| Rates of detection of prostate cancer (PCa) vary widely across the world, and PCa is the most common cancer in men in the Western countries. While improved early detection significantly decreased mortality, PCa still remains the second leading cause of cancer-related death in Western men. Although the incidence of prostate cancer was lower than the Western countries’, in recent years confirmed PCa, especially for advanced or metastatic PCa, represents an obviously rising trend in China with the aging and the diet habit changing.By2020, the incidence of PCa in China is rising to60-70/100,000. PCa has become one of the threats to men’s health in China.Features such as a high prevalence rate, a long latency period, and complicated pathogenesis have made PCa a desirable tumor to treat. Initially, PCa is androgen-dependent which depends largely on androgen receptor (AR) signaling for growth and maintenance. Androgen deprivation therapy has become the standard first-line treatment for hormone naive PCa, and approximately80%of patients undergo a "flare" response. Unfortunately, the development of hormone-refractory prostate cancer (HRPC) which associated with poor prognosis, high apoptosis-resistance and high mortality will be observed to occur within one or two years after androgen deprivation therapy. There are few efficacious treatment options available for curing, or even improving the survival and quality of life in patients with HRPC. Complicated factors and signals involves in the development of HRPC. Nevertheless, the underlie mechanism of PCa is still remains elusive. Therefore, novel strategies targeting the molecular basis of PCa progression and producing synergies of different signals are highly desirable.We present a large-scale, multi-omic interrogation on the alterations of genes in TCGA datasets, and identified a list of genetic disorders which is associated with malignant phenotypes such as invasion, metastasis, and drug resistance. We then took advantage of the profiles of Chinese Han PCa samples, and found AGR2overexpression was associated with the malignant phenotypes in Chinese Han PCa patients.Further study showed knockdown of AGR2caused DNA damage and resulted incellular senescence in PCa cells.Meanwhile we found natural product bisbibenzyls triggered DNA damage, leading to cellular senescence (at low concentration) and apoptotic cell death (at high concentration), which contributed to its antitumor effect in PCa cells.Part I Analysis of functional modules of AGR2based on PCa genomic data, and AGR2i-induced DNA damage is involved in regulation of cellular senescencePCa is the second most common cancer diagnosed in men worldwide. The prevalence of prostate cancer is growing, with an estimated1.1million men worldwide diagnosed in2012, accounting for15percent of the cancers diagnosed in men. In Stage IV prostate cancer, the5-year survival rate is about20%, and the10-year survival is above10%.There are more than200different types of cancer. It has been shown genome-wide genetic changes are correlated with specific types of cancer or the stages of tumor development. It is characterized by a progression of these changes at the cellular, genetic and epigenetic level that ultimately reprogram a cell to undergo uncontrolled cell division, thus forming a malignant mass. The Cancer Genome Atlas (TCGA) is a project to catalogue genetic mutations responsible for cancer, using genome sequencing and bioinformatics. We represented an effort in the War on Cancer that is applying high-throughput genome analysis techniques based on TCGA data to improve our ability to diagnose, treat, and prevent cancer through a better understanding of the genetic basis of this disease. Anterior gradient protein2homolog (AGR2) is one of candidate therapeutic target genes in PCa arising from the analysis of TCGA data. Current studies showed elevated levels of AGR2in adenocarcinomas of the prostate, breast, lung and pancreas. AGR2is an androgen-inducible protein expressed in normal prostate tissue, and its expression is significantly elevated in PCa tissue compared to benign prostatic epithelial tissue. Urine sediment tests determined AGR2transcript levels to be elevated, and AGR2has been proposed as one of the potential markers to detect malignant cells in the voided urine from PCa patients. In this study, we first observed that AGR2was overexpressed in Chinese Han PCa tissues, and AGR2expression profiles varied among cell lines, with PC3cells being the highest level, LNCaP and DU145relatively less, and hardly detectable in human normal prostate epithelial cell line RWPE1. Here, a potential role of AGR2in cellular senescence was first investigated as well.