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1.  Gambogic Acid Induces Apoptosis in Imatinib-Resistant Chronic Myeloid Leukemia Cells via Inducing Proteasome Inhibition and Caspase-Dependent Bcr-Abl Downregulation 
Purpose
Chronic myelogenous leukemia (CML) is characterized by the constitutive activation of Bcr-Abl tyrosine kinase. Bcr-Abl-T315I is the predominant mutation that causes resistance to imatinib, cytotoxic drugs, and the second-generation tyrosine kinase inhibitors. The emergence of imatinib resistance in patients with CML leads to searching for novel approaches to the treatment of CML. Gambogic acid, a small molecule derived from Chinese herb gamboges, has been approved for phase II clinical trial for cancer therapy by the Chinese Food and Drug Administration (FDA). In this study, we investigated the effect of gambogic acid on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl.
Experimental Design
CML cell lines (KBM5, KBM5-T315I, and K562), primary cells from patients with CML with clinical resistance to imatinib, and normal monocytes from healthy volunteers were treated with gambogic acid, imatinib, or their combination, followed by measuring the effects on cell growth, apoptosis, and signal pathways. The in vivo antitumor activity of gambogic acid and its combination with imatinib was also assessed with nude xenografts.
Results
Gambogic acid induced apoptosis and cell proliferation inhibition in CML cells and inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Our data suggest that GA-induced proteasome inhibition is required for caspase activation in both imatinib-resistant and -sensitive CML cells, and caspase activation is required for gambogic acid–induced Bcr-Abl downregulation and apoptotic cell death.
Conclusions
These findings suggest an alternative strategy to overcome imatinib resistance by enhancing Bcr-Abl downregulation with the medicinal compound gambogic acid, which may have great clinical significance in imatinib-resistant cancer therapy.
doi:10.1158/1078-0432.CCR-13-1063
PMCID: PMC3938960  PMID: 24334603
2.  Therapeutic Efficacy of an Fc-Enhanced TCR-Like Antibody to the Intracellular WT1 Oncoprotein 
Purpose
RMFPNAPYL (RMF), a WT1-derived CD8 T cell epitope presented by HLA-A*02:01, is a validated target for T-cell-based immunotherapy. We previously reported ESK1, a high avidity (Kd < 0.2nM), fully-human monoclonal antibody (mAb) specific for the WT1 RMF peptide/HLA-A*02:01 complex, which selectively bound and killed WT1+ and HLA-A*02:01+ leukemia and solid tumor cell lines.
Experimental Design
We engineered a second-generation mAb, ESKM, to have enhanced antibody dependent cell-mediated cytotoxicity (ADCC) function due to altered Fc glycosylation. ESKM was compared to native ESK1 in binding assays, in vitro ADCC assays, and mesothelioma and leukemia therapeutic models and pharmacokinetic studies in mice. ESKM toxicity was assessed in HLA-A*02:01+ transgenic mice.
Results
ESK antibodies mediated ADCC against hematopoietic and solid tumor cells at concentrations below 1µg/ml, but ESKM was about 5–10 fold more potent in vitro against multiple cancer cell lines. ESKM was more potent in vivo against JMN mesothelioma, and effective against SET2 AML and fresh ALL xenografts. ESKM had a shortened half-life (4.9 vs 6.5 days), but an identical biodistribution pattern in C57BL6/J mice. At therapeutic doses of ESKM, there was no difference in half-life or biodistribution in HLA-A*02:01+ transgenic mice compared to the parent strain. Importantly, therapeutic doses of ESKM in these mice caused no depletion of total WBCs or hematopoetic stem cells, or pathologic tissue damage.
Conclusions
The data provide proof of concept that an Fc-enhanced mAb can improve efficacy against a low-density, tumor-specific, peptide/MHC target, and support further development of this mAb against an important intracellular oncogenic protein.
doi:10.1158/1078-0432.CCR-13-2756
PMCID: PMC4119489  PMID: 24850840
3.  Monoclonal Antibody against Cell Surface GRP78 as a Novel Agent in Suppressing PI3K/AKT Signaling, Tumor Growth and Metastasis 
Purpose
The ER chaperone GRP78 translocates to the surface of tumor cells and promotes survival, metastasis, and resistance to therapy. An oncogenic function of cell surface GRP78 has been attributed to the activation of phosphoinositide 3-kinase (PI3K) pathway. We intend to use a novel anti-GRP78monoclonal antibody (MAb159) to attenuate PI3K signaling and inhibit tumor growth and metastasis.
Experimental Design
MAb159 was characterized biochemically. Anti-tumor activity was tested in cancer cell culture, tumor xenograft models, tumor metastasis models, and spontaneous tumor models. Cancer cells and tumor tissues were analyzed for PI3K activity. MAb159 was humanized and validated for diagnostic and therapeutic application.
