Tumor Microenvironment Remodeling in Gastrointestinal Cancer: Role of miRNAs as Biomarkers of Tumor Invasion

Gastrointestinal (GI) cancers are the most frequent neoplasm, responsible for half of all cancer-related deaths. Metastasis is the leading cause of death from GI cancer; thus, studying the processes that regulate cancer cell migration is of paramount importance for the development of new therapeutic strategies. In this review, we summarize the mechanisms adopted by cancer cells to promote cell migration and the subsequent metastasis formation by highlighting the key role that tumor microenvironment components play in deregulating cellular pathways involved in these processes. We, therefore, provide an overview of the role of different microRNAs in promoting tumor metastasis and their role as potential biomarkers for the prognosis, monitoring, and diagnosis of GI cancer patients. Finally, we relate the possible use of nutraceuticals as a new strategy for targeting numerous microRNAs and different pathways involved in GI tumor invasiveness.


Introduction
Gastrointestinal (GI) cancers are complex and heterogeneous diseases caused by the interaction of genetic and environmental factors. These types of tumors can affect several organs, such as the pancreas, liver, gallbladder, bile ducts, and colon. Because of the large cell mass and rapid turnover, GI cancers are among the most frequent neoplasms and are responsible for about half of all cancer-related deaths [1]. In fact, they account for 26% of global cancer incidence and 35% of all cancer-related deaths [2]. These neoplasms have different clinical features but share some similar characteristics; the most common is their poor prognosis meaning that most treatments can often be ineffective. Although much progress has been made in the early detection of colorectal cancer (CRC) through screening, the prognosis of other GI tumors tends to be unfavorable due to patients presenting late-stage diagnoses [3]. Current therapies involve adjuvant and neoadjuvant chemoradiotherapy, targeted therapy, or immunotherapy, which stimulate an antitumor immune response [3,4]. Despite these multidisciplinary approaches, a fair number of patients still develop distant metastasis and show resistance to therapy [5,6]. Indeed, metastasis is the leading cause of death from GI cancer, so it is of paramount importance to study the players and mechanisms that regulate tumor migration, invasion, and metastatic dissemination, in order to evaluate the possibility of using these substrates as new therapeutic targets. observed, which is considered a pre-neoplastic lesion. The pathogenesis of GISTs is determined by mutations in KIT or platelet-derived growth factor receptor A (PDGFRA) genes [45]. Surgery is the primary choice for resectable GISTs; however, the therapeutic treatment for unresectable metastatic GIST patients has been radically changed by the introduction of targeted therapy [46]. Administration of imatinib mesylate, together with other kinase inhibitors, has been associated with good improvement in clinical outcomes and survival [46]. Despite the many benefits, patients may develop resistance due to secondary mutations in KIT and PDGFRA that lead to the deregulation of crucial signaling pathways involved in cell proliferation and migration, such as RAS/RAF/MAPK and PI3K/Akt/mTOR [47].

Tumor Microenvironment in Promoting Cancer Cells Migration and Invasion
Over the past decade, several studies have shown that cancer growth is determined not only by tumor cells but also by the TME [48]. Indeed, it consists of a complex network of malignant and non-malignant cells that potentiate cancer progression and metastasis and modulate responses to therapy [48,49]. Specifically, the TME is composed of various elements, including tumor cells, immune cells such as T cells, B cells, dendritic cells (DC), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), CAFs, and blood and lymphatic vessels [50,51]. Intercellular communication is driven by a complex and dynamic network of cytokines, chemokines, growth factors, and inflammatory and matrix-remodeling enzymes, all of which can promote tumor progression and invasion ( Figure 1) [52]. In particular, the TME of GI tumors is characterized by the presence of CAFs, which promote tumor growth, angiogenesis, invasion, and metastasis, along with extracellular matrix (ECM) remodeling and even chemoresistance [53]. In addition, TME is defined by the presence of an immunosuppressive immune infiltrate mainly composed of M2 macrophages, MDSCs, and T regulatory cells (Tregs), and finally by the presence of soluble factors such as TGF-β, which has a primary role in promoting tumor migration and metastases ( Figure 1) [54]. Schematic representation of gastrointestinal TME and biological events leading to cell migration and metastasis formation. A pro-tumoral environment is established in the TME of GI tumors given by the presence of activated CAFs and immunosuppressive immune cells, leading to the production of cytokines, chemokines, and soluble factors. These are responsible for ECM remodeling and promotion of EMT that enhances tumor cell motility and subsequent metastasis formation. The figure was created with BioRender.com. Figure 1. Schematic representation of gastrointestinal TME and biological events leading to cell migration and metastasis formation. A pro-tumoral environment is established in the TME of GI tumors given by the presence of activated CAFs and immunosuppressive immune cells, leading to the production of cytokines, chemokines, and soluble factors. These are responsible for ECM remodeling and promotion of EMT that enhances tumor cell motility and subsequent metastasis formation. The figure was created with BioRender.com.