一、The genomic landscape of prostate cancer1. Dysregulation of transcriptome in PCa.To get a comprehensive overview on transcriptome in PCa, we analyzed RNA sequencing (RNAseq) profiles of256human tumor specimens and45normal specimens from TCGA. We identified24%genes that were up-regulated or down-regulated in the tumor specimen, including28.53%protein-coding gene (PCGene),27.64%long non-coding gene (lncRNA), and16.61%pseudogene.The results indicate complex dysregulation of the cellular transcriptome in PCa.2.Somatic DNA copy number aberrations in prostate cancer cell genome.We analyzed the somatic copy number alterations (SCNAs) across258tumor-normal paried samples. Data from high-resolution single nucleotide polymorphisms (SNP) microarray on258PCa specimens and the matching normal specimens were retrieved from TCGA.We found2448genes with high-frequency gain and loss, including961PCGenes,808lncRNAs, and679pseudogenes.3. Epigenetic analysis of PCa.To analyze DNA methylation alterations in PCa, we obtained DNA methylation microarray profiles (Infinium Human Methylation450BeadChip) on252human tumor and49normal control specimens from TCGA. We finally identified2617cancer-associated epigenetically silenced genes. The results indicatePCa is characterized by genome-wide methylation imbalance. Next, we performed analysis on miRNA (miRNA-seq) expression profiles of in259human tumor and50normal control specimens from TCGA. We identified36.90%miRNAs that were up-regulated or down-regulated in the tumor specimen compared to normal specimen.二、Analysis of expression and functional modules of AGR2based on bioinformatic and experimental data.1. Up-regulation of AGR2in PCa samples.AGR2is expressed in relatively high levels for prostate cancer patients based on TCGA data. DNA copy number and global methylation analysis showed there were no copy number variations or methylation dysregulation of AGR2in PCa samples. So enhanced mRNA stability and constitutive activation of transcription factor may be involved in upregulation of AGR2.2. AGR2was overexpressed in Chinese Han PCa tissues.An elevated expression for AGR2has been observed in prostate tumor tissues of Europe and USA men, however, little is known about the expression level in human tissues among the Chinese Han population. Immunohistochemical analysis of AGR2expression was performed in BPH tissues from41patients, PIN tissues from13patients, and PCa specimens from32patients. The data showed AGR2was overexpressed in Chinese Han PCa tissues as well. We then evaluate the differential expression of AGR2at the protein levels in a panel of various cell lines, and the resluts showedthe expression of AGR2was significantly up-regulated in PCa cells than normal prostate epithelial cells.3. Analysis of functional modules of AGR2based on TCGA datawe performed Guilt-by-Association (GBA) analysis for AGR2on RNASeq profiles in TCGA to characterize the biological functions and the molecular pathways influenced by AGR2-associated genes. The results showed AGR2correlates with cell cycle, DNA damage and repaired, and cell adhesion. 三、AGR2i-induced DNA damage is involved in regulation of cellular senescence in PCa.1. Knockdown of AGR2inhibited proliferation in PCa cells.Knockdown of AGR2by AGR2-targeting siRNA caused a decrease in cell viability in PC3cells, whereas forced expression of AGR2led to an increased cell proliferation of LNCaP and DU145cells.We further assessed the effect of AGR2in cell proliferation process by analysis of incorporation of BrdU into DNA after silencing the endogenous AGR2, and BrdU signaling in AGR2i-treated PCa cells was significantly decreased. Western blotting results showed inactivation of AKT and ERK was accordingly observed in AGR2i-treated cells.2.Knockdown of AGR2induced cell cycle arrest in PCa cells.AGR2was overexpressed in Chinese Han PCa tissues and had a positive correlation with Cyclin D1and p-Rb, but not with p16.AGR2-depletion resulted in accumulation of cells at the G0/G1phase.3. Knockdown of AGR2induced cellular senescence in PCa cells.AGR2siRNA treatment resulted in cellular senescence in all three PCa cell lines as indicated by an increase of flat, enlarged and senescence-associated β-galactosidase (SA-β-Gal) positive cells. Senescent response to AGR2-silencing was also evidenced by elevated fluorescent punctuate formation of tri-methyl-histone H3in AGR2-depleted cells.4. AGR2depletion-induced cellular senescence depends on CDKIs.Further studies indicated that LNCaP underwent a p21-dependent cellular senescence in response to AGR2depletion that requires inactivation of ERK signaling, while PC3was also p21-dependent, but involved in suppression of PI3K/Akt. Unlike LNCaP and PC3, senescent response of DU145was found to be mainly p27-dependent that may require upregulation of PTEN and inhibition of PI3K/Akt signaling.5. AGR2depletion-induced cellular senescence was accompanied with DNA damage.Senescence is considered as a DNA damage responseriggered by DNA hyper-replication or other cellular stress. Cells were stained with γH2AX-FITC antibodies and subjected to flow cytometryto quantitatively determine the expression levels of the DNA damage marker γH2AX. Senescent response to AGR2-silencing was evidenced by elevated