A Novel Pan-RAS Inhibitor with a Unique Mechanism of Action Blocks Tumor Growth in Mouse Models of GI Cancer.

Here we characterize a novel pan-RAS inhibitor, ADT-007, that potently and selectively inhibited the growth of histologically diverse cancer cell lines with mutant or activated RAS irrespective of the RAS mutation or isozyme. Growth inhibition was dependent on activated RAS and associated with reduced GTP-RAS levels and MAPK/AKT signaling. ADT-007 bound RAS in lysates from sensitive cells with sub-nanomolar EC 50 values but did not bind RAS in lysates from insensitive cells with low activated RAS. Insensitivity to ADT-007 was attributed to metabolic deactivation by UGT-mediated glucuronidation, providing a detoxification mechanism to protect normal cells from pan-RAS inhibition. Molecular modeling and experiments using recombinant RAS revealed that ADT-007 binds RAS in a nucleotide-free conformation to block GTP activation. Local injection of ADT-007 strongly inhibited tumor growth in syngeneic immune competent and xenogeneic immune deficient mouse models of colorectal and pancreatic cancer and activated innate and adaptive immunity in the tumor microenvironment. SIGNIFICANCE: ADT-007 is a novel pan-RAS inhibitor with a unique mechanism of action having potential to circumvent resistance to mutant-specific KRAS inhibitors and activate antitumor immunity. The findings support further development of ADT-007 analogs and/or prodrugs with oral bioavailability as a generalizable monotherapy or combined with immunotherapy for RAS mutant cancers. BACKGROUND: It is projected that colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDA) will cause 52,580 and 49,830 deaths in the US in 2023, respectively (1). The 5-year survival rates for CRC and PDA are 65% and 12%, respectively (1). Over 50% of CRC and 90% of PDA patients harbor mutations in KRAS genes that are associated with poor prognosis, making the development of novel KRAS inhibitors an urgent unmet medical need (2).

Metronomic Administration of Topotecan Alone and in Combination with Docetaxel Inhibits Epithelial-mesenchymal Transition in Aggressive Variant Prostate Cancers.

Prostate cancer is the second leading cause of noncutaneous cancer-related deaths in American men. Androgen deprivation therapy (ADT), radical prostatectomy, and radiotherapy remain the primary treatment for patients with early-stage prostate cancer (castration-sensitive prostate cancer). Following ADT, many patients ultimately develop metastatic castration-resistant prostate cancer (mCRPC). Standard chemotherapy options for CRPC are docetaxel (DTX) and cabazitaxel, which increase median survival, although the development of resistance is common. Cancer stem-like cells possess mesenchymal phenotypes [epithelial-to-mesenchymal transition (EMT)] and play crucial roles in tumor initiation and progression of mCRPC. We have shown that low-dose continuous administration of topotecan (METRO-TOPO) inhibits prostate cancer growth by interfering with key cancer pathway genes. This study utilized bulk and single-cell or whole-transcriptome analysis [(RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq)], and we observed greater expression of several EMT markers, including Vimentin, hyaluronan synthase-3, S100 calcium binding protein A6, TGFB1, CD44, CD55, and CD109 in European American and African American aggressive variant prostate cancer (AVPC) subtypes-mCRPC, neuroendocrine variant (NEPC), and taxane-resistant. The taxane-resistant gene FSCN1 was also expressed highly in single-cell subclonal populations in mCRPC. Furthermore, metronomic-topotecan single agent and combinations with DTX downregulated these EMT markers as well as CD44+ and CD44+/CD133+ "stem-like" cell populations. A microfluidic chip-based cell invasion assay revealed that METRO-TOPO treatment as a single agent or in combination with DTX was potentially effective against invasive prostate cancer spread. Our RNA-seq and scRNA-seq analysis were supported by in silico and in vitro studies, suggesting METRO-TOPO combined with DTX may inhibit oncogenic progression by reducing cancer stemness in AVPC through the inhibition of EMT markers and multiple oncogenic factors/pathways. Significance: The utilization of metronomic-like dosing regimens of topotecan alone and in combination with DTX resulted in the suppression of makers associated with EMT and stem-like cell populations in AVPC models. The identification of molecular signatures and their potential to serve as novel biomarkers for monitoring treatment efficacy and disease progression response to treatment efficacy and disease progression were achieved using bulk RNA-seq and single-cell-omics methodologies.

Integrating Pharmacogenomics Data-Driven Computational Drug Prediction with Single-Cell RNAseq to Demonstrate the Efficacy of a NAMPT Inhibitor against Aggressive, Taxane-Resistant, and Stem-like Cells in Lethal Prostate Cancer.

