The Study Of Anti-proliferative And Invasive Effects Of Inhibitor Of Growth 5 On Lung Cancer Cells And The Underlying Mechanisms

Lung cancer has become one of the leading causes of cancer death worldwide with increasing morbidity and mortality. Most lung cancer patients are diagnosed in late phase of cancer, with more than 50% of patients losing opportunity for operation. The effects of current treatments to most lung cancer patients are limited. Therefore, new therapeutic strategies are in urgent need. Researches have revealed that the initiation, development and metastasis of lung cancer are processes involving multiple genes and steps, among which the inactivation of tumor suppressors plays a crucial role in cancer growth and invasion.Members of Inhibitor of Growth belong to the candidate tumor suppressor family, including ING1, ING2, ING3, ING4 and ING5. Studies have shown that ING family proteins participate in many biological processes including regulation of cell growth, apoptosis and DNA damage repair. As a new member of the family, ING5 was first reported in 2003 that it interacted with P53 and activate the transactivation of P53, leading to cell cycle arrest and apoptosis. In addition, ING5 was component of two histone acetyltransferase(HAT)complexes and was involved in histone modification and chromatin remodeling. Thus, ING5 acts as a bridging molecule by connecting histones and HATs, suggesting that ING5 function as a tumor suppressor by regulating gene expression. Our previous studies have shown that ING5 interacts with INCA1, a cell cycle regulator, to inhibit proliferation of mouse embryonic fibroblast(MEF)cells and promote anti-CD95 antibody-induced apoptosis. However, these effects were not observed in INCA1 knockout MEF cells, suggesting that the anti-proliferative effects of ING5 might depends on its interaction with other proteins. However, how ING5 function as a tumor suppressor and what effects of ING5 in lung cancer progression are not clear.The malignant behavior of cancer includes activities of proliferation, migration and invasion, which are closely correlated with clinical tumor growth and metastasis. In the current study, based on stably established lung cancer cell lines with ING5 overexpression or knockdown, we would investigate the effects of ING5 on lung cancer proliferation and invasion and the possible mechanisms, in vitro and in vivo.MethodsExperiment oneLung cancer A549 and H1299 cells were used to construct A549-ING5 with ING5 overexpression and H1299-sh ING5 cells with ING5 knockdown via lentiviral infection. Proliferation assay and colony formation experiment were performed to study the effects of ING5 on lung cancer cell proliferation. Wound-healing assay, trans-well migration and invasion assay were carried out to study the effects of ING5 on lung cancer cell invasive ability.Experiment twoMale athymic nude mice(4 weeks old)were injected subcutaneously or through tail vein with A549-control cells or A549-ING5 cells, to establish mouse xenograft models. The effects of ING5 on tumor growth and metastaiss were evaluated in vivo.Experiment threeTo explore whether ING5 inhibited lung cancer cell invasive ability by suppressing epithelial-mesenchymal transition(EMT), cell morphological changes by ING5 overexpression in A549 cells were observed by inverted microscopy and scanning electronmicroscopy. The m RNA and protein levels of EMT-markers were further detected byq RT-PCR and Western Blot.ResultsExperiment one1. ING5 overexpression or knockdown cell lines were established. Compared with A549-control cells, ING5 expression was higher in A549-ING5 cells. ING5 expression was lower in H1299-sh ING5 cells compared with H1299-shcontrol cells. 2. ING5 overexpression significantly inhibited cell proliferation and colony formation abilities of A549 cells, whereas these effects were promoted by ING5 knock-down in H1299 cells. 3. ING5 overexpression inhibited migration of A549 cells as assessed by wound-healing assay and trans-well migration assay. The invasive ability of A549 cells was also inhibited by ING5 overexpression. As a confirmation, ING5 knock-down promoted cell migration and invasion in H1299 cells.Experiment two1. Results from mouse xenograft models showed that ING5 overexpression inhibited tumor formation and tumor growth. Tumor volumes were significantly decreased in A549-ING5 cells compared with A549-control cells. 2. Results from intravenous mouse xenograft models showed that all mice that were injected with A549-control cells developed multiple tumors in bilateral lungs, whereas 40% mice that were injected with A549-ING5 cells had lung tumors which were much less in quantity than control lung tumorsExperiment three1. ING5 overexpression in A549 cells displayed more cobblestone-like cell morphology and cluster formation, compared with A549 control cells which exhibited a more mesenchymal phenotype with elongated shape. Under scanning electron microscopy, the number of cell filopodia-like protrusions reduced significantly in ING5 over-expressing A549 cells.2. q RT-PCR and Western Blot results revealed that ING5 increased E-cadherin, and decreased N-cadherin, Snail and Slug at both m RNA and protein levels.Conclusions1. ING5 inhibits lung cancer cell proliferation, migration and invasion abilities in vitro. 2. ING5 suppresses growth and metastasis of lung cancer cells in xenograft mouse models. 3. ING5 inhibtis lung cancer cell aggressiveness via preventing EMT.