Various known IRXs have been found in human tumors, and are known to participate in suppressing or promoting cancer in various different tumor backgrounds. However, the roles IRXs play in NSCLC have rarely been reported. This study examined for the first time the expression of IRXs in NSCLC and their relationship with clinicopathological factors and prognosis from the level of transcription and protein expression. The results from this research could provide indications for more accurate diagnosis and prognosis predictions of NSCLC.
At present, IRX1 has been reported to act as a tumor suppressor gene in gastric cancer, bladder cancer, and head and neck squamous cell carcinoma（Bennett, et al.，2010，Kitchen, et al.，2016，Liu, et al.，2018）.It has also been reported to promote cancer in human glioma and osteosarcoma（Lu, Song, Tang, Zou, Han, Zhao, Yong, Yin, Xu, Xie, Kang, Lam, Yang, Shen, Wang，2015，P. Zhang, et al.，2018）. In NSCLC, the methylation level of IRX1 is thought to be positively correlated with the prognosis of patients with LUSC（Gao, Zhuang, Zhou, Ma, Zhao, Liu, Liu, Li, Feng, Sun，2019）, hypomethylation of IRX1 can promote tumor lung metastasis through CXCL14/NF-κB signaling（Lu, Song, Tang, Zou, Han, Zhao, Yong, Yin, Xu, Xie, Kang, Lam, Yang, Shen, Wang，2015）. In gastric cancer cells tudies, increasing IRX1 expression is able to inhibit peritoneal expansion and lung metastasis through anti-vasculogenic mimicry mechanisms（Jiang, et al.，2011）. In head and neck squamous cell carcinoma, IRX1 inhibits mitotic activity and promotes apoptosis through the TGF-β pathway（Bennett, et al.，2009）. In this study, expression of IRX1 mRNA in both LUAD and LUSC was significantly lower than that seen in normal tissues. We also found that patients with high expression of IRX1 had a higher ten years survival rate, and that this increased expression was negatively correlated with tumor stage, though there was no significant correlation between IRX1 and the prognosis or tumor stage of patients with LUSC.
Current research on IRX2 shows that it regulates MMP-2 and MMP-9 by activating the AKT pathway to promote tumor migration and invasion in osteosarcoma（Liu, et al.，2015，Liu, et al.，2014）. In soft tissue sarcoma, IRX2 affects development through the Wnt pathway（Adamowicz, et al.，2006）. Loss of IRX2 expression can also lead to early hematogenous spread in breast cancer（Werner, et al.，2015）. In NSCLC, it has been reported that methylation of IRX2 CpG islands in LUAD may be related to malignancy （Rauch, et al.，2012）. In this study it was found that IRX2, similar to IRX1, had low expression in both LUAD and LUSC cancer tissues, it is interesting that when patient’s prognoses were analyzed, LUAD patients with high IRX2 expression had higher survival rates, while LUSC patients with similar expression of IRX2 had lower rates of survival. Immunohistochemical information indicated that positive expression of IRX2 in LUSC was localized inside the nucleus, while mainly in cytoplasm in LUAD. It has been reported that IRX1is localized in the cytoplasm in normal brain tissue and low-grade gliomas, but was found in the nucleus in higher grade gliomas （Zhang, Liu, Xu, Wang, Cheng, Jin, Wang, Yang, Liu, Zhang, Tu，2018）. IRX5 is able to promote cancer cell proliferation through cyclinD1 in LUAD, and immunohistochemical data shows that it is localized in the nucleus in LUAD（Zhang, Qu, Ma, Zhou, Wang, Zhao, Zhang, Zhang, Wang, Zhang, Yu, Sun, Gao, Cheng, Guo, Huang, Zhou，2018）. From this, we can speculate that the effect of IRXs on the tumor process is related to cell localization, such that expression of IRX2 in the nucleus of LUSC cells determines a poor prognosis.
