Lung cancer is the leading cause of cancer death worldwide, and most of patients with lung cancer are non-small cell lung cancer (NSCLC). NSCLC is still very difficult to treat. Skp2 expression is identified to be a useful diagnosis marker of NSCLC patients. Skp2 participates in many cell processes in tumors, such as cell cycle, proliferation, metastasis, and apoptosis[6–8]. Since most of the Skp2 substrates are tumor suppressor proteins, it can be asserted that Skp2 functions as an oncogene[8, 31, 32]. AAA-237, a small molecule Skp2 inhibitor with methylene hydrazide skeleton, was firstly synthesized by our group. AAA-237 was found to have a significant inhibitory effect on different kinds of cancer cells, especially on NSCLC cells. In this study, we further investigated the effect of AAA-237 on NSCLC in vitro and in vivo and explored the underlying molecular mechanisms in details. Our results showed that AAA-237 inhibited proliferation, decreased DNA synthesis and inhibited colony formation of A549 and H1299 cells. AAA-237 also inhibited the migration and invasion by regulating Skp2/Twist axis. AAA-237 induced arresting of the cell cycle at the G0/G1 phase, which is regulated by Skp2-Cip/Kip and PI3K/Akt-FOXO1 signaling pathway. Furthermore, we found that treatment of high concentration AAA-237 could induce apoptosis in a mitochondria dependent manner, however, treatment of low concentration AAA-237 for long-term could induce senescence through DNA damage and exhibited a SASP phenotype. The same mechanism was also found in A549 xenograft nude mice model, AAA-237 dose-dependently inhibited the growth of tumor by inducing apoptosis and senescence. Taken together, these results suggest that Skp2 inhibitor AAA-237 could be used as apoptosis and senescence inducer with potential to be a novel anti-NSCLC agent.
Metastasis is the leading cause of mortality in human NSCLC. Tumor metastasis is a complicated process and EMT plays an important role in this process[34, 35]. EMT is a process which cancer cells lose epithelial features, cell shape changes and cells activate genes that help to define mesenchymal phenotype, what leads to an increased cell motility and dissemination of tumor to distant metastatic sites. When EMT occurs, the cells will lose the expression of E-cadherin, acquire the expression of N-cadherin and Vimentin and increase the expression of MMPs[35, 37]. The process of EMT is controlled by EMT-related transcription factor, such as twist [36, 38, 39]. Here, our results showed that the expression of E-cadherin was introduced and expression of MMP7, N-cadherin and Vimentin was reduced in NSCLC cells treated by AAA-237 through inhibiting the expression of transcription factor twist. AAA-237 may inhibit the migration and invasion of NSCLC by regulating Skp2/Twist axis.
The most famous substrates of Skp2 are the cell cycle inhibitors p21 Cip1 and p27 Kip1. Overexpression of Skp2 induced p21 Cip1 and p27 Kip1 ubiquitination and continuous degradation of p21 Cip1 and p27 Kip1, while low levels of Skp2 reduced their degradation. In our study, we found that after treatment of AAA-237, the expression of p21 Cip1 and p27 Kip1 in A549 and H1299 cells was increased. In addition, we enriched and analyzed the differential genes obtained from RNA-seq and found that the FOXO1 signaling pathway was involved in the regulation of cell cycle after treatment of AAA-237. It is known that the prominent role of transcription factor FOXO1 in arresting of cell cycle depended on the expression of its targets, such as p15 INK4B, p21 Cip1 and p27 Kip1 [40, 41], which initiated significant inhibition of cell proliferation. In our study, we further verified the regulatory effect of FOXO1 signaling pathway on arresting of cell cycle after AAA-237 treatment and demonstrated that AAA-237 increased the expression of nucleus FOXO1 by inhibiting the activation of PI3K/Akt signaling pathway, therefore improved its transcriptional activity and increased the expression of p15 INK4B, p21 Cip1 and p27 Kip1 at the transcription level and induced arrest of cell cycle.
One important finding in the study is that treatment of low concentration AAA-237 in cells for long time induced senescence of NSCLC cells. Senescence is an inevitable phenomenon in the survival and growth of organisms . DNA damage response (DDR) signaling is critical for DNA damage-induced senescence. The increased activity of γH2AX, the phosphorylation form of histone H2A member X at serine 139, in response to DDR critically regulates cellular senescence and has been considered as a biomarker of cellular senescence. Studies have revealed that Skp2 is also involved in DDR mechanisms[28, 29].In our study, we found that AAA-237 inhibited the expression of Skp2, which lead to the DNA damage. Accumulation of nuclear DNA damage stimulates the activity of γH2AX, and then triggers the initiation of cellular senescence.
Senescent cells are usually characterized by arrest of cell cycle and the production of SASP phenotype . The tumor suppressor protein p16 Ink4a is often transcriptionally activated in cells undergoing senescence and is one of the main regulators of this program. In senescent cells, arrest of cell cycle correlates with an augmented level of p16 INK4a . In our study, we found that the expression level of p16 Ink4a was increased after treatment of AAA-237, which indicated that DNA damage caused by AAA-237, may activate p16 INK4a. The p16 INK4a mediated senescence through activating p21 CIP1, which induced the arrest of cell cycle at G0/G1 phase.
In contrast to apoptotic cells, senescent cells are metabolically active and may affect other cells via secretion of multiple inflammatory proteins, described as the SASP. SASP molecules including CCL2, CCL5, IL7, and IL15 can recruit and activate innate and acquired immune cells, such as NK cells, monocytes, macrophages and T cells. In addition, these cells can transmit signals to immune cells, such as neutrophils, B cells, and dendritic cells after being activated, thereby establishing an immune activation microenvironment. Therefore, the induction of senescence can not only induce arrest of cell cycle, but also mobilize immune surveillance in tumor. Induction of senescence of cancer cell become a promising method in current cancer treatment. In our study, we demonstrated that AAA-237 induced senescence of NSCLC cells. AAA-237 increased the activity of SA-β-Gal and the expression of senescence markers γH2A.X and p16. The secretion of SASP-associated cytokines including IL-1A, IL-6, CXCL1, CXCL8, MMP1 and MMP10 was increased in cells treated by AAA-237 induced, which further promoted the infiltration levels of CD8+ T cells, monocytes and macrophages in lung cancer.
However, the accumulation of senescent cells in the tumor microenvironment would largely lead to tumor metastasis. The pro-inflammatory SASP produced by senescent cells greatly promotes the proliferation and migration of tumor cells. Therefore, long-existing senescent cells have brought challenges to the application of tumor senescence therapy. Senolytic drug is a kind of drug that selectively kills senescent cells[51, 52] . It can effectively eliminate senescent cells in the human body to reduce its harmful effects and maintain normal functions of the body. Thus, combining senescence-inducing therapy with senolytic drugs could be beneficial to short and long-term outcomes in cancer patients[42, 54]. Here, we demonstrated that AAA-237 induced senescence in some cases. Senolytic drug quercetin could selectively eliminate senescent A549 and H1299 cells. Thus, combining AAA-237-induced senescence therapy with senolytic drug quercetin could be beneficial to NSCLC patients.