Transforming Growth Factor-β Signaling Pathway in Colorectal Cancer and Its Tumor Microenvironment

Transforming growth factor-beta (TGF-β) signaling is one of the important cellular pathways that play key roles for tissue maintenance. In particular, it is important in the context of inflammation and tumorigenesis by modulating cell growth, differentiation, apoptosis, and homeostasis. TGF-β receptor type 2 (TGFBR2) mutations affected by a mismatch repair deficiency causes colorectal cancers (CRCs) with microsatellite instability, which is, however, associated with relatively better survival rates. On the other hand, loss of SMAD4, a transcription factor in the TGF-β superfamily signaling, promotes tumor progression. Loss of heterozygosity on chromosome 18 can case SMAD4-deficient CRC, which results in poorer patients’ survival. Such bidirectional phenomenon driven by TGF-β signaling insufficiency reflects the complexity of this signaling pathway in CRC. Moreover, recent understanding of CRC at the molecular level (consensus molecular subtype classification) provides deep insight into the important roles of TGF-β signaling in the tumor microenvironment. Here we focus on the TGF-β signaling in CRC and its interaction with the tumor microenvironment. We summarize the molecular mechanisms of CRC tumorigenesis and progression caused by disruption of TGF-β signaling by cancer epithelial cells and host stromal cells.


Introduction
Transforming growth factor-beta (TGF-β) signaling pathway plays critical roles in controlling tissue development, proliferation, differentiation, apoptosis, and homeostasis [1]. As such, disruption of this signaling pathway leads to several diseases including cancers. TGF-β signaling regulates many target genes-either positively or negatively-in a context-dependent manner [2]. Although TGF-β signaling inhibits epithelial growth in normal tissues, it promotes tumor cell progression in tissues with advanced cancer [3]. This phenomenon is known as TGFβ paradox. These context-dependent and paradoxical dynamics of the signaling complicate the understanding of the role of TGF-β signaling in cancer biology. As the effect on the colonic epithelial cells, TGF-β signaling exhibits reduction in cell proliferation, along with promotion of differentiation and apoptosis [4]. In addition to its effect on epithelial cells, TGF-β plays protective roles against luminal bacterial antigens by suppressing intestinal immune cells in the stroma and inducing immune tolerance [5,6]. Therefore, disruption of TGF-β signaling in the colon prompts tumor progression not only via epithelial cells transformation but also via tumor-stromal interactions [7][8][9][10][11].
Recent advances in DNA sequencing technology, such as next-generation sequencing and digital polymerase chain reaction (PCR), re-focuses the importance of DNA alteration in cancer cells. Recently, a novel classification for colorectal cancer (CRC) was advocated as consensus molecular subtype (CMS) based on the following molecular features: CMS1 (microsatellite instability (MSI)-immune) as motif is referred to as the SMAD binding element (SBE) where SMAD2/3/4 mainly bind. In addition, SMAD1/5/8 and SMAD2/3/4 can also bind the GC-rich element when activated [4,17]. R-SMAD molecules in the SMAD4-R-SMAD complex can also bind other transcription factors as partners to regulate their transcription. In addition to the canonical SMAD-dependent pathway, TGF-β superfamily ligands transduce non-canonical, SMAD-independent pathways such as various mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/Akt and Rho/Rhoassociated protein kinase (ROCK) pathways [18].

