Mechanism Of AGR2 Mediated Drug-resistance And Metastasis In Prostate Cancer And Coping Strategies Of Drug-resistance

As a kind of malignant tumor, prostate cancer (PCa) is a serious threat to the middle-aged and elderly men’s life. Its death rate is only second to lung cancer in North America. With the aging of population, changes of living environment and diet structure, the incidence of PCa grows rapidly in China. The proportion of middle-late patients is higher than that of abroad. Its death rate is in second place in Asia, only lower than that of Vietnam. Because of the high malignant degree, clinical lack of effective treatments, the study of the mechanism and treatment strategies for the malignant phenotype of PCa have been paid close attention to.As a kind of hormone dependent tumor, endocrine therapy is the most effective method for PCa. Unfortunately, the development of Castration-resistant prostate cancer (CRPC) has been observed to occur after endocrine therapy within 1 to 2 years. Chemotherapy is a major clinical response to CRPC. Including mitoxantrone and phosphorus nitrogen mustard, Docetaxel and Cabazitaxel are the first choice as the treatment for prostate cancer. As the derivative of the natural products, such drugs have been proved to be the only effective drug to prolong the life of patients with PCa. But statistics show that about 50% of patients with CRPC are not sensitive to docetaxel congenital, and produce tolerance or strong side effects after treatment with drugs. Similar to other antineoplastic drugs, it may induce tumor multi-drug resistant (Multidrug resistance, MDR) after treatment with Docetaxel, so as to cause the failure of chemotherapy. The mechanism of Docetaxel inducing MDR is complex, such as drug pump protein over-expression, microtubule structure-function change, antiapoptotic mechanisms change, MicoRNA regulation, epithelial-mesenchymal transition (EMT) of drug-resistant cell lines, etc. The docetaxel MDR reversal strategies are also positively explored. The affinity of Cabazitaxel is low to P-gp, and can be used as docetaxel mediated MDR reversal agents after treatment, which achieved good clinical effect. PCa MDR is signal mechanism of gene coexist. Single target resistance reversal is still limitated. So it is urgently needed to look for new treatment by further analyzing the mechanism of PCa malignant phenotype.Based genome sequencing data provided by The Cancer Genome Atlas (TCGA) plan, our group analyze genes which are closely related with malignant phenotype, such as CRPC installment, invasion and metastasis and drug resistance. Finally we select AGR2 as one of the candidate genes. There are close relationships between AGR2 and tumor occurrence and development. It is reported that AGR2 is highly expressed in prostate cancer and its high expression was related to low survival rates. As recently study show, AGR2 supports for drug resistance. AGR2 over-expression can contribute to the traditional chemotherapy drugs cisplatin, doxorubicin and gemcitabine tolerance in breast cancer, pancreatic cancer, French special ampullary cancer, lung cancer. Estrogen antagonist tamoxifen can induce AGR2 over-expression in breast cancer and resist to tamoxifen treatment. Endocrine treatment failure patient secreted less AGR2 to serum, meanwhile AGR2 was down-regulated in Doc resistant PCa cells. At the same time, based on the analysis of PC3 cells/Doc gene chip, we found that AGR2 was dramatically down-regulated. Further research suggested that lower expression of AGR2 in PCa cells can enhance protease activity, raise p65 expression, induce EMT and promote the invasion and metastasis of PCa. In addition, we also found that alkanes kauri type 2 compounds can inhibit PCa cell invasion and metastasis by down-regulating AGR2 expression and induce cell apoptosis by by increasing the PCa cells ROS level which causes DNA damage.Part I The decreased expression of AGR2 promotes drug-resistant in prostate cancer and the coping strategies of reversingWe found ubiquitin-proteasome pathway was highly correlated with AGR2 by bioinformatics methods analysis. Ubiquitin proteasome system (UPS) is one of protein quality control systems in eukaryotic cells. UPS works mainly through ubiquitination of the target proteins and degradation of abnormal proteins and short-lived proteins by proteasome. UPS plays an essential role in cell proliferation, cell cycle regulation, immune response, signal transduction and cell death. Recent studies have validated that proteasome activities in malignant tumors are much more active than those in non-malignant cells, confirming tumor cells are more dependent on the UPS to maintain cellular homeostasis. Therefore, tumor