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Supplementary Table 1 PDF file - 64K, Association of p-�-CaMKII IHC staining with osteosarcoma subtypes compared to normal bone

Supplementary Table 2 PDF file - 68K, Association of p-�-CaMKII IHC staining with osteosarcoma grade

Osteosarcoma is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. Despite improvements in osteosarcoma treatment, more specific molecular targets are needed as potential therapeutic options. One target of interest is α-Ca2+/calmodulin-dependent protein kinase II (α-CaMKII), a ubiquitous mediator of Ca2+-linked signaling, which has been shown to regulate tumor cell proliferation and differentiation. Here, we investigate the role of α-CaMKII in the growth and tumorigenicity of human osteosarcoma. We show that α-CaMKII is highly expressed in primary osteosarcoma tissue derived from 114 patients, and is expressed in varying levels in different human osteosarcoma (OS) cell lines [MG-63, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)/HOS, and 143B). To examine whether α-CaMKII regulates osteosarcoma tumorigenic properties, we genetically inhibited α-CaMKII in two osteosarcoma cell lines using two different α-CaMKII shRNAs delivered by lentiviral vectors and overexpressed α-CaMKII by retrovirus. The genetic deletion of α-CaMKII by short hairpin RNA (shRNA) in MG-63 and 143B cells resulted in decreased proliferation (50% and 41%), migration (22% and 25%), and invasion (95% and 90%), respectively. The overexpression of α-CaMKII in HOS cells resulted in increased proliferation (240%), migration (640%), and invasion (10,000%). Furthermore, α-CaMKII deletion in MG-63 cells significantly reduced tumor burden in vivo (65%), whereas α-CaMKII overexpression resulted in tumor formation in a previously nontumor forming osteosarcoma cell line (HOS). Our results suggest that α-CaMKII plays a critical role in determining the aggressive phenotype of osteosarcoma, and its inhibition could be an attractive therapeutic target to combat this devastating adolescent disease. Mol Cancer Res; 11(4); 349–59. ©2013 AACR.

Atherosclerosis prevalence is increased in chronic obstructive pulmonary disease (COPD) patients, independent of other risk factors. The etiology of the excess vascular disease in COPD is unknown, although it is presumably related to an underlying (if cryptic) systemic immune response. Autoantibodies with specificity for glucose-regulated protein 78 (GRP78), a multifunctional component of the unfolded protein response, are common in COPD patients and linked to comorbidities of this lung disease. We hypothesized anti-GRP78 autoreactivity might also be a risk factor for atherosclerosis in COPD patients. Carotid intima-medial thickness (cIMT) was measured in 144 current and former smokers by ultrasound. Concentrations of circulating IgG autoantibodies against full-length GRP78, determined by ELISA, were greater among subjects with abnormally increased cIMT (p < 0.01). Plasma levels of autoantibodies against a singular GRP78 peptide segment, amino acids 246–260 (anti-GRP78aa 246–260), were even more highly correlated with cIMT, especially among males with greater than or equal to moderate COPD (rs = 0.62, p = 0.001). Anti-GRP78aa 246–260 concentrations were independent of CRP, IL-6, and TNF-α levels. GRP78 autoantigen expression was upregulated among human aortic endothelial cells (HAECs) stressed by incubation with tunicamycin (an unfolded protein response inducer) or exposure to culture media flow disturbances. Autoantibodies against GRP78aa 246–260, isolated from patient plasma by immunoprecipitation, induced HAEC production of proatherosclerotic mediators, including IL-8. In conclusion, anti-GRP78 autoantibodies are highly associated with carotid atherosclerosis in COPD patients and exert atherogenic effects on HAECs. These data implicate Ag-specific autoimmunity in the pathogenesis of atherosclerosis among COPD patients and raise possibilities that directed autoantibody reduction might ameliorate vascular disease in this high-risk population.

TRAIL-activating therapy is promising in treating various cancers, including pancreatic cancer, a highly malignant neoplasm with poor prognosis. However, many pancreatic cancer cells are resistant to TRAIL-induced apoptosis despite their expression of intact death receptors (DRs). Protein O-GlcNAcylation is a versatile posttranslational modification that regulates various biological processes. Elevated protein O-GlcNAcylation has been recently linked to cancer cell growth and survival. In this study, we evaluated the role of protein O-GlcNAcylation in pancreatic cancer TRAIL resistance, and identified higher levels of O-GlcNAcylation in TRAIL-resistant pancreatic cancer cells. With gain- and loss-of-function of the O-GlcNAc-adding enzyme, O-GlcNActransferase (OGT), we determined that increasing O-GlcNAcylation rendered TRAIL-sensitive cells more resistant to TRA-8-induced apoptosis, while inhibiting O-GlcNAcylation promoted TRA-8-induced apoptosis in TRAIL-resistance cells. Furthermore, we demonstrated that OGT knockdown sensitized TRAIL-resistant cells to TRA-8 therapy in a mouse model in vivo. Mechanistic studies revealed direct O-GlcNAc modifications of DR5, which regulated TRA-8-induced DR5 oligomerization. We further defined that DR5 O-GlcNAcylation was independent of FADD, the adapter protein for the downstream death-inducing signaling. These studies have demonstrated an important role of protein O-GlcNAcylation in regulating TRAIL resistance of pancreatic cancer cells; and uncovered the contribution of O-GlcNAcylation to DR5 oligomerization and thus mediating DR-inducing signaling. The authors have demonstrated an important new role of protein O-GlcNAcylation in regulating pancreatic cancer TRAIL resistance; and uncovered the contribution of O-GlcNAcylation to TRAIL activation-induced oligomerization of death receptor 5 and apoptosis signaling. These findings support the potential use of O-GlcNAcylation inhibitors to enhance efficacy of TRAIL therapy.

