NOTCH and Esophageal Squamous Cell Carcinoma

Abstract

Esophageal squamous cell carcinoma (ESCC) is a deadly disease that requires extensive research on its mechanisms, prevention, and therapy. Recent studies have shown that NOTCH mutations are commonly seen in human ESCC. This chapter summarizes our current understanding of the NOTCH pathway in normal esophagus and in ESCC. In normal esophagus, NOTCH pathway regulates the development of esophageal squamous epithelium, in particular, squamous differentiation. Exposure to extrinsic and intrinsic factors, such as gastroesophageal reflux, alcohol drinking, and inflammation, down-regulates the NOTCH pathway, and thus inhibits squamous differentiation of esophageal squamous epithelial cells. In ESCC, NOTCH plays a dual role as both a tumor suppressor pathway and an oncogenic pathway. In summary, further studies are warranted to develop NOTCH activators for the prevention of ESCC and NOTCH inhibitors for targeted therapy of a subset of ESCC with activated NOTCH pathway.

Introduction

Esophageal cancer affected 17,290 adults and caused 15,850 deaths in the US in 2018 (Cancer Facts & Figures 2018). In the world, it is the seventh most prevalent cancer and the sixth leading cause of cancer-related death, with more than 572,000 new cases and 508,000 deaths each year (1, 2). Two main histological types of esophageal cancer exist, squamous cell carcinoma (SCC) and adenocarcinoma. Human esophageal squamous cell carcinoma (ESCC) develops from precancerous lesions, and its histopathology follows a step-wise pattern of hyperplasia, dysplasia, and SCC. The 5-year survival rate for ESCC is ~18%, a number that reflects late diagnosis, the aggressiveness of the disease, and a lack of effective treatment strategies (3, 4). Thus, there is a great need to further elucidate the molecular mechanisms and develop better preventive and therapeutic strategies for ESCC.

Recent technological advances in NextGen sequencing enabled the detection of gene mutations in human ESCC samples (5–14). Common genomic alterations included single nucleotide variants, copy number alterations and alterations in multiple signaling pathways. The overall mutation pattern appeared similar to that of head and neck SCC (HNSCC) but distinct from that of esophageal adenocarcinoma and lung SCC. Genes on the NOTCH pathway were frequently mutated in human ESCC (3, 4). This observation brings about interesting questions regarding the functional role of the NOTCH pathway in the development of ESCC.

NOTCH IN NORMAL ESOPHAGUS

NOTCH pathway is mediated through ligands (JAG1, JAG2, DLL1, DLL3, DLL4) binding to NOTCH receptors (NOTCH1, 2, 3, 4). These receptors are then cleaved to allow its intracellular domain (e.g., NICD1) to be released from the membrane and enter the nucleus to form a transcriptional complex with RBPJ. NICD1 displaces the repressive cofactors bound to RBPJ and recruits a transcriptional activator complex, which initiates transcription of NOTCH downstream effectors like HES1 (15). In the normal esophagus of rodents and humans, NOTCH1, NOTCH2, and NOTCH3 are highly expressed whereas NOTCH4 is expressed at a minimal level (16, 17). NOTCH3 expression was found to be subject to transcriptional regulation by NOTCH1, and loss of NOTCH signaling in mouse esophagus resulted in NOTCH3 silence (18). Nevertheless, NOTCH pathway is largely dispensable for maintaining the integrity of squamous epithelium. Although NOTCH1 is the major regulator of squamous differentiation among four NOTCH receptors, even in triple knockout mice (Notch1, Notch2, Notch3) the epidermis still formed almost normally except for the phenotypes of squamous hyperplasia and deficient barrier function (19).

Regulation of squamous differentiation by NOTCH:

NOTCH regulates squamous differentiation in the skin (20) and the esophagus (18, 21), particularly in the commitment of keratinocytes to terminal differentiation by a HES1-dependent mechanism (22, 23). Not surprisingly, NOTCH interacts with key regulators of squamous differentiation, such as P63 (24), IRF6 (25), NRF2 (26, 27), HPV8 E6 (28). Using gene microarray data of mouse esophagus at various developmental stages and ages, we found the NOTCH pathway participates in the development of mouse esophageal epithelium (29). Further studies have shown that NOTCH is required for the specification of esophageal progenitor cells from human progenitor stem cells. In agreement with these observations, genetic deficiency of NOTCH components (Rbpj, Jag1, Jag2) inhibited squamous differentiation in mouse esophagus (17).