Cancer-Associated Fibroblasts
CAFs constitute a dominant stromal component of the TME of GI cancers and contribute in many ways to tumor progression and resistance to therapy [53,55,56]. They originate predominantly from tissue-resident fibroblasts that are activated in response to signals from cancer cells and the TME [57]. In GI tumors, particularly in CRC and HCC, the presence of activated CAFs lead to increased expression of activated markers, such as α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP), and the production of large amounts of glycoproteins, including tenascin-C and collagen maturation enzymes, which are responsible for ECM remodeling [58,59]. Alterations in the biomechanical properties of the ECM can trigger cancer cell migration [53,60]. CAFs can generate gaps in stromal and basement membrane components that are connected through cell-cell junctions to promote EMT and mediate collective migration of tumor cells through matrix metalloproteases (MMP)-dependent or -independent mechanisms [61]. In addition, CAFs produce interleukin-(IL)-6 that induces the activation of the JAK2/STAT3 axis in GC and promotes EMT, increasing migration and invasion [62].
Finally, the accumulation of activated CAFs correlates with resistance to therapy. For example, the increase in FAP protein can cause resistance to chemotherapy, radiotherapy, and immune checkpoint blockade [55,70]. Furthermore, in experimental models of CRC, chemotherapy-stimulated CAFs increase the secretion of specific cytokines, such as IL-17A, which promotes chemoresistance through the NF-κB pathway and increases tumor invasion and growth in vivo. Of note, an increase in IL-17 was also observed in patients with therapy-resistant CRC [55,71].

Cytokines and Chemokines
Cytokines and chemokines are inflammatory mediators secreted by several components of the TME. Their action in promoting cancer development includes an antagonizing antitumor immune response, recruiting tumor-supportive stromal cells and immunesuppressive cells, inducing angiogenesis and metastasis, and altering the responses to therapeutic agents [72].
Several cytokines may contribute to tumor progression, particularly IL-1β, TNFα, and IL-6 play key roles in promoting and enhancing EMT. IL-6, secreted by CAFs, promotes STAT3 activation and, thus, tumor invasion and metastasis formation [73]. Similar to IL-6, IL-23 also acts by increasing the expression level of STAT3 and inducing EMT-mediated metastasis [74].
In addition to cytokines, several chemokines are also responsible for promoting tumor invasiveness. CXCL1 promotes the proliferation and migration of colon cancer cells and has a facilitating effect on tumor angiogenesis [75]. Increased CXCL1 promotes TAM2 migration by inhibiting the recall of CD4+ and CD8+ T cells at tumor sites [76]. CXCL5, in the GC TME, facilitates metastasis by promoting the invasion and migration of tumor cells through the activation of the ERK signaling pathway in cancer cells [77,78]. The CXCL12-CXCR4 axis is particularly important in tumor development to participate in CRC, pancreatic cancer, and HCC metastasis [79,80]. The binding of CXCL12 to its receptor CXCR4 on CRC cells causes pro-metastatic signaling through decreasing E-cadherin and inducing ICAM-1 expression [79]. CXCL12-CXCR4 also induces the Wnt/β-catenin pathway with increased MMP-2, MMP-9, and plasminogen activator and consecutive metastatic initiation in CRC [81]. As mentioned above, CAFs are responsible for the production of CXCL12; several studies have claimed that CXCL12 increases the proliferation, migration, and invasion of CRC cells through the induction of M2 polarization [65]. Another chemokine associated with the development of CRC and its liver metastasis is CXCL8, which promotes angiogenesis, proliferation, invasion, migration, and survival of tumor cells through the induction of EMT [82]. TGF-β promotes the generation of an immunosuppressive environment through the recall of TAM2, MDSC, and Tregs at the tumor site via activation of the Smad transcription factor [83][84][85][86]. TGF-β is a dominant effector in all GI cancers and mediates the conversion of fibroblasts to CAFs, promoting cell migration and tumor invasiveness through the induction of EMT [64,85]. In these tumors, TGF-β may have an antitumoral function in the early stage of tumor development by inducing apoptosis; on the contrary, in more advanced stages, it can support tumor progression by improving cell survival, EMT, migration, invasion, and metastasis [85]. In addition, TGF-β induces MMP-8 gene expression through PI3K/Akt/Rac1 signaling in HCC cells that promotes tumor cell EMT and malignant progression [87]. Finally, in addition to TGF-β, insulin-like growth factor-1 (IGF-1) can also promote cancer proliferation and survival, inducing EMT that contributes to tumor migration, invasiveness, and metastasis [88].

