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Aging is a risk factor for progressive fibrotic disorders involving diverse organ systems, including the lung. Idiopathic pulmonary fibrosis, an age-associated degenerative lung disorder, is characterized by persistence of apoptosis-resistant myofibroblasts. Here we demonstrate that sirtuin 3 (SIRT3), a mitochondrial deacetylase, is downregulated in the lungs of humans with idiopathic pulmonary fibrosis and in mice subjected to lung injury. Overexpression of Sirt3 cDNA via airway delivery restored the capacity for fibrosis resolution in aged mice, in association with activation of the forkhead box transcription factor FoxO3a in fibroblasts, upregulation of pro-apoptotic members of the Bcl2 family and recovery of apoptosis susceptibility. While transforming growth factor-β1 reduced levels of SIRT3 and FOXO3A in lung fibroblasts, cell non-autonomous effects involving macrophage-secreted products were necessary for SIRT3-mediated activation of FOXO3A. Together, these findings reveal a novel role of SIRT3 in pro-resolution macrophage functions that restore susceptibility to apoptosis in fibroblasts via a FOXO3A-dependent mechanism. Using a mouse model of lung fibrosis, the authors find that overexpression of SIRT3 in myofibroblasts lowers their threshold for apoptosis, allowing their clearance from tissues and restoration of the lost capacity for fibrosis resolution in aged mice.

The current treatment options for multiple myeloma (MM) osteolytic lesions are mainly combinations of chemotherapy and other small-molecule inhibitors, but toxic side effects still remain a major concern. Studies have shown that osteoclast activity is enhanced in MM patients through increased expression of receptor activator of nuclear factor κB ligand (RANKL), triggering RANK signaling on osteoclast precursors, which results in aggressive bone resorption. Furthermore, osteoprotegerin (OPG), a decoy receptor for RANKL, and the osteogenic potential of mesenchymal stem cells (MSCs) are significantly decreased in myeloma patients with multiple bone lesions. Thus, the use of OPG as a therapeutic molecule would greatly decrease osteolytic damage and reduce morbidity. However, in addition to inhibiting osteoclast activation, OPG binds to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), thereby rendering the tumor cells resistant to TRAIL-induced apoptosis and limiting the use of OPG for therapy. The present study developed a bone-disseminated myeloma disease model in mouse and successfully tested a cell therapy approach using MSCs, genetically engineered to express OPG variants that retain the capacity to bind RANKL, but do not bind TRAIL. Our results of skeletal remodeling following this regenerative stem cell therapy with OPG variants indicated a significant protection against myeloma-induced osteolytic bone damage in areas of major myeloma skeletal dissemination, suggesting the potential of this therapy for treating osteolytic damage in myeloma patients.

BACKGROUND—The antimicrobial peptide, LL-37 (leucine-leucine-37), stimulates proliferation, angiogenesis and cellular migration, inhibits apoptosis and is associated with inflammation. Since these functional processes are often exaggerated in cancer, the aim of the present study was to investigate the expression and role of LL-37 in prostate cancer (PCa) and establish its value as a therapeutic target. METHODS—We evaluated the expression of LL-37 and the murine orthologue, Cathelicidin Related Anti-Microbial Peptide (CRAMP) in human and murine prostate tumors, respectively. Compared to normal/benign prostate tissue, both LL-37 and CRAMP were increasingly overexpressed with advancing grades of primary prostate cancer and its metastasis in human tissues and in the Transgenic Adenocarcinoma Mouse Prostate (TRAMP) model, correspondingly. We subsequently knocked down CRAMP in the highly tumorigenic TRAMP-C1 cell line via a RNA interference (RNAi) strategy to examine the importance of CRAMP on cellular proliferation, angiogenesis, invasion, apoptosis, activation of signaling pathways and tumor kinetics. RESULTS—Abrogation of CRAMP expression led to decreased proliferation, invasion, type IV collagenase, and the amount of phosphorylated Erk1/2 and Akt signaling in vitro. These results were paralleled in vivo. Syngenic implantation of TRAMP-C1 cells subjected to CRAMP knockdown resulted in a decreased tumor incidence and size, and the down regulation of protumorigenic mechanisms. CONCLUSIONS—CRAMP knockdown in a murine prostate cancer model analogously demonstrated the tumorigenic contributions of LL-37 in PCa and its potential as a novel therapeutic target for the treatment of PCa and potentially, other cancers over-expressing the peptide.