Metastatic prostate cancer/PCa is the second leading cause of cancer deaths in US men. Most early-stage PCa are dependent on overexpression of the androgen receptor (AR) and, therefore, androgen deprivation therapies/ADT-sensitive. However, eventual resistance to standard medical castration (AR-inhibitors) and secondary chemotherapies (taxanes) is nearly universal. Further, the presence of cancer stem-like cells (EMT/epithelial-to-mesenchymal transdifferentiation) and neuroendocrine PCa (NEPC) subtypes significantly contribute to aggressive/lethal/advanced variants of PCa (AVPC). In this study, we introduced a pharmacogenomics data-driven optimization-regularization-based computational prediction algorithm ("secDrugs") to predict novel drugs against lethal PCa. Integrating secDrug with single-cell RNA-sequencing/scRNAseq as a 'Double-Hit' drug screening tool, we demonstrated that single-cells representing drug-resistant and stem-cell-like cells showed high expression of the NAMPT pathway genes, indicating potential efficacy of the secDrug FK866 which targets NAMPT. Next, using several cell-based assays, we showed substantial impact of FK866 on clinically advanced PCa as a single agent and in combination with taxanes or AR-inhibitors. Bulk-RNAseq and scRNAseq revealed that, in addition to NAMPT inhibition, FK866 regulates tumor metastasis, cell migration, invasion, DNA repair machinery, redox homeostasis, autophagy, as well as cancer stemness-related genes, HES1 and CD44. Further, we combined a microfluidic chip-based cell migration assay with a traditional cell migration/'scratch' assay and demonstrated that FK866 reduces cancer cell invasion and motility, indicating abrogation of metastasis. Finally, using PCa patient datasets, we showed that FK866 is potentially capable of reversing the expression of several genes associated with biochemical recurrence, including IFITM3 and LTB4R. Thus, using FK866 as a proof-of-concept candidate for drug repurposing, we introduced a novel, universally applicable preclinical drug development pipeline to circumvent subclonal aggressiveness, drug resistance, and stemness in lethal PCa.

Polygenic Ara-C Response Score Identifies Pediatric Patients With Acute Myeloid Leukemia in Need of Chemotherapy Augmentation.

PURPOSE: To establish a patient-specific polygenic score derived from cytarabine (ara-C) pathway pharmacogenomic evaluation to personalize acute myeloid leukemia (AML) treatment. MATERIALS AND METHODS: Single nucleotide polymorphisms (SNPs) in the ara-C-pathway genes were analyzed with outcome in patients from the multicenter-AML02 trial (N = 166). Multi-SNP predictor modeling was used to develop 10-SNP Ara-C_SNP score (ACS10) using top SNPs predictive of minimal residual disease and event-free survival (EFS) from the AML02-cohort and four SNPs previously associated with ara-C triphosphate levels in the AML97 trial. ACS10 was evaluated for association with outcomes in each clinical trial arms: the standard low-dose ara-C (LDAC, n = 91) and augmented high-dose ara-C (HDAC, n = 75) arms of AML02 and the standard Ara-C, daunorubicin and etoposide (ADE) (n = 465) and the augmented ADE + gemtuzumab ozogamicin (GO; n = 466) arms of AAML0531 trial. RESULTS: In the standard LDAC-arm of AML02 cohort, the low-ACS10 score group (≤ 0) had significantly worse EFS (ACS10 low v high hazard ratio [HR] = 2.81; 95% CI, 1.45 to 5.43; P = .002) and overall survival (OS; HR = 2.98; 95% CI, 1.32 to 6.75; P = .009) compared with the high-ACS10 group (score > 0). These results were validated in the standard-ADE arm of AAML0531, with poor outcome in the low-ASC10 group compared with the high-ACS10 group (EFS: HR = 1.35, 95% CI, 1.04 to 1.75, P = .026; OS: HR = 1.64, 95% CI, 1.2 to 2.22, P = .002). Within the augmented arms (AML02-HDAC and AAML0531-ADE + GO), EFS and OS did not differ between low- and high-ACS10 score groups. In both cohorts, patients with low-ACS10 consistently showed a 10-percentage point improvement in 5-year EFS with augmented therapy (AML02-HDAC or AAML0531-ADE + GO arms) than with standard therapy (AML02-LDAC or AAML0531-ADE arms). CONCLUSION: Patients with low-ACS10 score experienced significantly poor outcome when treated on standard regimen. Augmentation with either high-dose ara-C or GO addition improved outcome in low-ACS10 group. A polygenic ACS10 score can identify patients with unfavorable pharmacogenetic characteristics and offers a potential for an elective augmented therapy option.