A role for hereditary influences in the susceptibility for chronic obstructive pulmonary disease (COPD) is widely recognized. Cytotoxic lymphocytes are implicated in COPD pathogenesis, and functions of these leukocytes are modulated by interactions between their killer-cell immunoglobulin-like receptors (KIR) and human leukocyte antigen (HLA)-Class I molecules on target cells. We hypothesized HLA-Class I and KIR inheritance affect risks for COPD. HLA-Class I alleles and KIR genotypes were defined by candidate gene analyses in multiple cohorts of COPD patients (total n=392) and control smokers with normal spirometry (total n=342). Compared to controls, COPD patients had over-representations of HLA-C*07 and activating KIR2DS1, with under-representations of HLA-C*12. Particular HLA-KIR permutations were synergistic; e.g. the presence of HLA-C*07 + KIR2DS1 + HLA-C12null vs. HLAC*07null + KIR2DS1null + HLA-C12 was associated with COPD, especially among HLA-C1 allotype homozygotes (OR=18.5, 95%CI=3.7-90.9, p

Cellular calmodulin binds to the SH2 domain of Src kinase, and upon Fas activation it recruits Src into the death-inducing signaling complex. This results in Src-ERK activation of cell survival pathway through which pancreatic cancer cells survive and proliferate. We had proposed that the inhibition of the interaction of calmodulin with Src-SH2 domain is an attractive strategy to inhibit the proliferation of pancreatic cancer. Thus we have performed screening of compound libraries by a combination of methods and identified some compounds (initial leads) that target the calmodulin-binding region on the SH2 domain and inhibit the proliferation of pancreatic cancer cells in in vitro assays. Most of these compounds also exhibited varying degrees of cytotoxicity when tested against immortalized breast epithelial cell line (MCF10A). These initial leads are likely candidates for development in targeted delivery of compounds to cancer cells without affecting normal cells.

Rationale: Vascular calcification is a serious cardiovascular complication that contributes to the increased morbidity and mortality of patients with diabetes mellitus. Hyperglycemia, a hallmark of diabetes mellitus, is associated with increased vascular calcification and increased modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation). Objective: We sought to determine the role of protein O-GlcNAcylation in regulating vascular calcification and the underlying mechanisms. Methods and Results: Low-dose streptozotocin-induced diabetic mice exhibited increased aortic O-GlcNAcylation and vascular calcification, which was also associated with impaired aortic compliance in mice. Elevation of O-GlcNAcylation by administration of Thiamet-G, a potent inhibitor for O-GlcNAcase that removes O-GlcNAcylation, further accelerated vascular calcification and worsened aortic compliance of diabetic mice in vivo. Increased O-GlcNAcylation, either by Thiamet-G or O-GlcNAcase knockdown, promoted calcification of primary mouse vascular smooth muscle cells. Increased O-GlcNAcylation in diabetic arteries or in the O-GlcNAcase knockdown vascular smooth muscle cell upregulated expression of the osteogenic transcription factor Runx2 and enhanced activation of AKT. O-GlcNAcylation of AKT at two new sites, T430 and T479, promoted AKT phosphorylation, which in turn enhanced vascular smooth muscle cell calcification. Site-directed mutation of AKT at T430 and T479 decreased O-GlcNAcylation, inhibited phosphorylation of AKT at S473 and binding of mammalian target of rapamycin complex 2 to AKT, and subsequently blocked Runx2 transactivity and vascular smooth muscle cell calcification. Conclusions: O-GlcNAcylation of AKT at 2 new sites enhanced AKT phosphorylation and activation, thus promoting vascular calcification. Our studies have identified a novel causative effect of O-GlcNAcylation in regulating vascular calcification in diabetes mellitus and uncovered a key molecular mechanism underlying O-GlcNAcylation–mediated activation of AKT.

BackgroundThe processes that result in progression of idiopathic pulmonary fibrosis (IPF) remain enigmatic. Moreover, the course of this disease can be highly variable and difficult to accurately predict. We hypothesized analyses of body mass index (BMI), a simple, routine clinical measure, may also have prognostic value in these patients, and might provide mechanistic insights. We investigated the associations of BMI changes with outcome, plasma adipokines, and adaptive immune activation among IPF patients.MethodsData were analyzed in an IPF discovery cohort (n = 131) from the University of Pittsburgh, and findings confirmed in patients from the University of Alabama at Birmingham (n = 148). Plasma adipokines were measured by ELISA and T-cell phenotypes determined by flow cytometry.ResultsTransplant-free one-year survivals in subjects with the greatest rates of BMI decrements, as percentages of initial BMI (>0.68%/month), were worse than among those with more stable BMI in both discovery (HR = 1.8, 95%CI = 1.1-3.2, p = 0.038) and replication cohorts (HR = 2.5, 95%CI = 1.2-5.2, p = 0.02), when adjusted for age, baseline BMI, and pulmonary function. BMI decrements >0.68%/month were also associated with greater mortality after later lung transplantations (HR = 4.6, 95%CI = 1.7-12.5, p = 0.003). Circulating leptin and adiponectin levels correlated with BMI, but neither adipokine was prognostic per se. BMI decrements were significantly associated with increased proportions of circulating end-differentiated (CD28null) CD4 T-cells (CD28%), a validated marker of repetitive T-cell activation and IPF prognoses.ConclusionsIPF patients with greatest BMI decrements had worse outcomes, and this effect persisted after lung transplantation. Weight loss in these patients is a harbinger of poor prognoses, and may reflect an underlying systemic process, such as adaptive immune activation.