Mesenchymal stem cells (MSCs) are the most popular among the adult stem cells in tissue engineering and regenerative medicine. Since their discovery and functional characterization in the late sixties and early seventies, MSCs or MSC-like cells have been obtained from various mesodermal and non-mesodermal tissues, although majority of the therapeutic applications involved bone marrow derived MSCs. Based on its mesenchymal origin, it was predicted earlier that MSCs only can differentiate into mesengenic lineages like bone, cartilage, fat or muscle. However, varied isolation and cell culturing methods identified subsets of MSCs in the bone marrow which not only differentiated into mesenchymal lineages, but also into ectodermal and endodermal derivatives. Although, true pluripotent status is yet to be established, MSCs have been successfully used in bone and cartilage regeneration in osteoporotic fracture and arthritis respectively and in the repair of cardiac tissue following myocardial infarction. Immunosuppressive properties of MSCs extend utility of MSCs to reduce complications of graft versus host disease and rheumatoid arthritis. Homing of MSCs to sites of tissue injury, including tumor, is well established. In addition to their ability in tissue regeneration, MSCs can be genetically engineered ex vivo for delivery of therapeutic molecule(s) to the sites of injury or tumorigenesis as cell therapy vehicles. MSCs tend to lose surface receptors for trafficking and have been reported to develop sarcoma in long-term culture. In this article, we reviewed the current status of MSCs with special emphasis to therapeutic application in bone-related diseases.

Bone metastasis affects the majority of patients with advanced breast cancer and no adequate therapy exists. Bisphosphonates, like zoledronic acid, inhibit the osteolytic component of tumor growth in osseous tissues, but these drugs are not curative. The current study evaluated the combination of zoledronic acid with death receptor 5 agonists in an animal model of breast cancer bone metastasis.Female athymic nude mice (age 4-6 weeks, n=35) were inoculated with 200,000 luciferase-positive MDA- MB-435 cells by injection into the left ventricle. Animals were immediately imaged by bioluminescence technique and placed into one of the following therapy groups: Saline, hTRA8, hTRA8 + zoledronic acid, mTRA8, mTRA8 + zoledronic acid, or zoledronic acid monotherapy. DR5 agonists were given at 200 microg/dose and zoledronic acid 5 microg/dose, with mice treated biweekly for 4.5 weeks and imaged weekly.Combination therapy containing either hTRA8 or mTRA8 with zoledronic acid significantly reduced the number of secondary lesions (7.67+2.2 and 7.5+1.7 lesions/mouse, respectively) compared to saline treated controls (12.1+/-1.56 lesions/mouse) as assessed by bioluminescence imaging (p0.05). Additionally, monotherapy with hTRA8 resulted in a significant reduction in tumor number (8.3 +/- 2.9) compared to control animals. Total body tumor burden over time were significantly less in groups treated with hTRA8+zoledronic and mTRA8 + Zoledronic acid combination as compared with the saline control group. At day 33, both combination therapies and zoledronic acid monotherapy provided significant reduction in total tumor burden and tumor infiltration of hindlimbs by histomorphometry (p0.05).DR5 agonists in combination with bisphosphonates may be an acceptable combination therapy to reduce breast cancer growth in bone.