The Andrographolide Analogue 3A.1 Synergizes with Taxane Derivatives in Aggressive Metastatic Prostate Cancers by Upregulation of Heat Shock Proteins and Downregulation of MAT2A-Mediated Cell Migration and Invasion.

Conventional treatment with taxanes (docetaxel-DTX or cabazitaxel-CBZ) increases the survival rates of patients with aggressive metastatic castration-resistant prostate cancer (mCRPC); however, most patients acquire resistance to taxanes. The andrographolide analog, 19-tert-butyldiphenylsilyl-8,7-epoxy andrographolide (3A.1), has shown anticancer activity against various cancers. In this study, we investigated the effect of 3A.1 alone and in combination with DTX/CBZ against mCRPC and their mechanism of action. Exposure to 3A.1 alone exhibited a dose- and time-dependent antitumor activity in mCRPC. Chou-Talalay's combination index (CI) values of all 3A.1 + TX combinations were less than 0.5, indicating synergism. Co-treatment of 3A.1 with TX reduced the required dose of DTX and CBZ (P < 0.05). Caspase assay (apoptosis) results concurred with in vitro cytotoxicity data. RNA sequencing (RNAseq), followed by ingenuity pathway analysis (IPA), identified that upregulation of heat-shock proteins (Hsp70, Hsp40, Hsp27, and Hsp90) and downregulation of MAT2A as the key player for 3A.1 response. Furthermore, the top treatment-induced differentially expressed genes (DEGs) belong to DNA damage, cell migration, hypoxia, autophagy (MMP1, MMP9, HIF-1α, Bag-3, H2AX, HMOX1, PSRC1), and cancer progression pathways. Most importantly, top downregulated DEG MAT2A has earlier been shown to be involved in cell migration and invasion. Furthermore, using in silico analysis on the Cancer Genome Atlas (TCGA) database, this study found that MAT2A and highly co-expressed (r > 0.7) genes, TRA2B and SF1, were associated with worse Gleason score and nodal metastasis status in prostate adenocarcinoma patients (PRAD-TCGA). Immunoblotting, comet, and migration assays corroborated these findings. These results suggest that 3A.1 may be useful in increasing the anticancer efficacy of taxanes to treat aggressive PCa. SIGNIFICANCE STATEMENT: The andrographolide analogue, 19-tert-butyldiphenylsilyl-8,7-epoxy andrographolide (3A.1), showed anticancer activity against metastatic castration-resistant and neuroendocrine variant prostate cancers (mCRPC/NEPC). Additionally, 3A.1 exhibited synergistic anticancer effect in combination with standard chemotherapy drugs docetaxel and cabazitaxel in mCRPC/NEPC. Post-treatment gene expression studies revealed that heat shock proteins (Hsp70, Hsp40, Hsp27, and Hsp90) and MAT2A are important in the mechanism of 3A.1 action and drug response. Furthermore, DNA damage, cell migration, hypoxia, and autophagy were crucial pathways for the anticancer activity of 3A.1.

Identification and Characterization of Key Differentially Expressed Genes Associated With Metronomic Dosing of Topotecan in Human Prostate Cancer.

Repetitive, low-dose (metronomic; METRO) drug administration of some anticancer agents can overcome drug resistance and increase drug efficacy in many cancers, but the mechanisms are not understood fully. Previously, we showed that METRO dosing of topotecan (TOPO) is more effective than conventional (CONV) dosing in aggressive human prostate cancer (PCa) cell lines and in mouse tumor xenograft models. To gain mechanistic insights into METRO-TOPO activity, in this study we determined the effect of METRO- and CONV-TOPO treatment in a panel of human PCa cell lines representing castration-sensitive/resistant, androgen receptor (+/-), and those of different ethnicity on cell growth and gene expression. Differentially expressed genes (DEGs) were identified for METRO-TOPO therapy and compared to a PCa patient cohort and The Cancer Genome Atlas (TCGA) database. The top five DEGs were SERPINB5, CDKN1A, TNF, FOS, and ANGPT1. Ingenuity Pathway Analysis predicted several upstream regulators and identified top molecular networks associated with METRO dosing, including tumor suppression, anti-proliferation, angiogenesis, invasion, metastasis, and inflammation. Further, the top DEGs were associated with increase survival of PCa patients (TCGA database), as well as ethnic differences in gene expression patterns in patients and cell lines representing African Americans (AA) and European Americans (EA). Thus, we have identified candidate pharmacogenomic biomarkers and novel pathways associated with METRO-TOPO therapy that will serve as a foundation for further investigation and validation of METRO-TOPO as a novel treatment option for prostate cancers.