cells are more sensitive to UPS inhibitors. UPS has gradually become a new target for cancer therapy. Our studies found that Doc induced the degradation of AGR2 through autophagy. The decreased expression of AGR2 could increase proteasome activity, which indicated that Doc resistant PC3 may be sensitive to proteasome inhibitors.1. Doc promotes the degradation of AGR2 via induced autophagy.1) Doc decreased the expression of AGR2. Western blotting results showed that, AGR2 was down-regulated in PC3/Doc cell lines with Doc-induced resistance and in H460/RT cell lines with paclitaxel-induced resistance. It was detected by ELISA that serum from post and pro chemotherapy patients of non-small cell lung cancer showed the AGR2 contents in serum of patients after chemotherapy were reduced.2) Doc induced autophagy and promoted AGR2 degradation. Immunofluorescence showed that Doc could induce PC3 cells autophagy with a time-dependent manner. LC3 expression increased and cells gathered in spots, while intracellular AGR2 expression decreased. Western blotting results showed that with the extended time of Doc, the expression of LC3II increased and the expression of AGR2 and autophagy substrates p62 and CALCOCO2 persistently reduced. Co-immuno-precipitation proved that Doc could induce high level of AGR2 poly-ubiquitination. Using RNAi to silence the expression of LC3 in PC3 cells, the down-regulation of AGR2 induced by docetaxel was reversed.3) AGR2 lead to prostate cancer multidrug resistance downstream. MTT results showed that after the down-regulation of AGR2, PC3 cells emerged multidrug resistance to docetaxel, doxorubicin, cisplatin, vincristine. On the contrary, after RWPE1 cells over-expressing AGR2, it became sensitive to Doc, Dox, Cis and VCR.2. Decreased AGR2 reduces the sensitivity of prostate cancer to Doc1) The decreased AGR2 induced Doc-resistant in PC3 cells. According to the results of MTT assays, reducing the AGR2 protein level in PC3 cells could induce the resistance to Doc. Over-expression of AGR2 in RWPE1 cells enhanced the sensitivity to Doc.2) PC3/KD cells were resistant to Doc. MTT assays results showed that PC3/KD cells, which stably silenced the expression of AGR2, were resistance to Doc.3. AGR2 inhibits the activation of proteasome AGR2 inhibited proteasome activity. Through GO analysis of the expression between AGR2 and its related genes, it is found that the relevance between AGR2 protein and ubiquitin-proteasome pathway was the highest.1) Cell experiments confirmed that over-expression AGR2 of RWPE1 cells could significantly inhibited the activity of proteasome chymotrypsin-like proteases and peptide-based solution glutamic peptide, but it did not affect the activity of trypsin. Down-regulation of AGR2 in PC3 cells could increase proteasome chymotrypsin-like proteases and peptide-based glutamyl peptide activity. Western blotting results showed that the decreased or increased AGR2 expression can induce cellular total ubiquitination level to reduce or rise. The cellular total ubiquitination level in resistant cell lines also lowered.2) Vivo experiment confirmed AGR2 negatively regulated proteasome activity. Through tumor transplantation, immunohistochemistry and western blotting results proved that total ubiquitination level of tumor tissue reduced in AGR2 low expression group. Through measuring tumor proteasome activity, significant proteasome activity was found in decreased AGR2 expression group. However, trypsin was equally effective.4. Proteasome inhibitors can reverse the drug-resistance induced by decreased expression of AGR21) Low level of AGR2 enhanced the sensitivity of proteasome inhibitors in prostate cancer cells. MTT results showed that PC3 cells interfered the expression of AGR2, which could enhance the sensitivity of cells to proteasome inhibitor Bor. On the contrary, AGR2 over-expression could cause tolerance to those drugs.2) Xenograft tumor confirmed that after four times chemotherapy, the susceptibility of Doc in low AGR2 expression group was lower than that of control group. In contrast,Bortezomib had stronger inhibitory effect in low AGR2 expression group. Immunohistochemistry results demonstrated that through Bortezomib treatment, staining of Ki67 in low AGR2 expression group was remarkably lighter than that of control group. Besides, the potency of docetaxel is lower than Bortezomib.Part II AGR2 facilitates invasion and metastasis in prostate cancer by inducing angiogenesis and epithelial mesenchymal transitionBesides invasive and metastatic or chemotherapy drug resistant, multi-drug resistant and distal metastasis are also important biological characteristics inducing the death of CRPC patients. It is reported that AGR2 