CRC carcinogenesis is a complicated and multifactorial process resulting from many environmental exposures. This process involves the combined actions of multiple oncogenes and tumor suppressor genes . As a negative growth-control gene, GADD45B is implicated in DNA damage, cell cycle arrest, and apoptosis. According to reports in the literature, higher expressions of GADD45B are associated with a higher risk of recurrence based on a cluster analysis of patients with stage II/III colon cancer treated with surgery alone or surgery plus adjuvant fluorouracil plus leucovorin . Our study was consistent with the above research. To investigate the role of GADD45B in CRC, we first detected GADD45B expression in 64 pairs of CRC and ANCT by RT-qPCR. The results indicated that GADD45B was significantly up-regulated in CRC. Tissue microarrays are reliable tools for the clinicopathological characterization of cancer tissues [18, 19]. The IHC analysis in 152 CRC patients using tissue microarrays further confirmed the results of the RT-qPCR and also observed that up-regulation of GADD45B expression was correlated with relapse and death in CRC patients (P<0.05). The patients with higher GADD45B expression tended to have a higher risk of relapse and death. The Kaplan-Meier survival curves indicated that DFS was significantly worse in CRC patients that overexpressed GADD45B. A Cox multivariate analysis revealed GADD45B expression and TNM stage as significant factors affecting survival. GADD45B overexpression in patients and stage III patients were significantly associated with a poorer DFS (P<0.05). Therefore, we postulated that abnormal expression of GADD45B might be involved with the carcinogenesis of CRC and it showed potential for use as a prognostic marker in future.
Until now, the GADD45 gene family, including GADD45A, GADD45B and GADD45G, has been recognized as stress sensors that modulate the response of mammalian cells to genotoxic and physiological stresses and the process of tumor formation and progression [4, 20–22]. GADD45B shares the common functions of the GADD45 family, including the regulation of cell growth, cell apoptosis, cellular responses to DNA damage and anti-tumor immune responses [23–25]. The overexpression of GADD45B has been shown to inhibit cell growth in a variety of human tumor cell lines, including hepatocellular carcinoma cells, prostate cancer cells, breast cancer cells and others [26–28]. To investigate the function of GADD45B in CRC, we constructed a pEGFP-N1-GADD45B vector, synthesized SiRNA duplexes, and then respectively transfected them into three CRC cell lines. After successful transfection with high efficiency, we performed an analysis of cell apoptosis using the flow cytometric method. Compared with the control group, the cells transfected with pEGFP-N1-GADD45B had more apoptotic cells, whereas the cells transfected with Si-GADD45B had fewer apoptotic cells. We also investigated the effects of pEGFP-N1-GADD45B and Si-GADD45B on the expression of apoptosis-related proteins in CRC cells by Western blotting and found that the mitochondrial apoptotic pathway was activated by GADD45B. Therefore, as a tumor suppressor gene, GADD45B could induce apoptosis in CRC cell lines. According to the results of RT-qPCR and IHC, we presumed that GADD45B might lose its normal functions and promote carcinogenesis in CRC tissues. This inconsistency of GADD45B functions between in vitro cellular-level and in vivo tissue-level may be due to complex micro-environment in human body and the resulting abnormal gene expression. The potential mechanism needs for further exploration.
Because the colon and rectum are important digestive organs, the intestinal mucosa can be easily damaged by various factors [29–31]. The human CRC-prone disorders may contribute to the risk of CRC through the accumulation of specific products resulting from DNA damage. In our study, we found that CRC had a high expression of GADD45B, which is involved in the regulation of cell growth and apoptosis. This result suggested that CRC cells might accumulate a certain amount of DNA damage, leading to dysfunction and over-expression of GADD45B. In this scenario, under chronic genotoxic stress, the failure to appropriately repair DNA damage and control growth may contribute to colorectal carcinogenesis.
In human solid tumors, DNA damage and repair defects that lead to tumorigenesis usually involve multiple factors. Defects in functional p53, p21 (Cdkn1a), or growth suppression genes, such as those of the GADD45 gene family, could be involved . p53 is the most commonly mutated gene in cancer, exerting its activity by binding DNA response elements and regulating the transcription of specific genes in response to various stimuli, thus directing cells toward cell cycle arrest or apoptosis . Because p53 functions mainly as a transcription factor, it is important to explore the genes regulated by p53 that contribute to the regulation of apoptosis. In this study, we investigated how the changes of GADD45B expression influence apoptosis-related proteins expression. Our results showed that transfection with pEGFP-N1-GADD45B in SW480 led to an increase in P53, Bax and cleavage of PARP, as well as a decrease in Bcl-2 and unactivated caspase-3. We postulated that GADD45B-induced apoptosis may be associated with the p53-Bax mitochondrial apoptotic pathway. As apoptotic pathways are frequently altered in tumor progression, proteins associated with this pathway may have potential as prognostic biomarkers. Like other researches, we have shown pro-survival function of Bcl-2, pro-apoptotic function of Bax, and elevated expression of P53 and caspase 3 in CRC tissues and ANCT [34–36]. Determination of the tumors ability to undergo apoptosis holds immense potential as a new prognostic marker for CRC.
Cancer can be viewed as the result of a succession of genetic changes during which a normal cell is transformed into a malignant one. Apoptosis is intimately connected with the elimination of potentially malignant cells, hyperplasia and tumor progression . The abundance of literature suggests that defects along apoptotic pathways play a crucial role in carcinogenesis. GADD45B, as a tumor suppressor potentially through the p53-mediated apoptotic pathways, is paradoxically overexpressed in CRC and as such may play an unappreciated role in tumorigenesis. Defects in this gene might have broken some kind of balance of pro-apoptotic and anti-apoptotic proteins contributing to evasion of apoptosis and carcinogenesis.
Several studies have reported that GADD45B was decreased in human hepatocellular carcinoma (HCC) [8, 38]. Compared with the high levels of staining in colon cancer, breast cancer, prostate cancer, lymphoma, squamous cell carcinoma, and leiomyosarcoma, the under-expression of GADD45B was specific to liver cancer. As a novel pituitary tumor suppressor, microarray data showed the loss of GADD45B in gonadotrope tumors where its repression modulates cell proliferation, survival, and tumorigenicity . The hierarchical clustering of 19 pancreatic neuroendocrine tumors (PNETs) revealed that GADD45B was one of the most highly up-regulated genes in the malignant group of PNETs . Therefore, there is still controversy regarding the expression and function of GADD45B in various tumors. Abnormal expression of GADD45B is similar to tumor suppressor p53 and phosphatase and tensin homologue (PTEN), both of which showing high expression in some head and neck cancers, and mammary cancer [40–42]. Further investigations are needed to determine the underlying mechanisms behind the dysfunction of GADD45B and CRC tumorigenesis.