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Supplementary Fig. 1 Confirmation of cell synchronisation at the G1/S boundary. H1299 cells with or without prior transfection with plasmids expressing GFP-tagged CDC6 or GFP alone were transfected twice with the indicated miRNA mimics or control RNA. Untreated cells and those treated with the transfection reagent alone were used for comparison. Some 48 hrs after the final transfection, cells were synchronised at the G1/S boundary with mimosine, and the cell synchrony was determined by flow cytometry. Supplementary Fig. 2 MicroRNA-26a/b inhibit pre-replicative complex formation and cell cycle progression by suppressing CDC6 in human lung cancer cells. (A) SPAC cells with or without prior transfection with plasmids expressing GFP-tagged CDC6 or GFP alone were transfected twice with the indicated miRNA mimics or control RNA. Untreated cells and those treated with the transfection reagent alone were used for comparison. Some 48 hrs post transfection, cells were synchronized at the G1/S boundary with mimosine, and chromatin binding assay was performed with an aliquot of each cell sample. (B) The remaining cells were then released into fresh medium and harvested at different time points for FACS analysis.

Cancer is characterized by mutations, genome rearrangements, epigenetic changes, and altered gene expression that enhance cell proliferation, invasion, and metastasis. To accommodate deregulated cellular proliferation, many DNA replication-initiation proteins are overexpressed in human cancers. However, the mechanism that represses the expression of these proteins in normal cells and the cellular changes that result in their overexpression are largely unknown. One possible mechanism is through miRNA expression differences. Here, it is demonstrated that miR26a and miR26b inhibit replication licensing and the proliferation, migration, and invasion of lung cancer cells by targeting CDC6. Importantly, miR26a/b expression is significantly decreased in human lung cancer tissue specimens compared with the paired adjacent normal tissues, and miR26a/b downregulation and the consequential upregulation of CDC6 are associated with poorer prognosis of patients with lung cancer. These results indicate that miR26a/b repress replication licensing and tumorigenesis by targeting CDC6.Implications: The current study suggests that miR26a, miR26b, and CDC6 and factors regulating their expression represent potential cancer diagnostic and prognostic markers as well as anticancer targets.Visual Overview: http://mcr.aacrjournals.org/content/12/11/1535/F1.large.jpg.Mol Cancer Res; 12(11); 1535–46. ©2014 AACR.

Cancer is characterized by mutations, genome rearrangements, epigenetic changes, and altered gene expression that enhance cell proliferation, invasion, and metastasis. To accommodate deregulated cellular proliferation, many DNA replication-initiation proteins are overexpressed in human cancers. However, the mechanism that represses the expression of these proteins in normal cells and the cellular changes that result in their overexpression are largely unknown. One possible mechanism is through miRNA expression differences. Here, it is demonstrated that miR26a and miR26b inhibit replication licensing and the proliferation, migration, and invasion of lung cancer cells by targeting CDC6. Importantly, miR26a/b expression is significantly decreased in human lung cancer tissue specimens compared with the paired adjacent normal tissues, and miR26a/b downregulation and the consequential upregulation of CDC6 are associated with poorer prognosis of patients with lung cancer. These results indicate that miR26a/b repress replication licensing and tumorigenesis by targeting CDC6. Implications: The current study suggests that miR26a, miR26b, and CDC6 and factors regulating their expression represent potential cancer diagnostic and prognostic markers as well as anticancer targets. Visual Overview: http://mcr.aacrjournals.org/content/12/11/1535/F1.large.jpg. Mol Cancer Res; 12(11); 1535–46. ©2014 AACR.