Immune Infiltrating Cells
The immune cell populations within the TME play a significant role in patient prognosis and response to treatment [89]. The presence of a good immune infiltrate, such as tumorinfiltrating lymphocytes (TILs), correlates with a good prognosis in a large spectrum of solid tumors, including GI cancers [90,91]. However, pro-tumor immune populations, including TAM2, MDSCs, neutrophil N2, and Tregs, are highly present in these tumors [92][93][94]. Each one contributes to tumor aggressiveness through the secretion of inflammatory cytokines and chemokines, key effector molecules such as MMPs, prostaglandin E2 (PGE2), and TGF-β. The production of these molecules in the TME, as already described, is responsible for EMT and the promotion of tumor migration and invasiveness [95,96].
TAMs play an important role in tumor progression by promoting pro-angiogenic and immunosuppressive signaling [97]. Through the production of chemokines such as CCL17, CCL22, and CCL24, they lead to the recruitment of T helper 2 cells, Tregs, eosinophils, and basophils, inducing an immunosuppressive environment [98,99]. In addition, TAM2s produce inflammatory cytokines and TGF-β, which are associated with a more invasive tumor profile [99]. For these reasons, in GI tumors, especially in CRC, GIST, and HCC, they have a pro-tumor and pro-metastatic action, are associated with poor prognosis, and correlate with worse overall survival [100,101].
MDSCs play an important role in suppressing the immune response through a series of secretory factors such as arginases, nitrites, reactive oxygen species (ROS), immunosuppressive cytokines, and the expansion of immunosuppressive cells such as Tregs [102]. In GI tumors, MDSCs are strongly involved in the regulation of the immune system and act to dampen its response to tumors, promoting the escape of tumor cells from immunosurveillance and increasing both metastasis and recurrence [102]. For this reason, they are associated with poor prognosis and low survival [103]. MDSCs induce immunosuppressive effects through the production of several cytokines, such as IL-6, IL-10, PGE2, and TGF-β [104][105][106]. In addition, MDSCs promote tumor progression through the induction of MMP-9 and TGF-β, which are responsible for the establishment of a more invasive tumor phenotype [105].
Tumor-associated neutrophils (TANs) play a controversial role in tumor progression. Although they are known to have antitumor activity, many studies have shown that the presence of TANs is associated with the promotion of tumor metastatic potential and poor prognosis in many tumor types [107,108]. Especially, type 2 TANs have immunosuppressive action characterized by the production of chemokines, some having a role in cancer cell migration, such as CCL2, CCL3, CCL4, CCL8, CXCL8, and CXCL16 [109]. In HCC, TAN2 promotes tumor progression and metastasis by inducing the recruitment of TAM2 and Tregs into the TME [109].
Tregs are essential for the maintenance of immunological homeostasis and self-tolerance. In tumors, they may have extensive suppressive activity, secreting immunomodulatory cytokines and cytolytic molecules that allow them to regulate immune responses [110].
In GI tumors, high numbers of Tregs are often associated with poor prognosis and low survival rates [111,112]. It has been extensively studied that Tregs generate a strong proinflammatory environment in GI tumors through the secretion of inflammatory cytokines such as IL-12, TGF-β, and TNF-α promoting an immunosuppressive TME and, therefore, supporting tumor invasiveness and metastasis formation [113,114].

miRNA in Gastrointestinal Cancer
MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs, 17-25 nucleotides long that regulate gene-expression post-transcriptionally by recognizing complementary sites in the 3 untraslated region (UTR) of the target messenger RNA (mRNA). A single miRNA is able to target hundreds of mRNAs and influence the expression of many genes. A disruption of the miRNA-mediated gene expression control may lead to environmental stresses usually implicated in the development and progression of human cancer, such as starvation, hypoxia, oxidative stress, and DNA damage [115]. As a matter of fact, miRNAs are involved in cancer initiation, progression, and metastasis. Indeed, many miRNAs are found to be up-or down-regulated in cancer samples when compared to normal tissue ( Table 1). miRNAs can modulate the expression of mechanisms such as toll-like receptors, Wnt/β-catenin, Hedgehog, and Jak/Stat signaling pathways [116]. Of note, they can act as oncogenes or tumor suppressors through various mechanisms [117].