BACKGROUND Although anti-angiogenic therapy is a promising new line of therapy for prostate cancer, we recently reported that stable expression of endostatin arrested the progression of prostate cancer to poorly differentiated state and distant metastasis in TRAMP mice. However, the same therapy failed to provide any benefit when given either during or after the onset of metastatic switch. The present study determined the possible mechanisms behind the selective advantage of endostatin therapy in early-stage disease. METHODS Angiogenesis-related gene expression analysis was performed to identify target genes and molecular pathways involved in the therapy effects. Based on the results from in vivo studies, and recapitulation of the in vivo data in vitro using tumorigenic and non-tumorigenic human prostate cancer cells that are either androgen-sensitive or androgen-independent, analyses of possible mechanisms of the selective advantage of early treatment were performed using assays for cell proliferation, apoptosis, migration, and cell signaling. The identified mechanisms were further confirmed in vivo. RESULTS Results indicated that cells with high androgen receptor (AR) expression were more sensitive to endostatin treatment than androgen-independent cells with low or no AR expression. Endostatin was found to significantly downregulate the expression of growth factors, receptor tyrosine kinases, proteases, and AR both in vitro and in vivo only when the cells express high-levels of AR. Cell proliferation was not influenced by endostatin treatment but migration was significantly affected only in androgen-sensitive cells. Targeted downregulation of AR prior to endostatin treatment in androgen-sensitive cells and overexpression of AR in androgen-independent cells indicated that the effect of endostatin via AR downregulation is mediated by a non-genotropic mechanism on Ras and RhoA pathways, and independently of AR on MAPK/ERK pathway. CONCLUSIONS These data indicate that systemically stable endostatin expression delays the onset of metastatic switch by acting on multiple pathways involving AR. Prostate 69:1055–1066, 2009. © 2009 Wiley-Liss, Inc.

Purpose: The aims of this study were to investigate telomere function in normal and Barrett's esophageal adenocarcinoma (BEAC) cells purified by laser capture microdissection and to evaluate the effect of telomerase inhibition in cancer cells in vitro and in vivo. Experimental Design: Epithelial cells were purified from surgically resected esophagi. Telomerase activity was measured by modified telomeric repeat amplification protocol and telomere length was determined by real-time PCR assay. To evaluate the effect of telomerase inhibition, adenocarcinoma cell lines were continuously treated with a specific telomerase inhibitor (GRN163L) and live cell number was determined weekly. Apoptosis was evaluated by Annexin labeling and senescence by β-galactosidase staining. For in vivo studies, severe combined immunodeficient mice were s.c. inoculated with adenocarcinoma cells and following appearance of palpable tumors, injected i.p. with saline or GRN163L. Results: Telomerase activity was significantly elevated whereas telomeres were shorter in BEAC cells relative to normal esophageal epithelial cells. The treatment of adenocarcinoma cells with telomerase inhibitor, GRN163L, led to loss of telomerase activity, reduction in telomere length, and growth arrest through induction of both the senescence and apoptosis. GRN163L-induced cell death could also be expedited by addition of the chemotherapeutic agents doxorubicin and ritonavir. Finally, the treatment with GRN163L led to a significant reduction in tumor volume in a subcutaneous tumor model. Conclusions: We show that telomerase activity is significantly elevated whereas telomeres are shorter in BEAC and suppression of telomerase inhibits proliferation of adenocarcinoma cells both in vitro and in vivo.

Enhanced production of receptor activator of nuclear factor-kappaB ligand (RANKL) and its binding to RANK on the osteoclasts have been associated with osteolysis in breast cancer bone metastasis. Osteoprotegerin (OPG) is a decoy receptor that prevents RANKL-RANK interaction. This study determined the effects of sustained expression of OPG using a recombinant adeno-associated viral (rAAV) vector in mouse model of osteolytic breast cancer. Bone metastasis was established by intracardiac injection of the human breast cancer cell line MDA-MB-435. Following this, mice were administered a one-time intramuscular injection of rAAV encoding either OPG.Fc (OPG) or green fluorescent protein (GFP). Mice were killed 1 month later and the effects of therapy on tumor growth and bone remodeling were evaluated. Bioluminescence imaging showed significant reduction of tumor growth in bone of OPG.Fc-treated mice. Micro-computed tomography (microCT) analysis and histomorphometry of the tibia indicated significant protection of trabecular and cortical bones after OPG.Fc therapy. Despite the prevention of bone loss and tumor growth in bone, OPG.Fc therapy failed to provide long-term survival. OPG.Fc-treated mice developed more bone than age-matched normal mice, indicating a requirement for regulated transgene expression. Results of this study indicate the potential of rAAV-OPG therapy for reducing morbidity and mortality in breast cancer patients with osteolytic bone damage.