TGF-β Signaling in Clinical Situation of CRC Patients
Molecular classification of CRC can provide biological interpretability of CRC. In CMS1 CRC, MSI-H provides accumulation of many somatic gene mutations including TGFBR2, which results in the release of tumor neoantigens and activation of tumor immunity. It was reported that MSI-H was an independent favorable prognostic factor for Stage II/III (no distant metastasis) CRC patients after curative resection [15]. In fact, CMS1 CRC exhibits a low metastatic rate compared with other subtypes [22]. Although TGFBR2 mutation itself is not a favorable prognostic factor within MSI-H CRC population, MSI-H CRC patients exhibit better prognosis compared to microsatellite-stable ones [15,23]. However, once metastasized, CMS1 CRC exhibits poorer survival. One of the reasons of poor prognosis after metastases would be that MSI-H confers resistance to 5-fluorouracil (5-FU)-based chemotherapy [24].
MSI-H derived from deficient mismatch repair (dMMR) accounts for only 15% of CRCs. Alternatively, approximately 85% of invasive CRCs exhibit chromosomal instability (CIN) and loss of heterozygosity (LOH) in some chromatin areas [25]. CIN CRCs accumulate driver mutations such as adenomatous polyposis coli (APC), TP53, SMAD4, KRAS, and PI3K catalytic subunit-α (PIK3CA), which Vogelstein and colleagues advocated as the adenoma-carcinoma sequence [26]. In this sequence, TGF-β signaling pathway also plays a pivotal role in CRC progression. In fact, absent expression of SMAD4 (a key transcription factor for TGF-β signaling) is an independent poor prognostic factor after curative surgery for Stage II/III CRC and CRC liver metastases [15,[27][28][29]. Moreover, CMS4 carrying the mesenchymal phenotype with TGF-β-activated stroma showed the worst prognosis with low benefit from chemotherapy among all molecular classes [12,[30][31][32]. In this scenario, TGF-β signaling causes epithelial-to-mesenchymal transition (EMT) in cancer cells, resulting in an aggressive phenotype [18]. To this end, TGF-β signaling directs serrated adenomas to the mesenchymal CRC subtype, while TGFBR2 mutation impairs EMT [33,34]. Collectively, these basic and clinical data indicate that disruption of TGF-β signaling, especially in advanced CRC, results in an aggressive phenotype of CRC and, consequently, poor prognosis.

