CC is a commonly diagnosed malignant gynecological tumor. Globally, CC ranks second only to breast cancer in morbidity, while ranking first in incidence and mortality among females from regions with lower human development index . CC has a high potential for prevention, early detection, and cure . The dual prevention strategies of HPV vaccination and CC screening have reduced CC-related incidence and mortality; nevertheless, its incidence still ranks fourth among female-specific cancers in less developed regions of the world [28–30]. HPV-16 and HPV-18 together account for 70–75% of all CCs and 40–60% of CC precursor lesions . Although CC is preventable and curable in the very early stages of the disease, invasion and metastasis remain the main reasons for failure and death of treatment among patients with CC . Therefore, understanding the cellular and molecular mechanisms underlying CC-associated immune modulation is a prerequisite for the development of immunotherapy-based approaches to treat this deadly malignancy.
TIM-1 is preferentially expressed in Th2 cells, where it induces T cell activation and inhibits the development of peripheral tolerance [9, 33]. The expression of TIM-1 can convert a normal epithelial cell into a phagocyte, thus facilitating autophagy and the removal of apoptotic and necrotic cells [34, 35]. In cancer, the apoptotic program is compromised, leading to cell overgrowth and tumor formation . TIM-1 has been linked to immunomodulation, as polymorphisms in the human TIM-1 gene have been associated with various disorders, including autoimmune diseases, allergies, malignant tumors, and viral infections [7, 33]. Moreover, emerging evidence has shown that TIM-1 participates in the invasion and metastasis of tumor cells, and is involved in the occurrence and development of a variety of cancers [14, 18]. However, little is known about the biological roles of TIM-1 in CC.
In the present study, we found that TIM-1 is a mediator of the progression of CC. Furthermore, we discovered that TIM-1 plays a critical role in the malignant behavior of CC cells, as well as in the occurrence and development of CC. First, our study showed that TIM-1 expression is higher in CC tissues than that in CIN and normal cervical tissues, and is negatively correlated with vaginal involvement. The immunohistochemistry findings indicated that TIM-1 expression in CC tissues was markedly up-regulated compared to in CIN and normal cervical tissues. Our results also showed that TIM-1 expression in cervical cancer tissues was related to histological classification, FIGO stage, depth of infiltration, lymph node metastasis, nerve invasion, or vascular invasion compared to CIN tissues or normal cervical tissues. However, we found that the expression of TIM-1 in CC tissues showed no correlation with age, histology classification, degree of differentiation, FIGO stage, tumor diameter, depth of infiltration, lymph node metastasis, nerve invasion, or vascular invasion in CC cases; and there were no significant differences between HSIL and LSIL. This may have been due tothe small number of samples, and these findings may need to be further confirmed by large samples in future studies.
We also measured TIM-1 mRNA and protein levels in CC cell lines with different HPV phenotypes, and found that TIM-1 expression was low in HeLa (HPV-18-positive) and SiHa (HPV-16-positive) cells, and relatively high in C-33 A cells (HPV-negative). It should be noted that TIM-1 is expressed in three isoforms, and the intracellular TIM-1 protein may be secreted into the extracellular domain, indicating that intracellular protein levels do not increase as significantly as mRNA levels .
We also provided evidence that TIM-1 can promote the proliferation of CC cells, both in vitro and in vivo. CCK-8 and colony formation assays revealed that TIM-1 overexpression can greatly enhance the proliferative capacity of CC cells. Furthermore, we found that there were different mitotic abilities, and a reduction in the G1 population and an increase in the S population in both HeLa and SiHa cells overexpressing TIM-1, while an increase in the G2/M population was observed in HeLa cells overexpressing TIM-1. The role of p53 in the regulation of cell-cycle progression through the G1/S phase is well documented [38, 39]. Cyclins are divided into two groups, namely G1/S cyclins, which are essential for the control of the G1-to-S phase transition of the cell cycle and G2/M cyclins, which regulate the G2-to-M-phase transition [40, 41]. Cyclin D1 is a major positive regulator of the G1/S phase transition and therefore also of cell cycle progression, and its dysregulation can lead to abnormal cell growth and angiogenesis, as well as resistance to apoptosis [38, 41, 42]. In this study, the decrease in the proportion of cells in the G1 phase was accompanied by a decrease in p53 levels and an increase in cyclin D1 levels, a major downstream effector of p53 in mediating G1/S phase cell cycle transition [38, 42, 43]. These data suggested that TIM-1-induced cell cycle progression was correlated with down-regulation of p53 and up-regulation of cyclin D1 expression levels. We also showed that TIM-1 promotes CC progression in vivo in a mouse xenograft tumor model. Together, the above results suggest that TIM-1 may regulate CC cell proliferation and cell cycle progression via the p53 signaling pathway.