Acquired resistance to androgen receptor (AR)-targeted therapies compels the development of novel treatment strategies for castration-resistant prostate cancer (CRPC). Here, we report a profound effect of endostatin on prostate cancer cells by efficient intracellular trafficking, direct interaction with AR, reduction of nuclear AR level, and down-regulation of AR-target gene transcription. Structural modeling followed by functional analyses further revealed that phenylalanine-rich α1-helix in endostatin—which shares structural similarity with noncanonical nuclear receptor box in AR—antagonizes AR transcriptional activity by occupying the activation function (AF)-2 binding interface for coactivators and N-terminal AR AF-1. Together, our data suggest that endostatin can be recognized as an endogenous AR inhibitor that impairs receptor function through protein–protein interaction. These findings provide new insights into endostatin whose antitumor effect is not limited to inhibiting angiogenesis, but can be translated to suppressing AR-mediated disease progression in CRPC.

Osteolytic bone damage is a major cause of morbidity in several metastatic pathologies. Current therapies using bisphosphonates provide modest improvement, but cytotoxic side effects still occur prompting the need to develop more effective therapies to target aggressive osteoclastogenesis. Increased levels of receptor activator of NF–κB ligand (TNFSF11/RANKL), leading to RANKL-RANK signaling, remain the key axis for osteoclast activation and bone resorption. Osteoprotegerin (TNFRSF11B/OPG), a decoy receptor for RANKL, is significantly decreased in patients who present with bone lesions. Despite its potential in inhibiting osteoclast activation, OPG also binds to TNF-related apoptosis-inducing ligand (TNFSF10/TRAIL), making tumor cells resistant to apoptosis. Toward uncoupling the events of TRAIL binding of OPG and to improve its utility for bone remodeling without inducing tumor resistance to apoptosis, OPG mutants were developed by structural homology modeling based on interactive domain identification and by superimposing models of OPG, TRAIL, and its receptor DR5 (TNFRSF10B) to identify regions of OPG for rational design. The OPG mutants were purified and extensively characterized for their ability to decrease osteoclast damage without affecting tumor apoptosis pathway both in vitro and in vivo, confirming their potential in bone remodeling following cancer-induced osteolytic damage. Implications: OPG variants were developed that lack TRAIL binding, yet retain RANKL binding and suggest new possibilities for therapeutic targeting in osteolytic malignancies. Mol Cancer Res; 13(5); 819–27. ©2015 AACR.

Ovarian cancer remains difficult to treat mainly due to presentation of the disease at an advanced stage. Conditionally-replicating adenoviruses (CRAds) are promising anti-cancer agents that selectively kill the tumor cells. The present study evaluated the efficacy of a novel CRAd (Ad5/3-CXCR4-TIMP2) containing the CXCR4 promoter for selective viral replication in cancer cells together with TIMP2 as a therapeutic transgene, targeting the matrix metalloproteases (MMPs) in a murine orthotopic model of disseminated ovarian cancer. An orthotopic model of ovarian cancer was established in athymic nude mice by intraperitonal injection of the human ovarian cancer cell line, SKOV3-Luc, expressing luciferase. Upon confirmation of peritoneal dissemination of the cells by non-invasive imaging, mice were randomly divided into four treatment groups: PBS, Ad-ΔE1-TIMP2, Ad5/3-CXCR4, and Ad5/3-CXCR4-TIMP2. All mice were imaged weekly to monitor tumor growth and were sacrificed upon reaching any of the predefined endpoints, including high tumor burden and significant weight loss along with clinical evidence of pain and distress. Survival analysis was performed using the Log-rank test. The median survival for the PBS cohort was 33 days; for Ad-ΔE1-TIMP2, 39 days; for Ad5/3-CXCR4, 52.5 days; and for Ad5/3-CXCR4-TIMP2, 63 days. The TIMP2-armed CRAd delayed tumor growth and significantly increased survival when compared to the unarmed CRAd. This therapeutic effect was confirmed to be mediated through inhibition of MMP9. Results of the in vivo study support the translational potential of Ad5/3-CXCR4-TIMP2 for treatment of human patients with advanced ovarian cancer.

