A Review of Hedgehog Signaling in Radioresistant Esophageal Cancer: Potential Treatment Target

Abstract

Esophageal cancer is one of the most lethal malignancies worldwide. For locally advanced diseases, radiotherapy is the main treatment. The survival rates, however, are still appalling, which is partially due to radioresistance. In 6% of cases of esophageal cancer, the Hedgehog pathway is reactivated, and this pathway is crucial for the growth, progression, treatment resistance, and metastasis of esophageal cancer. Here, we conducted a comprehensive review of the current research on Hedgehog pathway in esophageal cancer.

Introduction

Esophageal cancer is one of the most lethal malignancies worldwide. ¹ There are 2 dominant subtypes: squamous cell cancer and adenocarcinoma. In China, more than 90% of them are esophageal squamous cell cancer (ESCC). ² Due to the nonspecific symptoms, most patients were initially diagnosed with locally advanced diseases. For inoperable locally advanced esophageal cancer, concurrent chemoradiotherapy was established as the standard treatment. Despite recent great progress in multiple treatment modalities, the outcome remains unsatisfactory with a 5-year overall survival rate of less than 20%. ³ After preoperative chemoradiotherapy, 57% to 71% of the disease persisted, according to earlier clinical trials, indicating that the esophageal cancer was extremely chemoradiotherapy-resistant.^4–6 Persistence of disease was found to be the most common treatment failure mode after radical radiotherapy. ⁷ Radioresistance could play a role in poor survival results.

Cancer stem cells are parts of the tumor cell populations that exist in the tumor, which is considered being correlated with cancer genesis and development, as well as the main reason for tumor recurrence and treatment resistance. ⁸ The Hedgehog (HH) pathway is one of the predominant members of the stem cell signaling network. ⁹ Targeting HH in basal cell, head and neck carcinoma, and cervical cancer has recently been successful. Here, we review the current research on the HH pathway in esophageal cancer.

Materials and Methods

PubMed and EMBASE were searched using the following keywords: Hedgehog pathway, Hedgehog, targeting Hedgehog, esophageal cancer, and radioresistance. Clinical studies, meta-analyses, reviews, and references from the articles were selected.

The HH Pathway and Its Ligands

The HH signaling pathway was originally identified by Nüsslein-Vollhard and Wieschaus in 1980. ¹⁰ It is highly expressed in early life and plays a pivotal role in the process of embryonic development. It also functions in tissue homeostasis and injury repairment postembryonically through effects on stem and progenitor cells, then gradually decreases in adulthood. The aberrant activation of HH signaling is associated with a variety of human malignancies.^11–13 According to earlier research, 6% of ESCCs reactivate the HH pathway. ¹⁴

The HH signaling pathway consists of 3 known ligands, Sonic Hedgehog (SHH), Indian Hedgehog, and Desert Hedgehog. The negative regulatory receptor patched (PTCH) is a 12-pass transmembrane protein, which represses the function of smoothened (SMO), a 7-transmembrane signal transduction protein. The downstream oncogenic transcription factors include glioma-associated oncogene homolog 1 (Gli1), Gli2, and Gli3. In the absence of ligand, HH pathway activity is repressed by PTCH1 and thereby restrains the activation and ciliary entry of SMO. When HH protein binds PTCH1, PTCH1 releases the repression of SMO. Then SMO accumulated in the primary cilium, thus causing activation of the Gli family of transcription factors. Gli proteins accumulate and translocate to the nucleus where they activate HH target genes such Wnt, Ptc, cyclin D1, and N-Myc. This further regulates cell growth and death, further mediating cell proliferation and apoptosis^15,16 (Figure 1).

Figure 1.

Hedgehog (HH) signaling pathway. The Hedgehog signaling pathway is inactive in the absence of sonic Hedgehog (SHH) ligand. In the presence of Shh, HH pathway can be activated. Ptch1-induced SMO repression is relieved, thereby allowing activation of SMO. This initiates Gli release from SUFU. Further promoting the increased Gli-target gene transcription.

HH Pathway and Esophageal Cancer

The ligand-independent activation pathway, the ligand-dependent autocrine activation pathway, and the ligand-dependent paracrine activation pathway are the 3 ways to activate the HH pathway. In the ligand-independent activation pathway, the Hh signal lacks a ligand, and the activation of the pathway is mainly through SMO activation mutation and PTCH1/SUFU inactivation mutation. The activation of autocrine signaling is cellular autonomy. The surrounding tumor cells produce and occupy Hh ligands. Most of these tumors showed ectopic expression of PTCH1 and Gli1, in addition to the overexpression of HH ligand. The paracrine Hh pathway plays a role in the development of numerous malignant cancers. Hh ligands are secreted by tumor cells, which bind to PTCH1 receptors on stromal cells, further activating the pathway. ¹⁷

