First and foremost, the HGF-MET pathway is the first reported and most well-studied signaling pathway of MACC1. The HGF-MET pathway is widely accepted as one of the key pathways in cancer development and tumor progression[18]. In 2009, Stein et al[1] first identified the oncogenic role of MACC1, including control of MET expression, motility and proliferation, and its involvement in the HGF-MET pathway. First, they found that overexpression of MACC1 mRNA in colon cancer cells led to the upregulation of HGF receptor MET expression at both the mRNA and protein levels. Furthermore, underexpression of MACC1 mRNA through the introduction of a specific small interfering RNA (siRNA) decreased MET expression. In contrast, MACC1 expression was not altered in non-metastatic or metastatic colon cancer cells when MET expression was inhibited. In addition, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) data indicated that the MET gene is a transcriptional target of MACC1. Based on these results, Stein et al[1] inferred that MACC1 controls MET expression via direct transcriptional regulation. In regards to the tumor-promoting functions of MACC1, Stein et al[1] showed that MACC1 protein expression strongly induced cell migration, invasion and colony formation compared to the control. Additionally, MACC1 siRNA and MET siRNA both decreased cell motility and proliferation, but double knockdown of MACC1 and MET had no additional tumor-suppressing effects. Stein et al[1] verified these results through in vitro experiments in colon cancer tissues. Nuclear MACC1 expression and high MET expression were predominant in metastatic colon cancer tissues, while MACC1 was expressed in the cytoplasm with moderate MET expression in non-metastatic tumors. Taken together, MACC1 is an important mediator of the HGF-MET signaling pathway that acts by binding to the MET promoter. Galimi et al[19] conducted genetic and expression analyses of MET, MACC1, and HGF in metastatic colorectal cancer (mCRC) using patient xenografts treated with an MET inhibitor. They examined the correlation between MET inhibition and pathology in a cohort of 103 consecutive liver metastases from colorectal carcinomas[19]. They explored whether copy number variations of MET, MACC1, and HGF and overexpression of those genes can predict tumor response to MET-targeted therapies. They also assessed whether genomic or transcriptional deregulation of those genes was correlated with the pathologic and molecular parameters of aggressive disease. As was observed by Stein et al[1], Galimi et al[19] found that MACC1 controls MET level as an upstream regulator of MET transcription; genomic gain of MACC1 led to increased expression of MET, regardless of gains of MET copy number. However, neither MET genomic gain nor overexpression affected tumor growth of mCRC. Additionally, their pathologic correlations and bioinformatics analyses showed that MACC1 contributes to CRC progression via mechanisms other than MET transcriptional upregulation.

In 2013, Stein’s[1] study group, the group that first identified MACC1, identified the MACC1 promoter using a promoter luciferase construct that directs transcription of MACC1[13]. They generated 5’ truncated deletion constructs and transfected them into colorectal cancer cell lines to determine the essential domains within this promoter region. Their results showed that the region from -426 to -18 constitutes the core promoter and harbors functional motifs for the binding of AP-1, Sp1, and C/EBP transcription factors; this finding was validated by site-directed mutagenesis studies. Knockdown of these transcription factors remarkably reduced MACC1 expression and resulted in decreased cell migration, which was rescued by ectopic overexpression of MACC1. Additionally, the results of studies in human colorectal tumors verified the correlation between MACC1 and c-Jun and Sp1 expression levels.

MicroRNA (miRNA) is well-accepted as one of the most important epigenetic regulators of gene expression. MACC1 is regulated by many miRNAs, including miR-1[14], miR-143[15], miR-200a[16] and miR-338-3p[17]. These miRNAs bind directly to the 3’ untranslated region (UTR) of MACC1 mRNA and inhibit MACC1 protein expression. In CRC, miR-1 downregulation and MACC1 upregulation collectively promote MET overexpression[14]. Migliore et al[14] found that miR-1 was downregulated in 84.6% of tumors studied and was significantly correlated with MET overexpression, particularly in metastatic tumors. Concurrent MACC1 upregulation and miR-1 downregulation are required to achieve increased MET expression. miR-1 can suppress MET expression in colon cancer cells and inhibit MET-induced invasive growth. miR-143 also targets MACC1, resulting in inhibition of cell invasion and migration of colorectal cancer cells[15]. Zhang et al[15] reported that miR-143 directly targets MACC1 in CRC. In colorectal cancer cells, the same effect of MACC1 knockdown, significant inhibition of cell growth, migration and invasion, is achieved by restoration of miR-143. Concurrent miR-143 overexpression and MACC1 suppression enhance those inhibitory effects. Conversely, inhibition of miR-143 promotes cell growth, migration and invasion in colorectal cancer cells. Furthermore, miR-143 expression is inversely correlated with MACC1 mRNA expression in CRC tissues. In addition to being targeted by miR-1 and miR-143 in colorectal cancer, MACC1 is targeted by other miRNAs in other types of cancers, such as miR-200a and miR-338-3p. miR-200a suppresses cell growth and migration by targeting MACC1 and predicts prognosis in hepatocellular carcinoma (HCC)[16]. Researchers have shown that miR-200a is markedly downregulated in HCC and has suppressive effects on tumor cell growth and metastasis. They found that miR-200a suppresses tumor growth and metastasis by directly targeting MACC1. Additionally, miR-338-3p inhibits EMT in gastric cancer (GC) cells by targeting MACC1/MET/AKT signaling[17]. As a tumor-suppressing miRNA, miR-338-3p inhibits the migration and invasion of GC cells in vitro, and knockdown of miR-338-3p in GC cells leads to mesenchymal-like changes. miR-338-3p influences the expression of EMT-associated proteins by upregulating the epithelial marker E-cadherin and downregulating the mesenchymal markers N-cadherin, fibronectin, and vimentin. Conversely, reintroduction of MACC1 reverses miR-338-3p-induced EMT suppression. miR-338-3p represses the MET/AKT pathway by directly targeting MACC1.