Purpose: Current evidence indicates that an osteoblast lesion in prostate cancer is preceded by osteolysis. Thus, prevention of osteolysis would reduce complications of bone metastasis. Bone marrow–derived mesenchymal stem cells have the ability to differentiate into osteoblast and produce osteoprotegerin, a decoy receptor for the receptor activator for nuclear factor κB ligand, naturally. The present study examined the potential of unmodified mesenchymal stem cells to prevent osteolytic bone lesions in a preclinical mouse model of prostate cancer. Experimental Design: The human prostate cancer cell line PC3 was implanted in tibiae of severe combined immunodeficient mice. After establishment of the tumor, either unmodified or genetically engineered mesenchymal stem cells overexpressing osteoprotegerin was injected at the site of tumor growth. The effects of therapy were monitored by bioluminescence imaging, micro–computed tomography, immunohistochemistry, and histomorphometry. Results: Data indicated significant (P < 0.001) inhibition of tumor growth and restoration of bone in mice treated with unmodified and modified mesenchymal stem cells. Detailed analysis suggested that the donor mesenchymal stem cell inhibited tumor progression by producing woven bone around the growing tumor cells in the tibiae and by preventing osteoclastogenesis. Conclusions: Overcoming the limitation of the number of mesenchymal stem cells available in the bone can provide significant amelioration for osteolytic damage without further modification. (Clin Cancer Res 2009;15(23):7175–85)

Adult stem cells represent a potential source for cell-based therapy of cancer. The present study evaluated the potential of bone marrow-derived mesenchymal stem cells (MSC), genetically modified to express interferon (IFN)-α, for the treatment of lung metastasis in an immunocompetent mouse model of metastatic melanoma. A recombinant adeno-associated virus (rAAV) 6 vector encoding IFN-α was used to transduce mouse bone marrow-derived MSC ex vivo. Expression and bioactivity of the transgenic protein from rAAV-transduced MSC were confirmed prior to in vivo studies. A lung metastasis model of melanoma was developed by i.v. injection of B16F10 cells into 8-week-old C57BL/6 mice. Ten days later, MSC transduced with rAAV-IFN-α or green fluorescent protein were intravenously injected. One cohort of mice was sacrificed to determine the effects of the therapy at an earlier time point, and another cohort was observed for long-term survival. Results indicated that systemic administration of MSC producing IFN-α reduced the growth of B16F10 melanoma cells and significantly prolonged survival. Immunohistochemistry analysis of the tumors from MSC-IFN-α-treated animals indicated an increase in apoptosis and a decrease in proliferation and blood vasculature. These data demonstrate the potential of adult MSC constitutively producing IFN-α to reduce the growth of lung metastasis in melanoma. Disclosure of potential conflicts of interest is found at the end of this article.

Antiangiogenic therapy is a promising alternative for prostate cancer growth and metastasis and holds great promise as an adjuvant therapy. The present study evaluated the potential of stable expression of angiostatin and endostatin before the onset of neoplasia and during the early and late stages of prostate cancer progression in transgenic adenocarcinoma of mouse prostate (TRAMP) mice. Groups of 5-, 10-, and 18-week-old male TRAMP mice received recombinant adeno-associated virus-6 encoding mouse endostatin plus angiostatin (E+A) by i.m. injection. The effects of therapy were determined by sacrificing groups of treated mice at defined stages of tumor progression and following cohorts of similarly treated mice for long-term survival. Results indicated remarkable survival after recombinant adeno-associated virus–(E+A) therapy only when the treatment was given at an earlier time, before the onset of high-grade neoplasia, compared with treatment given for invasive cancer. Interestingly, early-stage antiangiogenic therapy arrested the progression of moderately differentiated carcinoma to poorly differentiated state and distant metastasis. Immunohistochemical analysis of the prostate from treated mice indicated significantly lower endothelial cell proliferation and increased tumor cell apoptosis. Vascular endothelial growth factor receptor (VEGFR)-2 expression was significantly down-regulated in tumor endothelium after treatment but not VEGFR-1. Analysis of the neuroendocrine marker synaptophysin expression indicated that antiangiogenic therapy given at an early-stage disease reduced neuroendocrine transition of the epithelial tumors. These studies indicate that stable endostatin and angiostatin gene therapy may be more effective for minimally invasive tumors rather than advanced-stage disease. [Cancer Res 2007;67(12):5789–97]

