Individual and co-expression patterns of nerve growth factor and heme oxygenase-1 predict shorter survival of gastric carcinoma patients

Nerve growth factor (NGF) is a member of neurotrophin family, and the effects of NGF are mediated by its binding to two types of NGF receptors (NGFR): high affinity tropomyosin receptor kinase A (TrkA) and low affinity p75 neurotrophin receptor (p75^NTR) [1, 2]. However, in addition to the role of NGF in modulating proliferation, differentiation, and maturation of neuronal cells, NGF is a molecule of significant interest because of its involvement in the progression of various non-neuronal malignant tumors [1‐3]. Binding of NGF to TrkA promotes cell survival, proliferation, and differentiation via phosphatidylinositol 3-kinase (PI3K)/AKT and Ras/MAPK signaling pathways [1]. In contrast, binding of NGF or precursor of NGF (proNGF) to p75^NTR could induce cell survival or could be pro-apoptotic [1]; therefore, as a consequence, there are conflicting reports for the role of NGF-related signaling in human malignant tumors. An oncogenic role of NGF has been supported by reports that NGF and NGFR participate in the progression of various human cancers, such as breast cancer [4, 5], ovarian cancer [6], pancreatic cancer [7], oral carcinoma [8, 9], and salivary gland cancer [10]. In addition, the expression patterns of NGF or TrkA were associated with poor prognosis of tongue cancer [9], pancreatic cancer [7], and breast cancer [5, 11]. However, a tumor-suppressive role of p75^NTR has been reported in pancreatic cancer [7] and colorectal cancer [12].

Heme oxygenase-1 (HO1) is a rate-limiting enzyme degrading heme into carbon monoxide, free iron, and bilirubin [13]. Because of the responsiveness of HO1 to variable stressors such as hyperthermia, hypoxia, heavy metals, irradiation, inflammatory cytokines, and oxidative stress, HO1 demonstrates anti-inflammatory, anti-apoptotic, and immunomodulatory effects [14, 15]. However, despite the protective role of HO1, it has been reported that HO1 could have a role in tumorigenesis. The expression of HO1 has been demonstrated to be anti-apoptotic [16, 17], pro-angiogenetic [18] and associated with increased invasiveness of cancer cells [19] and chemoresistance [20]. However, there are also controversial reports that found that HO1 inhibited the proliferation and metastatic capacity of the hepatocellular carcinoma [21] and that inhibition of HO1 enhanced the chemosensitivity for cisplatin in squamous cell carcinoma cells [22]. Therefore, the role of HO1 in cancer progression remains controversial.

Gastric cancer is one of the major causes of cancer death and is particularly common in low- and middle-income regions [23]. However, despite extensive studies on the impact of NGF and HO1 in major human cancers, the studies on NGF and HO1 in gastric carcinoma are limited. Moreover, their roles in human cancers have been controversial. Although, it has been reported that NGF expression is associated with poor prognosis of gastric carcinomas [24], other reports found that the expression of NGF gene is higher in normal gastric tissue than in gastric cancer tissue [25, 26]. Regarding HO1 in gastric carcinoma, HO1 was shown to be anti-apoptotic in gastric cancer cells [16] and microsatellite polymorphism with higher promoter activity of HO1 was associated with higher risk of gastric cancer in Japanese woman [27]. In addition, because there is a close relation between NGF and HO1 in malignant tumors [5, 28, 29], this study investigated the expression and correlation of NGF and HO1 in human gastric carcinoma tissues. Thereafter, we evaluated the expression of NGF and HO1 as a prognostic indicator for gastric carcinoma patients.

In this study, we demonstrate that substantial cases of gastric carcinomas express NGF and HO1, and their expression patterns are significantly associated with each other. In addition, the expression patterns of NGF and HO1 were higher in gastric carcinomas compared with normal gastric mucosa (Fig. 1c). Similarly, a search of the Oncomine database (search condition: threshold p value; p < 0.001, threshold gene rank; top 10% gene, data type; mRNA) showed higher expression of HO1 mRNA in gastric carcinomas compared with normal gastric tissue (Fig. 5a) although the expression of NGF mRNA was controversial [36]. Especially, the expression patterns of NGF and HO1 in gastric carcinomas were significantly associated with shorter OS and RFS. These findings suggest that NGF-HO1 related pathways might have a role in the progression of gastric carcinomas. Similarly, higher expression of NGF mRNA was significantly associated with shorter OS of gastric cancer patients in search of OncoLnc database (Log-rank, p < 0.001) (Fig. 5b) [37]. In line with our results, the expression patterns of NGF and HO1 were associated with advanced clinicopathologic factors of breast carcinomas, and were independent indicators of poor prognosis of breast carcinoma patients [5]. In addition to the expression of NGF, the expression of NGFR was also associated with progressive factors of breast carcinomas, such as HER2 expression, estrogen receptor negativity, and higher histologic grade [38].