Serum response factor (SRF) plays vital roles in numerous cellular processes; however, the physiological function of SRF in skeletal tissue remains unknown. In several organ systems, SRF regulates the expression of insulin-like growth factor-1 (IGF-1), which is crucial for normal development of mineralized skeleton and bone remodeling throughout life. Here, we show that conditional deletion of SRF in osteoblasts by osteocalcin-Cre generated viable mice with normal body size and body weight. Compared with normal siblings, osteoblast-specific SRF-deficient adult mice exhibited a marked decrease in bone mineral density and bone formation rate. Deletion of SRF in primary mouse calvarial osteoblasts reduced cell differentiation and mineralization in vitro. This was accompanied by a decrease in IGF-1 expression and secretion. Addition of IGF-1 in the culture media enhanced osteoblast differentiation in control cells and partially restored the mineralization defect of SRF-deficient cells, supporting an important role of SRF in regulating IGF-1 and IGF-1-mediated osteoblast differentiation. IGF-1-induced Akt activation was inhibited in SRF-deficient calvarial cells and enhanced in the SRF overexpressed cells. In addition, SRF deficiency decreased the transcriptional activity of Runx2, the key transcription factor for osteogenesis. Overexpression of SRF induced Runx2 transactivity in control cells and restored Runx2 transactivity in the SRF-deficient cells. Taken together, we conclude that SRF is important for IGF-1-induced osteoblast differentiation and mineralization via regulating IGF-1 expression and Runx2 transactivity.

We have previously demonstrated that calmodulin (CaM) binds directly to c-FLIP(L) in a Ca(2+)-dependent manner. Deletion of the CaM-binding region (amino acid 197-213) results in reduced CaM binding, and increased Fas-mediated apoptosis and decreased tumorigenesis of cholangiocarcinoma cells. The present studies were designed to identify the precise amino acids between 197 and 213 that are responsible for CaM/FLIP binding, and their roles in mediating the anti-apoptotic function of c-FLIP(L). Sequence analysis of the CaM-binding region at 197-213 predicted three unique positively charged residues at 204, 207 and 209, which might be responsible for the CaM/FLIP binding. A point mutation at H204 of c-FLIP(L) was found to markedly reduce CaM binding, whereas point mutation at R207 or K209 did not affect c-FLIP(L) binding to CaM. Decreased CaM/FLIP binding was confirmed in cholangiocarcinoma cells overexpressing the H204 c-FLIP(L) mutant. Reduced CaM binding by the H204 mutant resulted in increased sensitivity to Fas-mediated apoptosis and inhibited tumor growth in mice compared with wild-type c-FLIP(L). Death-inducing signaling complex (DISC) analysis showed that the reduced CaM binding to H204 mutant resulted in less c-FLIP(L) recruited into the DISC. Concurrently, increased caspase 8 was recruited to the DISC, which resulted in increased cleavage and activation of caspase 8, activation of downstream caspase 3 and increased apoptosis. Therefore, these results demonstrate that the H204 residue is responsible for c-FLIP(L) binding to CaM, which mediates the anti-apoptotic function of c-FLIP(L), most likely through affecting recruitment of caspase 8 into the DISC and thus caspase 8 activation. These studies further characterized CaM/FLIP interaction and its function in regulating Fas-mediated apoptosis and tumorigenesis, which may provide new therapeutic targets for cancer therapy.

Pancreatic cancer remains a devastating malignancy with a poor prognosis and is largely resistant to current therapies. To understand the resistance of pancreatic tumors to Fas death receptor-induced apoptosis, we investigated the molecular mechanisms of Fas-activated survival signaling in pancreatic cancer cells. We found that knockdown of the Fas-associated protein with death domain (FADD), the adaptor that mediates downstream signaling upon Fas activation, rendered Fas-sensitive MiaPaCa-2 and BxPC-3 pancreatic cells resistant to Fas-induced apoptosis. By contrast, Fas activation promoted the survival of the FADD knockdown MiaPaCa-2 and BxPC-3 cells in a concentration-dependent manner. The pharmacological inhibitor of ERK, PD98059, abrogated Fas-promoted cell survival in FADD knockdown MiaPaCa-2 and BxPC-3 cells. Furthermore, increased phosphorylation of Src was demonstrated to mediate Fas-induced ERK activation and cell survival. Immunoprecipitation of Fas in the FADD knockdown cells identified the presence of increased calmodulin, Src, and phosphorylated Src in the Fas-associated protein complex upon Fas activation. Trifluoperazine, a calmodulin antagonist, inhibited Fas-induced recruitment of calmodulin, Src, and phosphorylated Src. Consistently, trifluoperazine blocked Fas-promoted cell survival. A direct interaction of calmodulin and Src and their binding site were identified with recombinant proteins. These results support an essential role of calmodulin in mediating Fas-induced FADD-independent activation of Src-ERK signaling pathways, which promote survival signaling in pancreatic cancer cells. Understanding the molecular mechanisms responsible for the resistance of pancreatic cells to apoptosis induced by Fas-death receptor signaling may provide molecular insights into designing novel therapies to treat pancreatic tumors.

