GDF-15 Signaling Leading to Epithelial-to-Mesenchymal Transition in Colorectal Cancer – a Literature Review

1. Introduction

Most colorectal cancers (CRCs) cases are sporadic and not passed down, even if inherited genetic predisposition plays a role in some cases. Early CRCs develop genetic and epigenetic alterations that allow for rapid cell growth, resulting in the invasive and metastatic characteristics of CRC (1).

Epithelial–mesenchymal transition (EMT) is a process by which epithelial cells differentiate into mesenchymal cells in response to normal or pathological stimuli (2). EMT has been shown to be strongly related with the dissemination of epithelial cell malignancies (3). Growth differentiation factor 15 (GDF-15), which appears in both intracellular and extracellular forms, is a divergent member of the transforming growth factor (TGF-β) superfamily. GDF-15 is broadly distributed in mammalian tissues and has several roles in a variety of illnesses, including inflammation, cancer, cardiovascular disease, and obesity (4).

Cancers with high GDF-15 levels, such as endometrial (5), prostate (6), pancreatic (7), and CRCs (8), may be linked to poor clinical outcomes. Although GDF-15 has been proposed as a target for CRC treatment (9, 10), its biological role remains undefined and findings regarding its biological functions and involvement in carcinogenesis are at times conflicting (11, 12).

2. Molecular mechanism of EMT in Colorectal Cancer

In EMT, epithelial cells acquire mesenchymal characteristics. Notable examples of EMT include embryonic development and organ formation (13), tissue repair and fibrosis (14), and cancer progression (15). Among the three types of EMT, the latter (type 3) is linked to the development of invasive or metastatic phenotype (16).

During EMT, tumor cells lose their apical– basal polarity, suffer disintegration of the tight junction, and reorganize their cytoskeletal architecture, allowing them to become invasive. Extracellular stimuli from the tumor microenvironment, such as growth hormones and inflammatory cytokines, as well as intra-tumor physical stressors such as hypoxia, are improperly regulated in cancer cells (17). Consequently, EMT programming allows tumor cells to efficiently metastasize by enabling them to readjust to the constantly shifting conditions of the tumor microenvironment.

The EMT process is executed by effector molecules, orchestrated by transcription factors (EMT core regulators), and initiated by external stimuli (EMT inducers) (18).

Most EMT effectors are subcellular structural proteins that dictate cellular phenotype. EMT is characterized by the downregulation of genes that encode cell junction proteins (Ecadherin, claudins and occludins) and activation of genes that produce mesenchymal adhesion-promoting proteins (vimentin, fibronectin and N-cadherin). Research suggests that stage III CRC with loss of E-cadherin expression has a dismal prognosis (19). Furthermore, overexpression of N-cadherin and fibronectin is associated with metastasis and a shorter overall survival in individuals with CRC (20, 21).

Developmental transcription factors that control epithelial and mesenchymal markers by influencing target genes or modulating their own expression are known as EMT core regulators. Increased expression of EMT-related transcription factors such as SNAIL, SLUG, TWIST1, TWIST2, ZEB, ZEB2, PROX1, FOXC2, FOXQ1, FOXM1, and FOXC1, has been correlated with CRC invasiveness and metastasis (1).

Several factors have been identified as potential EMT inducers in CRC. These inducers alter EMT-transcriptional factors directly or indirectly, triggering a cascade of intracellular signaling events. Hypoxia-inducible factor 1 alpha (HIF-1), myocyte enhancer factor 2D (MEF2D) and transmembrane protease/serine 4 (TMPRSS4) can induce EMT by activating ZEB transcription factors, while nucleotide binding protein-like (NUBPL) can upregulate SNAIL expression. The WNT/β -Catenin signaling pathway, which has been linked to EMT in CRC, can be induced by other proteins such as serine–threonine kinase receptor-associated protein (STRAP) and tumor suppressor candidate 3 (TUCS3). TGF-signaling and EMT are also promoted by neuropilin-2 (NRP2), GDF-15, and formin like2 (FMNL2). Eukaryotic initiation factor 5A2 (EIF5A2) overexpression may trigger EMT through an unidentified mechanism possibly involving C-Myc and metastasis-associated protein 1 (MTA-1) (1, 22) (Figure 1).