In the beginning of MACC1 research, Guo et al[34] reported the correlation of MACC1 protein expression and clinical features of GC. They performed an immunohistochemistry (IHC) assay to detect MACC1 protein expression in GC tissues. MACC1 and MET protein expression was positively correlated. In accordance with the metastasis-promoting properties of MACC1, MACC1 protein expression in GC tissues is significantly correlated with lymph node metastasis, peritoneal metastasis and hepatic metastasis, all of which contribute to a poor prognosis for GC patients. At nearly the same time, another publication confirmed the correlation between MACC1 and Met expression in GC and their correlation with clinical parameters and patient prognosis[35]. The results of that study also indicated that MACC1 and MET expression levels are highly increased and are significantly correlated in GC tissues. MACC1 and MET expression is associated with larger tumor size, deeper tumor invasion, lymph node metastasis, lymphatic involvement, venous invasion, distant metastasis and advanced clinical stage. Compared to patients with high levels of these proteins, patients harboring lower MACC1 and Met levels had a higher 5-year survival rate.

Those first two studies showed a correlation between high MACC1 expression and poor clinical outcome in GC patients, implying that MACC1 may be an independent prognostic indicator of GC. However, they both failed to explore the underlying mechanism through which MACC1 facilitates GC progression. However, Wang et al[2] managed to reveal important functions of MACC1 in GC. As with the first two studies, they first verified MACC1 expression in GC and its correlation with adverse clinical features. They studied fresh GC tissues from 361 GC patients (stages I-IV) by Western blot analysis of MACC1 and conducted a retrospective analysis of MACC1 protein expression by IHC. Then, they examined the correlation of MACC1 expression with the clinical parameters in GC patients. Consistent with previous studies, higher MACC1 expression was associated with higher frequencies of advanced disease, postoperative recurrence, metastases and a higher mortality rate. Clinical prognosis was significantly poorer for patients with tumors with high MACC1 expression. MACC1 overexpression significantly accelerated tumor proliferation, migration and invasion and facilitated metastasis of GC in vivo. Moreover, MACC1 mRNA expression was found to be significantly correlated with markers of EMT in patients with GC. MACC1 overexpression upregulated EMT factors and induced changes in the levels of EMT markers, and silencing of MACC1 reversed these changes. All these findings indicate that MACC1 modulates EMT in GC.

MACC1 also mediates acetylcholine-induced invasion and migration in GC. The neurotransmitter acetylcholine (ACh) is known to promote the growth and metastasis of several cancers by binding to M3 muscarinic receptors (M3Rs). By binding to M3Rs, ACh promotes GC cell invasion and migration and the expression of several markers of EMT. ACh upregulates MACC1 in GC cells, and MACC1 knockdown using siRNA attenuates the effects of ACh on GC cells. AMP-activated protein kinase (AMPK) serves as an intermediate signal between ACh and MACC1. MACC1 may mediate acetylcholine-induced invasion and migration in human GC cells by participating in the Ach/M3R/AMPK/MACC1 signaling pathway[36].

Considering that tumor metastasis requires a sufficient blood supply, which, in most cases, is achieved by neovascularization, angiogenesis is a well-recognized hallmark of cancer[37]. However, anti-angiogenesis therapy has no significant benefits for some cancer patients[37]. Hence researchers have focused on identifying alternative tumor blood supply sources independent of blood vessel endothelium, termed vasculogenic mimicry (VM)[38]. VM is formed by cancer cells and is strongly associated with EMT, TWIST1 activation and tumor progression.