TGFBR2 Mutation in Cancer Cells
For appropriate function of TGF-β signaling, active TGF-β receptors (both type 1 and 2 receptors) are mandatory [19]. TGFBR2 mutations are frequently found in MSI-H CRC [35]. MSI-H CRC cells carrying dMMR harbor silent expression of mismatch repair genes through either germline mutations of MMR genes such as MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MSH6, and Postmeiotic segregation increased 2 (PMS2), or MLH1 promoter hypermethylation [36,37]. Lynch syndrome is an autosomal dominant hereditary cancer syndrome that carries germline mutations of one of these 4 MMR genes, resulting in the development of many types of cancers including CRC, endometrial, ovarian, gastric, small intestine, pancreatic, and urothelial tract cancers [38]. Lynch syndrome accounts for approximately 3% of CRCs, whereas approximately 12-15% of CRCs is sporadic MSI-H CRC, resulting from hypermethylation of the MLH1 promoter [39]. Because TGFBR2, carrying a 10-adenine repeat, is only one of the genes affected by dMMR, it is possible that TGFBR2 mutation is merely a bystander event [13,40]. However, according to several mouse studies, TGFBR2 mutation itself does have the potential to transform normal colonic epithelial cells to malignant cells [41,42]. In addition to TGFBR2 mutation, BMP receptor type 2 (BMPR2) can be also affected by dMMR as it also carries a 7-adenine repeat that is also affected by dMMR [43].
MSI-H CRC exhibits high immune response that can be targeted for immune checkpoint inhibitors [44]. Although high immune response in MSI-H CRC is the result of tumor neoantigen load caused by hypermutation, TGFBR2 impairment can also directly promote inflammation in the tumor microenvironment of CRC. TGFBR2 deficiency in an APC-deleted mouse model of intestinal adenoma increased inflammatory burden and promoted tumor progression via producing tumor necrosis factor-α (TNF-α), interleukin (IL)-8, and TGF-β1 as well as suppressing of anti-inflammatory cytokines such as IL-10 and interferon (IFN)-γ, which resulted in increased infiltration of CD11b + Gr1 + granulocyte population into the tumor microenvironment ( Figure 2) [45]. Moreover, TGFBR2 disruption in combination with inflammation in the colon causes invasive CRC via tumor-associated macrophage (TAM) infiltration [46]. TGFBR2 inactivation in CRC cells contributes to the malignant phenotype via multiple pathways such as Wnt-β-catenin, Hippo, and MAPK [47]. In a mouse model designed to conditionally knockdown TGFBR2 in the proximal colon, TGFBR2 impairment in combination with Wnt-β-catenin pathway activation promoted the upregulation of Gasdermin C, which stimulated the proliferation of CRC cells [48].
Glycosylation is an important post-translational protein modification and also affects malignant phenotype of cancer cells [49]. TGF-β signaling can also modulate the protein glycosylation pattern in MSI-H CRC. TGFBR2 impairment in MSI-H CRC cell line, HCT116, can upregulate some of glycosylation-related genes and affect important cell signaling pathways such as Notch [50]. In this cell line model, Nectin-3 (a cell surface glycoprotein that modulates cancer cell invasion and metastasis) was also upregulated when TGFBR2 was impaired, and reconstitution of TGFBR2 upregulated growth differentiation factor-15 (GDF15; one of the ligands of TGF-β superfamily signaling) in a cell line model [51]. TGFBR2 mutation in CRC can also cause changes in the components secreted by cancer cells. For example, in vitro experiments with MSI-H TGFBR2-deficient HCT116 cells revealed that extracellular matrix and nucleosome-related proteins were upregulated, while proteasome-associated proteins in the extracellular vesicles were downregulated [52,53]. Clinical implications of these cell line-based experiments are, however, not fully understood. adenoma increased inflammatory burden and promoted tumor progression via producing tumor necrosis factor-α (TNF-α), interleukin (IL)-8, and TGF-β1 as well as suppressing of anti-inflammatory cytokines such as IL-10 and interferon (IFN)-γ, which resulted in increased infiltration of CD11b + Gr1 + granulocyte population into the tumor microenvironment ( Figure 2) [45]. Moreover, TGFBR2 disruption in combination with inflammation in the colon causes invasive CRC via tumor-associated macrophage (TAM) infiltration [46]. TGFBR2 inactivation in CRC cells contributes to the malignant phenotype via multiple pathways such as Wnt-β-catenin, Hippo, and MAPK [47]. In a mouse model designed to conditionally knockdown TGFBR2 in the proximal colon, TGFBR2 impairment in combination with Wnt-β-catenin pathway activation promoted the upregulation of Gasdermin C, which stimulated the proliferation of CRC cells [48].
Glycosylation is an important post-translational protein modification and also affects malignant phenotype of cancer cells [49]. TGF-β signaling can also modulate the protein glycosylation pattern in MSI-H CRC. TGFBR2 impairment in MSI-H CRC cell line, HCT116, can upregulate some of glycosylation-related genes and affect important cell signaling pathways such as Notch [50]. In this cell line model, Nectin-3 (a cell surface glycoprotein that modulates cancer cell invasion and metastasis) was also upregulated when TGFBR2 was impaired, and reconstitution of TGFBR2 upregulated growth differentiation factor-15 (GDF15; one of the ligands of TGF-β superfamily