Objective— Clinical and experimental studies demonstrate the important roles of vascular smooth muscle cells (VSMC) in the pathogenesis of atherosclerosis. We have previously determined that the osteogenic transcription factor Runx2 is essential for VSMC calcification. The present study characterized Runx2-regulated signals and their potential roles in vascular calcification. Methods and Results— In vivo studies with atherogenic apolipoprotein E −/− mice demonstrated that increased oxidative stress was associated with upregulation of Runx2 and receptor activator of nuclear factor κB ligand (RANKL), which colocalized in the calcified atherosclerotic lesions and were juxtaposed to infiltrated macrophages and osteoclast-like cells that are positively stained for an osteoclast marker, tartrate-resistant acid phosphatase. Mechanistic studies using RNA interference, a luciferase reporter system, chromatin immunoprecipitation, and electrophoretic mobility shift assays indicated that Runx2 regulated the expression of RANKL via a direct binding to the 5′-flanking region of the RANKL. Functional characterization revealed that RANKL did not induce VSMC calcification, nor was RANKL required for oxidative stress–induced VSMC calcification. Using a coculture system, we demonstrated that VSMC-expressed RANKL induced migration as well as differentiation of bone marrow-derived macrophages into multinucleated, tartrate-resistant acid phosphatase–positive osteoclast-like cells. These effects were inhibited by the RANKL antagonist osteoprotegerin and with VSMC deficient in Runx2 or RANKL. Conclusion— We demonstrate that Runx2 directly binds to the promoter and controls the expression of RANKL, which mediates the crosstalk between calcifying VSMC and migration and differentiation of macrophages into osteoclast-like cells in the atherosclerotic lesions. Our studies provide novel mechanistic insights into the regulation and function of VSMC-derived RANKL in the pathogenesis of atherosclerosis and vascular calcification.

Cholangiocarcinoma is a highly malignant tumor with limited therapeutic options. We have previously reported that tamoxifen (TMX) induces apoptosis of cholangiocarcinoma cells and reduces cholangiocarcinoma tumorigenesis in mice. In the present studies, we determined the effect of combination therapy of TMX and gemcitabine (GMT), another chemotherapeutical reagent for many cancers, on cholangiocarcinoma tumorigenesis and investigated the responsible mechanisms. GMT inhibited cell growth and induced apoptosis of cholangiocarcinoma cells in a concentration-dependent manner. TMX enhanced GMT-induced apoptosis of cholangiocarcinoma cells. Consistently, GMT (15 mg/kg) inhibited cholangiocarcinoma tumorigenesis in nude mice by 50%. TMX (15 mg/kg) enhanced the inhibitory effect of GMT on tumorigenesis by 33%. The inhibition of tumor growth correlated with enhanced apoptosis in tumor tissues. To elucidate the mechanisms underlying the additive effects of TMX on GMT-induced apoptosis, we determined the activation of caspases in cholangiocarcinoma cells exposed to GMT, TMX, or both. Activation of caspases 9 and 3, as well as cytochrome c release to the cytosol, was demonstrated in cells exposed to both reagents. In contrast, TMX activated caspase 2, whereas GMT had no effect. Inhibition of caspase 2 activation decreased TMX-, but not GMT-, induced activation of caspase 3 and apoptosis of cholangiocarcinoma cells. Similarly, activation of caspase 2 was found in tumors from TMX-treated mice, but not GMT-treated mice. Therefore, the enhanced effect of TMX on GMT-induced cholangiocarcinoma cell death is partially mediated by activation of caspase 2. TMX and GMT both induce apoptosis and inhibit cholangiocarcinoma tumorigenesis, which may be attributed to the activation of distinct apoptosis signals by TMX and GMT. Our studies provide in vivo evidence and molecular insight to support the use of TMX and GMT in combination as an effective therapy for cholangiocarcinoma.

Osteosarcoma is the most frequent type of primary bone cancer in children and adolescents. These malignant osteoid forming tumors are characterized by their uncontrolled hyperproliferation. Here, we investigate the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in the growth of human osteosarcoma. We show that alpha-CaMKII is expressed in human osteosarcoma cell lines and in primary osteosarcoma tissue derived from patients. The pharmacologic inhibition of CaMKII in MG-63 and 143B human osteosarcoma cells by KN-93 resulted in an 80 and 70% decrease in proliferation, respectively, and induced cell cycle arrest in the G(0)/G(1) phase. The in vivo administration of KN-93 to mice xenografted with human osteosarcoma cells significantly decreased intratibial and subcutaneous tumor growth. Mechanistically, KN-93 and alpha-CaMKII siRNA increased p21((CIP/KIP)) gene expression, protein levels, and decreased the phosphorylation of retinoblastoma protein and E2F transactivation. Furthermore, the inhibition of CaMKII decreased membrane-bound Tiam1 and GTP-bound Rac1, which are known to be involved in p21 expression and tumor growth in a variety of solid malignant neoplasms. Our results suggest that CaMKII plays a critical role in the growth of osteosarcoma, and its inhibition could be an attractive therapeutic target to combat conventional high-grade osteosarcoma in children.

Pancreatic cancer is the fourth leading cause of cancer deaths in the United States. It is generally a fatal cancer with poor prognosis. Once pancreatic cancer becomes metastatic, it is uniformly fatal with an overall average survival of typically 6 months. The only potentially curative therapy for pancreatic cancer is surgical resection. Unfortunately, the majority of pancreatic cancer patients have advanced and inoperable disease at the time of diagnosis. Studies from our group and others have supported that modulating tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis provides potential novel avenues for cancer therapy. However, resistance of pancreatic cancer to TRAIL therapy remains a large clinical hurdle. Thus, better understanding of the molecular events regulating pancreatic cancer cell apoptosis would facilitate the development of novel strategies to enhance the efficacy of TRAIL therapy.