Results
MAb159 specifically recognized surface GRP78, triggered GRP78 endocytosis, and localized to tumors but not normal organs in vivo. MAb159 inhibited tumor cell proliferation and enhanced tumor cell death both in vitro and in vivo. In MAb159 treated tumors, PI3K signaling was inhibited without compensatory MAPK pathway activation. Furthermore, MAb159 halted or reversed tumor progression in the spontaneous PTEN loss driven prostate and leukemia tumor models, and inhibited tumor growth and metastasis in xenograft models. Humanized MAb159, which retains high affinity, tumor specific localization, and the anti-tumor activity, was non-toxic in mice and had desirable pharmacokinetics.
Conclusions
GRP78 specific antibody MAb159 modulates PI3K pathway and inhibits tumor growth and metastasis. Humanized MAb159 will enter human trials shortly.
doi:10.1158/1078-0432.CCR-13-1106
PMCID: PMC4151476  PMID: 24048331
Surface GRP78; monoclonal antibody; targeted cancer therapy; PI3K
4.  Mast Cells in Tumor Microenvironment Promotes the in vivo Growth of Pancreatic Ductal Adenocarcinoma 
Purpose
Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer death. No effective therapy is currently available for PDAC because of the lack of understanding of the mechanisms leading to its growth and development. Inflammatory cells, particularly mast cells have been shown to play key roles in some cancers. We carried out this study to test the hypothesis that mast cells in the tumor microenvironment are essential for PDAC tumorigenesis.
Experimental Design
The presence of inflammatory cells at various stages of PDAC development was determined in a spontaneous mouse model of PDAC (K-rasG12V). The importance of mast cells was determined using orthotopically implanted PDAC cells in mast cell-deficient Kitw-sh/w-sh mice and further confirmed by reconstitution of wild-type bone marrow-derived mast cells. Clinical relevance was assessed by correlating the presence of mast cells with clinical outcome in patients with PDAC.
Results
In the spontaneous mouse model of PDAC (K-rasG12V), there was an early influx of mast cells to the tumor microenvironment. PDAC tumor growth was in mast cell-deficient Kitw-sh/w-sh mice, but aggressive PDAC growth was restored when PDAC cells were injected into mast cell-deficient mice reconstituted with wild-type bone marrow-derived mast cells. Mast cell infiltration into the tumor microenvironment was predictive of poor prognosis in patients with PDAC.
Conclusions
Mast cells play an important role in PDAC growth and development in mouse models and are indicative of poor prognosis in humans, which makes them a potential novel therapeutic target.
doi:10.1158/1078-0432.CCR-11-0607
PMCID: PMC4089502  PMID: 21976550
Pancreatic cancer; mast cell; inflammation
5.  Impact of Ischemia and Procurement Conditions on Gene Expression in Renal Cell Carcinoma 
Purpose
Previous studies have shown that ischemia alters gene expression in normal and malignant tissues. There are no studies that evaluated effects of ischemia in renal tumors. This study examines the impact of ischemia and tissue procurement conditions on RNA integrity and gene expression in renal cell carcinoma.
Experimental Design
Ten renal tumors were resected without renal hilar clamping from 10 patients with renal clear cell carcinoma. Immediately after tumor resection, a piece of tumor was snap frozen. Remaining tumor samples were stored at 4C, 22C and 37C and frozen at 5, 30, 60, 120, and 240 minutes. Histopathologic evaluation was performed on all tissue samples, and only those with greater than 80% tumor were selected for further analysis. RNA integrity was confirmed by electropherograms and quantitated using RIN index. Altered gene expression was assessed by paired, two-sample t-test between the zero time point and aliquots from various conditions obtained from the same tumor.
Results
One hundred and forty microarrays were performed. Some RNA degradation was observed 240 mins after resection at 37C. The expression of over 4,000 genes was significantly altered by ischemia times or storage conditions. The greatest gene expression changes were observed with longer ischemia time and warmer tissue procurement conditions.
Conclusion
RNA from kidney cancer remains intact for up to 4 hours post surgical resection regardless of storage conditions. Despite excellent RNA preservation, time after resection and procurement conditions significantly influence gene expression profiles. Meticulous attention to pre-acquisition variables is of paramount importance for accurate tumor profiling.
doi:10.1158/1078-0432.CCR-12-2606
PMCID: PMC3658320  PMID: 23136194
Ischemia; gene expression microarrays; tissue procurement; renal cell carcinoma
6.  ONCOGENIC BRAF(V600E) PROMOTES STROMAL CELL-MEDIATED IMMUNOSUPPRESSION VIA INDUCTION OF INTERLEUKIN-1 IN MELANOMA 
Purpose
In this study, we assessed the specific role of BRAF(V600E) signaling in modulating the expression of immune regulatory genes in melanoma, in addition to analyzing downstream induction of immune suppression by primary human melanoma tumor-associated fibroblasts (TAFs).