The aberrant activation of the HH signal pathway is related to the development and progression of many cancers.^11–13 Similar findings have been demonstrated in esophageal cancer. SMO, PICH1, SUFU, and GLI1/2/3 mutation frequencies in ESCC were reported to be 1%, 4%, 1%, and 6%, respectively. ¹⁸ Maryam et al used real-time PCR to detect the expression of Gli1 mRNA in 49 ESCC tissues. They found that the expression of Gli1 in ESCC and adjacent tissues was significantly higher than that in normal tissues. ¹⁹ Similar to this, Min et al discovered the increased expression of Gli1 in ESCC cell lines, which is correlated with the invasiveness of ESCC. The application of Gli agonist purmorphamine can enhance the ability of metastasis and infiltration of ESCC cells. Moreover, high levels of Gli1 expression could inhibit the expression of E-cadherin and increase the expression of Vimentin and Snail, suggesting that Gli may mediate the occurrence of epithelial–mesenchymal transition. ²⁰ These findings suggested the pivotal role of the HH signaling pathway in ESCC survival and proliferation. Additional underlying mechanisms for HH involve phosphoinositide-3 kinase/protein kinase B (AKT) and the mitogen-activated protein kinase signaling, ¹⁹ cross talk between HH and stemness signaling pathway, ²¹ associated fibroblasts-secreted exosomes, ²² as well as noncoding RNA.^17,23

According to a meta-analysis, Gli1 overexpression is a significant predictor of a worse outcome in the majority of solid malignancies. ²⁴ In ESCC, the expression of Gli1 was reported to be associated with lymph node metastasis and tumor progression.^19,25 A Japanese retrospective study evaluated 69 ESCC patients treated with neoadjuvant chemoradiotherapy (nCRT). The results showed that patients with Gli1 nuclear-positive cancers had a significantly poorer prognosis than those without (mean disease-free survival [DFS]: 250 vs 1738 months, 2-year DFS 0 vs 54.9%, P = .009; mean overall survival [OS]: 386 vs 1742 months, 2-year OS 16.7 vs 54.9%, P = .001). ²⁶ Wei and Xu reviewed 35 samples from esophageal cancer patients and found that the OS of patients with Gli1-positive tumors was considerably lower than that of patients with Gli1-negative tumors (P = .003). ¹⁹

Hedgehog Pathway and Radioresistance in Esophageal Cancer

Radioresistance is one of the dominant causes of local failure after radiation therapy. HH pathway has been implicated in esophageal cancer radiation resistance. ²⁷ After repeated low-dose irradiation (cumulative dose 60Gy) of human esophageal cancer cell line Eca109, Fei et al established a radiation-resistant cell line Eca109R. They demonstrated that the expression levels of Gli1, Shh, Ptch, and Smo protein in Eca109R cell line were significantly higher than that in Eca109 cell line. Radiation resistance is strongly correlated with Gli1 overexpression in parental cell lines. Knockout of Gli1 gene can increase the sensitivity of radiation-resistant cell lines. It is suggested that Gli1 may be related to the occurrence and development of radiation resistance in esophageal cancer. ²⁸ One research from MD Anderson cancer center explored HH in esophageal tumor xenografts after preoperative chemoradiotherapy. They found Shh and Gli-1 were highly expressed in persistent tumors after chemoradiotherapy and during tumor regrowth. When blocking the Hh pathway, the radiation cytotoxicity of esophageal cancer cells is enhanced. These results showed Hh signaling may induce chemoradiation resistance in esophageal cancers. ²⁹ Teichman et al found that radiotherapy combined with SMO inhibitor LDE225 significantly delayed tumor growth in esophageal adenocarcinoma xenotransplantation model with sustained upregulation of HH, compared with SHH antibody 5E1 alone, but not in the model where HH not significantly upregulated. This result indicated that HH signaling pathway mediates radiotherapy response, and inhibition of this pathway may increase the radiosensitivity of HH-dependent tumors. ³⁰ Another research from MD Anderson cancer center validated in esophageal cancer that patients with higher Gli-1 labeling indices had a lower rate of pathologic complete response after neoadjuvant chemoradiation (P < .0001). Plus, their preclinical experiment also demonstrated that Gli-1 mediates radioresistance. ³¹ The polycomb group (PcG) family BMI1, acting downstream of the HH pathway, was reported to be involved in ESCC radioresistance. After preoperative CRT, patients harboring BMI1-positive esophageal squamous cancer cells showed a poorer DFS than those BMI1-negative patients (mean time 16.8 vs 71.2 months; 3y DFS 13.3% vs 49.9%, P = .002) as well as of OS (mean time 21.8 vs 76.6 months; 3-year OS 16.2% vs 54.9%, P = .0005). ³² In vitro and in vivo experiments, high CD155 expression was also found to promote radioresistance in esophageal cancer by triggering the YAP/TAZ-Hippo signaling pathway. ³³