Mutations and Deletions of SMAD4 (Co-SMAD) in CRC Cells
LOH is one of the common features of carcinogenesis that causes cancer cells to lose tumor suppressor genes and acquire malignant phenotype [54]. Chromosome 18q21 is frequently affected by LOH in microsatellite-stable CRC. There are many genes in this chromosomal region including SMAD2, SMAD4, and deletion of colon cancer (DCC) that may contribute to form malignant phenotype of CRC. Among the genes on chromosome 18q21, SMAD4 is the established tumor suppressor gene, and loss of SMAD4 disrupts canonical TGF-β signaling because it is a transcription factor for the signaling [54]. Moreover, The Cancer Genome Atlas (TCGA) database revealed that SMAD4 is one of the most frequently mutated genes in CRC [55].
SMAD4 is one of the key driver genes that contribute to CRC progression and metastasis [56,57]. Consequently, SMAD4 loss onto APC mutation in intestinal epithelial cells causes malignant invasive phenotype in mouse models [58,59]. Loss of SMAD4 protein expression is found approximately 20-40% of human CRCs [8][9][10][11]60,61]. Although LOH can be the main cause of SMAD4 loss in CRC, there are other proposed mechanisms that contribute to SMAD4 defect in post-transcriptional and post-translational regulation: ubiquitylation, sumoylation, and mircoRNA interference [62,63].
SMAD4 acts as a transcription factor by forming trimers with R-SMAD components and directly regulates target genes. R-SMAD-SMAD4 complexes can also associate with DNA-binding partners to act as a transcription co-factor [4]. Therefore, there are many target genes regulated by SMAD4, indicating that several changes in cancer cells happen when R-SMAD-SMAD4 complexes are disrupted [64][65][66]. RNA sequencing comparing SMAD4-proficient and SMAD4-deficient colonic epithelial cells in mice revealed upregulation of many inflammation-related genes [66]. Among them, Ccl9, one of the C-C motif chemokine ligands, is upregulated in a SMAD4-deficient intestinal tumor mouse model [59]. In this mouse model, chemokine expression recruits myeloid cells with corresponding receptors, Ccr1, to promote tumor invasion and metastases [59,67,68]. Similar interactions between SMAD4-deficient CRC cells and surrounding myeloid-derived cells via chemokine signaling was observed in human CRC samples [8][9][10][11]. Namely, loss of SMAD4 from CRC cells promotes upregulation of C-C motif chemokine CCL15 (human orthologue of mouse Ccl9) to recruit CCR1 + myeloid-derived suppressor cells (MDSC) [8][9][10]69]. SMAD4-deficient CRC cells also produce C-X-C motif chemokine ligand CXCL1/8 to recruit its corresponding receptor CXCR2 + tumor-associated neutrophils (TAN) [11]. MDSC accumulation is characteristic of CMS4 immune contexture [70]. These experimental and observational results suggest that SMAD4-deletion in CRC causes both cancer cell phenotype and tumor microenvironment to switch to the more aggressive cancer phenotype.
TGF-β signaling plays a crucial role in angiogenesis in the tumor microenvironment [71,72]. SMAD4 can also regulate the expression of vascular endothelial growth factor (VEGF)-A and VEGF-C, the main angiogenic factors for tumor angiogenesis and lymphangiogenesis. Disruption of SMAD4 promotes upregulation of these angiogenic factors, resulting in promoting angiogenesis and lymphangiogenesis in CRC [73,74]. TGF-β signaling also plays a critical role in the differentiation of epithelial cells. Loss of SMAD4 promotes β-catenin expression, and simultaneous SMAD4 loss and Wnt activation in the intestinal epithelium trigger the acquisition of stem cell properties and lead to de-differentiation and rapid adenoma formation in the differentiated intestinal epithelium of the Cre-driven conditional mouse model [75,76].
SMAD4 loss-induced changes in vivo and in vitro were summarized in Tables 1 and 2. Table 1. SMAD4 loss-induced changes in vivo (animal models).

Non-Canonical TGF-β Signaling Pathways in CRC
Non-canonical TGF-β signaling pathways also modulate important cellular physiology. Disruption of non-canonical TGF-β pathways as well as canonical ones is also frequently found in CRC. Although loss of SMAD4 may lead to the blockade of canonical TGF-β signaling, it alters BMP signaling via non-canonical pathway to promote CRC metastasis through activation of Rho/ROCK pathway, leading to EMT, migration, and invasion [81]. Loss of SMAD4 also activates alternative MEK/ERK pathways to promote cell mortality, migration, and invasion [82,83].

Cancer tissues contain abundant stromal cells in addition to cancer epithelial cells. Interaction between cancer cells and surrounding stromal cells can either promote or inhibit cancer progression.
In the tumor microenvironment, various cytokine/chemokine networks play pivotal roles in controlling the interaction between cancer cells and stromal cells [84,85]. As described above, CMS4 mesenchymal phenotype with poor prognosis characterizes prominent TGF-β activation [12]. These observations suggest that TGF-β activation in the tumor microenvironment can promote the tumor-stromal interaction to induce a malignant CRC phenotype and poorer prognosis (Figure 3).

