Prognostic values of apoptosis-stimulating P53-binding protein 1 and 2 and their relationships with clinical characteristics of esophageal squamous cell carcinoma patients: a retrospective study

Esophageal carcinoma is the eighth most prevalent cancer and the seventh leading cause of cancer-related death worldwide [1] and is the fourth leading cause of cancer-related death in China [2]. The most common histological type of esophageal carcinoma in China is esophageal squamous cell carcinoma (ESCC) [1]. The 5-year overall survival (OS) rate of ESCC patients after esophagectomy has significantly increased over the past several decades but remains approximately 20% [3–5]. The late appearance of symptoms, rapid progression, local recurrence, and metastasis are the main reasons for the low survival rate [6–9]. However, the molecular mechanism of ESCC genesis and progression is not well understood, and biomarkers for predicting clinical outcome of ESCC patients are unavailable. Therefore, there is an urgent need to identify sensitive and specific biomarkers for ESCC.

The well-known tumor suppressor protein P53 has been reported to be involved in the development of many types of cancer, including esophageal carcinoma [10]. Over 10 years ago, two proteins both containing a large number of ankyrin (Ank) repeat sequences, an Src-homology 3 (SH3) domain, and a proline-rich domain were identified as activators for P53 apoptotic function and were thus named apoptosis-stimulating P53-binding protein 1 (ASPP1) and 2 (ASPP2) [11, 12]. ASPP1 and ASPP2 have been reported to play important roles in the developement, progression, metastasis, and prognosis of cancer. Samuels-Lev et al. [11] and Slee et al. [12] have demonstrated a specific effect of ASPP1 and ASPP2 in breast cancer on the apoptotic and transactivation functions of P53 for the expression of proapoptotic targets, such as Bax, P53 up-regulated modulator of apoptosis (PUMA), and P53-induced gene 3 (PIG3), through combination with P53 via the Ank sequence and SH3 domain. However, ASPP1 and ASPP2 failed to affect the cell cycle arrest function of P53 under the same conditions. ASPP1 and ASPP2 could mediate the cell fate transition between life and death. Sgroi et al. [13] found that invasive and metastatic breast cancer tissue exhibited reduced ASPP2 expression compared with normal breast tissue, suggesting a possible role of ASPP2 in breast cancer progression and metastasis. Mak et al. [14] reported the down-regulation of ASPP2 in choriocarcinoma, which contributed to the increased migratory potential of the tumor cells. High ASPP2 expression has been reported to predict good prognosis in some tumors, including breast cancer [15], non-small cell lung cancer [16], diffuse large B cell lymphoma, and follicular center lymphoma [17]. Moreover, a recent study showed that ASPP2 also induced apoptosis through a P53-independent mechanism [18]. Additionally, ASPP2 plays an important role in regulating the nuclear factor kappa B subunit 1 (NFκB1) inflammatory pathway [19] and in modulating autophagy [18, 20] and cell polarity [21, 22].

Although ASPP1 and ASPP2 have been reported to play crucial roles in the development of several malignancies, their involvement in ESCC development remains undetermined. In this study, we detected the expression of ASPP1, ASPP2, and P53 in ESCCs using immunohistochemistry to investigate their prognostic values and relationships with clinical characteristics of ESCC patients.

In the present study, we demonstrated that ASPP2 expression was significantly increased in ESCC and was associated with histological differentiation and age. ASPP2 was an independent predictor of OS and DFS of ESCC patients. Although the expression of ASPP1 was not associated with overall prognosis, it was associated with the prognosis of patients with stage III disease.

