HMGB1 is a nuclear protein that is highly expressed in tumor cells. In the nucleus, HMGB1 binds specific chromatin DNA and plays an important role in DNA recombination, DNA repair, gene transcription regulation, cell differentiation, and other biological processes. In recent years, studies have revealed that mechanically damaged and necrotic cells can release HMGB1 from the nucleus into the surrounding environment. Once HMGB1 is secreted out of the cell, it can induce inflammation in the body and have a complex effect on the immune system, thereby promoting the occurrence of tumors. Understanding the mechanisms of HMGB1 in the development and progression of cancer may make way for a novel molecular targeted therapy for cancer. In this study, we aimed to investigate the effect of HMGB1 on the apoptosis and activation of tumor or immune cells, and explain the molecular mechanism of HMGB1 in lung cancer occurrence.
Firs of all, to verify the presence of HMGB1 protein in tumor and immune cells, we used western blot analysis to detect cell lysates without treatment and found that HMGB1 content in these cells was abundant. In patients, tumor cells often rupture due to the effects of drugs and self-apoptosis. Therefore, in this study, we used TCL to simulate tumor cell rupture to release HMGB1, and then studied the impact of HMGB1 releasing on nearby tumor and immune cells.
Next, on one hand, we used HMGB1 inhibitor to directly inhibit the expression of HMGB1 in Lewis lung cancer cells. On the other hand, we used TCL containing low HMGB1 to co-incubate with tumor cells, monocyte macrophages, and isolated mouse spleen cells. Then flow cytometry or CCK8 assay were used to detect the growth of various cells and the expression of CD69 on the surface of immune cells. For tumor cells, we found that inhibiting the expression of HMGB1 in Lewis lung cancer cells will lead to a decrease in cell proliferation and increase in apoptosis. In contrast, the removal of HMGB1 from the TCL can promote the proliferation of lung cancer cells, and inhibit apoptosis.
For immunocytes, inhibition of apoptosis was observed when the above TCL was applied to mouse macrophage cell line RAW264.7 and mouse spleen lymphocytes. After inhibiting HMGB1, the apoptotic induction of TCL on immune cell was weakened. This indicates that HMGB1 released by ruptured tumor cells can promote the apoptosis of macrophages and mouse spleen cells in vitro. In addition, we found that TCL contain normal HMGB1 promotes CD69 expression on immune cells, and TCL containing low HMGB1 can further upregulate CD69 expression on the surface of immune cells. CD69 is one of the earliest surface antigens expressed after immune cell activation. When expressed, it can be used as a costimulatory signal to promote further cell activation and proliferation [12]. Therefore, when macrophages and mouse spleen cells are stimulated by normal TCL, the number of CD69 + cells increase, which meaning TCL has a promotional effect on the activity of immune cells. After reducing HMGB1 expression, the number of CD69 + cells significantly increased. This shows that HMGB1 secreted by tumor cells has an inhibitory effect on the activity of immune cells [13].
Following that, to demonstrate activated immune cells have good immune activity, we used ELISA to detect the secretion of cytokines by immune cells. The results showed that in mouse spleen cells, the removal of HMGB1 from TCL increased IL-2 and IL-6 secretion in immune cells but IL-4 secretion did not change significantly. IL-2 and IL-4 are mainly produced by activated T cells. IL-2 mainly promotes the proliferation and differentiation of lymphocytes, in particular, the differentiation of CD8 + T cells, which in turn promotes the production of cellular immune responses and triggers antitumor immunity [14]. IL-4 exerts immunomodulatory effects on B cells, T cells, and macrophages, especially to stimulate B cell proliferation, synthesize and secrete antibodies, and eliminate pathogenic microorganisms [15]. Results of the IL-2 and IL-4 secretion indicate that decreasing the release of HMGB1 from ruptured tumor cells can significantly affect cellular immune responses, making it more conducive to the elimination of tumor cells. For macrophages, we detected high IL-6 and TNF-α levels in the low HMGB1 TCL group, suggesting that macrophages were activated when HMGB1 was inhibited. As a result of the increased secretion of cytokines IL-2, TNF-α, and IL-6, it confirmed that the removal of HMGB1 from TCL is favorable for the enhancement of immune cell activity to antitumor.
By combining flow cytometry analysis and cytokine detection, we found that HMGB1 expression in tumor can enhance the proliferation of tumor cells; however, when HMGB1 is released from the rupture tumor cells, it’s inhibitory effect on immune cells is observed. This shows that HMGB1 can promote the occurrence of tumors in two ways. First, HMGB1 directly acts as an “activating factor” in tumor cells to induce their division and proliferation, which is favorable for tumorigenesis. Second, HMGB1 enters the tumor microenvironment through ruptured tumor cells as an “inhibitor” and inhibits the activity of immune cells, rendering them unable to effectively eliminate tumor cells. Therefore, tumor cells orchestrate an “immune escape” which affects their growth, leading to the development of more tumors and even metastasis [16–18]. Notably, HMGB1 released through ruptured tumor cells can induce tumor cell apoptosis after acting on the cells themselves.
The effect of HMGB1 in TCL on tumor and immune cells is different; this may be related to the activation of different HMGB1 related signaling pathways in cells [19]. In this study, we focused on the changes in the NF-кB signaling pathway in Lewis lung cancer and immune cells.
We found that after the HMGB1 content in TCL decreased, the expression levels of NF-κB, Bcl-2, and MMP-9 proteins in the immune cells were all upregulated while Casp9 protein expression was downregulated. Bcl-2 is a classic antiapoptotic molecule [20] and MMP-9 is an important protein related to cell proliferation [21]. Casp9 expression is often closely related to the occurrence of cell apoptosis [22]. MMP-9 is located upstream of the NF-κB pathway, and it can act on Bcl-2 or Casp9 downstream to conduct cell activation or inhibition signals. Our results indicated that immune cells had no contact with HMGB1, they were less prone to apoptosis and their immune activity would be enhanced. It is also proving once again that HMGB1 released as a results of tumor cell necrosis and rupture has an inhibitory effect on immune cells, and this effect is produced by affecting signal transduction molecules such as Bcl-2, MMP-9, and Casp-9. In addition, we found that in low HMGB1 TCL group, the RAGE proteins expression of immune cells was downregulated. Because RAGE is a cell-surface receptor of HMGB1, we believe that HMGB1 regulates the activity of immune cells by binding to RAGE and inducing the NF-κB signaling pathway [25].
However, in contrast to HMGB1 released by tumor cell necrosis and rupture, HMGB1 expressed in tumor cells themselves can promote tumor cell proliferation through the NF-κB signaling pathway. This may be caused by the different chemical modifications in the molecular structure of HMGB1 in normal tumor cells as well as ruptured and necrotic tumor cells [23–24]. The specific reasons for these underlying mechanisms need to be further elucidated.
In summary, our research found that HMGB1 present in lung cancer cells can promote their own proliferation, while HMGB1 released as a result of tumor cell necrosis and rupture has an inhibitory effect on the activity of immune cells. Therefore, lung cancer cells can avoid attack by immune cells, which in turn enhances their ability to divide and proliferate. This immunological effect of HMGB1 is accomplished by affecting the NF-κB signaling pathway. In general, HMGB1 hinders the normal function of immune cells and promotes the occurrence of lung cancer.