Relationship between Tumor Budding and Partial Epithelial-Mesenchymal Transition in Head and Neck Cancer

Abstract

Tumor budding (TB), a microscopic finding in the stroma ahead of the invasive fronts of tumors, has been well investigated and reported as a prognostic marker in head and neck squamous cell carcinoma (HNSCC). Epithelial-mesenchymal transition (EMT) is a crucial step in tumor progression and metastasis, and its status cannot be distinguished from TB. The current understanding of partial EMT (p-EMT), the so-called halfway step of EMT, focuses on the tumor microenvironment (TME). Although this evidence has been investigated, the clinicopathological and biological relationship between TB and p-EMT remains debatable. At the invasion front, previous research suggested that cancer-associated fibroblasts (CAFs) are important for tumor progression, metastasis, p-EMT, and TB formation in the TME. Although there is biological evidence of TB drivers, no report has focused on their organized functional relationships. Understanding the mechanism of TB onset and the relationship between p-EMTs may facilitate the development of novel diagnostic and prognostic methods, and targeted therapies for the prevention of metastasis in epithelial cancer. Thus far, major pieces of evidence have been established from colorectal cancer (CRC), due to a large number of patients with the disease. Herein, we review the current understanding of p-EMT and TME dynamics and discuss the relationship between TB development and p-EMT, focusing on CAFs, hypoxia, tumor-associated macrophages, laminin-integrin crosstalk, membrane stiffness, enzymes, and viral infections in cancers, and clarify the gap of evidence between HNSCC and CRC.

Keywords: cancer-associated fibroblast; head and neck squamous cell carcinoma; partial epithelial–mesenchymal transition; tumor budding; tumor microenvironment.

Figure 1

Representative biomarkers of TB and p-EMT. Of note, SNAIL2 amplification is strongly specific to the p-EMT status. Although several markers for these statuses have been reported, there is a lack of verification of the specificity of these markers to distinguish TB and p-EMT status.

Figure 2

Role of hypoxia in the formation of TB. Hypoxia in advanced TME promotes TB development. Genes whose expression is induced by HIF-1α activation by hypoxia promote EMT. YAP is activated in a hypoxic TME and was abrogated by the knockdown of ME1 which promotes the Warburg effect in cancer cells and induces EMT. YAP activation is a significant factor that promotes the EMT phenotype and is deeply involved in the progression of the tumor. In a hypoxic situation, YAP activation is responsible for the upregulation of GPRC5A by binding to HIF-1α. Then, activated YAP also stabilizes HIF-1α and enhances its action. The expression of MMP9 and MMP7 is also upregulated by HIF-1α activation. Moreover, hypoxic TME can induce tumor cells to secrete enhanced amounts of TEVs. The dynamic intercellular crosstalk that is mediated by TEVs mobilizes oncogenic factors, relocalizes CAFs to tumor sites, p-EMT and APT development, and TB formation which sustains cancer progression and metastasis.

Figure 3

In hepatocellular carcinoma, CAF-derived CCL5 promotes metastasis by binding to specific receptors, CCR5, and stabilizing HIF-1α under normoxia, upregulating one of the EMT genes ZEB1 and promoting TB development. In CRC, CCL5 blockade reduces tumor growth, decreases migration of tumor cells, reduces metastases, and decreases infiltration of T_regs in the tumor. CCL5 can stabilize PD-L1 in vitro and in vivo. CCR5 can also modulate TGF-β activity, which subsequently promotes an EMT. TB cells secrete high levels of CCL5, which recruits fibroblasts through CCR5–SLC25A24 signaling and leads to the development of a characteristic fibroblast cluster around TB cells at the invasive front of CRC. This further facilitates tumor angiogenesis and collagen synthesis, recruitment of CAFs, and promotes malignant progression. CCR5 can also modulate TGF-β activity, which subsequently promotes an EMT and increases tumor cell migration via the activation of the NF-κB pathway.

Figure 4

The role of TAM, laminin–integrin interaction, basement membrane stiffness, and F. nucleatum as a TB driver. TAMs located in the surrounding of the tumor mass induce loss of tight junction proteins at tumor cell–cell contacts, and cause TB from the colonosphere bulk of CRC. This is due to the MMP7 secretion by activating the NF-κB pathway. Moreover, CAF-derived exosomes serve as chemoattractants, which recruit various immune cells, including monocytes, promoting CRC progression and the release of cancer cell-derived exosomes. In PDAC, high-grade TB cases display lower M1 macrophages in the stroma and increased M2 macrophages in the tumor tissue, and displayed fewer CD3+, CD4+, and CD8+ T cells. Inversely, FOXP3+ T_regs were found to be elevated in high-grade TB cases. CAFs also recruit granulocyte-macrophage colony-stimulating factor and IL-6, promote the differentiation of monocytes into M2 macrophages and activate them to release chemokines and exosomes, which promote TB formation. Softening and enhanced remodeling of the basement membrane also promote TB development in stratified epidermis via the activation of focal adhesion kinase and YAP, while stiffening of the basement membrane promotes folding, and the laminin–integrin crosstalk in the basement membrane plays a key role to generate TBs. Moreover, F. nucleatum upregulated the expression of p-EMT-related genes in HNSCC cells with an epithelial phenotype. F. nucleatum-infected HNSCC cells had upregulated MMP1, MMP9, and IL-8. The expression of cell survival markers MYC, JAK1, and STAT3 and EMT markers ZEB1 and TGF-β were also significantly elevated and promoted TB development. These mediators also recruit CAFs in the TME and promote TB formation.