Experimental Design
Primary human melanocytes and melanoma cell lines were transduced to express WT or V600E forms of BRAF, followed by gene expression analysis. The BRAF(V600E) inhibitor vemurafenib was used to confirm targets in BRAF(V600E)-positive melanoma cell lines and in tumors from melanoma patients undergoing inhibitor treatment. TAF lines generated from melanoma patient biopsies were tested for their ability to inhibit the function of tumor antigen-specific T-cells, prior to and following treatment with BRAF(V600E)-upregulated immune modulators. Transcriptional analysis of treated TAFs was conducted to identify potential mediators of T-cell suppression.
Results
Expression of BRAF(V600E) induced transcription of IL-1α and IL-1β in melanocytes and melanoma cell lines. Furthermore, vemurafenib reduced the expression of IL-1 protein in melanoma cell lines and most notably in human tumor biopsies from 11 of 12 melanoma patients undergoing inhibitor treatment. Treatment of melanoma-patient-derived TAFs with IL-1α/β significantly enhanced their ability to suppress the proliferation and function of melanoma-specific cytotoxic T cells, and this inhibition was partially attributable to upregulation by IL-1 of COX-2 and the PD-1 ligands PD-L1 and PD-L2 in TAFs.
Conclusions
This study reveals a novel mechanism of immune suppression sensitive to BRAF(V600E) inhibition, and suggests that clinical blockade of IL-1 may benefit patients with BRAF wild-type tumors and potentially synergize with immunotherapeutic interventions.
doi:10.1158/1078-0432.CCR-12-1632
PMCID: PMC3463754  PMID: 22850568
Melanoma; BRAF(V600E); interleukin-1; tumor-associated fibroblasts (TAFs); cytotoxic T lymphocytes (CTL)
7.  Activated MET is a molecular prognosticator and potential therapeutic target for malignant peripheral nerve sheath tumors 
Purpose:
MET-signaling has been suggested a potential role in malignant peripheral nerve sheath tumors (MPNSTs). Here, MET function and blockade were preclinically assessed.
Experimental Design:
Expression levels of MET, its ligand HGF, and phosphorylated MET (pMET) were examined in a clinically annotated MPNST tissue microarray incorporating univariable and multivariable statistical analyses. Human MPNST cells were studied in vitro and in vivo; WB and ELISA were used to evaluate MET and HGF expression, activation, and downstream signaling. Cell culture assays tested the impact of HGF-induced MET activation and anti-MET-specific siRNA inhibition on cell proliferation, migration, and invasion; in vivo gelfoam assays were used to evaluate angiogenesis. Cells stably transduced with anti-MET shRNA constructs were tested for growth and metastasis in SCID mice. The effect of the tyrosine kinase inhibitor XL184 (Exelixis) targeting MET/VEGFR2 on local and metastatic MPNST growth was examined in vivo.
Results:
All three markers were expressed in MPNST human samples; pMET expression was an independent prognosticator of poor patient outcome. Human MPNST cell lines expressed MET, HGF, and pMET. MET activation increased MPNST cell motility, invasion, angiogenesis, and induced MMP2 and VEGF expression; MET knockdown had inverse effects in vitro and markedly decreased local and metastatic growth in vivo. XL184 abrogated human MPNST xenograft growth and metastasis in SCID mice.
Conclusions:
Informative prognosticators and novel therapies are crucially needed to improve MPNST management and outcomes. We demonstrate an important role for MET in MPNST, supporting continued investigation of novel anti-MET therapies in this clinical context.
doi:10.1158/1078-0432.CCR-11-0193
PMCID: PMC3117912  PMID: 21540237
Malignant peripheral nerve sheath tumor; MET; Biomarker; Therapeutic target; XL184
8.  The Biphasic Role of NF-κB in Progression and Chemoresistance of Ovarian Cancer 
Purpose
NF-κB is a transcription factor known to promote tumorigenesis. However, NF-κB is also known to be proapoptotic and may potentially function as a tumor suppressor, although such a functional role has not been extensively investigated in human cancer.
Experimental Design
A dominant-negative mutant of IκBα with mutations at S32A and S36A was used to inhibit the function of NF-κB in ovarian cancer cell lines. The transcription ability, tumorigenesis, apoptosis, and drug sensitivity were examined in derivative cell lines in comparison with parental cells. We also analyzed the association of nuclear expression of NF-κB p65 with patient survival in an ovarian cancer tissue array.