Fig. 1. Schematic of EMT induction

Adapted from “Regulation of EMT in Colorectal Cancer: A Culprit in Metastasis” by Vu T and Datta PK. Cancers (Basel). 2017 Dec 16;9(12):171 (1)

3. Oncogenic role of GDF-15 in Colorectal Cancer

Growth/differentiation factor-15, also known as macrophage inhibitory cytokine 1, nonsteroidal anti-inflammatory drug-activated gene-1, placental bone morphogenetic protein, prostate-derived factor or placental transforming growth factor (PTGFB), was identified more than two decades ago as a distant member of the transforming growth factor (TGF) superfamily (23). In situ hybridization revealed that the human GDF-15 gene is positioned on chromosome 19p13.1–13.2. The GDF-15 genes in humans, rats, and mice are comprised of two exons and one intron that interrupts the coding sequences in the pre-prodomain of their respective proteins (4).

GDF-15 has been shown to suppress early tumor promotion under normal physiological settings. However, aberrant expression of this gene in advanced malignancies results in proliferation, invasion, metastasis, cancer stem cell production, immune evasion, and a diminished response to treatment (24). GDF-15 has been shown to promote tumor cell proliferation, survival, invasion, metastasis, and chemoresistance (12), and has been associated with the development of several malignancies, including cervical cancer, colorectal cancer, and multiple myeloma (8).

A recent study examined the impact of an inflammatory microenvironment on colon cancer invasion and metastasis in vitro and in vivo. Anti-GDF-15 antibody therapy inhibited colon cancer cell invasion and migration in vitro, demonstrating that GDF-15 participates in inflammation-induced invasiveness. Lipopolysaccharide-induced inflammation elevated GDF-15 production in vivo, while c-Fos deletion inhibited colon cancer cell lung metastasis in a mouse model. Similarly, colon cancer metastases and TNM stages were shown to be linked with c-Fos expression in patient samples. As a result, the study established that GDF-15 stimulated colon cancer invasion and metastasis through the extracellular signal-regulated kinase 1/2 (Erk1/2)/cFos signaling pathway (25).

The results of a multivariate Cox analysis revealed that serum GDF-15 is an independent prognostic factor for CRC. Cancer-specific survival was higher for individuals with lower GDF-15 serum levels, according to the results of another study (26).

Conversely, other findings suggest that GDF-15 may function as a tumor suppressor gene in the context of colon cancer (27).

4. Circulating GDF-15 as a biomarker

The AJCC TNM classification is the best predictor of CRC prognosis. Other prognostic indicators include poorly differentiated or high histological grade, vascular or lymphatic invasion, intestinal obstruction or perforation at diagnosis, and increased preoperative serum carcinoembryonic antigen presence (CEA) (28). However, due to the great degree of individual heterogeneity, an individual’s chance of recurrence following therapy cannot be predicted properly (29). Allele loss of chromosome 18, microsatellite instability-deficient mismatch repair (MSI/dMMR) , and mutations in KRAS have been shown to negatively affect prognosis in cancer patients (30, 31). According to the research on EMT biomarkers, low expression of E-cadherin (32) and high expression of N-cadherin (21), Slug, and Vimentin (33) have been associated with a worse prognosis in CRC (34).

Several studies have attributed GDF-15 plasma levels to CRC diagnosis and death (11, 35, 36). High plasma levels of GDF-15 before diagnosis of CRC are related with an increased risk of CRC incidence and CRC-specific mortality, according to two significant cohorts evaluated in a prospective study, even after adjusting for other inflammatory indicators, risk factors, and clinical and pathological features (age, sex, tumor location, polyp history, tumor grade, or disease stage) (37).

Another study revealed that surgical excision of CRC decreases serum GDF-15, which was elevated in the context of tumor recurrence (26). Moreover, monitoring serum levels after surgery might help predict cancer recurrence. Further examination of the clinical significance of GDF-15 showed that serum levels are complementary to CEA and that GDF-15 is associated with the development or recurrence of CRC with liver metastases. However, GDF-15 blood concentrations were shown to be extremely variable in individuals with benign illness, indicating that serum levels should be read with caution (38), especially given the possibility of acute inflammation in CRC patients during chemotherapy (39).