ASPP2 is commonly considered a tumor suppressor, and its expression is often reduced in malignant tumors [14, 24, 25]. However, inconsistent with previous reports, our results indicated that ASPP2 expression was significantly increased in ESCCs as compared with noncancerous tissues, suggesting that ASPP2 might be involved in ESCC tumorigenesis. Interestingly, the expression of the well-known tumor suppressor p53 was also reported to be increased in several types of cancer, including ESCC, and its high expression was related with a mutation [26, 27]. It is well known that protein phosphorylation and dephosphorylation serve as “signal switch” and are the most important mechanisms of signaling pathway regulation. Wild-type (unphosphorylated) P53 is usually undetectable using immunohistochemistry, whereas mutant (phosphorylated) P53 with dominant-negative activities and oncogenic properties can be detected because its half-life is much longer [28–32]. High expression of mutant (phosphorylated) P53 has been observed in many malignancies [11, 29, 33–35]. Protein expression is highly cell environment-specific and is regulated both genetically and epigenetically. Similar to P53, ASPP2 is also a tumor suppressor that is up-regulated in ESCC. We hypothesized that high ASPP2 expression in ESCC may be due to epigenetic alterations, such as phosphorylation and methylation.

Moreover, some proteins, such as nuclear factor erythroid-2 related factor 2 (NRF2), play important roles in protecting normal cells from tumorigenesis or disease development, but can be hijacked by cancer cells upon neoplasm initiation [36, 37]. It has been reported that NFR2 functions as a suppressor in carcinomas through autophagy-related [38] and inflammation-related cancer signaling pathways [39, 40]. Mounting evidence has suggested that ASPP2 also plays an antitumor role in malignancies through autophagy- [18] and inflammatory-related pathways independent of P53 [19]. Thus, we suggest that ASPP2 may function through similar pathways to NRF2. However, the underlying mechanism requires further investigation.

Furthermore, our results showed that high ASPP2 expression was associated with high degree of differentiation of ESCC, suggesting that ASPP2 may repress the progression of ESCC. We also found that high P53 expression was associated with large tumor size, indicating that mutant P53 expression may be involved in tumor progression, which is consistent with a recent study [23]. Although we did not detect a difference in ASPP1 expression between ESCCs and noncancerous tissues, high ASPP1 expression was associated with high degree of histological differentiation and shallow invasion of ESCCs, suggesting that high ASPP1 expression may inhibit tumor progression.

According to our results, low ASPP2 expression independently predicted poor prognosis of ESCC patients, which is consistent with previous reports of other cancers [15–17]. More aggressive therapy modality (e.g., including chemotherapy and radiotherapy) and close follow-up may be necessary for patients with low ASPP2 expression. Our study confirmed that high P53 expression was an independent predictor of short DFS, which was consistent with the results reported in literature [23, 41, 42]. Similar to ASPP2, patients with high P53 expression need more aggressive therapy modality and close follow-up to reduce the chance of relapse or metastasis. Although high ASPP1 expression was not associated with the prognosis of the whole cohort, it was associated with prolonged OS and DFS of patients with stage III disease. We hypothesize that ASPP1 mainly suppress tumor pregression in patients with advanced ESCC.

As their names “apoptosis-stimulating protein of P53 1 and 2” suggest, ASPP1 and ASPP2 were initially reported to function in a P53-dependent manner. Samuels-Lev et al. [11] found ASPP2 down-regulation in tumors with wild-type P53 but not in those with mutant P53. Iwabuchi et al. [43] suggested that ASPP1 and ASPP2 exerted their function of inhibiting tumorigenesis only in the presence of wild-type P53. However, later studies showed that ASPP2 played tumor-suppressing roles independent of P53, such as enhancing colorectal cancer cell apoptosis through autophagy inhibition [18], inhibiting cell migration through Src inactivation [14], and stimulating Ras-induced senescence through direct interactions with Ras-GTP [44]. Additionally, we did not find any correlation among ASPP1, ASPP2, and P53, indicating that they may all function independently. However, in this study, we only detected ASPP1 and ASPP2 expression using immunohistochemistry and the results need further experiments to verify and explore.

ASPP1 may mainly function in advanced ESCCs rather than the gamut or early-stage ESCCs. ASPP2 may be an independent prognostic biomarker in ESCC, and patients with low ASPP2 expression may need more aggressive therapy modality and close follow-up. In the future, we will conduct in vivo and in vitro experiments to verify the immunohistochemistry results and further explore the biological mechanism of ASPP1’s and ASPP2’s functions in ESCC.