Glutaminase inhibitor CB-839 synergizes with carfilzomib in resistant multiple myeloma cells.

Curative responses in the treatment of multiple myeloma (MM) are limited by the emergence of therapeutic resistance. To address this problem, we set out to identify druggable mechanisms that convey resistance to proteasome inhibitors (PIs; e.g., bortezomib), which are cornerstone agents in the treatment of MM. In isogenic pairs of PI sensitive and resistant cells, we observed stark differences in cellular bioenergetics between the divergent phenotypes. PI resistant cells exhibited increased mitochondrial respiration driven by glutamine as the principle fuel source. To target glutamine-induced respiration in PI resistant cells, we utilized the glutaminase-1 inhibitor, CB-839. CB-839 inhibited mitochondrial respiration and was more cytotoxic in PI resistant cells as a single agent. Furthermore, we found that CB-839 synergistically enhanced the activity of multiple PIs with the most dramatic synergy being observed with carfilzomib (Crflz), which was confirmed in a panel of genetically diverse PI sensitive and resistant MM cells. Mechanistically, CB-839 enhanced Crflz-induced ER stress and apoptosis, characterized by a robust induction of ATF4 and CHOP and the activation of caspases. Our findings suggest that the acquisition of PI resistance involves adaptations in cellular bioenergetics, supporting the combination of CB-839 with Crflz for the treatment of refractory MM.

FDA Approval Summary: Lenvatinib for Progressive, Radio-iodine-Refractory Differentiated Thyroid Cancer.

The FDA approved lenvatinib (Lenvima, Eisai Inc.) for the treatment of patients with locally recurrent or metastatic, progressive, radioactive iodine-refractory (RAI-refractory) differentiated thyroid cancer (DTC). In an international, multicenter, double-blinded, placebo-controlled trial (E7080-G000-303), 392 patients with locally recurrent or metastatic RAI-refractory DTC and radiographic evidence of disease progression within 12 months prior to randomization were randomly allocated (2:1) to receive either lenvatinib 24 mg orally per day (n = 261) or matching placebo (n = 131) with the option for patients on the placebo arm to receive lenvatinib following independent radiologic confirmation of disease progression. A statistically significant prolongation of progression-free survival (PFS) as determined by independent radiology review was demonstrated [HR, 0.21; 95% confidence interval (CI), 0.16-0.28; P < 0.001, stratified log-rank test], with an estimated median PFS of 18.3 months (95% CI, 15.1, NR) in the lenvatinib arm and 3.6 months (95% CI, 2.2-3.7) in the placebo arm. The most common adverse reactions, in order of decreasing frequency, observed in the lenvatinib-treated patients were hypertension, fatigue, diarrhea, arthralgia/myalgia, decreased appetite, decreased weight, nausea, stomatitis, headache, vomiting, proteinuria, palmar-plantar erythrodysesthesia syndrome, abdominal pain, and dysphonia. Adverse reactions led to dose reductions in 68% of patients receiving lenvatinib at the 24 mg dose and 18% of patients discontinued lenvatinib for adverse reactions leading to residual uncertainty regarding the optimal dose of lenvatinib.

RRM1 and RRM2 pharmacogenetics: association with phenotypes in HapMap cell lines and acute myeloid leukemia patients.

BACKGROUND: Ribonucleotide reductase catalyzes an essential step in the cellular production of deoxyribonucleotide triphosphates and has been associated with clinical outcome in cancer patients receiving nucleoside analog-based chemotherapy. MATERIALS & METHODS: In the current study, we sequenced the genes RRM1 and RRM2 in genomic DNA from HapMap cell lines with European (Utah residents with northern and western European ancestry [CEU]; n = 90) or African (Yoruba people in Ibadan, Nigeria [YRI]; n = 90) ancestry. RESULTS: We identified 44 genetic variants including eight coding SNPs in RRM1 and 15 SNPs including one coding SNP in RRM2. RRM1 and RRM2 mRNA expression levels were significantly correlated with each other in both CEU and YRI lymphoblast cell lines, and in leukemic blasts from acute myeloid leukemia (AML) patients (AML97, n = 89; AML02, n = 187). Additionally, RRM1 expression was higher among patient features indicative of a high relapse hazard. We evaluated SNPs within the RRM1 and RRM2 genes in the HapMap lymphoblast cell lines from CEU and YRI panels for association with expression and cytarabine chemosensitivity. SNPs of potential significance were further evaluated in AML patients. RRM1 SNPs rs1042919 (which occurs in linkage disequilbrium with multiple other SNPs) and promoter SNP rs1561876 were associated with intracellular 1-ß-D-arabinofuranosyl-CTP levels, response after remission induction therapy, risk of relapse and overall survival in AML patients receiving cytarabine and cladribine. CONCLUSION: These results suggest that SNPs within ribonucleotide reductase might be helpful predictive markers of response to nucleoside analogs and should be further validated in larger cohorts.