Anti-angiogenic gene therapy is a rational alternative for inhibiting tumor growth and metastasis and holds greater promise as an adjuvant therapy. Since this form of therapy targets tumor vasculature and not tumor cells directly, for sustained tumoristatic effects, stable expression of anti-angiogenic factors at a therapeutic level needs to be maintained. In the present study, we evaluated the potential of recombinant adeno-associated virus (rAAV)-mediated stable expression of angiostatin and endostatin during early and late stages of prostate cancer progression in TRAMP mice. Cohorts of 8 week- old and 18 week-old male TRAMP mice received either no virus or 1.2 |[times]| 1011 genomic particles of rAAV encoding green fluorescent protein (GFP) or mouse endostatin plus angiostatin by intramuscular (i.m.) injection. The effects of therapy were determined by periodic immunohistochemical analyses of the prostate for apoptotic index, endothelial cell growth and tumor proliferation. The levels of endostatin and angiostatin in systemic circulation were measured by ELISA and survival index was recorded as the endpoint.

Abnormalities in androgen-androgen receptor (AR) axis have long been recognized for their roles in promoting prostate tumor growth and metastasis. Conventional therapies targeting androgen ablation show transient tumor regression, however, when the tumor recurs during castration-refractory metastatic stage, the current treatment options are very limited. Although the precise mechanism by which prostate cancer (PCa) cells circumvent the androgen deprivation is unclear, recurring tumors have been shown to retain AR activity and upregulation of AR-target gene expression such as prostate specific antigen (PSA), suggesting that targeting AR remains a key component of developing novel therapeutic strategies. Here, we show a profound effect of endostatin, an endogenous angiogenesis inhibitor, on proliferation and invasion of AR-positive PCa cells by targeting AR function. The present study identified that intracellular trafficking of endostatin and direct interaction with AR disrupts AR nuclear translocation and the consequent transcriptional activation of PSA gene. In addition, our structural modeling and site-directed mutagenesis studies suggest that the binding mechanism may include the interaction of bulky side chains (F31 and F34) present in endostatin with the co-activator binding interface (AF-2) in AR ligand-binding domain (LBD). Regarding drug resistance to the second-generation androgen antagonists in patients with metastatic PCa, recent studies revealed that a missense mutation in AR LBD (F876L) and the consequential antagonist-to-agonist switch confers continued AR activity. In this context, our study suggests that endostatin can be recognized as an endogenous AR inhibitor where its molecular interaction with AR may prevent from agonistic switch of AR function by fully masking the AF-2 subdomain. Overall, the current finding provides new insights into endostatin whose anti-cancer activity is not only limited to inhibiting angiogenesis, but can also be expanded to suppressing AR-mediated PCa progression by disrupting AR transactivation. Citation Format: Joo Hyoung Lee, Tatyana Isayeva, Matthew Larson, Diptiman Chanda, Igor Chesnokov, Selvarangan Ponnazhagan. Endostatin is a novel inhibitor of androgen receptor function in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 612. doi:10.1158/1538-7445.AM2014-612

Our recent studies indicated a significant up-regulation of anterior gradient protein-2 (AGR-2) in bone metastatic human prostate cancer cells, PC3, following growth in bone marrow conditioned medium. Evidence indicates that AGR2 belongs to the family of protein disulfide isomerases and has been linked to intestinal mucus production, tumor growth and metastasis of various adenocarcinomas and poor survival of patients with breast and prostate cancers. Few recent reports have also reported the regulatory elements of AGR2 function, but its specific role(s) in tumorigenesis and metastasis is still unclear. In the present study, we have analyzed the role of AGR2 in cancer progression and metastasis using prostate cancer as a model. Human prostate cancer tissue microarrays and autopsy tissues, obtained from University of Alabama at Birmingham consortiums were immunostained for AGR-2 expression. Results were also confirmed in a spontaneously developing transgenic adenocarcinoma of mouse prostate (TRAMP) model. Further characterization of AGR-2 function was determined in PC3 cells with targeted silencing of AGR-2 expressions for tumor growth and metastases in vitro and in vivo. Results of our study found AGR-2 expression mainly in the prostate luminar epithelial cells, but no difference was observed between normal prostate gland, BPH and early stages of prostate cancer in both human and TRAMP mouse. Significant reduction in AGR-2 expression was observed in grade IV & V prostate cancers. Interestingly, although declining AGR-2 was evident in higher grade prostate tumors, significantly elevated AGR-2 expressions were observed in the metastatic sites of the same individuals. AGR-2-silenced PC3 cells formed tumors in SCID mice similar to control cells whereas their ability to remain attached to various ECM proteins were greatly compromised. Several key integrins, which mediates ECM binding, were also found to be down-regulated in the AGR-2 silenced PC3 cells, suggesting loss of AGR-2 may be associated with their detachment from the primary tumor and initiation of metastasis. AGR-2 is further up-regulated in metastatic sites indicating its necessity for restoration of integrins for attachment in a new microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5173. doi:1538-7445.AM2012-5173