Results
We show that NF-κB functions as a tumor suppressor in four ovarian cancer cell lines, but it functions as an oncogene in their aggressive chemoresistant isogenic variants. NF-κB can exert its proapoptotic or antiapoptotic effect by activating or repressing mitogen-activated protein kinase (MAPK) phosphorylation in parental or aggressive chemoresistant variant cell lines. We also show that the nuclear accumulation of p65 in epithelial cancer tissue is associated with a good response to chemotherapy and can predict longer overall survival for patients with ovarian cancer.
Conclusions
Our data provide strong evidence that NF-κB can function as a biphasic regulator, either suppressing or enhancing ovarian cancer growth through the regulation of MAPK and cellular apoptosis.
doi:10.1158/1078-0432.CCR-10-3265
PMCID: PMC3152795  PMID: 21339307
9.  IQGAP1 Plays an Important Role in the Invasiveness of Thyroid Cancer 
Purpose
This study was designed to explore the role of IQGAP1 in the invasiveness of thyroid cancer and its potential as a novel prognostic marker and therapeutic target in this cancer.
Experimental Design
We examined IQGAP1 copy gain and its relationship with clinicopathological outcomes of thyroid cancer and investigated its role in cell invasion and molecules involved in the process.
Results
We found IQGAP1 copy number gain ≥ 3 in 1/30 (3%), 24/74 (32%), 44/107 (41%), 8/16 (50%), and 27/41 (66%) of benign thyroid tumor, follicular variant papillary thyroid cancer (FVPTC), follicular thyroid cancer (FTC), tall cell PTC, and anaplastic thyroid cancer, respectively, in the increasing order of invasiveness of these tumors. A similar tumor distribution trend of copy number ≥ 4 was also seen. IQGAP1 copy gain was positively correlated with IQGAP1 protein expression. It was significantly associated with extrathyroidal and vascular invasion of FVPTC and FTC and, remarkably, a 50–60% rate of multifocality and recurrence of BRAF mutation-positive PTC (P = 0.01 and 0.02, respectively). siRNA knockdown of IQGAP1 dramatically inhibited thyroid cancer cell invasion and colony formation. Co-immunoprecipitation assay demonstrated direct interaction of IQGAP1 with E-cadherin, a known invasion-suppressing molecule, which was up-regulated when IQGAP1 was knocked down. This provided a mechanism for the invasive role of IQGAP1 in thyroid cancer. In contrast, IQGAP3 lacked all these functions.
Conclusions
IQGAP1, through genetic copy gain, plays an important role in the invasiveness of thyroid cancer and may represent a novel prognostic marker and therapeutic target for this cancer.
doi:10.1158/1078-0432.CCR-10-1627
PMCID: PMC3005072  PMID: 20959410
thyroid cancer; IQGAP1; gene copy gain; E-cadherin; BRAF mutation
10.  NF1 INACTIVATION IN ADULT ACUTE MYELOGENOUS LEUKEMIA 
Purpose
This study was conducted to identify novel genes with importance to the biology of adult acute myelogenous leukemia (AML).
Experimental design
We analyzed DNA from highly purified AML blasts and paired buccal cells from 95 patients for recurrent genomic microdeletions using ultra-high density Affymetrix SNP 6.0 array-based genomic profiling.
Results
Through fine mapping of microdeletions on 17q, we derived a minimal deleted region of ~0.9Mb length that harbors 11 known genes; this region includes Neurofibromin 1 (NF1). Sequence analysis of all NF1 coding exons in the 11 AML cases with NF1 copy number changes identified acquired truncating frameshift mutations in 2 patients. These NF1 mutations were already present in the hematopoetic stem cell compartment. Subsequent expression analysis of NF1 mRNA in the entire AML cohort using FACS sorted blasts as a source of RNA identified 6 patients (one with a NF1 mutation) with absent NF1 expression. The NF1 null states were associated with increased Ras-bound GTP, and shRNA-mediated NF1 suppression in primary AML blasts with wild type NF1 facilitated colony formation in methylcellulose. Primary AML blasts without functional NF1, unlike blasts with functional NF1, displayed sensitivity to rapamycin-induced apoptosis, thus identifying a dependence on mTOR signaling for survival. Finally, colony formation in methylcellulose ex vivo of NF1 null CD34+/CD38− cells sorted from AML bone marrow samples was inhibited by low dose rapamycin.
Conclusions
NF1 null states are present in 7/95=7% of adult AML and delineate a disease subset that could be preferentially targeted by Ras or mTOR-directed therapeutics.
doi:10.1158/1078-0432.CCR-09-2639
PMCID: PMC2921448  PMID: 20505189
AML; genomic microdeletions; NF1 mutations
11.  CXCR2 Promotes Ovarian Cancer Growth through Dysregulated Cell Cycle, Diminished Apoptosis, and Enhanced Angiogenesis 
Purpose
Chemokine receptor CXCR2 is associated with malignancy in several cancer models; however, the mechanisms involved in CXCR2-mediated tumor growth remain elusive. Here, we investigated the role of CXCR2 in human ovarian cancer.