Purpose: Activating extrinsic apoptotic pathways targeting death receptors (DR) using agonistic antibodies or TNF-related apoptosis-inducing ligand (TRAIL) is promising for cancer therapy. However, most pancreatic cancers are resistant to TRAIL therapy. The present studies aimed to identify combination therapies that enhance the efficacy of TRAIL therapy and to investigate the underlying mechanisms. Experimental Design: A xenograft model in nude mice was used to determine pancreatic cancer tumorigenesis and therapeutic efficacy of TRA-8, a monoclonal agonistic antibody for DR5. Pancreatic cancer cells were used to characterize mechanisms underlying PARP-1 regulation of TRA-8–induced apoptosis in vitro. Results: PARP-1 was found highly expressed in the TRA-8–resistant PANC-1 and Suit-2 cells, compared with TRA-8–sensitive BxPc-3 and MiaPaca-2. Inhibition of PARP-1 with a pharmacologic inhibitor sensitized PANC-1 and Suit2 cells to TRA-8–induced apoptosis in a dose-dependent manner. Furthermore, siRNAs specifically knocking down PARP-1 markedly enhanced TRA-8–induced apoptosis in vitro and augmented the efficacy of TRA-8 therapy on tumorigenesis in vivo. PARP-1 knockdown increased TRA-8–induced activation of caspase-8 in the death-induced signaling complex (DISC). Immunoprecipitation with DR5 antibody identified the recruitment of PARP-1 and PARP-1–mediated protein poly-ADP-ribosylation (pADPr) modification in the DR5-associated DISC. Further characterization revealed that PARP-1–mediated pADPr modification of caspase-8 inhibited caspase-8 activation, which may contribute to its function in regulating TRA-8 resistance. Conclusions: Our studies provide molecular insights into a novel function of PARP-1 in regulating the extrinsic apoptosis machinery and also support interventions combining PARP-1 inhibitors with DR agonists for pancreatic cancer therapy. Clin Cancer Res; 19(17); 4750–9. ©2013 AACR.

Osteosarcoma is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. Despite improvements in osteosarcoma treatment, more specific molecular targets are needed as potential therapeutic options. One target of interest is α-Ca2+/calmodulin-dependent protein kinase II (α-CaMKII), a ubiquitous mediator of Ca2+-linked signaling, which has been shown to regulate tumor cell proliferation and differentiation. Here, we investigate the role of α-CaMKII in the growth and tumorigenicity of human osteosarcoma. We show that α-CaMKII is highly expressed in primary osteosarcoma tissue derived from 114 patients, and is expressed in varying levels in different human osteosarcoma (OS) cell lines [MG-63, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)/HOS, and 143B). To examine whether α-CaMKII regulates osteosarcoma tumorigenic properties, we genetically inhibited α-CaMKII in two osteosarcoma cell lines using two different α-CaMKII shRNAs delivered by lentiviral vectors and overexpressed α-CaMKII by retrovirus. The genetic deletion of α-CaMKII by short hairpin RNA (shRNA) in MG-63 and 143B cells resulted in decreased proliferation (50% and 41%), migration (22% and 25%), and invasion (95% and 90%), respectively. The overexpression of α-CaMKII in HOS cells resulted in increased proliferation (240%), migration (640%), and invasion (10,000%). Furthermore, α-CaMKII deletion in MG-63 cells significantly reduced tumor burden in vivo (65%), whereas α-CaMKII overexpression resulted in tumor formation in a previously nontumor forming osteosarcoma cell line (HOS). Our results suggest that α-CaMKII plays a critical role in determining the aggressive phenotype of osteosarcoma, and its inhibition could be an attractive therapeutic target to combat this devastating adolescent disease. Mol Cancer Res; 11(4); 349–59. ©2013 AACR.

Objective— Smad4 is a central mediator of transforming growth factor-β/bone morphogenetic protein signaling that controls numerous developmental processes as well as homeostasis in the adult. The present studies sought to understand the function of Smad4 expressed in vascular smooth muscle cells (VSMC) in vascular development and the underlying mechanisms. Methods and Results— Breeding of Smad4 flox/flox mice with SM22α-Cre mice resulted in no viable offspring with SM22α-Cre ; Smad4 flox/flox genotype in a total of 165 newborns. Subsequent characterization of 301 embryos between embryonic day (E) 9.5 and E14.5 demonstrated that mice with SM22α-Cre ; Smad4 flox/flox genotype died between E12.5 and E14.5 because of decreased cell proliferation and increased apoptosis in the embryonic heart and arteries. Additionally, deletion of Smad4 more specifically in SMC with the inducible smooth muscle myosin heavy chain (SMMHC)-Cre mice, in which decreased cell proliferation was observed only in the artery but not the heart, also caused lethality of the knockout embryos at E12.5 and E14.5. The Smad4 -deficient VSMC lacked smooth muscle α-actin filaments, decreased expression of SMC-specific gene markers, and markedly reduced cell proliferation, migration, and attachment. Using specific pharmacological inhibitors and small interfering RNAs, we demonstrated that inhibition of transforming growth factor-β signaling and its regulatory Smad 2/3 decreased VSMC proliferation, migration, and expression of SMC-specific gene markers, whereas inhibition of bone morphogenetic protein signaling only affected VSMC migration. Conclusion— SMC-specific deletion of Smad4 results in vascular defects that lead to embryonic lethality in mice, which may be attributed to decreased VSMC differentiation, proliferation, migration, as well as cell attachment and spreading. The transforming growth factor-β signaling pathway contributes to VSMC differentiation and function, whereas the bone morphogenetic protein signaling pathway regulates VSMC migration. These studies provide important insight into the role of Smad4 and its upstream Smads in regulating SMC function and vascular development of mice.