miRNA and Tumor Microenvironment
As previously described, TME is a key factor in the progression and proliferation of tumor cells and drug resistance [139]. Recently, attention was given to the role of miR-NAs in the modulation of different types of cells in the TME, such as immune cells and CAFs [139][140][141]. Aberrant expression of miRNAs leads to the reprogramming of cells in CAFs in HCC and GC. In detail, miR-21 converts hepatic stellate cells in activated CAFs by regulating TGF-β signaling and inducing phosphatase and tensin homolog (PTEN) down-regulation and consequent up-regulation of the PI3K/Akt signaling pathway in HCC [127,142]. miR-1247-3p converts normal fibroblast in CAFs promoting HCC progression [143]. miR-27a acts as an oncogene inducing the reprogramming of normal fibroblast in CAFs promoting cancer proliferation and metastasis in GC [130]. Moreover, miR-106b directly targets the PTEN gene in CAFs promoting proliferation, migration, and invasion of GC cells [144].
CAFs-derived miR-493-5p plays a role in the progression and cell growth in CCA. In fact, miR-493-5p expression levels are higher in extracellular vesicles derived from CAFs than the ones from normal fibroblasts [141]. miRNAs secreted by CAFs also play a role in drug resistance. For example, GC cells' resistance to cisplatin treatment is due to the expression of miR-522 derived from CAFs [145]. miRNAs secreted by CAFs also play an important role in CRC. Jiang et al. highlighted the correlation between the expression of exosomal miR-181b-3p derived from CAFs and CRC development and progression [146]. A recent study by Liu et al. demonstrated that exosomal miR-29a targets and reduces proteins expressed on the vascular endothelial cells in CRC, facilitating metastasis [147].
Like CAFs, the activation of TAMs also plays a role in the modulation of TME. In GC, exosomal miR-21 derived from TAMs leads to resistance to cisplatin through suppression of apoptosis and activation of the PI3K/Akt signaling pathway in cancer cells [148]. Other miRNAs promote cancer progression and metastasis regulating immune cell responses, such as miR-192-5p, which modulates tumor-infiltrating Tregs in GC activity [149].

miRNAs as Potential Biomarkers in Gastrointestinal Cancers
As mentioned above, miRNA levels are altered in different pathological processes of several tumors, including GI cancers. This deregulation may act as a specific tumor signature and could be useful in differential diagnosis and in correlation with specific clinical characteristics. miRNA expression levels could be associated with a better or worse prognosis. For example, miR-451 is associated with a worse prognosis in GC and CRC [150]. Therefore, the idea of using miRNAs as potential biomarkers for GI tumor screening, prognosis, diagnosis, and disease monitoring is increasingly dominant [151,152]. Profiling of miRNAs has advantages over mRNA and protein, such as their stability in body fluids and human formalin-fixed paraffin-embedded (FFPE) tissues, their small size, and their regulatory function on different target molecules and pathways [153].

miRNAs as a Biomarker in Gastric Cancer
Usually, oncogenic miRNAs are up-regulated in GC, while tumor-suppressor miRNAs are down-regulated [151]. An alteration of miRNA expression can induce changes in cell proliferation, cell cycle progression, apoptosis, cell migration, and invasion that may lead to GC (Table 2). Several studies demonstrated that miRNAs are able to promote migration and invasion of cancer cells. Hu et al. studied the over-expression of miR-532 in GC tissues and cells compared to normal stomach tissue and surrounding non-cancer tissue [154]. Wound healing and transwell assay demonstrated that miR-532 induces cell migration and invasion in the GC by targeting Nkd1 and inhibiting the Wnt/β-catenin pathway [154]. Numerous miRNAs affect cell migration and invasion in GC, such as miR-215, targeting FOXO1 and up-regulating activated leukocyte cell adhesion molecule (ALCAM) [151] or miR-186, which modulates Twist1 (Table 2) [155,156]. miR-192-5p expression facilitates cell proliferation, the EMT process, and cell invasion in GC by regulating Wnt, TGF-β, and PI3K/Akt signaling pathways [157,158]. Shayimu et al. studied the role of miR-922 on normal gastric epithelial and GC cell lines. This study highlighted the capability of miR-922 to induce cell invasion and migration by promoting MMP-2 and MMP-9 while suppressing SOCS1 expression [138]. Moreover, miR-922 is able to block apoptosis and promote cell proliferation in GC cell lines [138]. The capability of miR-210 to induce, alone or in synergy with long non-coding RNA MIR210HG, cell migration and metastasis was also studied [137]. In particular, c-myc activates miR-210 and MIR210HG, resulting in the induction of cell migration by promoting MMPs [137]. As noted by Zhang et al., miR-21 has an important role in tumor invasion and metastasis in GC. It is up-regulated in GC, and its main target is RECK, a protein involved in the modulation of MMP-2, MMP-9, and MMP-14 expression [159]. Moreover, miR-21 modulates PTEN and Programmed Cell Death 4 (PDCD4) expression, supporting invasion, migration, EMT, and metastasis. These processes are also promoted by the modulation of several EMT proteins, such as vimentin, SNAIL, ACTA2, and TWIST1 [160]. Other studies reported that miR-196a/b over-expression increases radixin (RDX) protein levels and down-regulates MAX dimerization protein 1 (MDX1), promoting cell migration, invasion, and metastasis [161,162]. Other miRNAs, such as miR-370, interfere with the TGF-β signaling pathway, involved in cell migration, and with the Ubiquinol-cytochrome c reductase core protein 2 (UQCRC2) axis regulating EMT [163,164]. On the other side, decreased expression of tumor suppressor miRNAs, such as miR-218 and let-7, promote tumor invasiveness in GC by eliminating repression of the Robo1 pathway and increasing high mobility group AT-hook 2 (HGMB2) expression, respectively [165,166]. In addition, miR-200 family members increase EMT by reducing the expression of E-cadherin repressors [167]. The down-regulation of miR-335 and miR-153 promotes invasion and metastasis processes targeting BCL-w and SNAIL [168,169]. Furthermore, some miRNAs, such as miR-9, miR-10b, and miR-223, have a dual role in GC progression, acting as both tumor promoters and suppressors according to their target genes [151]. Table 2 lists the principal miRNAs that are deregulated in GC.