Comprehensive genetic analysis of cytarabine sensitivity in a cell-based model identifies polymorphisms associated with outcome in AML patients.

A whole-genome approach was used to investigate the genetic determinants of cytarabine-induced cytotoxicity. We performed a meta-analysis of genome-wide association studies involving 523 lymphoblastoid cell lines (LCLs) from individuals of European, African, Asian, and African American ancestry. Several of the highest-ranked single-nucleotide polymorphisms (SNPs) were within the mutated in colorectal cancers (MCC) gene. MCC expression was induced by cytarabine treatment from 1.7- to 26.6-fold in LCLs. A total of 33 SNPs ranked at the top of the meta-analysis (P < 10(-5)) were successfully tested in a clinical trial of patients randomized to receive low-dose or high-dose cytarabine plus daunorubicin and etoposide; of these, 18 showed association (P < .05) with either cytarabine 50% inhibitory concentration in leukemia cells or clinical response parameters (minimal residual disease, overall survival (OS), and treatment-related mortality). This count (n = 18) was significantly greater than expected by chance (P = .016). For rs1203633, LCLs with AA genotype were more sensitive to cytarabine-induced cytotoxicity (P = 1.31 × 10(-6)) and AA (vs GA or GG) genotype was associated with poorer OS (P = .015), likely as a result of greater treatment-related mortality (P = .0037) in patients with acute myeloid leukemia (AML). This multicenter AML02 study trial was registered at www.clinicaltrials.gov as #NCT00136084.

Clinical significance of CD33 nonsynonymous single-nucleotide polymorphisms in pediatric patients with acute myeloid leukemia treated with gemtuzumab-ozogamicin-containing chemotherapy.

PURPOSE: The purpose of this study was to evaluate clinical implications of CD33 single-nucleotide polymorphisms (SNP) in pediatric patients with acute myeloid leukemia (AML) treated with gemtuzumab-ozogamicin (GO)-based therapy. EXPERIMENTAL DESIGN: We genotyped four CD33 SNPs: rs35112940 (G>A; Arg304Gly), rs12459419 (C>T; Ala14Val), rs2455069 (A>G; Arg69Gly), and rs1803254 (G>C; 3'UTR) in pediatric patients undergoing induction chemotherapy containing GO (COG-AAML03P1 trial; n = 242) or not containing GO (St. Jude AML02 trial; n = 172). RESULTS: CD33 SNPs were correlated significantly with clinical characteristics and treatment outcome. The coding SNPs, rs35112940 and rs12459419, were significantly associated with clinical endpoints in COG-AAML03P1 but not in the St. Jude AML02 trial. Specifically, among white patients in COG-AAML03P1, the 3-year overall survival (OS) rate from remission was 84% ± 8% for those homozygous (GG) for rs35112940 versus 68% ± 15% for the other genotypes (P = 0.018); these patients also had a lower relapse risk and superior disease-free survival. Likewise, patients homozygous for variant allele (TT) for rs12459419 were more likely to have favorable risk disease than CC and CT genotypes (52% vs. 31%, P = 0.034) and significantly lower diagnostic blast CD33 expression than other genotypes (P < 0.001). CONCLUSION: Our data suggest that genetic variations in CD33 could impact clinical outcome of GO-based therapy in pediatric AMLs.

Pathway-based pharmacogenomics of gemcitabine pharmacokinetics in patients with solid tumors.

AIM: The aim of this study was to evaluate the association of gemcitabine pathway SNPs with detailed pharmacokinetic measures obtained from solid tumor patients receiving gemcitabine-based therapy. MATERIALS & METHODS: SNPs within nine gemcitabine pathway genes, namely CDA, CMPK, DCK, DCTD, NT5C2, NT5C3, SLC28A1, SLC28A3 and SLC29A1 were analyzed for association with gemcitabine pharmacokinetics. RESULTS: Significant association of gemcitabine clearance with SNPs in NT5C2 was identified. Clearance of 2´,2´-difluorodeoxyuridine, a gemcitabine metabolite was significantly predicted by CDA, SLC29A1 and NT5C2 SNPs. This study reports an association of formation clearance of 2´,2´-difluoro-2´-deoxycytidine triphosphate, an active form of gemcitabine with SNPs within uptake transporters SLC28A1, SLC28A3 and SLC29A1. CONCLUSION: Genetic variation in gemcitabine pathway genes is associated with its pharmacokinetics and hence could influence gemcitabine response. Our study identified pharmacogenetic markers that could be further tested in larger patient cohorts and could open up opportunities to individualize therapy in solid tumor patients.