Cancer-Associated Fibroblast
Cancer-associated fibroblast (CAF) is one of the major components of the tumor microenvironment and act as an important factor in tumor progression and metastasis [86][87][88]. TGFβ activation in the tumor microenvironment promotes differentiation of mesenchymal stem cells (MSCs) to CAFs, activating phosphorylation of signal transducer and activator of transcription 3 (STAT3) and nuclear localization of p-STAT3 via Janus kinase (JAK)/STAT pathway [86]. Normal fibroblasts or endothelial cells can also be converted to CAFs by stimulation of TGF-β superfamily ligands, such as nodal or TGF-β2, to support tumor growth [89,90]. TGF-β ligands also directly activate CAFs; activated CAFs produce chemokine CXCL12 and interact CXCR4 + CRC cells, or IL-11 to activate CRC cells through GP130/STAT signaling to metastasize to distant organs [91][92][93]. CAFs secrete TGF-β ligand from themselves to accelerate malignant phenotype of CRC in the hypoxic condition that normally occurs in vivo within the tumor microenvironment [94,95].

Natural Killer Cell
Natural killer (NK) cells can be expanded ex vivo, activated and transferred to cancer patients to effectively kill cancer cells. However, highly immunosuppressive tumor microenvironment caused, in part, by excessive TGF-β signaling, may limit the activity of NK cells. Exposure to TGF-β ligand decreases the ability of activated NK cells to kill cancer cells ex vivo, while inhibition of TGF-β signaling in the tumor microenvironment preserves the function of highly activated, in vitro expanded NK cells in CRC in vivo [96].

Cancer-Associated Fibroblast
Cancer-associated fibroblast (CAF) is one of the major components of the tumor microenvironment and act as an important factor in tumor progression and metastasis [86][87][88]. TGF-β activation in the tumor microenvironment promotes differentiation of mesenchymal stem cells (MSCs) to CAFs, activating phosphorylation of signal transducer and activator of transcription 3 (STAT3) and nuclear localization of p-STAT3 via Janus kinase (JAK)/STAT pathway [86]. Normal fibroblasts or endothelial cells can also be converted to CAFs by stimulation of TGF-β superfamily ligands, such as nodal or TGF-β2, to support tumor growth [89,90]. TGF-β ligands also directly activate CAFs; activated CAFs produce chemokine CXCL12 and interact CXCR4 + CRC cells, or IL-11 to activate CRC cells through GP130/STAT signaling to metastasize to distant organs [91][92][93]. CAFs secrete TGF-β ligand from themselves to accelerate malignant phenotype of CRC in the hypoxic condition that normally occurs in vivo within the tumor microenvironment [94,95].

Natural Killer Cell
Natural killer (NK) cells can be expanded ex vivo, activated and transferred to cancer patients to effectively kill cancer cells. However, highly immunosuppressive tumor microenvironment caused, in part, by excessive TGF-β signaling, may limit the activity of NK cells. Exposure to TGF-β ligand decreases the ability of activated NK cells to kill cancer cells ex vivo, while inhibition of TGF-β signaling in the tumor microenvironment preserves the function of highly activated, in vitro expanded NK cells in CRC in vivo [96].

TANs
Peripheral neutrophil-to-lymphocyte ratio (NLR) in patients with CRC predicts the prognosis after treatment [97][98][99][100]. Although higher neutrophil level could be just bystander in patients with poor prognosis, neutrophils may contribute directly to the tumor progression. In the tumor microenvironment of SMAD4-deficient CRC, TANs are recruited via the chemokine CXCL1/8-CXCR2 axis [11]. Tumor microenvironment contains at least two types of neutrophils: N1 with an anti-tumoral phenotype and N2 with a protumoral phenotype [101][102][103]. TGF-β signaling in the tumor microenvironment regulates phenotypical changes in neutrophils to N2 protumoral ones, while TGF-β blockade with a small molecule inhibitor results in the recruitment and activation of N1 antitumoral TANs [101].