Prostate cancer has the highest rate of incidence among all cancers in men in the United States and the second leading cause of death behind lung cancer. The human homologue of the frog (Xenopus) anterior gradient protein (AGR-2) has gained considerable attention in recent years for its putative role in neoplastic growth, metastasis and poor survival of patients with breast and prostate cancers. AGR-2 is reported to be androgen dependent and expressed only during the early stages of prostate cancer progression, whereas significant decline in AGR-2 levels in higher grades tumors coincided with metastatic disease. Human primary prostate cancer tissues and regional metastasis were immunostained for AGR-2 expression. Results indicated that AGR-2 was localized in the glandular epithelium of the prostate gland. No significant difference in AGR-2 staining was observed between normal, BPH and well-differentiated prostate cancers, except in normal prostate, where AGR-2 was mostly concentrated in the nuclei. Similar results were observed in a spontaneously developing transgenic adenocarcinoma of mouse prostate (TRAMP) model. AGR-2 expressions was reduced significantly in moderately and poorly-differentiated tumors in both human and TRAMP mice. Higher AGR-2 levels were found in the metastatic tissue albeit lower expressions in the highest grades prostate tumors within the same individual, suggesting further requirement of AGR-2 for growth at the metastatic sites. Targeted silencing of AGR-2 in a bone metastatic prostate cancer cell line, PC3, resulted in significantly reduced cellular adhesion to various extracellular matrix proteins and a failure to form acinar structures following 3-dimensional growth on matrigel. We also determined that AGR-2 silencing led to down-regulation of crucial integrins in PC3 cells, which possibly explains mechanism by which AGR-2 maintains polarized morphology of epithelial cells through promotion of cell adhesion. Based on our findings, we hypothesize that loss of AGR-2 in prostate cancer cells allows detachment from the basement membrane of the primary site and initiates metastasis. AGR-2 as well as integrin levels are restored during passage through circulation and at the metastatic site(s), helping them re-attach to the new microenvironment. Citation Format: Diptiman Chanda, Anandi Sawant, Jonathan Adam Hensel, Tatyana Isayeva, Stephanie D. Reilly, Gene P. Siegal, Claire Smith, Selvarangan Ponnazhagan. Loss of anterior gradient protein in high-grade prostate cancer is associated with loss of tumor cell adhesion and epithelial cell polarity [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B52.

Skeletal metastasis is common in late stage prostate cancer, which leads to significant decline in the quality of life and mortality. In a recent study, we identified over-expression of anterior gradient protein (AGR2) in the PC3, bone metastatic prostate cancer cells following in vitro growth in bone marrow-conditioned medium. AGR2 is a secreted protein and originally reported for its role in the formation of forebrain and mucus-secreting cement gland in the frog Xenopus laevis. Role of AGR2 in carcinogenesis and metastasis is recently being investigated and reports indicate it is over-expressed in various adenocarcinomas and linked to poor survival. But no study has elucidated the role of AGR2 in bone metastasis. When AGR2 expression was compared between human prostate cancer cell lines obtained from bone (PC3), lymph node (LnCap) and brain (Du145) by RT-PCR and Western blot, highest level of AGR2 was observed in PC3 cells. To further confirm, PC3 cells expressing firefly luciferase were injected in athymic nude mice via intra-cardiac route and cells were retrieved from bone, adrenal, lung and heart 3 weeks later following organ dissemination. RT-PCR analysis indicated highest AGR2 expression in PC3 cells isolated from bone microenvironment, suggesting that AGR2 may have a potential role in prostate cancer bone metastasis. To correlate the relevance of this finding in humans, AGR2 immunohistochemistry was performed on human primary and metastatic prostate cancer tissue sections. Preliminary results indicate higher AGR2 expression in metastasized bone sections compared to primary prostate cancers. Additionally, AGR2 was stably silenced in the PC3 cells utilizing a short hairpin RNA and growth kinetics of the cell line was tested in vivo following intra-cardiac injection in a preclinical mouse model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5261. doi:10.1158/1538-7445.AM2011-5261