Rationale: Vascular calcification is a hallmark of atherosclerosis, a major cause of morbidity and mortality in the United States. We have previously reported that the osteogenic transcription factor Runx2 is an essential and sufficient regulator of calcification of vascular smooth muscle cells (VSMC) in vitro. Objective: To determine the contribution of osteogenic differentiation of VSMC to the pathogenesis of vascular calcification and the function of VSMC-derived Runx2 in regulating calcification in vivo. Methods and Results: SMC-specific Runx2-deficient mice, generated by breeding SM22α-Cre mice with the Runx2 exon 8 floxed mice, exhibited normal aortic gross anatomy and expression levels of SMC-specific marker genes. Runx2 deficiency did not affect basal SMC markers, but inhibited oxidative stress-reduced expression of SMC markers. High-fat-diet-induced vascular calcification in vivo was markedly inhibited in the Runx2-deficient mice in comparison with their control littermates. Runx2 deficiency inhibited the expression of receptor activator of nuclear factor κB ligand, which was accompanied by decreased macrophage infiltration and formation of osteoclast-like cells in the calcified lesions. Coculture of VSMC with bone marrow–derived macrophages demonstrated that the Runx2-deficient VSMC failed to promote differentiation of macrophages into osteoclast-like cells. Conclusions: These data have determined the importance of osteogenic differentiation of VSMC in the pathogenesis of vascular calcification in mice and defined the functional role of SMC-derived Runx2 in regulating vascular calcification and promoting infiltration of macrophages into the calcified lesion to form osteoclast-like cells. Our studies suggest that the development of vascular calcification is coupled with the formation of osteoclast-like cells, paralleling the bone remodeling process.

Objective— Vascular calcification is prevalent in patients with atherosclerosis, diabetes mellitus, and end stage renal disease, and it increases morbidity and mortality of these patients. Dramatic vascular calcification has been demonstrated in several experimental diabetic models, including the streptozotocin-induced mouse model of diabetes. Using low-dose STZ, we demonstrated that STZ-induced hyperglycemia was associated with increased protein O-link glycosylation (O-GlcNAc) modification in the mouse vasculature. The present studies sought to determine the role of O-GlcNAc modifications in regulating vascular calcification. Methods and Results— We found that Thiamet-G, a specific inhibitor of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc, dramatically increased O-GlcNAc modification in primary mouse vascular smooth muscle cells (VSMC). Non-toxic concentrations of Thiamet-G (10nM - 10μM) induced calcification of VSMC in a dose-dependent manner. Thiamet-G-induced VSMC calcification was associated with increased expression of osteogenic genes, including the key regulator for VSMC calcification Runx2, osteocalcin, alkaline phosphatase, and collagen I (Runx2=2.97±0.31, OC=4.85±0.10, ALP=2.35±0.33, ColIa=1.86±0.20, p Furthermore, OGA shRNA markedly increased O-GlcNAc modification in VSMC. A 3.7-fold increase in calcification, as indicated by calcium content, was demonstrated in OGA knockdown VSMC compared with control VSMC. Increased calcification was associated with a 4.8-fold increase in expression of osteocalcin, a Runx2-regulated osteogenic gene. In addition, OGA knockdown enhanced and sustained phosphorylation/activation of AKT, an upstream signal that we have reported to regulate Runx2 activity. Conclusion— We have found that increased O-GlcNAc modification in VSMC enhances calcification of VSMC, which may be attributed to increased activation of AKT and Runx2 signals. These results provide novel mechanistic insights into the role of protein O-GlcNAc modification in regulating vascular calcification in diabetes.

Pancreatic cancer is the fourth leading cause of cancer death in the USA. The 5-year survival rate has only improved from 2% to 6% in the past 35 years. TNF-related apoptosis-inducing ligand (TRAIL) or agonistic antibodies bind to death receptor 4 or 5 and induce apoptosis in a wide variety of tumors. TRA-8, a humanized activating antibody for the death receptor 5 (DR5), is a potent apoptosis-inducing reagent for cancer cells. However, several pancreatic cell lines are resistant to TRA-8-induced apoptosis. The goal of our studies is to indentify therapeutic agents that sensitize resistant pancreatic cancer cells to TRA-8-induced apoptosis and determine the underlying mechanism. Among an array of compounds, we identified that trifluoperazine (TFP) and tamoxifen (TMX), two calmodulin (CaM) antagonists, enhanced TRA-8-induced cell death of TRA-8 resistance pancreatic cells. Both TFP and TMX increased TRA-8-induced apoptosis of resistant PANC-1 cells in dose-dependent manners. TMX treatment enhanced the efficacy of TRA-8 therapy on tumorigenesis of PANC-1 cells in a mouse xenograft model by 5 fold (n=7, p Citation Format: Kaiyu Yuan, Tong Zhou, Jay McDonald, Yabing Chen. Augmentation of DR5 expression by calmodulin antagonists sensitizes TRA-8-induced apoptosis in pancreatic 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 2268. doi:10.1158/1538-7445.AM2014-2268