Studies of IRX3 in nephroblastoma have shown that it can inhibit tumor growth through the classic Wnt/β-catenin pathway（Holmquist Mengelbier, et al.，2019）. There are also reports that IRX3 interacts with the NOTCH pathway and Rho signaling during the formation of renal tumors（Mengelbier, et al.，2010，Scarlett, et al.，2015）. IRX3, as the target of mir-377, can also promote the occurrence of liver cancer（Wang, et al.，2016）, however, there is no related research on the role of IRX3 in lung cancer. Here, the expression of IRX3 in LUAD tumor tissue was found to be higher than that in normal tissue, while expression in tumor tissue of LUSC patients was slightly lower. When analyzing the relationship of expression with tumor stage, the expression of IRX3 in LUSC patients was significantly different between tumor stages. However, prognostic statistics show that IRX3 was positively correlated with the prognosis of LUAD patients, which leads to the opposite conclusion of the expression pattern data. In order to determine why these differences exist, the composition of the IRX3 protein structure was examined (Supplementary Figure1), and it was found that the structure of IRX3 and IRX1 are very similar, both have a homeobox TALE-type domain and a homeobox KN domain in the same location. The difference between the two is that the lenghth of IRX3 protein is 501aa, while IRX1 is 480aa, a leucine to proline change exists in the 422nd amino acid of IRX3 protein, along with a glutamine to histidine change at the 479th. From this, we can speculate that the different roles of IRX1 and IRX3 in NSCLC are not related to their homeobox TALE-type and homeobox KN domains, but should be related to their different N-terminal structures, though the detailed mechanism behind this differential behavior must be studied further
IRX4 has been reported to inhibit the growth of prostate cancer cells by interacting with vitamin D receptors (VDR)（Nguyen, et al.，2012）. Differential gene analysis of NSCLC showed that IRX4 may also act as a tumor-promoting gene in NSCLC（Zhang, Qu, Ma, Zhou, Wang, Zhao, Zhang, Zhang, Wang, Zhang, Yu, Sun, Gao, Cheng, Guo, Huang, Zhou，2018）. Here, IRX4 was scarcely expressed in LUAD patients, but was expressed significantly higher in tumor tissues of LUSC patients. Interestingly, the expression of IRX4 was correlated to a poor prognosis in LUAD, but was not significantly correlated with the prognosis in LUSC. Therefore, although the expression of IRX4 in LUAD patients is low, it may still act as a good prognostic indicator. It is important to note, however, that the above results were all based on mRNA expression, as there was a lack of protein expression information available, because of this, the expression and function of IRX4 in NSCLC still needs further study.
IRX5 can inhibit tumor development via the Hippo and non-classical Wnt pathways in nephroblastoma（Holmquist Mengelbier, Lindell-Munther, Yasui, Jansson, Esfandyari, Karlsson, Lau, Hui, Bexell, Hopyan, Gisselsson，2019）. In prostate cancer, IRX5 plays a cancer-promoting role, and its deletion can cause P21 increase, G2/M phase cell arrest, induce apoptosis, and promote P53 expression（Myrthue, et al.，2008）. IRX5 promotes cell migration through the RhoA/Rock1 pathway in colon cancer（Zhu, et al.，2019）, and has also been reported to promote hepatocarcinogenesis（Zhu, et al.，2018）. In LUAD, IRX5 can cause G1 phase cell arrest and promote cell proliferation through cyclinD1（Zhang, Qu, Ma, Zhou, Wang, Zhao, Zhang, Zhang, Wang, Zhang, Yu, Sun, Gao, Cheng, Guo, Huang, Zhou，2018）. In this study, the expression of IRX5 in LUAD tumor tissues was found to be higher than that in normal tissues, which is consistent with known reports. Interestingly, IRX5 expression patterns were significantly different from different tumor stages in LUSD patients, with a significant increase seen in stage IV patients, suggesting further that high expression of IRX5 indicates a malignant outcome with LUSC. IRX5 is similar in structure to IRX2, whose role in cancer is mostly related to cell migration and distant spread, so it is reasonable to speculate that high expression of IRX5 in patients with stage IV LUSC may be affected by migration invasion as well.
IRX6 has rarely been studied in tumors, it was reported, however, that IRX6 plays a role as a oncogene in colorectal cancer, and that its expression suggests a poor prognosis（Zuo, et al.，2019）. Here, IRX6 expression in tumor tissues of LUAD patients was found to be lower than that in normal tissues and patients with high expression had better prognoses, suggesting that it plays a tumor-suppressive role in LUAD. The known immunohistochemical data also shows that IRX6 is expressed in the cytoplasm in tumor cells of lung cancer patients, which is consistent with our previous conjectures, the members of the IRX family seem to play a suppressive role in cancer when located in the cytoplasm, but may play a role in promoting cancer when localized to the nucleus.
This study demonstrates important insights into the expression patterns and prognostic analysis of IRX members in NSCLC. We believe that in NSCLC, IRX1 and IRX2 play a tumor suppressive role in LUAD and may become prognostic indicators. IRX4 seems to have a carcinogenic role in LUAD, and may become a potential target for LUAD treatment. Differential localization of IRXs in lung cancer cells was also found to have opposing effects in terms of tumor development, the discovery of this phenomenon still needs more study in order to gain a deeper understanding of the mechanism of IRX action and the mechanism of nuclear access，which would be helpful in terms of understanding how to regulate these proteins to change the malignant biological behavior of tumor cells.