Pancreatic cancer is characterized by its extremely poor prognosis and it is the 4th leading cancer death in both men and women. The limited scientific knowledge and lack of effective treatment of this cancer are critically urging to an extensive research development in the field. Emerging evidence shows that pancreatic cancer cells exhibit aberrant activation of class I phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling. To understand the molecular mechanism underlying the relationship between signaling activation and pancreatic cancer cell growth, we investigated the anti-cancer effect of a novel dual inhibitor of PI3K/mTOR, PI-103 {3-(4-(4-Morpholinyl)pyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl)phenol}, on pancreatic cancer cells. The cell proliferation experiment assessed by MTT assay showed that PI-103 (0.1-10μM) effectively inhibits the growth of p53-mutant pancreatic cancer cells including Panc-1, MIA paca-2, BxPC-3 and AsPC-1 cells. The growth inhibitory effect of PI-103 was further supported by clonogenic assay showing a complete blockage of colony formation of Panc-1 cells after 7 days treatment. Our results demonstrated that PI-103 induces autophagy in Panc-1 cells characterized by a significant increase of the conversion of LC3-1 to LC3-II, which was then attenuated by an autophagy inhibitor, 3-methyladenine (3-MA), and augmented using a thiol proteinase inhibitor, E-64d, by blocking the degradation of autolysosomes. Knockdown of the expression of beclin1 with siRNA attenuated LC3 conversion and the growth inhibitory effect of PI-103 as well as colony formation, suggesting a possible linkage of autophagy and cell growth inhibition. Furthermore, cell cycle analysis and apoptotic assay with Annexin V staining showed that PI-103 induced G0/G1 arrest significantly without an effective induction of apoptosis. Western blotting revealed that PI-103 regulated the expression of cell-cycle relevant proteins, including increased p27 and decreased CDK1 and cyclin B. In addition to the effect of PI-103 on autophagy and cell cycle arrest, to our surprise, a striking appearance of cytoplasmic vacuoles ranging from 1-4μm in diameter was observed in the cells treated with PI-103 at as early as 3 hrs. The origin of the vacuoles was explored using the markers with GFP-LAMP1 for late endosome and lysosome, and DsRed-FYVE-labeling for early endosome, respectively. The results showed the large vacuoles are LAMP1-positive lysosomes or late endosomes, but not early endosomes, which is leading to future studies. Taken altogether, our data show that PI-103 is a potent compound for inhibiting pancreatic cancer cell growth, and the anti-cancer effect may operate through the induction of autophagy and cell cycle arrest. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2497.

Loss of cellular zinc accumulation is one of the characteristic features of prostate cancer, compared to the high zinc level in normal prostate epithelial cells. We previously demonstrated that zinc exposure induces apoptosis in human prostate malignant cells via a mitochondrial pathway in both in vitro and in vivo models, suggesting an important role of zinc homeostasis in growth control of prostate cancer. However, it remains unclear whether the apoptotic effect of zinc via the targeting of mitochondrial function is the only mechanism leading to growth inhibition in prostate cancer. Recent progress in autophagy indicates that this evolutionarily conserved catabolic process mediates the degradation of cellular components including damaged mitochondria and could promote either cell survival or cell death. The goal of this study was to ascertain the zinc effect on autophagy and its relationship to prostate cancer cell growth. Zinc induced autophagy was characterized in human prostate cancer PC-3 cells by Western blot analysis of LC-3, sub-cellular localization of ectopically expressed GFP-LC3, and transmission electron microscopy (TEM). Reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) were measured by fluorescence labeling and FACS analysis. Cell viability was assessed by MTT assay. Our results showed that zinc (15µM) significantly increased the conversion of LC3-I to LC3-II in a time-dependent manner (2.5 fold increase at 24 hrs), and induced the formation of punctuate GFP-LC3 structures in the cytoplasm as revealed by a fluorescence microscopy. Blockade of autophagy flux by inhibiting autolysosomal degradation using a thiol proteinase inhibitor, E-64d, greatly augmented zinc-induced LC3-II conversion. Electron microscopy observation revealed typical morphologic ultra-structures for autophagy including the formation of autophagosomes and autophagosome-related cytoplasmic structures. Inhibition of class III PI3K by 3-MA (3-methyladenine) attenuated zinc-induced autophagy and the cell death as quantified by MTT assay. Furthermore, zinc treatment led to a striking increase in ROS production and alteration in MMP, which were then abrogated by anti-oxidative N-acetyl-cysteine (NAC) associated with a significant suppression of zinc-induced autophagy and cell death. Our data indicate that zinc exposure induces autophagy in prostate malignant cells and leads to autophagy-related cell death, which is most likely due to direct targeting of mitochondrial function by zinc. Thus, we have provided evidence that the inhibitory role of zinc in prostate cancer cell growth may operate through the convergence of apoptosis and autophagy pathways, both of which are downstream from mitochondrial stress signaling leading to nuclear degradation and cellular metabolic alteration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4856.