miRNAs as Biomarker in Cholangiocarcinoma
The aberrant expression of several miRNAs (Table 3) modulates the expression of genes involved in the processes of invasion and cell proliferation in CCA. The up-regulation of miR-10a-5p induces proliferation in CCA cell lines by targeting and modifying PTEN and phospho-Akt (ser473) expression [125]. Zhang et al. also demonstrated that the aberrant expression of miR-30a-5p can inhibit CCA cell apoptosis and promote CCA progression by targeting the SOCS3 gene [189]. In 2018, Wan et al. showed that CCA tissues presented an elevated expression of miR-383 compared to normal hepatic tissue samples [190]. Wan et al. demonstrated that the over-expression of miR-383 negatively regulates the interferon regulatory factor-1 (IRF1), involved in the cell cycle, inhibiting its tumor suppressor role and inducing cell proliferation and migration [190]. It was demonstrated that several miRNAs were identified as prognostic and diagnostic biomarkers in CCA. For instance, Kishimoto et al. demonstrated miR-21 over-expression, thus indicating that it may be used as a biomarker able to distinguish cancer patients from healthy ones. Notably, miR-21 targets NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase (15-PGDH/HPGD), promoting cell growth and up-regulates EMT-related KLF4, N-cadherin, vimentin, Akt and ERK1/2, inducing EMT and invasion [191][192][193]. In addition, the over-expression of miR-27a promotes cell proliferation, migration, and invasion. Effectively, high levels of miR-27a are associated with lymph node metastasis and a poor prognosis in CCA patients [194]. Many studies reported an abnormal expression of miR-29b in several human cancers. miR-29b was down-regulated and associated with poor overall survival in CCA cells and tissues [195]. Proliferation tests, together with flow cytometry analysis, demonstrated that miR-29b influences both cell cycle and apoptosis in CCA cell lines [195].
Sheng et al. reported the up-regulation of Yes-associated protein 1 (YAP1), a transcriptional coactivator of the tumor-suppressive Hippo pathway, and implicated in CCA pathogenesis. Moreover, it was discovered that YAP is the target of miR-16, which results in suppressed CCA determining proliferation, invasion, and metastasis [196].

miRNAs as Biomarkers in Hepatocellular Carcinoma
Recently, numerous studies have analyzed the role of miRNAs in the development and progression of HCC [210]. miRNA profiles have proven that they are an important source for potential biomarkers as they made possible the distinction of liver cancer cells from hepatocytes [144] (Table 5). The WNT/β-catenin pathway is frequently up-regulated, and it is involved in the proliferation, migration, invasion, and survival of liver cancer cells. Moreover, it was discovered that several miRNAs implicated in this deregulation, including miR-21, miR-106b, miR-135, and miR-315, are overexpressed, and miR-122, miR-145, miR-214 are down-regulated [144]. Additionally, the down-regulation of miR-221/-222, miR-30, and miR-148a contribute to the E-cadherin loss, reducing cell-cell adhesion and promoting EMT [211]. Down-regulation of miR-23b and miR-34a plays a role in HCC progression by inducing cell proliferation, migration, invasion, and metastasis [212].
It has been demonstrated that down-regulation of different miRNAs, such as miR-199a-5p or miR-29c, promotes tumorigenesis and increases cell invasion in HCC by modulating MMP activities and regulating the cell cycle, respectively [213,214]. Instead, miR-9 overexpression promoted migration and invasion in HCC cells, likewise in other types of GI cancer such as colon cancer and GC [123,124,215,216]. Sun et al. studied the connection between the over-expression of miR-1246 and the putative target CADM1, involved in cell-cell interaction, and the capacity of the miRNA to induce metastasis in HCC [217]. Various miRNAs, in addition, enhance HCC progression mediating the EMT event, such as miR-330-3p and miR-192 [218].
About nineteen deregulated miRNAs are implicated in angiogenesis, invasion, and metastasis, such as miR-122, which enhance these processes by inhibiting the p53 signaling pathway and modulating disintegrin and metalloprotease 17 (ADAM17) [219]. miR-139 and miR-151 were also involved in the ADAM17 pathway facilitating angiogenesis, invasion, and metastasis [220].