In addition to enhanced proliferation, resistance to apoptosis is also a hallmark of cancer cells. Apoptosis plays an important role in both carcinogenesis and cancer treatment . We also found that overexpression of TIM-1 reduced the proportions of apoptotic HeLa and SiHa cells. The p53 gene functions as a tumor suppressor through its proapoptotic activities [39, 44]. Generally, the regulation of this form of cell death involves the activity of the p53 and Bcl-2 family genes . The proteins of the Bcl-2 family are known to be important regulators of apoptosis and involve anti- or pro-apoptotic members, such as Bcl-2 and BAX [36, 44]. p53 has been reported to positively regulate BAX expression and negatively regulate Bcl-2 transcription . Our findings demonstrated that up-regulation of TIM-1 significantly decreased the expression of p53 and BAX, and increased that of Bcl-2 and mTOR in CC cells. mTOR is involved in cell growth, proliferation, apoptosis, and many other biological processes mainly through the PI3K/AKT/mTOR signaling pathway . The mTOR pathway also reduces the BAX/Bcl-2 ratio . Taken together, our study showed that p53 and BAX activities were reduced, while those of mTOR and Bcl-2 increased. These results suggest that up-regulation of TIM-1 inhibited CC cell apoptosis through the regulation of p53 and mTOR activation, thus modulating CC pathogenesis and progression.
An increased migratory capability is another prominent characteristic of cancer cells. Wound healing and Transwell migration assays showed that both the migration capacities and migration rates of HeLa and SiHa cells were enhanced with TIM-1 overexpression. MMP-2 belongs to the matrix metalloproteinase family of proteins that can hydrolyze the extracellular matrix and promote invasion of tumor cells . Furthermore, VEGF is the strongest and most specific tumor angiogenesis-promoting factor currently known and can promote tumor angiogenesis, proliferation, and migration . In the present study, we found that the levels of MMP-2 and VEGF were increased in HeLa and SiHa cells that overexpression TIM-1. Taken together, these observations suggest that TIM-1 can enhance the migratory capacity of CC cells.
Tumor cell invasion and metastasis are among the main reasons for the poor prognosis of patients with CC . We found that overexpression of TIM-1 enhanced the invasive ability of CC cells. EMT is closely related to tumor invasion and metastasis  and is necessary for tumor cells to leave the site of the primary tumor, invade surrounding tissues, and establish distant metastases. Key effector molecules of EMT include E-cadherin, N-cadherin, and vimentin [22, 50]. N-cadherin and vimentin are two important mesenchymal cell markers and are highly expressed in mesenchymal cells ; the loss of E-cadherin is considered a vital event in EMT, a process that is regulated by several transcription factors, including Snail, Slug, and members of the Zeb family. In this study, western blotting revealed that TIM-1 overexpression decreased the expression of E-cadherin while simultaneously increasing that of N-cadherin, vimentin, Snail1, MMP-2, and VEGF. Taken together, our results suggest that TIM-1 can enhance the migratory and invasive abilities of CC cells, as well as induce their EMT by up-regulation of N-cadherin, vimentin, Snail1, MMP-2, and VEGF, and down-regulation of E-cadherin.
The PI3K/AKT/mTOR pathway is known to play a central role in the growth and proliferation of CC cells[43, 53, 54]. Studies have showed that TIM-1-mediated T cell stimulation can recruit the p85 adapter subunit of PI3K, thus promoting T cell activation via the PI3K pathway . Recent, studies showed that TIM-1 knockdown inhibited the biological behaviors of NSCLC and glioma cells through the inactivation of PI3K/Akt pathway [14, 16]. Consequently, we investigated whether the PI3K/AKT/mTOR pathway was involved in the TIM-1-mediated regulation of CC cell functions. Western blotting analysis revealed that the expression levels of PI3K, p-AKT, and mTOR were significantly higher in the TIM-1-overexpressing group than in the MOCK and NC groups, whereas the total AKT protein levels remained unchanged. This demonstrated that TIM-1 can promote the growth and progression of CC by activating the PI3K/AKT pathway, and these effects were positively associated with the expression of cyclin D1, Snail1, N-cadherin, vimentin, Bcl-2, MMP-2, mTOR, and VEGF, and negatively associated with the expression of p53, E-cadherin, and BAX. The PI3K/AKT pathway has recently been reported to inhibit the transcriptional activity of p53 as well as its pro-apoptotic functions [25, 26].
It has been shown that p53 can regulate the expression of MMP-2 . p53 signaling can also affect Snail, Slug, and Twist levels to negatively regulate EMT , while loss of function or mutation of p53 has been reported to promote cancer cell EMT by derepressing Snail1 protein expression and activity . Furthermore, there is growing evidence that p53 can negatively regulate VEGF expression . As mentioned above, TIM-1 influenced the p53/cyclin D1-mediated regulation of the CC cell cycle and proliferation, and inhibited apoptosis through its regulatory effects on p53 expression and mTOR activation. Taken together, the results of our study and those of the above-mentioned studies demonstrate that TIM-1 is capable of enhancing the proliferative, invasive, and metastatic potential of CC cells, while also inhibiting their apoptosis, through activation of the PI3K/AKT/p53 and PI3K/AKT/mTOR signaling pathways (Figure 9).
In conclusion, we provide convincing evidence that TIM-1 expression is markedly increased in CC tissues compared to normal and CIN tissues, and that TIM-1 is also expressed in CC cell lines (C-33 A, HeLa, and SiHa) with different HPV phenotypes. Furthermore, we provided novel evidence that TIM-1 overexpression induced EMT, promoted cell migration and invasion, and inhibited cell apoptosis in CC through modulation of the PI3K/AKT/p53 and PI3K/AKT/mTOR signaling pathways. These results suggest that TIM-1 may play an important role in the occurrence and progression of CC, and may be a valuable biomarker and a potential therapeutic target for CC treatment.