Experimental Design
CXCR2 expression was silenced by stable small hairpin RNA in ovarian cancer cell lines T29Gro-1, T29H, and SKOV3. Western blotting, immunofluorescence, enzyme-linked immunosorbent assay, flow cytometry, electrophoretic mobility shift assay, and mouse assay were used to detect CXCR2, interleukin-8, Gro-1, cell cycle, apoptosis, DNA binding of NF-κB, and tumor growth. Immunohistochemical staining of CXCR2 was done in 240 high-grade serous ovarian carcinoma samples.
Results
Knockdown of CXCR2 expression by small hairpin RNA reduced tumorigenesis of ovarian cancer cells in nude mice. CXCR2 promoted cell cycle progression by modulating cell cycle regulatory proteins, including p21 (waf1/cip1), cyclin D1, CDK6, CDK4, cyclin A, and cyclin B1. CXCR2 inhibited cellular apoptosis by suppressing phosphorylated p53, Puma, and Bcl-xS; suppressing poly(ADP-ribose) polymerase cleavage; and activating Bcl-xL and Bcl-2. CXCR2 stimulated angiogenesis by increasing levels of vascular endothelial growth factor and decreasing levels of thrombospondin-1, a process likely involving mitogen-activated protein kinase, and NF-κB. Overexpression of CXCR2 in high-grade serous ovarian carcinomas was an independent prognostic factor of poor overall survival (P < 0.001) and of early relapse (P = 0.003) in the univariate analysis.
Conclusions
Our data provide strong evidence that CXCR2 regulates the cell cycle, apoptosis, and angiogenesis through multiple signaling pathways, including mitogen-activated protein kinase and NF-κB, in ovarian cancer. CXCR2 thus has potential as a therapeutic target and for use in ovarian cancer diagnosis and prognosis.
doi:10.1158/1078-0432.CCR-10-0483
PMCID: PMC2930833  PMID: 20505188
12.  TRAIL and doxorubicin combination induces pro-apoptotic and anti-angiogenic effects in soft tissue sarcoma in vivo 
Purpose
Novel therapeutic approaches for complex karyotype soft tissue sarcoma (STS) are crucially needed. Consequently, we assessed the efficacy of tumor necrosis factor-related apoptosis- inducing ligand (TRAIL), in combination with chemotherapy, on local and metastatic growth of human STS xenografts in vivo.
Experimental Design
TRAIL was evaluated alone and combined with low dose doxorubicin in two human STS SCID mouse xenograft models utilizing fibrosarcoma (HT1080; wild-type p53) and leiomyosarcoma (SKLMS1; mutated-p53), testing for impact on local growth, metastasis, and overall survival. MRI was used to evaluate local growth and bioluminescence was used to longitudinally assess lung metastases. Tissues were evaluated via immunohistocemistry and TUNEL staining for treatment effects on tumor cell proliferation, apoptosis, angiogenesis, angiogenic factors, and TRAIL receptor expression. qRTPCR angiogenesis array was utilized to assess therapy-induced gene expression changes.
Results
TRAIL/doxorubicin combination induced marked STS local and metastatic growth inhibition in a p53 independent manner. Significantly increased (p<0.001) host survival I was also demonstrable. Combined therapy induced significant apoptosis, decreased tumor cell proliferation, and increased TRAIL receptor (DR4 and DR5) expression in all treated tumors. Moreover, decreased microvessel density was observed, possibly secondary to increased expression of the anti-angiogenic factor CXCL10 and decreased pro-angiogenic IL-8 cytokine in response to TRAIL/doxorubicin combination, as was also observed in vitro.
Conclusions
Given the urgent need for better systemic approaches to STS, clinical trials evaluating TRAIL in combination with low dose chemotherapy are potentially warranted.
doi:10.1158/1078-0432.CCR-09-2443
PMCID: PMC2967255  PMID: 20406839
Soft tissue sarcoma; TRAIL; doxorubicin; apoptosis; angiogenesis; Therapy
13.  Phase I Study of GTI-2040, an Antisense to Ribonucleotide Reductase, in Combination with High-Dose Cytarabine in Patients with Acute Myeloid Leukemia 
Purpose
Inhibition of ribonucleotide reductase reduces the availability of the endogenous pool of deoxycytidine and may increase cytarabine (AraC) cytotoxicity. We performed a phase I dose escalation trial of AraC combined with GTI-2040, a 20-mer antisense oligonucleotide shown in preclinical studies to decrease levels of the R2 subunit of ribonucleotide reductase, to determine the maximum tolerated dose in adults with relapsed/refractory acute myeloid leukemia.
