. Differences between groups in terms of MACC1 transcript levels in plasma were calculated by using non-parametric (exact) Wilcoxon-Mann–Whitney tests. We performed all calculations with SPSS, version 21.ResultsIn patient tissues, there was a statistically significant difference in MACC1 expression in normal vs. adenoma samples (p = 0.004) and normal vs. cancer samples (p < 0.001). There was however no major difference in MACC1 expression between adenoma vs. cancer cases or tubular adenomas vs tubulovillous adenomas. The area under the curve for both normal vs. adenoma and normal vs. cancer cases were 70 and 67 %, respectively. MACC1 expression was not correlated to age, gender or anatomical sample location. In patient plasma, MACC1 transcripts in adenoma patients were significantly higher than in plasma from normal patients (p = 0.014). However, the difference between normal and hyperplastic plasma MACC1 transcripts was not statistically significant.ConclusionMetastasis-associated in colon cancer 1 is expressed at early stages of colorectal oncogenesis within the affected colonic tissue in this patient cohort. The plasma transcripts can be used to stratify African American patients at risk for potential malignant colonic lesions.

Increased MACC1 expression indicates a poor prognosis independent of MET expression in gastric adenocarcinoma

Metastasis-associated in colon cancer-1 (MACC1), a newly identified oncogene, promotes tumor proliferation and invasion via the MET pathway. The purpose of this study is to determine the clinical significance of MACC1 and MET expression in the long-term survival and recurrence in a large cohort of gastric carcinoma patients following curative resection. We evaluated the prognostic value of MACC1 and MET expression using immunohistochemistry in 331 gastric adenocarcinoma patients. MACC1-positive patients had lower overall survival (OS) or event-free survival (EFS) rates than MACC1-negative patients (P=0.039 and P=0.044, respectively), while MET positivity itself was not associated with either OS or EFS. Multivariate analysis identified the expression level of MACC1 protein as an independent negative prognostic factor for OS or EFS (P=0.021 and P=0.016, respectively). This study suggests that MACC1 is an independent prognostic factor in gastric adenocarcinoma and that the prognostic impact of MACC1 may be associated with MACC1 partners other than MET.

Metastasis-Associated in Colon Cancer-1 Associates With Poor Prognosis and Promotes Cell Invasion and Angiogenesis in Human Cervical Cancer.

The aim of this study is to investigate the clinicopathologic significance and potential role of metastasis-associated in colon cancer-1 (MACC1) in the progression of cervical cancer. MACC1 expression was examined in cervical cancer cell lines, 6 matched cervical cancer tissues, and adjacent noncancerous tissues using Western blotting and real-time reverse transcriptase polymerase chain reaction. MACC1 protein expression and localization were determined in 181 paraffin-embedded archived cervical cancer samples using immunohistochemistry. Statistical analyses were applied to evaluate the clinicopathologic significance. The effects of MACC1 on cell migration, invasion, and angiogenesis were examined using migration assay, wound healing assay, 3-dimensional morphogenesis assay, and chicken chorioallantoic membrane assay. Western blotting was performed to examine the impact of MACC1 on the Akt and nuclear factor κB signaling pathways. Both protein and messenger RNA levels of MACC1 was up-regulated in cervical cancer cell lines and cervical cancer tissues, as compared with normal tissues. High MACC1 expression was detected in 96 (53%) of 181 of the cervical cancer tissues. In addition, high MACC1 expression correlated significantly with aggressiveness of cervical cancer, including International Federation of Gynecology and Obstetric stage (P = 0.001), pelvic lymph node metastasis (P = 0.004), recurrence (P = 0.037), and poor survival (P = 0.001). Moreover, enforced expression of MACC1 in cervical cancer cell lines significantly enhanced cell migration, invasion, and angiogenesis. Conversely, knockdown of MACC1 caused an inhibition of cell migration, invasion, and angiogenesis. Up-regulation of MACC1 increased, but knockdown of MACC1 decreased the expression of matrix metalloproteinase-2 and matrix metalloproteinase-9. Furthermore, enforced expression of MACC1 could enhance, but knockdown of MACC1 could reduce AKT and nuclear factor κB pathway activity. Our findings suggest that MACC1 protein, as a valuable marker of cervical cancer prognosis, plays an important role in the progression of human cervical cancer cells.

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.