Potential Treatment Targeting HH Pathway in Esophageal Cancer

Itraconazole is a well-known systemic antifungal drug. Recent studies have found that it could function as an important antagonist of the HH signal pathway. ³⁵ It exerts a series of antitumor effects by reducing the release of Gli1 via acting on SMO of HH pathway, including promoting apoptosis of cancer cells, inhibiting cell proliferation through indirect inhibition of NF-κB pathway and inflammation, facilitating the expression of cell cycle-dependent kinase inhibitors, and inhibiting the expression of target genes such as bcl-2 and cyclin-2 of GLI1 transcription. In addition, itraconazole can increase Bnip3 and eventually lead to the dissociation of Beclin-1/bcl-2 complex and promote autophagy of cancer cells. ³⁶

The inhibitory effect of itraconazole on tumor growth was also observed in vitro. Ronan divided Barrett's esophagus rat model into 2 groups, experiment group was treated with itraconazole, whereas the control group was injected with saline. The results showed that the incidence of esophageal adenocarcinoma in the itraconazole group was significantly lower than that in the control group (8% vs 32%, P = .033). The expression level of SHH in the itraconazole group was slightly lower than that in the control group. It is confirmed that itraconazole can reduce the SHH expression and occurrence of adenocarcinoma in Barrett's esophagus in the animal model, showing its antitumor activity. ³⁷ The phase II study (NCT04018872) is currently ongoing attempting to evaluate the effect of itraconazole as a neoadjuvant therapy following nCRT in the treatment of locoregional esophageal and gastroesophageal junction carcinomas. We also launched a phase II study (NCT04481100) to evaluate the efficacy and safety of concurrent chemoradiotherapy combined with itraconazole in patients with locally advanced esophageal squamous cancer.

Vismodegib, sonidegib, and glasdegib, the oral small molecular inhibitors of SMO, have so far been approved for the treatment of basal cell carcinoma (vismodegib and sonidegib)^38–41 and acute myeloid leukemia (glasdegib). ⁴² Mostly, the efficacy of SMO inhibitors has been demonstrated in vivo and in vitro experiments, for example, in lung cancer cells, ⁴³ chronic myeloid leukemia, ⁴⁴ uterine leiomyosarcoma cells, ⁴⁵ and ameloblastoma cell lines, ⁴⁶ which showed a reduction in cell proliferation and cell cycle arrest. However, its function in esophageal has not been established. In nCRT resistance esophageal cancer tissues (little or no response to nCRT, Mandard tumor regression grade 4 and 5), vismodegib was observed to lead to a decrease in cancer stemness and both radiation and carboplatin resistance. ⁴⁷ MD Anderson has initiated a phase IB/II trial to explore the toxicity and response rate of Taladegib, a novel SMO inhibitor, with standard preoperative chemoradiation in patients with localized nuclear Gli-1 expressing adenocarcinoma of the esophagus or gastroesophageal junction (NCT02530437) (Figure 2).

Figure 2.

Potential target therapy of hedgehog signaling pathway regulating radioresistance in esophageal cancer. Vismodegib, sonidegib, glasdegib, and Taladegib suppress HH pathway by repressing the function of SMO. Itraconazole reduces the release of Gli1 and further inhibits HH pathway.

Most of the recent clinical trials which combined radiotherapy with chemotherapy and target therapy resulted in negative results.^48–50 It may be partly due to a lack of precision targets and predictive markers. Gli and other targets may become the potential direction of new drugs. The abovementioned clinical trials (NCT04018872, NCT04481100, NCT02530437) are worth looking forward to (detailed in Table 1).

Table 1.

Ongoing Clinical Trials Targeting Hedgehog Pathway Combined With Radiation Therapy in Esophageal Cancer.

NCT no.	Regimen	Disease	Intervention mode	Phase	Estimated enrollment	Primary endpoint
04018872	Itraconazole: 300 mg bid from completion of neoadjuvant chemoradiation until esophagectomy for 6-8 weeks	Locoregional esophageal and gastroesophageal junction carcinomas	Single group assignment	2	78	Percentage of pathological complete response
04481100	Itraconazole: 100 mg bid for 6 weeks concurrent with chemoradiation	Locally advanced esophageal squamous cancer	Single group assignment	2	38	Objective response rate
02530437	Phase 1b:taladegib qd on days 1-38 with concurrent cheoradiotherapy Phase 2: taladegib qd for 7 days, followed by regimen as phase 1	Localized esophageal or gastroesophageal junction cancer	Single group assignment	1b-2	66	Safety(1b) pCR (2)

Conclusions

Due to the unsatisfied outcome of locally advanced esophageal cancer, the new combination of treatment warrants exploration. With the development of tumor molecular biology research, targeted therapy has become a new direction to improve efficacy. Hedgehog pathway plays an important role in the occurrence, development, treatment resistance, and metastasis of esophageal cancer. It is expected that the ongoing clinical trials and explorations inhibiting the HH pathway can further improve the prognosis of locally advanced esophageal cancer.