miRNAs as Biomarkers in Colorectal Cancer
As mentioned above, CRC cells are characterized by a high capability of proliferation, invasion, and metastasis. Among the factors that are involved in CRC development, epigenetic modifications have been reported. CRC pathogenesis is characterized by a stage-specific miRNA expression. Table 6 lists miRNAs involved in the development and progression of CRC. Let-7, miR-21, miR-29a, and cluster miR-17-92 are relevant [254]. Let-7 is deregulated in several GI cancers and modulates Ras and Myc expression controlling tumor progression and metastasis in CRC [255]. The increased expression level of miR-21 is associated with invasion and liver and lymph node metastasis [256]. miR-29a is a metastasis-promoter due to its tumor suppressor target gene Kruppel-like factor 4 (KLF4) that up-regulates MMP-2 expression and, at the same time, down-regulates E-cadherin expression [257]. Cluster miR-17-92 is typically amplified and has several target genes involved in CRC progression and metastasis. Especially, miR-17 targets the MYC family, increasing tumor progression and invasion [258,259]. Sun et al. demonstrated the role of miR-103a-3p in the promotion of cell invasion and metastasis by regulating the glycolysis process mediated by the Hippo pathway [134]. MiR-152-3p also promotes cell adhesion and metastases. In particular, miR-152-3p negatively regulates the Aquaporin-11 (AQP11) protein, which is usually involved in the repression of cell growth and adhesion [135]. Moreover, Zhu et al. suggested that inhibition of miR-152-3p could stop the progression of CRC [135]. The aberrant expression of miR-23a-3p is also involved in CRC development. This miRNA, in fact, inhibits the expression of NDRG4 in cancer cells with the consequent increase in cell proliferation, migration, and invasion [129]. miRNAs are also implicated in drug resistance phenomena. It has been demonstrated that miR-93-5p is up-regulated in CRC cells that are resistant to chemotherapeutics and may regulate proteins involved in multidrug resistance and target cyclin-dependent kinase inhibitors [133]. Additionally, miR-101 was reported as a tumor suppressor that is down-regulated in CRC, and its direct target is Cyclooxygenase-2 (COX-2), whose high expression contributes to cell growth and invasion [153].
Recent studies have demonstrated that the tumor suppressor miR-137-3p is downregulated in several cancers, such as non-small cell lung cancer (NSCLC), liver cancer, breast cancer, and CRC. miR-137-3p promotes migration, EMT, and invasion of CRC cells in a lysine-specific demethylase 1 (LDS-1)-dependent manner [260].
Finally, Volker et al. investigated miR-192, miR-17, and miR-200c down-regulation in CRC invasion and metastatic process. These three miRNAs negatively control target genes associated with ECM remodeling in fibroblasts. Notably, miR-192 targets integrins, such as ITGB1 and ITGAV, inhibiting cell adhesion and metastasis; miR-200c modulates the EMT process via E-cadherin repressors, ZEB1 and ZEB2 [259]. In fact, in this study, it was shown that the enhancement of miR-192, miR-17, and miR-200c suppresses the invasiveness and metastasis of CRC cells.

miRNAs as Biomarkers in Gastrointestinal Stromal Tumors
A growing number of research has shown that miRNAs play an important role not only in tumorigenesis but also in the risk stratification of GIST [272]. Table 7 briefly summarizes miRNAs of major significance in invasion, EMT, and metastasis in GIST.
miR-196a is one of the most relevant miRNAs involved in tumor progression. In fact, a positive correlation was found between the over-expression of miR-196a and tumor progression from metaplasia to adenocarcinoma [272]. In particular, miR-196a up-regulates annexin A1 (ANXA1), involved in the invasion process, increasing the risk of metastasis formation [273]. miR-186 inhibition is associated with the over-expression of genes implicated in GIST metastasis by modulating the Met/Akt signaling pathway [274]. Therefore, miR-196a over-expression and low expression of miR-186 are associated with poorer prognosis in GIST patients [47].
Yamamoto et al. demonstrated that the down-regulation of miR-133b determines the over-expression of Fascin-1, an actin-binding protein important in the regulation of cell adhesion and migration. In addition, they discovered the correlation between overexpression of Fascin-1 and worse prognosis in GIST patients. For this reason, Fascin-1 should be a useful biomarker to predict cancer aggressiveness [275].
It was found that the down-regulation of miR-137 can control EMT in GIST via TWIST1 inhibition [276]. Liu et al. demonstrated that miR-152 plays a tumor suppressor role by targeting genes associated with cell proliferation, migration, and invasion, and it results in down-regulated in GIST [277]. Recently, a study on miR-218 showed that its downregulation inhibits cell proliferation, migration, and invasion through direct targeting of the KIT gene [278]. Many different tumors exhibit a down-regulation of let-7 family members, which is also implied in GIST migration, invasion, and metastasis [272].