Prostate cancer commonly metastasizes to the skeleton in advanced stages and leads to significant decline in life expectancy. In a recent study we demonstrated enhanced proliferation of the human prostate cancer cell line PC3 when cultured in the presence of bone marrow conditioned medium (BMCM). In order to identify key molecules involved in increasing proliferation rate, total RNA was isolated from PC3 cells grown in either BMCM or regular medium and subjected to cDNA microarray analysis. Results indicated a significant up-regulation of anterior gradient homolog 2 (AGR2) in the PC3 cells cultured in BMCM compared to those grown in regular medium. AGR2 is a secreted protein and originally reported for its involvement in the formation of forebrain and mucus-secreting cement gland in the frog Xenopus laevis. AGR2 is also highly expressed in human adenocarcinomas of the prostate, breast, esophagus and the pancreas and a study indicated poor survival of prostate cancer patients with higher AGR2 expression. We compared AGR2 gene expression by RT-PCR in different metastatic prostate cancer cell lines, isolated from the bone (PC3 and C4-2B), brain (Du145) and the lymph node (LnCap). Highest AGR2 expression was observed in bone metastatic cell lines, mainly PC3 cells and least expression was observed in Du145 cells. PC3 cells expressing firefly luciferase were also injected via intra-cardiac route into 6 week-old SCID mice and widespread metastases were observed in the lung, heart, adrenal and bone. Three weeks after the inoculation of the PC3 cells, the mice were sacrificed, tumor cells were harvested from various metastatic sites, total RNA isolated and subjected to RT-PCR analysis for AGR2 expression. Interestingly, highest AGR2 expression was observed in PC3 cells isolated from bone microenvironment, suggesting that AGR2 may have a potential role in prostate cancer bone metastasis. To test this hypothesis, we developed a subclone of PC3 cells with targeted knock down of AGR2 transcripts using lentivirus shRNA vector and tested the cell clone in vitro by implanting intra-tibially or subcutaneously in SCID mice. Results of this study indicated a failure of tumor development in AGR2 knocked down PC3 clones. 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 231.

We recently reported that stable expression of endostatin and angiostatin arrested the progression of well differentiated prostate carcinoma to poorly-differentiated state and distant metastasis in TRAMP mice when given prior to the onset of metastasis switch. Analysis of the expression of angiogenesis-related genes in the prostate tissue of endostatin treated mice indicated a significant downregulation of genes involved in cell motility, proliferation, metastasis and angiogenesis when therapy was initiated at early stage-disease. In vitro experiments demonstrated that recombinant endostatin treatment significantly downregulated the expression of growth factors, receptor tyrosine kinases and proteases in androgen sensitive cells. A similar pattern was confirmed in prostate tissues obtained from in vivo studies. Targeted downregulation of androgen receptor (AR) prior to endostatin treatment indicated that the effect of endostatin through AR downregulation is by a non-genotypic mechanism on MAPK/ERK pathway, and independently of Src kinase. Further analysis indicated the kinetics of endostatin internalization for androgen sensitive prostate cancer cells to be much higher than in androgen independent cells. Co-immunoprecipitation studies in human prostate cancer cells, and yeast two-hybrid studies with endostatin and AR demonstrated a direct binding of endostatin protein with AR. Based on the results so far, we predict that direct interaction of endostatin with AR possibly eliminates ligand binding during early stages of prostate cancer arresting and delaying the progression of the disease. However, once metastatic switch occurs, when the cells grow in androgen independent manner, a limitation in endostatin internalization or binding of non-androgen ligands at other domains in AR impairs the tumoristatic ability of endostatin. Ongoing studies are aimed in identifying interaction domains of endostatin with AR by NMR as well as testing the combination of endostatin therapy with therapies targeting AR signaling. 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 1246.