γH2AX, which means AGR2i-induced DNA damage may be involved in regulation of cellular senescence. Part Ⅱ DNA damage response indeuced by bisbibenzyls is involved in regulation of cellular senescence and apoptosis in prostate cancerWe found AGR2depletion induced DNA damage and resulted in cellular senescence in PCa cells, and cellular senescence was shown to be one of the key determinants of tumor response to therapy in vitro and in vivo.The concept of pro-senescence therapy has emerged over the past few years as a novel therapeutic approach to treat cancers. Damage to DNA, the prime target of anticancer therapy, triggers programmed cellular responses. In addition to apoptosis, therapy-mediated cellular senescence has been identified as another drug-responsive program that impacts the outcome of cancer therapy.One of the most efficient chemical reagents used in cancer chemotherapy are DNA damage inducers, which can cause a variety of DNA lesions via multiple mechanisms and resulted in cellular senescence and apoptosis. For example, camptothecin and etoposide can trigger single-strand breaks (SSBs) or double-strand DNA breaks (DSBs) by trapping topoisomerase-DNA covalent complexes, subsequently leading to the cell death; cisplatin and its derivatives forms DNA cross links as monoadduct, interstrand crosslink, intrastrand crosslink or DNA protein crosslink, thus induced DNA damage; cyclophosphamide alkylates the N-7position of guanine and results in frameshift mutation. Cells exposed to chemicals are prone to acquire multiple sites of bulky DNA. Moreover, DNA damaging agents can damage other biomolecules such as proteins, carbohydrates, lipids, and RNA. The accumulation of damage, to be specific, double-strand breaks or adducts stalling the replication forks, are among known stimulation signals for a global response to DNA damage. The global response to damage is an act directed toward the cells’ own preservation and triggers multiple pathways of macromolecular repair, lesion bypass, tolerance, senescence, or apoptosis. The common features of global response are induction of multiple genes, cell cycle arrest, and inhibition of cell division.Depending on the type of DNA lesions, specific cell cycle checkpoints and cellular cascades are activated by DNA-damaging agents. Checkpoint activation pauses the cell cycle and gives the cell time to repair the damage before continuing to divide. As widely accepted, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3related (ATR) signaling pathways play important roles in response to DNA damage response. ATM responds mainly to DSBs, and initiates phosphorylation of downstream targets such as Chk2, BRCA1, and NBS1proteins at the site of DNA damage. These factors act together to induce G1, S, and G2cell cycle arrests, DNA repair, and/or activation of cell death pathways. While ATR is activated in response to replication stress, it triggers the activation of Chkl, which in turn leads to the phosphorylation of Cdc25and prevents the activation of CDK1/Cyclin B and mitotic entry. Upon DSBs, the process of DSBs end joining involves numerous proteins and enzymes through nonhomologous end joining (NHEJ) and homologous recombination (HR) repair mechanisms. For example, the Ku70/86heterodimer is critical in NHEJ, since it binds to the broken DNA ends and recruits repair-related proteins including DNA-dependent protein kinase, XRCC4, and DNA Ligase Ⅳ. The rate of DNA repair is dependent on many factors, including the cell type, the age of the cell, and the extracellular environment. A cell that has accumulated a large amount of DNA damage, or one that no longer effectively repairs damage incurred to its DNA, can enter one of three possible states:an irreversible state of dormancy, known as senescence, or cell suicide, also known as apoptosis.一、Low concentrations of RD triggered p21dependent cellular senescence.1. Low concentrations of RD induced cellular senescence.0.5μmole/L RD treatment caused growth arrest at G0/G1phase with features of cellular senescence phenotype such as enlarged and flattened morphology, increased senescence-associated-beta-galactosidase staining cells, and decreased cell proliferation in PCa cells. Induction of cellular senescence by RD occurred through activation of DNA damage response including increases in the γH2AX,inactivation of Chk1/2, and suppression of repair-related Ku70/86and phosphor-BRCA1in PCa cells.2. RD-triggered cellular senescence is p21denpendent.It has been demonstrated that the p21-Rb signaling pathway, activated by DNA damage response, plays an important role in the activation of G0/G1checkpoint response and cellular senescence.We then detected the activation of p21in PCa cells, and found the expression of the p21protein was markedly enhanced. We next explored a link between the enhanced p21 expression and senescence induction in response to RD by genetic knockdown of p21. The results revealed that deletion of p21by targeting siRNA impaired