Other Biomarkers in Gastrointestinal Cancers
In addition to the use of miRNAs as possible biomarkers, several molecules have emerged over the years as biomarkers helpful for the diagnosis, follow-up, and response to treatment in GI cancers. To date, the most widely used biomarker in the clinic for GI cancer is carcinoembryonic antigen (CEA), a protein found in CRC and CCA patients that plays an important role in cell adhesion and intracellular signaling [280]. It is used as a prognostic, diagnostic, and response-to-therapy biomarker and as a target for cancer immunotherapy (DOTAP) [281]. Indeed, high levels of CEA in the blood of patients with CRC are associated with disease progression, while a decrease is found in patients after surgery [282]. Moreover, tumor-associated antigens, such as CA19-9, a marker in the diagnosis and follow-up of some GI cancers such as pancreatic cancer, CRC, GC, and CCA [283][284][285], are extensively used. The first is found at high levels in the serum of CRC patients, while the second is considered the first tumor marker for GC [286][287][288]. Alpha-fetoprotein (AFP) is a glycoprotein capable of binding to different types of membrane receptors and intracytoplasmatic proteins, blocking or enhancing the responses of intracellular signaling pathways. It is an important biomarker used for early diagnosis and prognosis of HCC patients [289].

Nutraceuticals
Nowadays, conventional anticancer therapy implicates the use of radiotherapy and chemotherapy, but such treatments are still expensive and inefficient, especially due to drug resistance and severe adverse events. To date, there are many experimental studies showing the usefulness of nutraceuticals as a complementary approach to standard therapy, thanks to their low toxicity and multiple biological activities [290,291]. Nutraceuticals are also known as bioactive compounds isolated from plants or food and include dietary fibers, antioxidants, phytochemicals, polyunsaturated fatty acids, amino acids, prebiotics and probiotics, and other types of natural food [292].
Many of them have multiple therapeutic properties, such as antioxidant, anti-inflammatory and anticancer, and they seem to be very interesting in the management or treatment of malignancies, cardiovascular diseases, diabetes, obesity, osteoporosis, and the immune system, used alone or in combination with other drugs [293].
Much evidence has revealed that numerous amounts of bioactive substances or extracts from medical plants play an important role in the treatment of different types of GI tumors [290].
Berberine (BBR) is an alkaloid used in Chinese plant-based medicines. It has a wide spectrum of therapeutic actions, such as inhibition of cell proliferation, migration, induction of cellular death, and the enhancement of chemosensitivity through the modulation of NF-κB, PI3K/Akt, and the MAPK signaling pathways. In addition, it is involved in immunity, inflammation [295,296], and, in some cases, in reversing gastric cancerogenesis [297].
Particularly, BBR up-regulates E-cadherin expression and down-regulates N-cadherin, fibronectin, and vimentin expressions to modulate the EMT. Furthermore, BBR suppresses migration and invasion of tumor cells via the IL-6/JAK2/STAT3 signaling pathway and inhibiting MMP-9 protein levels.
BBR up-regulates miR-203 expression, a tumor-suppressive miRNA that binds at the 3 -UTR of Bcl-w oncogene, also resulting in a decrease in chemoresistance [298].
Oleanolic acid (OA) is a pentacyclic triterpene isolated from several plants, including Olea europaea [299]. An antitumoral effect has been demonstrated through the overexpression of miR-98-5p involved in the regulation of Treg/Th17 balance in GC tissues [300].
Paeoniflorin is a bioactive substance of Radix Paeoniae Rubra and is potentially used as a novel therapeutic agent in GC TME. In fact, it improves the immune microenvironment through the up-regulation of miR-149 in gastric CAFs, inhibiting the secretion of IL-6 and leading to the inactivation of the IL-6/STAT3/MMP signaling axis [301].
Sulforaphane is an isothiocyanate derived from the Brassicaceae family, including cabbage and broccoli. It has several activities, including chemopreventive and chemotherapeutic, in different tumors such as lung, bladder, and CRC. As an anticancer drug, Sulforaphane affects all three stages of carcinogenesis. Many studies revealed that it is able to suppress progression and angiogenesis processes in CRC by the inhibition of HIF-1α and VEGF expression [302,303].
Resveratrol (RV) is a polyphenol found in red wine, grapes, peanuts, and other food products that has been reported to have antineoplastic activity on different malignancies [304]. It could present an antitumor potentiality in CCA. In fact, RV can inhibit the secretion of IL-6 from CAFs, preventing the induction of EMT and cancer cell migration. Furthermore, it strongly promotes E-cadherin expression while suppressing N-cadherin, thus, resulting in a reverse phenomenon of the EMT and a reduction in invasiveness and metastasis in cancer cells [305].
Curcumin (CUR) is a non-flavonoid polyphenol purified from the rhizome of the Curcuma longa. It is a pleiotropic molecule with anti-inflammatory, antioxidant, immunomodulatory, and antimicrobial properties [306]. Recent studies have shown that CUR has an antitumor effect on several malignancies, including GI cancers [307].
CUR is a multitarget drug capable of decreasing the expression of molecules involved in angiogenesis, such as VEGF, and tumor invasion, such as intracellular adhesion molecule-1 (ICAM-1), MMP-2, and MMP-9 in CCA [308].
Furthermore, CUR can suppress proliferation, migration, and invasion processes promoting apoptosis of cancer cells in HCC by targeting the circ_0078710/miR-378b/PRIM2 signaling pathway [309] and up-regulating the miR-200 family, involved in the EMT suppression [310].
CUR down-regulates miR-21 expression in CRC and GC. As mentioned above, miR-21 plays an important role in cancer cell migration and invasion through the activation of the PI3K/Akt/mTOR pathway and modulation of MMP-2, MMP-9, and MMP-14 [159,310].
Several plant-and food-derived compounds have a high potential to treat GI tumors in line with relatively low toxicity to normal cells. Indeed, they could be used in combination with conventional anticancer drugs in order to have a potential synergistic effect in cancer therapy [295,311].