contributes to the invasion and metastasis in prostate cancer, oral epithelial carcinoma, pancreatic carcinoma, thyroid carcinoma and lung adenocarcinoma, but the mechanism is still unclear. In this paper we discuss the function of AGR2 that facilitates invasion and metastasis in prostate cancer, and come to a conclusion that AGR2 can be a target for prostate cancer treatment.The growth and metastasis of malignant tumor depend on abundant nutritional support. However, the angiogenesis in local tissue provides tumors with the oxygen and other essential nutrients. Tumors induce blood vessel growth (angiogenesis) by secreting various growth factors. Vascular endothelial growth factor (VEGF), a diffusible glycoprotein, which is closely related to tumor genesis, development and metastasis, is a widely over-expressed pro-angiogenic factor in most solid cancers and plays a critical role in angiogenesis, leading to the proliferation, migration of endothelial cells and tube formation. VEGF secreted by tumor cells interacts with VEGF receptors (VEGFRs) in endothelial cells and stimulates downstream signaling molecules to promote the growth, survival and migration of endothelial cells.The epithelial-mesenchymal transition was first recognized as a feature of embryogenesis in multicellular organisms. Besides, EMT participates in the process of tumour and the incidence of many chronic diseases, such as renal fibrosis. EMT is a process in which epithelial cells lose their cell polarity and adhesion, at the same time gain migratory and invasive properties to become mesenchymal stem cells, plays an critical role in the invasion and metastasis of the malignant tumors as a result of enhance of anti-apoptotic property. During the process E-Cadherin which is the marker of epithelial cells decreased and N-Cadherin increased. Many signaling pathways, a variety of growth factors, extracellular matrix components may induced EMT by regulating the transcription factors such as Snail, Slug, ZEB1, ZEB2 which can bind to E-Cadherin promoter whereas factors such as Twist, Foxc1, Foxc2, NF-κB repress E-cadherin indirectly. Our paper showed AGR2 could increase the expression of VEGFR2 and p65 to induce angiogenesis and EMT involved PCa invasive and metastatic.1. AGR2 facilitaes invasion and metastasis in PCa.1) AGR2 promoted invasion and metastasis in cells in vitro. Wound healing assays and matrigel-coated transwell assays demonstrated that down-regulate the expression of AGR2 in PC3 cells which are high expressed AGR2, the number of cells through the matrigel and the number of cells through the cell Migration significantly decreased, indicated that the ability of the wound healing weakened compared to control cells. Over-expression of AGR2 in RWPE1 cell significantly enhanced the invasiveness and migratory capabilities compared to normal control cells.2) AGR2 facilitated invasion and metastasis in PCa cells in vivo. Orthotopic transplantation models of human prostate carcinoma in nude mice and biolumine- scence measurement in vivo demonstrated that over-expression of AGR2 in PCa tissue, promoted the tumor cells metastasis to the liver, armpits, and neck.2. AGR2 promotes Angiogenesis via increasing the expression of VEGFR21) The photos comparison from Animal experiments revealed that the color of tumor tissue with high level of AGR2 group were rich in blood vessels and appeared more red compared to the normal group. Immunohistochemistry showed an increasing number of angiogenesis markers CD34 and VEGFR2 in the AGR2 over-expression group.2) AGR2 promoted angiogenesis mechanism. Western blotting showed that, down-regulation of AGR2 could inhibit the expression of VEGFR2 and reduce downstream PI3K-AKT phosphorylation levels, whereas over-expression of AGR2, on the contrary, VEGFR-AKT pathway was activated. Vessel formation assays confirmed that, human umbilical vein endothelial cells (HUVEC) with AGR2 over-expression formed significantly more small tubes than control. The matrigel-coated transwell assays showed that the secretion of AGR2 facilitied HUVEC cells pass through the well and enhanced the invasion and metastasis capacity.3. AGR2 facilitates the occurrence of EMT by inhibiting activity of proteasome and decreasing the degradation of p651) AGR2 facilitated the occurrence of EMT in prostate cancer. We observed a remarkable change in the morphology of PC3 cells that high expression of AGR2 leads to increasing cell-cell contacts. While control RWPE1 cells display cobble-stone-like, typical epithelial cell morphology, AGR2 over-expressed RWPE1 cells show spindle shaped, fibroblast-like morphology with loss of cell-cell contacts and increased cell scattering. Western blotting and immunofluorescence