RD-induced senescent cells in all three PCa cells, indicating that RD-induced cellular senescence was, at least in part, in a p21-dependent manner.3. RD triggers cellular senescence in PC3xenograft.To determine whether RD could induce tumor cell senescence in vivo, as observed in cultured cells, human PC3xenografts were developed in male nude mice. Administration of RD had no effect on body weight but significantly reduced tumor growth of tumor-bearing mice.To examine whether decreases in tumor mass and size by RD were associated with DNA damage-mediated cellular senescence, tissue samples were prepared for immunohistochemical analysis. Consistent with results in culture cells, senescent cells were pronounced in RD-treated tumor tissues compared to the placebo treatment, as evidenced by increased SA-P-Gal staining, associated with substantial accumulation of ting nand positive stained tri-Me-K9-H3which serve as a senescence-associated heterochromatic foci. Meanwhile, RD-mediated alterations in the expressions of p21were similar to the observations shown in culture cells. These data demonstrated that RD triggered DNA damage and cellular senescence that contributed to its observed antitumor effect.二、RD triggered DNA damage and inhibited DNA repair in PCa cells.1. RD caused remarkable DNA damage in PC3cells.After24h treatment with10μmol/L RD, cell cycle was significantly arrested in G2/M phase in PC3cells. In addition to interference with cell cycle progression, RD was able to noticeably induce apoptosis as well. As evidenced by induction of yH2AX foci, micronuclei, and DNA fragmentation in Comet assay, we found RD caused remarkable DNA damage in PC3cells. Time kinetic and dose-dependent studies showed that ATM/Chk2and ATR/Chkl signaling pathways were sequentially activated in response to RD. Blockage of ATM/ATR signaling led to the attenuation of RD-inducedyH2AX, and to the partial recovery of cell proliferation.2. RD inhibited DNA repair in PC-3cellsWestern blotting data showed RD exposure resulted in the inactivation of BRCA1which is a critical molecule in the initial recruitment of other repair proteins/enzymes at the DNA breaks. To determine the effects of RD on DSBs repair, the ability of DNA repair was evaluated with HR and NHEJ reporters. Positive signal significantly declined in either NHEJ or HR repair systems in cells treated with RD, indicating that DSBs repair was impaired in response to RD. Based on the observations above, we further clarified the role of Ku70/Ku86in response to RD-induced DNA damage. DNA end-binding activity of Ku70/Ku86displayed that, compared with the untreated cells, the binding activities of both Ku70/Ku86in treated cells were significantly decreased after12h treatment. Microarray analysis revealed that genes related to damaged-DNA binding, DNA repair, cell cycle, and apoptosis were significantly changed in RD-treated cells.3. RD triggers apoptosis associated with the induction of DNA damage in PC3xenograft.Administration of RD to xenograft mice reduced tumor growth, and coordinately caused alterations in the expression of genes involved in DNA damage and repair, along with cell apoptosis.Changes of molecular markers associated with DNA damage in treated mice displayed that RD caused DNA-damage response via ATM/ATR-mediated signaling as evidenced by substantial accumulation of yH2AX, abrogation of phosphor-BRCAl, and Ku70/Ku86abundance. The data demonstrated that RD triggered DNA damage and impaired repair proteins, leading to apoptotic cell death, which contributed to its antitumor effect.Part III The molecular mechanism of Bisbibenzyls-induced apoptosis in androgen dependent prostate cancer cellsIn the last half of the century, advances in tumor cell biology and molecular biology of cancer have been responsible for achievements in cancer therapy. Currently, docetaxel is as an active agent to moderately improve quality of life and survival conditions in patients with metastatic HRPC. However, only about50%of patients treated with docetaxel have a prostate-specific antigen response. Hence, many efforts have been made to investigate novel mechanisms of action of bioactive chemicals with less toxicity and high efficacy to treat HRPC. Macrocyclic bisbibenzyls are a series of phenolic natural products isolated from liverwort, which exert a variety of biological activities, including antifung, antioxidation and cytotoxicity. Our previous research demonstrates its inhibitory effect on PCa cells.Currently, PI3K pathway is considered a key regulator of progression to androgen-independent prostate cancer, while in androgen-dependent prostate cancer androgen receptor (AR) signaling pathway plays key role in maintaining development, proliferation and differentiation in prostate gland. AR, a nuclear receptor, dissociated from heat shock protein when it bind its ligand, which in turn promotes AR nuclear location and dimerization. Activated AR binds to AR response elements (AREs) and