Conclusions and Perspective
In summary, this review systematically describes aspects related to the mechanisms responsible for tumor migration and invasiveness in GI cancers. Metastasis is the leading cause of death from GI cancer, so it is of paramount importance to study the mechanisms that regulate tumor migration, invasion, and metastasis to propose novel therapeutic targets and biomarkers. The migration of tumor cells and their ability to form metastases are regulated by mechanisms driven by the TME. In GI tumors, the TME is composed of different cell types which cooperate to promote and trigger metastasis formation. Specifically, CAFs play a pivotal role in tumor progression by enhancing EMT and, thus, promoting migration and metastasis formation. These processes are further supported by the presence of an immunosuppressive infiltrate, mainly composed of Treg, MDSC, and TAM2 cells, and by the presence of chemokines and cytokines such as CXCL12, IL-6, and TGF-β.
Recent studies have also demonstrated the critical role of miRNAs in promoting tumor progression and metastasis formation in GI tumors. They may modulate signaling pathways involved in migration and tumor invasion, such as Wnt/β-catenin, Hedgehog, and Jak/Stat. Many miRNAs are found to be up-or down-regulated in tumor tissues compared to healthy tissues, and to date, they are being considered as potential predictors for prognosis, monitoring, and diagnosis of GI cancer patients. Of note, recent studies have shown that the use of nutraceuticals, bioactive compounds isolated from plants or foods, would be useful in GI cancers as a complementary approach to standard therapy. Indeed, they are able to target miRNAs and other molecules that regulate several mechanisms involved in tumor migration and invasiveness. This could pave the way for new targeted therapies for GI cancer treatment.
The high aggressiveness and late diagnosis in patients with GI tumors often result in a poor prognosis. Therefore, it is necessary to develop new tools to improve early diagnosis and prognosis for these patients. A growing number of studies are highlighting the important role of miRNAs as potential markers in many neoplasms. The use of miRNAs as biomarkers offers the great advantage of developing minimally invasive methods due to their stability, which renders them easy to detect in different body fluids, such as blood.
On the other hand, the main disadvantage of their use in large-scale therapeutic applications could be the difficulty of predicting the overall effect of the miRNAs of interest due to the possible high number of target genes. Especially in a therapeutic context, their application could represent a risk because of the occurrence of unknown and unpredictable side effects, possibly triggering physiological function disorders or the development of additional diseases. For this reason, Zhang et al. proposed the design of new delivery systems for miRNA that recognize specific features at the site of the malignant lesion [312].
Further investigations are, therefore, needed for the application of miRNAs as tumor biomarkers. In fact, they could offer important opportunities for the development of new diagnostic and prognostic strategies that would help to improve the clinical outcome of patients with GI tumors.

Conflicts of Interest:
The authors declare no conflict of interest.  Signal transducer and activator of transcription  Tregs  T regulatory cells  TGF-β  Transforming growth factor-β  TME  Tumor microenvironment  TAMs  Tumor-associated macrophages  TANs  Tumor-associated neutrophils  TILs Tumor-infiltrating lymphocytes TWIST Twist family bHLH transcription factor 1 KIT Tyrosine kinase UQCRC2