Genetic variants in cytosolic 5'-nucleotidase II are associated with its expression and cytarabine sensitivity in HapMap cell lines and in patients with acute myeloid leukemia.

Cytosolic 5'-nucleotidase II (NT5C2) is involved in the development of 1-ß-d-arabinofuranosylcytosine (ara-C) resistance and has been associated with clinical outcome in patients receiving ara-C-based chemotherapy. NT5C2 inactivates ara-C by dephosphorylating ara-C monophosphate to ara-C. In this study, we sequenced NT5C2 in genomic DNA samples from International HapMap project panels with European [Centre d'Etude du Polymorphisme Humain (CEU); n = 90] or African [Yoruba people in Ibadan, Nigeria (YRI); n = 90] ancestry. We identified 41 genetic variants [one insertion-deletion and 40 single nucleotide polymorphisms (SNPs)], including three nonsynonymous SNPs (Y3A, K47R, and Q136R). Twenty-five SNPs were novel and 16 overlapped with the HapMap data. Subjects with African ancestry had NT5C2 mRNA expression levels that was significantly higher than those with European ancestry (p = 0.005). Furthermore, there was a correlation between NT5C2 mRNA expression and ara-C sensitivity in CEU but not in YRI cell lines. None of the nonsynonymous SNPs demonstrated any effect on NT5C2 activity. The genotypes of several SNPs were significantly associated with NT5C2 mRNA expression and/or ara-C sensitivity in CEU cell lines, but very few were significant in YRI cell lines. Of most interest, SNPs (linkage disequilibrium group CEU.12) in the 5'-untranslated region were associated with NT5C2 expression and ara-C sensitivity in HapMap cell lines and with NT5C2 mRNA expression and ara-C sensitivity in diagnostic leukemic blasts from pediatric patients with acute myeloid leukemia. Functional genomics analysis demonstrated that the promoter SNP rs11191612 was associated with altered luciferase activation in reporter assays and altered DNA-protein binding in gel shift assays. These results suggest that genetic variations in NT5C2 influence its expression and, potentially, cellular responses to nucleoside analogs.

Angiotensin II-induced upregulation of AT(1) receptor expression: sequential activation of NF-kappaB and Elk-1 in neurons.

It has been clearly established that increased circulating angiotensin II (ANG II) with concurrent upregulation of brain and peripheral ANG II type 1 receptors (AT(1)R) are important mediators in the pathophysiology of several diseases characterized by sympatho-excitation. In an effort to further understand the regulation of AT(1)R expression in neurons, we determined the role of sequential activation of the transcription factors nuclear factor-kappaB (NF-kappaB) and Ets-like protein 1 (Elk-1) in AT(1)R upregulation. We used CATH.a neurons as our neuronal cell model. Cells were treated with ANG II (100 nM) over a preset time course. Following ANG II activation, there was a temporal increase in the p65 subunit of NF-kappaB that was observed at 30 min, peaked at 1 h, and was sustained up to 24 h. There was a concomitant decrease of IkappaB and increased IkappaK expression. We also observed an increase in AT(1)R expression which followed the temporal increase of NF-kappaB. The activation of NF-kappaB was blocked by using the inhibitors parthenolide or p65 small interfering RNA (siRNA) which both led to a decrease in AT(1)R expression. The expression of Elk-1 was upregulated over a time period following ANG II activation and was decreased following NF-kappaB inhibition. p65-DNA binding was assessed using electrophoretic mobility shift assay, and it was shown that there was a time-dependent increased binding that was inhibited by means of parthenolide pretreatment or siRNA-mediated p65 gene silencing. Therefore, our results suggest a combined role for the transcription factors NF-kappaB and Elk-1 in the upregulation of AT(1)R in the CATH.a cell neuronal model. These data imply a positive feedback mechanism that may impact neuronal discharge sensitivity in response to ANG II.

Insulin-like growth factor-1 induces phosphorylation of PI3K-Akt/PKB to potentiate proliferation of smooth muscle cells in human saphenous vein.