Experimental Design
Twenty-three adults (ages 18–59 years) were enrolled in this dose escalation phase I trial, receiving high-dose AraC twice daily combined with infusional GTI-2040. An ELISA-based assay measured plasma and intracellular concentrations of GTI-2040. R2 protein changes were evaluated by immunoblotting in pretreatment and post-treatment bone marrow samples.
Results
The maximum tolerated dose was 5 mg/kg/d GTI-2040 (days 1–6) and 3 g/m2/dose AraC every 12 hours for 8 doses. Neurotoxicity was dose limiting. Eight patients (35%) achieved complete remission. Mean bone marrow intracellular concentration of GTI-2040 were higher at 120 hours than at 24 hours from the start of GTI-2040 (P = 0.002), suggesting intracellular drug accumulation over time. Reductions in bone marrow levels of R2 protein (>50%) were observed at 24 and 120 hours. Higher baseline R2 protein expression (P = 0.03) and reductions after 24 hours of GTI-2040 (P = 0.04) were associated with complete remission.
Conclusions
GTI-2040 and high-dose AraC were coadministered safely with successful reduction of the intended R2 target and encouraging clinical results. The clinical efficacy of this combination will be tested in an upcoming phase II study.
doi:10.1158/1078-0432.CCR-08-0109
PMCID: PMC2993318  PMID: 18559610
14.  Fine mapping of chromosome 6q23-25 region in familial lung cancer families reveals RGS17 as a likely candidate gene 
We have previously mapped a major susceptibility locus influencing familial lung cancer risk to chromosome 6q23–25. However, the causal gene at this locus remains undetermined. In this study, we further refined this locus to identify a single candidate gene, by fine mapping using microsatellite markers and association studies using high-density SNPs. This region-wide scan across 6q23-25 found significant association between lung cancer susceptibility and three SNPs in the first intron of the RGS17. Association of two SNPs, rs4083914 and rs9479510, was further confirmed in two independent familial lung cancer populations. Quantitative RT-PCR analysis of matched tumor and normal human tissues found that RGS17 transcript accumulation is highly increased in sporadic lung tumors. Human lung tumor cell proliferation is inhibited upon knockdown of RGS17 transcript and enhanced upon overexpression of RGS17. Our findings indicate that RGS17 may influence familial susceptibility to lung cancer through its affects on cell proliferation.
doi:10.1158/1078-0432.CCR-08-2335
PMCID: PMC2746091  PMID: 19351763
linkage; haplotype; association; polymorphism; proliferation; tumor
15.  DNA Repair Gene Polymorphisms and Risk of Pancreatic Cancer 
Purpose
The current research was undertaken to examine the association between genetic variations in DNA repair and pancreatic cancer risk.
Experimental Design
We analyzed nine single nucleotide polymorphisms (SNPs) of seven DNA repair genes (LIG3, LIG4, OGG1, ATM, POLB, RAD54L, and RECQL) in 734 patients with pancreatic adenocarcinoma and 780 healthy controls using the Taqman method. Information on cigarette smoking, alcohol consumption, medical history, and other risk factors was collected by personal interview.
Results
The homozygous mutant genotype of LIG3 G-39A (odds ratio [OR], 0.23; 95% confidence interval [CI] = 0.06-0.82; P = 0.027) and ATM D1853N (OR, 2.55; 95% CI = 1.08-6.00; P = 0.032) was significantly associated with altered risk for pancreatic cancer. A statistically significant interaction of ATM D1853N and LIG4 C54T genotype with diabetes on the risk of pancreatic cancer was also detected. Compared to non-diabetics with the ATM D1853N GG genotype, non-diabetics with the GA/AA, diabetics with the GG, and diabetics with the GA/AA genotypes, respectively, had ORs (95% CI) of 0.96 (0.74-1.24), 1.32 (0.89-1.95), and 3.23 (1.47-7.12) (Pinteraction = 0.032, likelihood ratio test). The OR (95% CI) was 0.91 (0.71-1.17), 1.11 (0.73-1.69), and 2.44 (1.34-4.46) for non-diabetics carrying the LIG4 CT/TT genotype, diabetics with the CC genotype, and diabetics carrying the CT/TT genotype, respectively, compared to non-diabetics carrying the CC genotype (Pinteraction= 0.02).
Conclusions
These observations suggest that genetic variations in DNA repair may act alone or in concert with other risk factors on modifying a patient's risk for pancreatic cancer.
doi:10.1158/1078-0432.CCR-08-1607
PMCID: PMC2629144  PMID: 19147782
pancreatic cancer; DNA repair; oxidative stress; genetic polymorphisms; single nucleotide polymorphism (SNP)
16.  Successful Radiotherapy of Tumor in Pretargeted Mice by 188Re-Radiolabeled Phosphorodiamidate Morpholino Oligomer, a Synthetic DNA Analogue 
Purpose
Pretargeting has been attracting increasing attention as a drug delivery approach. We recently proposed Watson-Crick pairing of phosphorodiamidate morpholino oligomers (MORF) for the recognition system in tumor pretargeting. MORF pretargeting involves the initial i.v. injection of a MORF-conjugated antitumor antibody and the subsequent i.v. injection of the radiolabeled complement. Our laboratory has reported on MORF pretargeting for diagnosis using 99mTc as radiolabel. We now report on the use of MORF pretargeting for radiotherapy in a mouse tumor model using 188Re as the therapeutic radiolabel.