TAMs
TAMs in the tumor microenvironment were known to release pro-inflammatory cytokines there to form inflamed environment, which engages in bidirectional interaction between cancer cells and host immune cells [104]. SMAD4 deletion in CRC cells decreased the number of S100A8 + monocytes or CD68 + TAMs in the tumor microenvironment; this is associated with unfavorable prognoses in CRC patients [105,106]. Recruited TAM in the CRC microenvironment produces TGF-β ligand to promote proliferation and invasion via EMT or VEGF [107,108]. In compound mutant mice that have mutations in Apc and Tgfbr2 in the intestinal epithelial cells, TAM infiltrates into the invasive tumors express membrane-type1-matrix metalloproteinase (MMP), causing MMP2 activation [46]. Although TAMs can be one of the sources of TGF-β ligand expression, the effects of TGF-β signaling on TAMs in CRC is not fully understood [108].

T Lymphocyte
As NLR can predict CRC patients' prognosis, low levels of T cell infiltration or low activity of type 1 T-helper cells (T H 1) can also predict poor outcome of CRC patients [109]. Infiltrating T cells are activated through both signals from major histocompatibility complex (MHC)-presented immunogenic peptide antigens to the T cell receptor (TCR) and signals from CD80/86 on the antigen-presenting cells (APC) to T cell surface receptor CD28. However, once activated, T cell expressed co-inhibitory receptors, such as cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death 1 (PD1), and become exhausted T cell [110]. Abundant TGF-β in the tumor microenvironment promotes T cell exclusion and prevents them to acquire T H 1-effector phenotype [95]. Inhibition of TGF-β using a small molecule inhibitor, galunisertib, unleashed a potent and enduring cytotoxic T cell response against CRC cells and rendered tumors susceptible to anti-PD-L1-PD-1 therapy.
Regulatory T cells (T-regs) are a subpopulation of T cells that modulate immune system and self-antigen tolerance. T-regs express biomarkers such as CD4, forkhead box P3 (FOXP3), and CD25, and differentiates from naïve CD4 + T cells by TGF-β stimulation [111]. In the tumor microenvironment, T-regs play a critical role in suppressing tumor immunity and promoting cancer progression [112]. Therefore, enriched TGF-β in the tumor microenvironment of CRC promotes phenotypical changes of T cells to T-regs and tumor progression [113]. A meta-analysis comparing T-reg infiltration and patients' prognosis reported that high FOXP3 + T-reg density was associated with poor overall and disease-free survival in patients with all types of cancer [114]. However, the prognostic role of T-reg infiltration is tumor-dependent, and, among CRC patients, a higher number of tumor-infiltrating FOXP3 + T-regs predicts favorable outcome of CRC patients [114,115].

Discussion
CRC is the second to third most common cause of cancer-related deaths worldwide [116,117]. Recent developments in surgical technology (e.g., laparoscopic or robotic surgery) and novel therapeutic compounds (small molecule inhibitors and molecular targeted antibodies) are promising, and the mortality rate of CRC is gradually decreasing in some western countries [116,117]. However, once metastasized, treatment strategies can be limited, and, if unresectable, outcomes in CRC patients are unfavorable. TGF-β signaling may contribute to this unfavorable outcome in CMS4 and metastasized CMS1 subpopulations. Therefore, comprehensive understanding of TGF-β signaling in both tumor cells and the tumor microenvironment is mandatory for the construction of novel therapeutic strategies.
As discussed above, the disruption of TGF-β signaling in CRC cells generally promotes tumor formation in the early stage, while its activation may promote cancer invasion and metastasis. Moreover, its activation in the tumor microenvironment generally suppresses tumor immunity and supports cancer cell survival. The bidirectional function of TGF-β signaling within cancer cells and multi-directional functions between cancer cells and their microenvironment make effective drug discovery for CRC treatment difficult. This is because simple blockade of TGF-β signaling can activate tumor immunity in the tumor microenvironment but may alter cancer cell phenotypes to more aggressive ones.
Author Contributions: K.K. and Y.S. conceptualize this study. Y.I. and K.K. wrote the manuscript. All authors reviewed the manuscript for important intellectual content.
Funding: This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K.K. and Y.I.).

Conflicts of Interest:
The authors declare no conflict of interest.