regulates gene transcription, such as PSA. Previous report displayed that AR promoted development from hormone-dependent prostate cancer to horomone-independent prostate cancer. Abnormal androgen signaling due to aberrant expression mutations, or dysregulation of the AR gene has been linked to prostate tumorigenesis, and progression of prostate cancer into advanced, castration-resistant disease. Treatment of androgen-dependent LNCaP cells with macrocyclic bisbibenzyl compound Riccardin C (RC), Marchantin M (MM) or Riccardin D (RD) caused significantly cell cycle arrest and apoptosis. AR is a key factor for cell proliferation in LNCaP cells, so we investigated whether bisbibenzyl compound involved in inhibiting AR function and triggering apoptosis in LNCaP cell.一、Macrocyclic bisbibenzyl compounds induced apoptosis by inhibiting AR expression and function in LNCaP cells.1. Exposure of androgen-denpendent cell line LNCaP to RC, MM, or RD caused markedly suppression of cell proliferation.Treatment of LNCaP cells with RC, MM or RD caused cell cycle arrest in G0/G1phase associated with moderate increases of p53, and p21protein levels, and significantly triggered apoptosis with induction of a higher ratio of Bax/Bcl-2, and elevated cleavage of PARP.2. Induction of apoptosis was accompanied with the decreased expression and transcriptional activity of the AR.As demonstrated by western-blotting analysis, AR expression was significantly repressed in LNCaP cells exposed to RC, MM and RD. Luciferase activity assays showed RC, MM and RD treatment markedly suppressed androgen-mediated expression of the AR promoter activity. We further examined the impact of bisbibenzyls on AR transcriptional activity by monitoring the activity of the promoter of prostate specificantigen (PSA), a well-known AR target gene, and found PSA promoter was dramatically repressed by RC, MM and RD in LNCaP cells. We then examined SRC-1/AR interactions using co-immunoprecipitation (co-IP), and declined SRC-1proteins were noticeably observed in cells treated with RC, MM and RD.Thus, inhibitory effect of bisbibenzylson cell proliferation was associated with the downregulation of AR expression,leading to the abolishment of AR.3. RC, MM and RD targeting proteasome activity promotes autophagy inLNCaP cells.Proteasome activity assays with purified proteasome in the presence of bisbibenzyls were performed, and the results revealed that RC, MM and RDsignificantly inhibited the proteasome. Werstern blotting and co-IP results demonstrated that the levels of polyubiquitinated-AR were noticeably increased in LNCaP cells treated with RC, MM or RD. We hypothesized that bisbibenzyl-mediated proteasome inhibition simultaneously activates autophagy in LNCaP cells, which in turn is involved in degrading the polyubiquitinated-AR. In our study, induction of LC3B expression and conversion was found in LNCaP cells treated with the compounds which demonstrated the ability of autophagy induction by these agents. We then performed immunofluorescence co-location analysis on AR and LC3or Ub, and the results showed a co-localization of AR and LC3or Ub, suggesting that activation of autophagy by RC, MM and RD might be involved in the process of ubiquitinated ARdegradation.Part IV Conclusions and Innovation一、Conclusions1. A map of prostate cancer genome variation revealed characteristic differences between normal prostate and prostate cancer.2. AGR2overexpressed in PCa, and knockdown of AGR2caused cellular senescence in PCa cells.4. Riccardin D induced DNA damage-mediated cellular senescence and apoptosis in prostate cancer.5. Macrocyclic bisbibenzyl compounds induced apoptosis by inhibiting AR expression and function in androgen-denpendent cell line LNCaP.二、Innovation1. With the multi-omic profiles and well-annotated clinical and pathological information on large-scaled clinical samples, we presented the genomic landscape of PCa and provided a powerful resource to improve our ability to diagnose, treat, and prevent cancer through a better understanding of the genetic basis of this disease.2. We firstly reported AGR2was overexpressed in Chinese Han PCa tissues, and knockdown of AGR2caused cellular senescence in PCa cells.3. We firstly reported macrocyclic bisbibenzyl compounds prevented AR expression and function, and promoted apoptosis in LNCaP cells. Since AR in LNCaP cells was mutant, the characteristics of bisbibenzyl will be beneficial for the treatment of prostate cancer expression constitutive activation of the AR.4. We firstly reported RD triggered DNA damage and inhibited DNA repair, thus induced cellular senescence or apoptosis in prostate cancer. Significantly, DNA repair contributes to the drug-resistant phenotype, so that RD may reverse the drug-resistant phenotype by inhibiting repair of chemotherapy-induced genotoxic damage. This finding provides the foundation for the use of RD as a chemotheraputic candidate. |
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