Experimental Design
An initial tracer study was done to estimate radiation dose, and was followed by the radiotherapy study at 400 μCi per mouse with three control groups (untreated, MORF antibody alone, and 188Re complementary MORF alone).
Results
Tracer study indicated rapid tumor localization of 188Re and rapid clearance from normal tissues with a tumor area under the curve (AUC) about four times that of kidney and blood (the normal organs with highest radioactivity). Tumor growth in the study group ceased 1 day after radioactivity injection, whereas tumors continued to grow at the same rate among the three control groups. At sacrifice on day 5, the average net tumor weight in the study group was significantly lower at 0.68 ± 0.29 g compared with the three control groups (1 24 ± 0.31g, 1 25 ± 0.39 g, and 1 35 ± 0.41g; Ps <0.05), confirming the therapeutic benefit observed by tumor size measurement.
Conclusions
MORF pretargeting has now been shown to be a promising approach for tumor radiotherapy as well as diagnosis.
doi:10.1158/1078-0432.CCR-06-0844
PMCID: PMC1587615  PMID: 16914585
17.  Essential role of aldehyde dehydrogenase 1A3 (ALDH1A3) for the maintenance of non-small cell lung cancer stem cells is associated with the STAT3 pathway 
Purpose
Lung cancer stem cells (CSCs) with elevated aldehyde dehydrogenase (ALDH) activity are self-renewing, clonogenic and tumorigenic. The purpose of our study is to elucidate the mechanisms by which lung CSCs are regulated.
Experimental Design
A genome-wide gene expression analysis was performed to identify genes differentially expressed in the ALDH+ vs. ALDH− cells. RT-PCR, western blot and Aldefluor assay were used to validate identified genes. To explore the function in CSCs we manipulated their expression followed by colony and tumor formation assays.
Results
We identified a subset of genes that were differentially expressed in common in ALDH+ cells, among which ALDH1A3 was the most upregulated gene in ALDH+ vs. ALDH− cells. ShRNA-mediated knockdown of ALDH1A3 in NSCLCs resulted in a dramatic reduction in ALDH activity, clonogenicity and tumorigenicity, indicating that ALDH1A3 is required for tumorigenic properties. By contrast, overexpression of ALDH1A3 by itself it was not sufficient to increase tumorigenicity. The ALDH+ cells also expressed more activated Signal Transducers and Activators of Transcription 3 (STAT3) than ALDH− cells. Inhibition of STAT3 or its activator EZH2 genetically or pharmacologically diminished the level of ALDH+ cells and clonogenicity. Unexpectedly, ALDH1A3 was highly expressed in female, never smokers, well differentiated tumors, or adenocarcinoma. ALDH1A3 low expression was associated with poor overall survival.
Conclusion
Our data show that ALDH1A3 is the predominant ALDH isozyme responsible for ALDH activity and tumorigenicity in most NSCLCs, and that inhibiting either ALDH1A3 or the STAT3 pathway are potential therapeutic strategies to eliminate the ALDH+ subpopulation in NSCLCs.
doi:10.1158/1078-0432.CCR-13-3292
PMCID: PMC4438754  PMID: 24907115
Lung cancer; cancer stem cells; ALDH1A3; STAT3; Stattic
18.  Real-Time Immune Monitoring to Guide Plasmid DNA Vaccination Schedule Targeting Prostatic Acid Phosphatase (PAP) in Patients with Castration-Resistant Prostate Cancer 
BACKGROUND
We have previously reported that a DNA vaccine encoding prostatic acid phosphatase (PAP) could elicit PAP-specific T cells in patients with early recurrent prostate cancer. In the current pilot trial we sought to evaluate whether prolonged immunization with regular booster immunizations, or “personalized” schedules of immunization determined using real-time immune monitoring, could elicit persistent, antigen-specific T cells, and whether treatment was associated with changes in PSA doubling time (PSA DT).
METHODS
16 patients with castration-resistant, non-metastatic prostate cancer received six immunizations at two-week intervals, and then either quarterly (Arm 1) or as determined by multi-parameter immune monitoring (Arm 2).