Coronary revascularization by coronary artery bypass grafting (CABG) is recommended in patients with recurrent myocardial ischemia. However, the long-term results of CABG using saphenous vein (SV) graft, compared to internal mammary artery (IMA) graft, have not been satisfactory. The SV graft failure is due to the development of intimal hyperplasia, a process characterized by abnormal migration and proliferation of smooth muscle cells (SMCs) in the intimal layer of the vein graft. Insulin growth factor 1 (IGF-1) is a major mitogenic growth factor released at the site of the shear stress-induced graft injury. This study, for the first time, compares the extent of IGF-1-PI3K-Akt activation in isolated human bypass graft conduits. Human SV and IMA vessels were collected and SMCs isolated and cultured. In cultured SMCs, effect of IGF-1 was examined on total and phosphorylated PI3K, Akt and IGF-1R by Western blot analysis. Cell proliferation was measured using BrdU ELISA. There was no significant difference in the basal expression of phosphorylated PI3K, Akt and IGF-1R in SV and IMA SMCs from human bypass conduits. However, we observed an upregulation of IGF-1 receptors in the SV SMCs in response to IGF-1 stimulation with no effect in IMA SMCs. Furthermore, the immunoblotting and cellular activation of signaling ELISA (CASE) assay demonstrated a significantly higher activity of both PI3K and Akt in IGF-1-stimulated SV SMCs than IMA. This was inhibited by an IGF-1R blocking antibody. IGF-1 induced proliferation in both SV and IMA SMCs was inhibited by a PI3K inhibitor, wortmannin. These data demonstrate differential activity of IGF-1-induced PI3K-Akt activation, which was quantitatively and temporally greater in SV SMCs than in the IMA. This, at least in part, could explain the greater propensity of the SV conduits than the IMA to undergo intimal hyperplasia following CABG.

Temporal PTEN inactivation causes proliferation of saphenous vein smooth muscle cells of human CABG conduits.

Abstract Internal mammary artery (IMA) coronary artery bypass grafts (CABG) are remarkably resistant to intimal hyperplasia (IH) as compared to saphenous vein (SV) grafts following aorto-coronary anastomosis. The reason behind this puzzling difference still remains an enigma. In this study, we examined the effects of IGF-1 stimulation on the PI3K-AKT/PKB pathway mediating proliferation of smooth muscle cells (SMCs) of IMA and SV origin and the specific contribution of phosphatase and tensin homologue (PTEN) in regulating the IGF-1-PI3K-AKT/PKB axis under these conditions. Mitogenic activation with IGF-1, time-dependently stimulated the phosphorylation of PI3K and AKT/PKB in the SV SMCs to a much greater extent than the IMA. Conversely, PTEN was found to be significantly more active in IMA SMCs. Transient overexpression of PTEN in SMCs of SV and IMA inhibited AKT/PKB activity and upstream of AKT/PKB, caused a reduction of IGF-1 receptors. Downstream, PTEN overexpression in SV SMCs induced the transactivation of tumour suppressor protein p53 by down-regulating the expression of its inhibitor MDM2. However, PTEN overexpression had no significant effect on MDM2 and p53 expression in IMA SMCs. PTEN overexpression inhibited IGF-1-induced SMC proliferation in both SV and IMA. PTEN suppression, induced by siRNA transfection of IMA SMCs diminished the negative regulation of PI3K-PKB signalling leading to greater proliferative response induced by IGF-1 stimulation. Thus, we show for the first time that early inactivation of PTEN in SV SMCs leads to temporally increased activity of the pro-hyperplasia PI3K-AKT/PKB pathway leading to IH-induced vein graft occlusion. Therefore, modulation of the PI3K-AKT/PKB pathway via PTEN might be a novel and effective strategy in combating SV graft failure following CABG.

Regulation of cell cycle entry by PTEN in smooth muscle cell proliferation of human coronary artery bypass conduits.