Current chemotherapy and radiotherapy are not curative for pancreatic cancer. Tumor necrosis factor-related apoptosis-inducing ligand or agonist antibodies for death receptor 4/5 have been shown to be a promising cancer therapy. TRA-8, a humanized activating antibody for the death receptor 5 (DR5), is a potent apoptosis-inducing reagent for cancer cells. However, several pancreatic cell lines are resistant to TRA-8-induced apoptosis. We have previously demonstrated that calmodulin (CaM) antagonists enhance the Fas death receptor-mediated apoptosis pathway. Therefore, we investigated whether inhibition of CaM might affect DR5-induced apoptotic signaling in pancreatic cancer cells. We found that CaM antagonists, trifluoperazine (TFP) and tamoxifen (TMX), dose dependently enhanced TRA-8-induced apoptosis of the TRA-8-resistant PANC-1 pancreatic cells. Consistently, TFP and TMX increased TRA-8-induced activation of caspase 8, caspase 9 and caspase 3. Pretreatment of PANC-1 with Z-IETD-FMK, the caspase-8 inhibitor, decreased TFP-enhanced apoptosis by 2 fold, and markedly decreased caspase 3 activation. Immunoprecipitation analysis of the DR5-induced death inducing signaling complex (DISC) identified the interaction of CaM with DR5. Activation of DR-5 by TRA-8 increased the recruitment of CaM into the DISC, which was inhibited by TFP. In addition, TFP decreased the recruitment of the adaptor protein, FADD, and other DISC proteins that regulate cell survival, including Src and the caspase-8 inhibitory protein FLIP. Collectively, these results support an important role for CaM in DR5-induced DISC formation, thus regulating DR5-mediated survival and apoptotic signaling in pancreatic cancer cells. In summary, we have demonstrated that CaM antagonists sensitize pancreatic cancer cell to TRA 8-induced apoptosis, and that CaM binding to DR5 regulates DR5-induced DISC formation. Defining the function of CaM in regulating DR5-induced signaling pathways may lead to novel therapeutic strategies including the use of CaM antagonists with TRA-8 as combination therapy for pancreatic cancer. Citation Format: Kaiyu Yuan, Tong Zhou, Jay McDonald, Yabing Chen. Inhibition of calmodulin enhances TRA-8-induced apoptosis in pancreatic cancer cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2953. doi:10.1158/1538-7445.AM2013-2953

Pancreatic cancer is a highly malignant tumor with limited therapy and poor prognosis. Activation of death receptors (DR)-induced apoptotic pathways using antibodies or agonists for the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapy. However, most pancreatic tumors are resistant to TRAIL therapy. The present studies aimed to identify combination therapies that enhance the efficacy of TRAIL therapy by sensitizing resistant pancreatic cancer cells to TRAIL-induced apoptosis, and to investigate the underlying mechanisms. Using TRA-8, a monoclonal agonistic antibody specific for DR5, we demonstrated that TRA-8 induced apoptosis of 60% and 83% of BxPc-3 and MiaPaCa-2 pancreatic cancer cells in 24 hours. By contrast, only 20% and 24% of PANC-1 and Suit-2 cells underwent apoptosis in response to TRA-8 (n=3), despite the fact that they express high levels of the DR5 receptor. The expression of poly(ADP-ribose) polymerase-1 (PARP-1) was higher in the PANC-1 and Suit-2 cells than that BxPc-3 and MiaPaCa-2 cells, which may contribute to their resistance to TRA-8-induced apoptosis. Inhibition of PARP-1 with a pharmacologic inhibitor (PJ-34) sensitized PANC-1 to TRA-8 induced apoptosis in a dose-dependent manner. Furthermore, small interfering RNAs specifically knocking down PARP-1 in PANC-1 cells enhanced TRA-8-induced apoptosis in vitro by 2.5 fold, as well as the efficacy of TRA-8 therapy on tumorigenesis of PANC-1 cells in a mouse xenograft model by 3.8 fold (n=8, p Citation Format: Kaiyu Yuan, Yong Sun, Tong Zhou, Jay McDonald, Yabing Chen. PARP-1 regulates death receptor 5-mediated apoptosis in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2947. doi:10.1158/1538-7445.AM2013-2947

Targeting the extrinsic apoptosis pathway initiated by death receptors has been shown to be a promising therapy for pancreatic cancer. TRA-8, a humanized activating antibody for the death receptor 5 (DR5), is a potent therapeutic reagent for pancreatic cancer. However, several pancreatic cell lines are resistant to TRA-8-induced cell death. We have previously demonstrated that calmodulin (CaM) is recruited into the death-inducing signaling complex (DISC) by Fas death receptor activation, and that antagonists of CaM regulate DISC formation. Therefore, we determined the function of CaM in DR5-induced apoptosis of pancreatic cancer cells. We found that trifluoperazine (TFP), a CaM antagonist, enhanced TRA-8-induced cell death of pancreatic cells, PANC-1 and S2VP10, by 55% and 45% respectively. Consistent with the increased apoptosis, TFP increased TRA-8-induced activation of caspase 8 and caspase 3 in these cells. To determine the underlying molecular mechanisms, we analyzed the DR5-induced DISC. Interaction of CaM and DR5 was demonstrated by co-immunoprecipitation by using DR5 antibody. Activation of DR-5 by TRA-8 increased recruitment of CaM into the DISC which was inhibited by treatment with TFP. In addition, TFP decreased recruitment of the adaptor protein, FADD, and recruitment of other DISC proteins that regulate cell survival, including Src and FLICE-like inhibitory protein (FLIP), an inhibitor of caspase-8. These results suggest an important role for CaM in DR5-induced DISC formation in pancreatic cancer cells, regulating DR5-mediated survival and apoptotic signaling. In summary, we have demonstrated that CaM antagonism sensitizes pancreatic cancer cell to TRA 8-induced cell death, and that CaM binding to DR5 regulates DR5-induced DISC formation. Defining the function of CaM in regulating DR5-induced signaling pathways may lead to novel therapeutic strategies for pancreatic cancer therapy including possible combination therapy with TRA-8 and calmodulin antagonists. 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 246. doi:1538-7445.AM2012-246