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

Effects of small interfering RNA silencing MACC-1 expression on cell proliferation, cell cycle and invasion ability of cervical cancer SiHa cells

To investigate the expression of metastasis-associated in colon cancer-1 (MACC-1) mediated by siRNA, and to study the effects of its downregulation on cell proliferation, cell cycle and invasion ability of cervical cancer SiHa cells. MACC-1 siRNA and control siRNA were transfected into cervical cancer SiHa cells, and the expression of MACC-1 protein after transfection with MACC-1 siRNA was detected by Western blotting. The changes of cell proliferation, cell cycle and invasion ability of the SiHa cells were determined by CCK-8 kit, flow cytometry and Boyden chamber assay. The expressions of cell cycle- and invasion-related proteins were analyzed by Western blotting. Compared with the untreated group (0.317 ± 0.023) and control siRNA group (0.309 ± 0.021), the expression of MACC-1 protein was downregulated in the MACC1 siRNA group (0.041 ± 0.006) (P < 0.05), and its downregulation significantly suppressed the cell proliferation, altered the cell cycle distribution and reduced the cell invasion ability of the SiHa cells (P < 0.05). Compared with the untreated group (0.217 ± 0.025 and 0.215 ± 0.024) and the control siRNA group (0.222 ± 0.023 and 0.207 ± 0.027), the expression of cyclin D1 and Cdk2 proteins were significantly decreased in the MACC1 siRNA group (0.076 ± 0.010 and 0.039 ± 0.007) (P < 0.05). Compared with the untreated group (0.099 ± 0.007) and control siRNA group (0.105 ± 0.012), the expression of p21 protein was significantly increased in the MACC1 siRNA group (0.676 ± 0.044) (P < 0.05). The downregulation of MACC-1 expression also evoked a decrease of expressions of MMP-2 and MMP-9 proteins and an increase of E-cadherin protein expression (P < 0.05). MACC-1 downregulation-mediated inhibition of proliferation and decreased invasion ability of tumor cells may be closely associated with the alterations of expressions of cell cycle- and invasion-related proteins.

Metastasis-Associated in Colon Cancer 1 Is a Novel Survival-Related Biomarker for Human Patients with Renal Pelvis Carcinoma

Metastasis-associated in colon cancer 1 (MACC1) has recently been identified as a novel independent prognostic indicator for metastasis occurrence, overall survival and cancer-free survival for patients with colon cancer and other solid tumors. In this study, we investigated the role of MACC1 in the development and progression of renal pelvis carcinoma, a form of upper tract urothelial carcinomas. MACC1 protein has been found in the cytoplasm as well as in the nucleus of the transitional epithelial cells of the normal renal pelvis in immunohistochemical (IHC) assays. Quantitative IHC examinations revealed that MACC1 abnormal abundance in cancerous tissues might represent a biological indicator clinically suggestive of tumor malignancy in the renal pelvis. Furthermore, investigation of the association of MACC1 protein levels with clinicopathological parameters in this study has suggested a correlation of MACC1 expression with tumor-node-metastasis stage and histopathological grade of patients with renal pelvis carcinoma, with elevated MACC1 protein levels frequently associated with higher aggressiveness of the disease. Moreover, both disease-free survival and overall survival for the patients in the high MACC1 expression group were significantly lower than those in the low expression group. Multivariate analysis with a Cox proportional-hazards model suggested that MACC1 is indeed an independent prognostic indicator of overall survival and cancer-free survival for patients with renal pelvis carcinoma. Thus, MACC1 may represent a promising prognostic biomarker candidate, as well as a potential therapeutic target for this disease.

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.

Downregulated expression of metastasis associated in colon cancer 1 (MACC1) reduces gallbladder cancer cell proliferation and invasion

Metastasis associated in colon cancer 1 (MACC1), a key regulator of the hepatocyte growth factor (HGF)/MET signaling pathway, has been implicated in multiple human cancers. However, little is known regarding its expression and biological function in human gallbladder cancer (GBC). In this study, we focused on the clinical significance and biological functions of MACC1 in GBC and found that MACC1 protein overexpression was frequently detected in GBC tissues. Patients with MACC1-positive tumors had worse overall survival than patients with MACC1-negative tumors. Furthermore, treatment of GBC lines with MACC1-targeting small interfering RNA oligonucleotides (MACC1-siRNA) significantly reduced the proliferation of GBC-SD and OCUG-1 cell lines and diminished both anchorage-independent growth on soft agar and cell migration. These data indicate that MACC1 acts as a putative oncogene in GBC and could be a novel diagnostic and therapeutic target for GBC.

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.