showed knockdown of AGR2 inhibited EMT occurrence in PC3 cells, whose phenomenons are down-regulated expression of EMT marker protein E-Cadherin and up-regulated expression of Vimentin. When up-regulate AGR2 in PC3, the consequences are on the contrary, indicating that AGR2 facilitates cells transform from epithelium to mesenchym.2) AGR2 induced the occurrence of EMT by decreasing the degradation of p65, which was the functional subunit of NF-κB pathway. Immunofluorescence, western blotting and RT-PCR experiments showed that AGR2 enhanced the expression of p65 on the protein level rather than the level of transcription, at the same time up-regulated the expression of matrix metalloproteinase MMP16, MMP2, MMP9 in the downstream of p65. Since AGR2 can inhibit proteasome activity, we found AGR2 can prolong half-life of p65 by actidione (ActD). While knockdown the p65 in PC3 cell lines, western blotting showed that E-Cadherin, TCF8, MMP16 expression increased and Vimentin expression decreased. The opposite results were observed by over-expression of p65. Immunohistochemistry experiments in RWPE1 and PC3 cell lines demonstrated that the expression level of p65 is consistent with the AGR2. So we draw a conclusion that AGR2 facilitates the occurrence of EMT by regulating the expression of p65.Prart Ⅲ The effect of JA and JB on AGR2 and apoptosis of prostate cancer cellsThe development of CRPC has been observed to occur within a few years after hormonal deprivation therapy and is associated with poor prognosis, high apoptosis-resistance and high mortality. There are few efficacious treatment options available for curing, or even improving the survival and quality of life in patients with CRPC. Although Doc is the most effective chemotherapy drugs for PCa, development of resistance is inevitable, and patients’disease will eventually progress. Since complicated factors and signals involve in the development of CRPC, monotherapy regimens or chemotherapy drugs targeting a particular signaling pathway are difficult to obtain a satisfactory result. Thus, novel natural compounds that are able to induce prostate cancer cell death through various signal pathways are highly desirable, ent-Kauranes diterpenoids are a series of diterpenoid natural products isolated from liverwort, which exert inhibitory effect on PCa cells. The present study was designed to uncover the cellular/molecular basis by which they exerted their antitumor effect.1. JA and JB inhibit cell proliferation and induce apoptosis1) Exposure of PC3 cell to JA or JB caused markedly suppression of cell proliferation. As demonstrated by MTT analysis, JA and JB both exhibited strong anti-proliferative effect on many cancer cells. It appeared that all tested cell lines of PCa showed response to both JA and JB, and non-neoplastic prostate epithelial RWPE1 cells were resistant to the treatments with higher IC50 values compared with PCa cells. We monitored the cell growth in response to the treatments by the Reaf-Time Cell Analyzer SP Instrument, the results revealed that either JA or JB time-dependently reduced the viability of cells.2) JA and JB induced apoptosis in PC3 cells. As shown in flow cytometry results, both JA and JB promoted apoptosis in PC3 cells in a time-dependent fashion. However, the apoptosis rate was observed much faster by the treatment of JA. Western blotting analysis displayed that the induction of caspase 3 cleavage to proteolytic fragment in PC3 cells led to JA/JB induced death. To investigate the role of apoptosis in JA-and JB-induced cell death in PC3 cells, z-VAD-fmk, a pan inhibitor of caspase was pretreated in presence and absence of JA and JB. The data showed z-VAD-fmk dramatically rescued cell death in PC3 cells.2. JA and JB affect the expression of AGR21) ent-Kauranes diterpenoids targeted AGR2. Western blotting demonstrated that JA reduced the expression of AGR2, but JB had no effect on AGR2. Both JA and JB could reduce the levels of MMP16 and increase the expression of E-Cadherin in PC3 cells in a time-dependent manner.2) JA and JB inhibited prostate cancer invasion and metastasis. Wound healing assays and matrigel-coated transwell assays showed that the compounds both JA and JB could inhibit PC3 cells invasion and metastasis.3. Induction of ROS by JA and JB contributes to their effect on apoptosis via mitochondrial and DNA damage1) JA and JB caused ROS accumulation in PC3 cells. As shown in flow cytometry analysis, a rapid and significant increase in ROS was observed in PC3 cells after 2 h treatment with JA, the high level of ROS sustained during the prolonged treatment period. Elevated ROS was also markedly accumulated in JB-treated cells at 2 h, and maintained up to 12 h, which was