Regarding the role of the NGF signaling pathway in tumorigenesis, an association between the expression of NGFR TrkA and p75^NTR has been reported in various types of cancers. NGF is involved in the proliferation of breast cancer cells through the NGFR TrkA-MAPK pathway and it prevents apoptosis by activating the NGFR p75^NTR-NFκB pathway [39]. Overexpression of TrkA promoted growth and metastatic potential of xenografted breast cancer cells by activating the MEK and PI3K pathways and enhancing resistance to anoikis [4]. In pancreatic cancer, mRNA expression of TrkA was associated with an aggressive tumor phenotype with perineural invasion and a higher degree of pain [7]. However, this study found mRNA expression of p75^NTR to be an indicator of favorable prognosis [7]. When considering NGF-NGFR as a therapeutic target of human malignant tumors, NGF might confer chemoresistance of cancer cells due to the role of NGF in the resistance to oxidative stress and apoptosis [28, 29]. In addition, proNGF produced by cancer cells is involved in the invasiveness of breast cancer cells [40]. Therefore, inhibition of proNGF, NGF, or NGFR has been proposed as therapeutic stratagems for human malignant tumors [40‐42]. Small interfering RNA against NGF or proNGF, anti-NGF antibodies, and inhibitors of NGFR were pro-apoptotic and anti-proliferative for cancer cells and inhibited tumor growth and invasiveness [40‐42]. Moreover, since there is a possibility that NGF selectively influences the proliferation of breast cancer cells but not normal breast epithelial cells, NGF might be an optimal therapeutic target for particular cancer types [43]; the effect of NGF on cancer cells differs according to the expression status of TrkA and/or p75^NTR and varies with the use of chemotherapeutic agents [44]. Despite some limitations, these finding suggest that the NGF pathway could be a useful therapeutic target for the treatment of malignant human tumors. Although, the study for the NGF as a therapeutic target of gastric carcinoma is limited, our results suggest that NGF-related pathways are important in the progression of gastric carcinomas by presenting NGF expression as a prognostic marker of gastric carcinoma. Similarly, a recent report has shown that the immunohistochemical expression of NGFR TrkA was associated with advanced clinicopathological factors of gastric carcinomas, such as lymph node metastasis and distant metastasis, and predicted shorter disease-specific survival and RFS [24]. However, there are also reports that seem to contradict these. The mRNA levels of NGF and TrkA were lower in gastric cancer compared with normal gastric mucosa [25]. Therefore, further study is needed to clear the role of NGF in gastric carcinogenesis.

In our study, the expression patterns of NGF and HO1 in gastric cancer tissue were significantly associated with each other. 45 of 67 (67%) cases of NGF-positive gastric carcinomas were positive for HO1 and 60 of 100 NGF-negative cases were also negative for HO1. When we compared the immunohistochemical staining scores between the expression of NGF and HO1, the expression of NGF significantly correlated with the expression of HO1 (Pearson’s correlation coefficient; r = 0.329, p < 0.001). This result might be related to the fact that NGF induces expression of HO1. NGF stimulates gene expression of HO1 and causes resistance to oxidative stress through MEK- and a PI3K-dependent mechanisms [28, 29]. The relationship between NGF and HO1 in the resistance to stress might be a mechanism for the progression of malignant human tumors. Supportive of this hypothesis, we found that the expressions of both NGF and HO1 were significantly associated with shorter survival of gastric carcinoma patients. Especially, the individualized HO1 expression and co-expression pattern of NGF and HO1 were independent indicators of poor prognosis of gastric carcinoma. The NGF⁺/HO1⁺ subgroup showed the shortest survival and the NGF⁻/HO1⁻ subgroup of gastric carcinoma patients had the longest survival time. We published similar results in breast carcinomas. The expression of NGF, HO1, and the combined NGF/HO1 expression patterns were independent indicators of poor prognosis of breast carcinomas [5]. Similarly, the expression of HO1 predicted shorter survival of non-small cell lung cancer patients [19]. These findings suggest that evaluation of individual and co-expression pattern of NGF and HO1 in human cancer tissue might be helpful for prediction of outcome. In addition, higher expression of HO1 was associated with resistance to apoptosis of gastric cancer cells [16] and increased invasiveness of non-small cell lung cancers [19]. HO1 also promotes tumor progression by inducing angiogenesis in pancreatic cancer [45] and urothelial carcinoma [18]. Moreover, therapeutic efficacy of the inhibition of HO1 was presented in pancreatic cancer cells. The mRNA and protein levels of HO1 were higher in pancreatic cancer cells compared with normal pancreatic tissue and suppression of HO1 with siRNA inhibited proliferation and sensitize tumors to chemo- and radio-therapy [46]. However, there are also discrepancies in reports on the role of HO1 in tumorigenesis. In a small study that analyzed 55 colorectal cancer patients, HO1 expression was associated with fewer lymph node metastases and favorable survival of patients [47]. Inhibition of HO1 with siRNA increased the proliferation and invasiveness of androgen-sensitive prostate cancer cells and breast cancer cells [48, 49]. In addition, overexpression of HO1 inhibited proliferation and invasion via down-regulation of MMP9 in pancreatic and breast cancer cells [48, 49]. These conflicting results might be related with the varied roles of the NGF-HO1 pathway in the resistance to stress. As we have seen with the expression of DNA damage response molecules, resistance to the stresses could be tumor suppressive or induce resistance to anti-cancer therapy [33, 35, 50]. Defects in BRCA1/2 promote the early development of breast and ovarian carcinomas, but also make cancer cells sensitive to DNA damage-inducing anti-cancer therapy and anti-PARP therapy [50, 51]. When stress-resistant signaling occurs, continued sub-lethal stresses could induce the accumulation of aberrant tumorigenic signaling and eventually cause the development and progression of cancers. It has been also reported that HO1 might be anti-tumorigenic by protecting healthy normal cells, but also protecting already established cancer [52]. Therefore, additional study is needed to elucidate the role of the NGF-HO1 pathway and its potential as a therapeutic target for human cancers.

In conclusion, this study demonstrated that the expression of NGF and HO1 might be helpful for the estimation of prognosis of gastric carcinoma patients. In addition, this study suggests that regulation of the NGF-HO1 pathway might be a new therapeutic strategy for the treatment of gastric carcinoma patients with significant risk factors.