Cholangiocarcinoma is a highly malignant tumor with limited therapeutic options. Gemcitabine (GMT) is currently used as a first line chemotherapeutical reagent for many cancers, including cholangiocarcinoma. However, it is often used in combination with other therapeutical strategies, due to high toxicity of GMT. We have previously reported that antagonists of calmodulin (CaM), including tamoxifen (TMX), trifluoperazine (TFP) and W7, induce apoptosis of cholangiocarcinoma cells and reduce cholangiocarcinoma tumorigenesis in mice. In the present studies, we determined the effect of combination therapy of TMX and GMT on cholangiocarcinoma tumorigenesis and investigated the mechanisms responsible for their efficacy. The effect of TMX and GMT on cholangiocarcinoma cell growth and apoptosis in vitro was determined by MTS and Annexin V staining analysis. The expression/activation of apoptotic signaling molecules were assessed by Western blot analysis. A nude mouse xenograft model was utilized for in vivo cholangiocarcinoma tumorigenesis as we previously reported. TMX (15mg/kg), GMT (15mg/kg) and combination of both were administrated by intraperitoneal injection. Tumor growth was measured every 3 days. Apoptosis in xenograft tumors was determined by TUNEL and cleaved caspase 3 staining. GMT inhibited cell growth and induced apoptosis of cholangiocarcinoma cells in a concentration-dependent manner. TMX enhanced GMT-induced apoptosis and GMT inhibition of cell growth in cholangiocarcinoma cells. GMT (15mg/kg, every third day) inhibited cholangiocarcinoma tumorigenesis in nude mice by 50%. TMX (15mg/kg, two out of three days) enhanced the inhibitory effect of GMX on tumorigenesis by 33%. The inhibition of tumor growth correlated with enhanced apoptosis in tumor tissues. To elucidate the mechanisms underlying the additive effects of TMX on GMT-induced apoptosis, we determined activation of caspases in cholangiocarcinoma cells exposed to GMT, TMX or both. Activation of caspase 9 and caspase 3, as well as cytochrome c release to the cytosol were demonstrated in cells exposed to either reagent alone. By contrast, TMX but not GMT activated caspase 2. Pharmacologic inhibition of caspase 2 activation decreased TMX-, but not GMT-, induced activation of caspase 3 and apoptosis of cholangiocarcinoma cells. Similarly, activation of caspase 2 was found in tumors from TMX-treated mice, but not GMT-treated mice. Therefore, the enhanced effect of TMX on GMT-induced cholangiocarcinoma cell death is partially mediated by activation of caspase 2. TMX and GMT both induce apoptosis and inhibit cholangiocarcinoma tumorigenesis, which may be attributed to the activation of distinct apoptosis signals by TMX and GMT. Our studies provide in vivo evidence and molecular insight to support the use of TMX and GMT in combination as an effective therapy for cholangiocarcinoma. 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 4089. doi:10.1158/1538-7445.AM2011-4089

Pancreatic cancer remains a devastating malignancy with poor prognosis, which is largely resistant to current therapies. Death receptor-initiated apoptotic pathways have been implicated both in pancreatic cancer pathogenesis and therapy. To understand the resistance of pancreatic tumor to Fas death receptor-induced apoptosis, we investigated the molecular mechanisms of Fas-activated survival signaling in pancreatic cancer cells. Using lentivirus-mediated RNA interfering strategy, we generated pancreatic cancer cell lines MiaPaCa-2 and BxPc-3 with stable knockdown of the Fas associated protein with death domain (FADD), the adaptor that mediates downstream signaling upon Fas activation. Knockdown of FADD rendered these Fas-sensitive pancreatic cells resistant to apoptosis induced by the Fas agonist antibody, CH-11. Consistently, CH-11 induced recruitment of caspase-8 into the Fas-activated death inducing signaling complex was blocked in FADD knockdown cells, which was associated with no activation of caspase-8 and caspase-3. By contrast, CH-11 promoted the survival of the FADD knockdown MiaPaCa2 and BxPc3 cells in a concentration-dependant manner, as measured by MTS assay. Increased phosphorylation of ERK was demonstrated in the FADD knockdown cells in response to CH-11 treatment. The pharmacological inhibitor of ERK, PD98059, abrogated CH-11-promoted cell survival in FADD knockdown MiaPaCa-2 and BxPC-3 cells. Furthermore, increased phosphorylation of Src was demonstrated in FADD knockdown cells in response to CH-11. The Fas-induced activation of Src was blocked by the specific Src inhibitor, PP2, which further inhibited the Fas-induced activation of ERK and cell survival of the FADD knockdown cells. To elucidate the mechanisms underlying Fas-induced Src activation, we analyzed Fas-associated proteins by immunoprecipitaion of Fas in the FADD knockdown cells. Increased Src and phosphorylated Src was indentified in the Fas-associated protein complex activation by CH-11. Concomitantly, increased recruitment of calmodulin (CaM) into the Fas-associated protein complex was demonstrated as well. Trifluoperazine, a CaM antagonist, inhibited the Fas-induced recruitment of CaM, Src and phosphorylated Src. Consistently, trifluoperazine inhibited Fas-induced activation of Src and blocked Fas-promoted cell survival in the FADD knockdown cells. These results demonstrate that Fas induces FADD-independent survival signaling in pancreatic cancer cells via CaM-mediated activation of Src-ERK signaling pathways. Understanding the molecular mechanisms responsible for the resistance of pancreatic cells to apoptosis induced by Fas-death receptor signaling may provide molecular insights into designing novel therapies to treat pancreatic tumors. 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 4776. doi:10.1158/1538-7445.AM2011-4776