followed by a slight decrease after 24 h treatment. We then introduced vitamin C (Vc) to block ROS to investigate the role of ROS in cells exposed to the agents. As results showed, Vc significantly prevented the JA-or JB-induced ROS, importantly JA-triggered inhibitory effect was pronouncedly reversed, from 50.14% by JA alone to 13.65% by JA combined with Vc, highlighting the importance of ROS in JA-mediated cytotoxicity. Similar observations were also shown in JB-treated cells in the presence of Vc from 52.64% by JB alone to 31.98% by JB combined with Vc.2) JA and JB induced mitochondrial damage in PC3 cells. Considering the relevance of ROS production and mitochondria, the change in structure of mitochondria was examined by transmission electron microscopy. The results clearly revealed that both JA and JB caused dramatic mitochondrial swelling, leading to disorganized cristae and vacuolar structure.3) JA and JB induced DNA damage in PC3 cells. Neutral comet assay indicated that DNA tail was detectable after 4 h treatment by JA, or following 8 h exposure by JB, and became more pronounced with prolonged treatment, consistent with the observations above that JA was more potent than that of JB. Western blotting data showed provided additional evidence that DNA damage response including increases in the yH2AX, inactivation of Chkl/2, suppression of repair-related Rad51, and increased repair-related Ku70/80 at the early time and then decreased.4. Suppression of c-Myc and activation of JNK are required for blockade of cell cycle by JA and JB1) JA and JB contributed to cell cycle arrest in PC3 cells at different phases. As indicated by flow cytometry assays, the results indicated that treatment of cells with JA caused a dose-dependent accumulation of cells in the G0/G1 phase, the cells in the Go/G1 phase increased by 56.74%,69.63%, and 76.67% at concentrations of 0,0.75, and 1.5 μM of JA, respectively. However, unlike the JA, JB treatment caused a dose-dependent accumulation of cells in the G2/M phase, the increased number of cells in the G2/M phase was 10.58%,13.58%, and 27.66% at the concentrations of 0,2.5, and 5μM of JB, respectively. Western blotting data showed Cyclin DU Cyclin E and CDK4 were reduced after treat-ment with JA in a time-dependent manner. Meanwhile, p21CIP1, an important cyclin-dependent kinase inhibitor, was up-regulated following 4 h treatment with JA. As to JB, Cyclin B1 and phospho-Cdc2 were reduced, p21CIP1 was up-regulated at the same time.2) Suppression of c-Myc and activation of JNK are required for blockade of cell cycle by JA and JB at different phases. As indicated by western blotting assays, the protein level of c-Myc significantly decreased after treatment with JA. We performed rescue experiments to determine the role of c-Myc in JA-induced cell cycle arrest byincreasing the abundance of c-Myc in cells transfected with a c-Myc plasmid Ectopic expression of c-Myc resulted in disruption of the cells arrested in Go/G1 phase by JA, the cells in the Go/G1 phase were 72.03% and 75.43% in the absence and presence of JA. By comparison, the percentage of cells in the G0/G1 phase increased from 74.54% to 81.23% after JA treatment. We studied the PI3K/AKT、p38 and JNK signaling pathways, and found phosphor-JNK level was up-regulated in JB-treated cells. Knockdown the endogenous expression of JNK1/2 by JNKl/2-targeting siRNA significantly disrupted the G2/M phase blockade induced by JB,9.02% to 13.56% compared 4.88% to 6.72%. Part IV Conclusion, Innovation and Deficiencies1. Conclusion1) Doc promotes AGR2 degradation via autophagy pathway.2) The decreased of AGR2 contributes to Doc-resistance in prostate cancer.3) Effect of AGR2 on angiogenesis and epithelial mesenchymal transition contributes to invasion and metastasis in prostate cancer.4) ent-Kauranes diterpenoids, exert ROS and cell cycle-dependent effects on apoptosis of prostate cancer cells.2. Innovation1) We firstly reported AGR2 inhibited the proteasome activities. Moreover, Doc-resistance cell lines were sensitive to proteasome inhibitors.2) We firstly reported Doc can promote the degradation of AGR2 via autophagy.3) AGR2 prevented the degradation of p65 to regulate epithelial mesenchymal transition in prostate cancer. This finding provides a new mechanism for AGR2 promoting invasion and metastasis.3. Deficiencies1) We have not indicted the mechanism of inhibition the proteasome activity by AGR2. We will complete this study in the future.2) As a secreted protein, AGR2 may affect the activity of VEGF pathway in extracellular. We will study the mechanism as the next assignment3) Compound JB may act as a better drug to treatment drug-resistant PCa compared to Doc, but we have not confirmed this.