NOTCH mutations in normal esophagus:

It is surprising that NOTCH1, NOTCH2, and NOTCH3 mutations occurred much more often in physiologically normal human esophageal epithelia (66.2% samples) than in ESCC (15% samples), and distribution pattern of the mutation sites was similar in normal and ESCC samples. Human subjects with ESCC risk factors (alcohol drinking, tobacco smoking, aging) were more likely to carry NOTCH mutations than those without these risk factors (30–32). However, few of the mutations were present in all the cells of the normal clones, and many of the NOTCH mutations were found in spatially distinct subclones. These data suggest that NOTCH mutations are not sufficient to drive carcinogenesis, and some other mutations are needed. NOTCH mutations can be either driver or passenger mutations in human ESCC (33, 34).

Why normal esophageal epithelial cells are susceptible to NOTCH mutations? A recent report based solely on cancer genome sequencing and epidemiological data estimated that DNA replication errors may contribute to 38.9% gene mutations in ESCC, while hereditary mutations and environmental factors contribute to 0.5% and 60.6% mutations, respectively (35). This was believed to be due to a relatively high rate of stem cell division in the esophagus although debates still remain (36, 37). Interestingly, there was a significant decrease in the rate of stem cell division in the human esophagus with age. In contrast, only a small decrease was observed in the mouse esophagus. These data provide a plausible explanation for the enigmatic age-dependent deceleration in cancer incidence in very old humans but not in mice (38).

Response of the NOTCH pathway to gastroesophageal reflux:

Regurgitation of gastric and duodenal contents (e.g., acid and bile acids) into the esophagus causes heartburn and leads to substantial impairment of quality of life and work productivity. Some subjects with gastroesophageal reflux are further complicated with Barrett’s esophagus (BE) when histologic evidence of intestinal metaplasia is present in the human esophageal epithelium. Acid and bile acids inhibited the NOTCH pathway in esophageal squamous epithelial cells (39–41), supporting an essential role of the NOTCH pathway in squamous differentiation. Moreover, inhibition of the NOTCH pathway favored goblet cell differentiation, which is diagnostic of human BE. Treatment of a rat model of reflux-induced BE with a γ-secretase inhibitor converted the proliferative Barrett’s epithelial cells into terminally differentiated goblet cells, whereas the squamous epithelium remained intact (42). When human esophageal squamous epithelial cells were stably transfected with an intestinal transcription factor, CDX2, these cells formed crypt-like structures, overexpressed differentiation markers of intestinal columnar epithelial cells and goblet cells, and downregulated NOTCH pathway genes (43). These data support NOTCH inhibition as one of the molecular mechanisms of human BE as a result of exposure to gastroesophageal reflux (44).

Response of the NOTCH pathway to alcohol drinking:

NOTCH pathway was inhibited by ethanol in the pancreas and smooth muscle cells (45, 46). Mechanistically, ethanol suppressed the NOTCH pathway through inhibition of γ-secretase proteolytic activity (47). In the esophagus, we first found out that ethanol exposure downregulated PAX9 expression in human esophageal epithelial cells in vitro and mouse forestomach and tongue in vivo (48). More importantly, PAX9 was found to be a downstream effector of the NOTCH pathway in esophageal squamous epithelial cells, and ethanol exposure inhibited the NOTCH pathway as well (unpublished data). Consistent with the function of NOTCH in squamous differentiation, we also showed that Pax9 deficiency in mouse esophagus promoted cell proliferation and delayed cell differentiation, and PAX9 was downregulated in human ESCC (48).

Response of the NOTCH pathway to inflammation:

Inflammatory cytokines (IL4, IL5, IL1, TNFα) suppressed NOTCH-dependent transcription, NOTCH ligands and NOTCH1 target genes in human esophageal epithelial cells. These changes contributed to the development of eosinophilic esophagitis and possibly inflammation-associated ESCC (49).

NOTCH in ESCC

NOTCH mutations in ESCC:

Based on the original data from two studies of 227 cases of human ESCC (7, 13), NOTCH1, NOTCH2, and NOTCH3 mutations took place in 8%, 3% and 1.9% of human ESCC, respectively. Point mutations tended to cluster in the EGF-like repeats and thus potentially resulted in the loss of function (Figure 1A, B, C). These mutations tended to be mutually exclusive (Figure 1D). RBPJ (a key repressor of canonical NOTCH pathway) and FBXW7 (the substrate-recognition subunit of an SCF-type ubiquitin ligase complex targeting NOTCH1) were also frequently mutated (50, 51). It was interesting that NOTCH1 mutation was mutually exclusive with PIK3CA mutation. NOTCH1 mutation was associated with well-differentiated, early-stage malignancy, and less metastasis to regional lymph nodes. Patients with NOTCH mutations tended to have a worse prognosis than those without (Figure 2). In contrast, patients with PIK3CA mutations had better response to chemotherapy and longer survival time than those without (52). Moreover, lower expression of NOTCH1 was associated with poorer prognosis than higher expression after adjustment for age, sex, tumor stage, smoking and alcohol consumption (10).