Considering that MACC1 induces EMT and is associated with poor prognoses of patients with GC, Wang et al[5] aimed to determine whether MACC1 promotes VM and regulates the TWIST signaling pathway, which is closely related to the EMT process in GC. To prove this theory, they investigated MACC1 expression and VM by IHC in 88 patients with stage IV GC and investigated the role of TWIST1 and TWIST2 in MACC1-induced VM both in GC xenografts and in GC cell lines. As expected, the VM density was significantly increased in tumor tissues from patients who died of GC and was positively correlated with MACC1 immunoreactivity. Furthermore, the patients with high VM density had a significantly poorer survival rate. The nuclear expression of MACC1, TWIST1, and TWIST2 is upregulated in GC tissues compared with normal tissues, and overexpression of MACC1 increased TWIST1/2 expression and induced typical VM in GC xenografts and GC cell lines. Nuclear translocations of MACC1, TWIST1, and TWIST2 were induced by HGF, and an MET inhibitor reversed those effects. These findings indicate that MACC1 promotes VM in GC by regulating the HGF/MET-TWIST1/2 signaling pathway, allowing GC cells to gain an increased blood supply and grow and possibly migrate faster.

In addition to EMT, lymph node metastasis is another common feature of GC and is one of the major causes of GC death[39]. Lymphangiogenesis is the first and most critical step of lymph node metastasis[40]. According to the aforementioned studies, MACC1 expression is associated with lymph node metastasis. Sun et al[4] reported that MACC1 promotes lymphangiogenesis and that MACC1 and lymphangiogenesis are positively correlated both in vivo and in vitro. In that study, an indirect co-culture system was constructed to determine whether MACC1 promotes lymphangiogenesis. The study involved collecting supernatant from MACC1 overexpressed/knockdown GC cells and treating human lymphatic endothelial cells (HLECs) with the collected supernatant. The results showed that supernatant from MACC1 overexpressed GC cells enhanced the proliferative and migratory capacities of HLECs. Additionally, MACC1 overexpression in xenografts promoted lymphatic vessel formation. In regards to the underlying mechanism, MACC1 significantly increases the expression of VEGF-C/D, which are important factors in the regulation of lymphangiogenesis and the promotion of lymphatic metastasis in vivo and in vitro[41].

In addition to vascular supply, tumor metastasis relies on sufficient metabolic supply to a large extent. In cancer cells, glycometabolism is reprogrammed to better accommodate the increased energy demand. Unlike normal cells, the main metabolic process of cancer cells is glycolysis, known as the Warburg effect, and this allows cancer cells to better adapt to and resist metabolic stress[42]. MACC1 has been reported to be involved in metabolic reprogramming in GC. MACC1 expression is significantly upregulated via adenosine monophosphate-activated protein kinase (AMPK) signaling in response to glucose deprivation-induced metabolic stress[3]. The results of that study showed that MACC1 expression is positively correlated with the maximum standardized uptake value of ¹⁸F-deoxyglucose in GC patients and that MACC1 enhances ¹⁸F-deoxyglucose uptake in GC cells and xenografts. The underlying mechanism of this MACC1 effect is promotion of the Warburg effect through upregulation of the activities and expression of a series of glycolytic enzymes, including hexokinase, pyruvate dehydrogenase kinase and lactate dehydrogenase, in GC cells. This metabolic shift enhances cell viability and resistance to apoptosis by facilitating ATP generation, reducing reactive oxygen species production and stabilizing mitochondrial membrane potential. In contrast, MACC1 silencing, which blocks the Warburg effect, causes GC cells to be more vulnerable to metabolic stress.

MiRNAs are well-recognized as one of the key epigenetic regulators of the EMT process and are associated with metastasis in GC[43]. Huang et al[17] found that miR-338-3p directly binds to MACC1 3’ UTR to regulate MACC1 expression. Because miR-338-3p was previously reported to be aberrantly expressed in GC, it was the first miRNA that they examined via bioinformatics analysis as a potential MACC1 regulator. Then, they showed that miR-338-3p inhibited the migration and invasion of GC cells and that inhibition of miR-338-3p in GC cells resulted in mesenchymal-like changes. MiR-338-3p also upregulated the epithelial marker E-cadherin and downregulates the mesenchymal markers, N-cadherin, fibronectin, and vimentin. Furthermore, reintroduction of MACC1 reversed miR-338-3p-induced EMT inhibition. MACC1 was proven to be directly targeted by miR-338-3p, and the Met/Akt pathway was repressed by MACC1 inhibition. Consistently, the expression of miR-338-3p and MACC1 is inversely correlated in human GC tissue samples.

After determining the diagnostic and prognostic value of circulating MACC1 in CRC, the study group of Stein et al[1] conducted a prospective study of GC and found that circulating MACC1 is a diagnostic and prognostic biomarker for GC[44]. Compared to tumor-free volunteers, the levels of circulating MACC1 transcripts were increased in GC patients of all disease stages: tumor without metastasis, tumor with synchronous metastasis, and tumor with metachronous metastasis. Both the sensitivity and specificity of MACC1 as a diagnostic and prognostic biomarker for GC were statistically significant. Importantly, GC patients with high circulating plasma MACC1 transcript levels demonstrated significantly shorter survival compared to patients with low MACC1 levels. Furthermore, GC patients with high circulating plasma transcript levels of MACC1 and S100A4 had significantly shorter survival compared to patients with low levels of both biomarkers and patients with elevation of only one biomarker.