Numerous articles have established that GDF-15 plays a critical function in tumor growth suppression, although they did not specifically study CRC.

One study tested the serum level of GDF-15 in 260 healthy blood donors as well as in 193 patients with adenomatous polyps or colon cancer. The study found that the TumorNode-Metastasis (TNM) stage was associated with the level of GDF-15 serum (4, 40).

Both the pro-domain and mature domains of GDF-15 may have various biological functions, depending on the context in which they are expressed (41, 42). It’s possible that during different phases of tumor evolution, these diverse forms may have contradictory purposes. In addition, GDF-15 may have varied roles in cancer depending on its intracellular location.

5. Resistance to chemotherapy

The conventional treatment for colon cancer is chemotherapy based on 5-fluorouracil (5-FU). Resistance to 5-FU is a major problem in clinical treatment, with much still unknown about the processes that allow colon cancer cells to become resistant to 5-FU treatment.

An investigation into whether 5-FU-resistant colon cancer cells undergo EMT and apoptosis, as well as the involvement of GDF-15 in controlling these processes revealed that 5-FU resistance in colon cancer was induced by suppression of GDF-15 and the downstream Smad signaling pathway. 5-FU-resistant human colon cancer cells/FU cells were created by exposing them to steadily increasing doses of 5-FU over time. Subsequently, HCT-15/FU cells resistant to 5-FU could be made susceptible to 5-FU by overexpressing GDF-15. Moreover, HCT-15/FU with decreased GDF-15 expression showed a more robust proliferation ability and metastatic propensity in vivo.

The findings suggest that the suppression of the Smad signaling pathway caused EMT in HCT-15/FU cells (via the modification of Ncadherin, E-cadherin, and MMP14 expression). The lack of E-cadherin in the HCT-15/FU group was further verified by in vivo tests (27).

Abbreviations

5-FU – 5-fluorouracil

AJCC-American Joint Committee on Cancer

CEA – carcinoembryonic antigen

CRC – colorectal cancer

EIF5A2 – eukaryotic initiation factor 5A2

EMT – epithelial-to-mesenchymal transition

Erk1/2 – extracellular signal-regulated kinase 1/2

FMNL2 – formin-like2).

GDF-15 – growth/differentiation factor-15

HIF-1 – hypoxia-inducible factor 1 alpha

MEF2D – myocyte enhancer factor 2D

MIC-1 – macrophage inhibitory cytokine 1

MSI/dMMR – microsatellite instability-deficient mismatch repair

MTA-1 – metastasis-associated protein 1

NAG-1 – nonsteroidal anti-inflammatory drug-activated gene-1

NRP2 – neuropilin-2

NUBPL – nucleotide binding protein-like

PDF – prostate-derived factor

PLAB – placental bone morphogenetic protein

PTGFB – placental transforming growth factor

STRAP – serine–threonine kinase receptor-associated protein

TGF – transforming growth factor

TMPRSS4 – transmembrane protease/serine 4

TNM – Tumor, Node, Metastasis

TUCS3 – tumor suppressor candidate 3

6. Conclusion and outlook

The EMT is one of the most widely acknowledged of the pathways that lead to metastasis, which accounts for the majority of cancer-related fatalities. To complete the process of EMT, a complex network of molecular signaling channels and regulators must operate together.

Although GDF-15 has antitumor action in the early stages of carcinogenesis, it has been shown to significantly increase the rate of cancer invasion and metastasis in advanced stages of cancer. Functional investigations of GDF-15’s role in malignancy are scarce and disputed: on one hand, the literature demonstrates overexpression of GDF-15 in cancers; on the other, GDF-15 has been found to act as a tumor suppressor.

To create optimal treatment methods for CRC, it is necessary to understand the fundamental underlying mechanisms behind these transformations, especially regarding how CRC cells gain invasive and metastatic features. Cancer treatment might benefit from a better comprehension of the EMT regulating mechanism. As yet, the involvement of GDF-15 in CRC metastasis and EMT is unclear.

Statements

Author Contributions: CL and CVL conceived of the presented idea. EAM, SMD, SR collected the data. CL and CVL drafted the manuscript. DS performed critical revision of the article.

Consent for publication: As the corresponding author, I confirm that the manuscript has been read and approved for submission by all co-authors

Funding:This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector.

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

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