Figure 1. NOTCH mutations in human ESCC.

These plots are downloaded from the TCGA database (www.cbioportal.org) based on the original data from two studies of 227 cases of human ESCC (7, 13). EGF-LR, epidermal growth factor-like repeat; LNR, LIN12-NOTCH repeat; NOD and NODP, NOTCH protein domain; ANK, Ankyrin repeats; DUF, domain of unknown function.

Figure 2. NOTCH mutations and prognosis of human ESCC.

The plot is downloaded from the TCGA database (www.cbioportal.org) based on the survival data from one study of 88 cases of human ESCC (13).

NOTCH as a tumor suppressor pathway:

In the normal esophagus, NOTCH functions as a tumor suppressor (53). Exposure to an oro-esophageal carcinogen, 4-nitroquinoline 1-oxide (4NQO), caused loss of NOTCH1 expression in the basal cells of normal esophageal squamous epithelium, as well as Notch1 mutations. Loss of Notch1 in the squamous epithelial cells caused spontaneous SCC in the skin, but not the esophagus. However, loss of Notch1 promoted 4NQO-induced oro-esophageal SCC (54, 55). Similarly, NOTCH inhibition in mouse esophagus increased the number and size of tumors following exposure to an esophageal carcinogen, diethylnitrosamine (56).

Using the lineage tracing technique in mice carrying a conditional dominant-negative mutant of Maml1 (a transcriptional coactivator for NOTCH), Alcolea et al found that NOTCH inhibition prevented differentiation of mutant progenitor cells and promoted differentiation of neighboring wild-type progenitor cells in mouse esophagus (56). Such combined effects led to clonal expansion with mutant cells eventually replacing the entire epithelium, supporting the idea that NOTCH mutation promotes field change in the human esophageal epithelium (30–32).

NOTCH as an oncogenic pathway:

However, the NOTCH pathway plays a dual role in carcinogenesis, both oncogenic and tumor suppressor, depending on the cellular and genetic context (53, 57–60). It is believed that NOTCH pathway turns to be oncogenic during the process of carcinogenesis. In ESCC, cellular senescence checkpoint functions (e.g., P16-Rb, P14, P53) determined differential NOTCH1-dependent oncogenic and tumor-suppressor activities (61). Activated NOTCH1 was detected in a small subset of cancer cells at the invasive front in human ESCC, which correlated with higher tumor aggressiveness. NOTCH1 facilitated not only epithelial-mesenchymal transition but also TGFβ-mediated tumor initiation by increasing the number of cancer stem cells (62).

Similar to ESCC, NOTCH activity is contextual and NOTCH in HNSCC is considered to have a dual role as a tumor suppressor and an oncogene (63), at least in a subset of HNSCC based on genomics data (60). NOTCH4-HEY1 pathway induced epithelial-mesenchymal transition in cultured cells (64). Both deficiency and activation of Notch1 promoted oral squamous cell carcinogenesis in a genetic model driven by HPV E6/E7 and Kras^G12D (59). Inactivation of the NOTCH pathway by a dominant-negative form of Maml1 promoted HNSCC induced by 4NQO, especially in the presence of p53 mutation or HPV16 E6/E7 oncoproteins (54). On the other hand, RBPJ acted as a tumor-promoting function in HNSCC (65).

CONCLUSION

Considering its function as a tumor suppressor, NOTCH activators may be used for the prevention of ESCC. Chemical NOTCH activators, e.g., resveratrol, valproic acid, chrysin, hesperetin, thiocoraline, N-methylhemeanthidine chloride (66–71), may be further tested for their protective effect on ESCC. Other than chemical NOTCH activator, a NOTCH3 antibody is also known to activate NOTCH (72). NOTCH activation may also be achieved through inhibition of negative regulators of the NOTCH pathway, such as FBXW7 or NUMB. On the other hand, NOTCH inhibition needs to be explored as a potential therapy for a subset of ESCC with activated NOTCH pathway.

It should be noted that the NOTCH pathway is complicated in the esophagus just like in many other organs. NOTCH pathway in the normal esophagus behaves differently from that in ESCC. NOTCH target genes in the normal esophagus and those in ESCC need to be identified for functional characterization of the NOTCH pathway in these contexts. Moreover, it remains to be elucidated how NOTCH pathway discriminates between distinct ligands as well as receptors in these contexts (73). If we further consider NOTCH pathway in the tumor microenvironment (74) and non-canonical NOTCH pathway (75), it is obvious that a lot more studies are warranted to elucidate the sophisticated role of the NOTCH pathway in ESCC.

Abbreviations:

4NQO

4-nitroquinoline 1-oxide

BE

Barrett’s esophagus

ESCC

esophageal squamous cell carcinoma

HNSCC

head and neck squamous cell carcinoma

SCC

squamous cell carcinoma