Proliferation of smooth muscle cells (SMCs) is the key event in the pathogenesis of intimal hyperplasia (IH) leading to coronary artery bypass graft (CABG) occlusion. The saphenous vein (SV) conduits are often affected by IH, while the internal mammary artery (IMA) conduits remain remarkably patent. SMC proliferation is mediated by the cell cycle, under the control of cyclin-dependent kinases (cdks), cdk-inhibitors and the retinoblastoma protein (Rb). Early passage of the SMCs through the cell cycle involves crossing the non-reversible G(1) checkpoint, the restriction (R) point. In this study, we investigated the effect of mitogenic insulin-like growth factor (IGF)-1 stimulation on the R-point and its relationship with the phosphorylation of Rb protein and the cdk inhibitors p21 and p27 in SV and IMA SMCs. We observed no change in the R-point following IGF-1 activation in either SV or IMA SMCs. However, Rb-phosphorylation occurred much earlier and was quantitatively greater in SV SMCs than IMA. Overexpression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in SV SMCs followed by IGF-1 activation significantly decreased the expression of cyclin E and pRb and induced p27 expression in SV SMCs, while, pRb levels were markedly decreased and p27 levels were significantly increased in IMA SMCs. Silencing the PTEN gene by siRNA transfection of IMA SMCs significantly induced the expression of pRb and inhibited p27 expression, while, the expression levels of cyclin E, pRb, p21 and p27 were unaffected by the silencing of PTEN in SV SMCs. These results demonstrate that the PTEN plays a critical role in regulating cell cycle entry. Therefore, overexpression of PTEN possibly by means of gene therapy could be a viable option in regulating the cell cycle in SV SMCs in the treatment of vein graft disease.

Gene therapy of fibroproliferative vasculopathies: current ideas in molecular mechanisms and biomedical technology.

Intimal hyperplasia occurs primarily as a part of the pathogenesis of coronary artery disease or secondary to therapeutic intervention in relieving vascular occlusion. Intimal hyperplasia involving vascular smooth muscle cells is found in atherosclerosis, post-balloon angioplasty restenosis, in-stent restenosis and vein graft disease, predominantly involving the use of saphenous vein conduits in coronary artery bypass grafting procedures. One potentially exciting area is that of gene therapy. Gene and protein expression patterns at the site of vasculoproliferative lesions have been widely studied and several target areas have been identified on the basis of whether the gene has an antiproliferative, proapoptotic, matrix degrading or endothelial protective action. Blood vessels are easily accessible for the delivery of the gene product, and experimental studies using animal models have used catheter-delivered gene products at the site of vascular injury. Currently, the application of antisense technology and adenoviral vector-mediated delivery has shown significant promise, albeit in in vitro or animal model settings. In this review, we discuss the current knowledge in the application of gene therapy in fibroproliferative vasculopathies. We examine some of the cellular mechanisms and intermediaries which could be potential candidates for gene targeting. We also present some of the advances in biomedical technology that might provide useful vehicles for pinpoint delivery of the gene product. Could the future of restenosis treatment be in gene therapy or is it misplaced enthusiasm?

Cellular, molecular and immunological mechanisms in the pathophysiology of vein graft intimal hyperplasia.

Coronary artery disease, leading to myocardial infarction and ischaemia, affects millions of persons and is one of the leading causes of morbidity and mortality worldwide. Invasive techniques such as coronary artery bypass grafting are used to alleviate the sequelae of arterial occlusion. Unfortunately, restenosis or occlusion of the grafted conduit occurs over a time frame of months to years with a gradual reduction in patency, especially in vein grafts. The events leading to intimal hyperplasia (IH) formation involve numerous cellular and molecular components. Various cellular elements of the vessel wall are involved as are leucocyte-endothelial interactions that trigger the coagulation cascade leading to localized thrombus formation. Subsequent phenotypic modification of the medial smooth muscle cells and their intimal migration is the basis of the lesion formation that is thought to be propagated by an immune-mediated reaction. Despite intense scrutiny, the pathophysiology of IH remains an enigma. Although several growth factors, cytokines and numerous other biomolecules have been implicated and their relationship to prohyperplasia pathways such as the phosphatidyl-inositol 3-kinase (PI3K)-Akt pathway has been established, many pieces of the puzzle are still missing. An in-depth understanding of early vein graft adaptation and progression is necessary to improve the long-term prognosis and develop more effective therapeutic measures. In this review, we have critically evaluated and summarized the literature to elucidate and interlink the numerous established and emerging factors that play a key role in the development of IH leading to vein graft restenosis.

Cells, cytokines and cellular immunity in the pathogenesis of fibroproliferative vasculopathies.

Atherosclerosis and restenosis are the result of vascular injury followed by an inflammatory and fibroproliferative response that involves a large number of growth factors, cytokines, and cellular elements. Platelet activation and leukocyte recruitment into the arterial intima play a crucial role, initiating a whole spectrum of reactions leading to vascular smooth muscle cell hyperplasia and intimal migration. The roles of macrophages and lymphocytes and mast cells as mediators of inflammation and immune response is discussed, as are the roles of growth factors and cytokines. New light on the 'old' problems will help us to devise newer and better therapeutic strategies to combat these clinical entities.