RESULTS
Patients were on study a median of 16 months; four received 24 vaccinations. Only one event associated with treatment > grade 2 was observed. 6/16 (38%) remained metastasis-free at 2 years. PAP-specific T cells were elicited in 12/16 (75%), predominantly of a Th1 phenotype, which persisted in frequency and phenotype for at least one year. IFNγ-secreting T-cell responses measured by ELISPOT were detectable in 5/13 individuals at one year, and this was not statistically different between study arms. The overall median fold change in PSA DT from pre-treatment to post-treatment was 1.6 (range 0.6–7.0, p=0.036).
CONCLUSIONS
Repetitive immunization with a plasmid DNA vaccine was safe and elicited Th1-biased antigen-specific T cells that persisted over time. Modifications in the immunization schedule based on real-time immune monitoring did not increase the frequency of patients developing effector and memory T-cell responses with this DNA vaccine.
doi:10.1158/1078-0432.CCR-14-0169
PMCID: PMC4102643  PMID: 24850844
DNA vaccine; prostate cancer; prostatic acid phosphatase; clinical trial; immune monitoring
19.  Old drug new use - Amoxapine and its metabolites as potent bacterial β-glucuronidase inhibitors for alleviating cancer drug toxicity 
Purpose
Irinotecan (CPT-11) induced diarrhea occurs frequently in cancer patients and limits its usage. Bacteria β-glucuronidase (GUS) enzymes in intestines convert the non-toxic metabolite of CPT-11, SN-38G, to toxic SN-38, and finally lead to damage of intestinal epithelial cells and diarrhea. We previously reported amoxapine as potent GUS inhibitor in vitro. To further understand the molecular mechanism of amoxapine and its potential for treatment of CPT-11 induced diarrhea, we studied the binding modes of amoxapine and its metabolites by docking and molecular dynamics simulation, and tested the in vivo efficacy on mice in combination with CPT-11.
Experimental Design
The binding of amoxapine, its metabolites, 7-hydroxyamoxapine and 8-hydroxyamoxapine, and a control drug loxapine with GUS was explored by computational protocols. The in vitro potencies of metabolites were measured by E. Coli GUS enzyme and cell-based assay. Low dosage daily oral administration was designed to use along with CPT-11 to treat tumor-bearing mice.
Results
Computational modeling results indicated that amoxapine and its metabolites bound in the active site of GUS and satisfied critical pharmacophore features: aromatic features near bacterial loop residue F365’ and hydrogen bond toward E413. Amoxapine and its metabolites were demonstrated as potent in vitro. Administration of low dosages of amoxapine with CPT-11 in mice achieved significant suppression of diarrhea and reduced tumor growth.
Conclusions
Amoxapine has great clinical potential to be rapidly translated to human subjects for irinotecan induced diarrhea.
doi:10.1158/1078-0432.CCR-14-0395
PMCID: PMC4079752  PMID: 24780296
amoxapine; β-glucuronidase; irinotecan (CPT-11); molecular docking and dynamics simulation; drug reposition
20.  Niclosamide inhibits androgen receptor variants expression and overcomes enzalutamide resistance in castration resistant prostate cancer 
Purpose
Enzalutamide, a second-generation antiandrogen, was recently approved for the treatment of castration-resistant prostate cancer (CRPC) in patients who no longer respond to docetaxel. Despite these advances that provide temporary respite, resistance to enzalutamide occurs frequently. AR splice variants such as AR-V7 have recently been shown to drive castration resistant growth and resistance to enzalutamide. This study was designed to identify inhibitors of AR variants and test its ability to overcome resistance to enzalutamide.
Experimental Design
The drug screening was conducted using luciferase activity assay to determine the activity of AR-V7 after treatment with the compounds in the Prestwick Chemical Library, which contains about 1120 FDA-approved drugs. The effects of the identified inhibitors on AR-V7 activity and enzalutamide sensitivity were characterized in CRPC and enzalutamide-resistant prostate cancer cells in vitro and in vivo.
Results
Niclosamide, an FDA-approved anti-helminthic drug, was identified as a potent AR-V7 inhibitor in prostate cancer cells. Niclosamide significantly downregulated AR-V7 protein expression by protein degradation through a proteasome dependent pathway. Niclosamide also inhibited AR-V7 transcription activity and reduced the recruitment of AR-V7 to the PSA promoter. Niclosamide inhibited prostate cancer cell growth in vitro and tumor growth in vivo. Furthermore, the combination of niclosamide and enzalutamide resulted in significantly inhibition of enzalutamide-resistant tumor growth, suggesting that Niclosamide enhances enzalutamide therapy and overcomes enzalutamide resistance in castration resistant prostate cancer cells.
Conclusions
Niclosamide was identified as a novel inhibitor of AR variants. Our findings offer preclinical validation of niclosamide as a promising inhibitor of androgen receptor variants to treat, either alone or in combination with current antiandrogen therapies, advanced prostate cancer patients, especially those resistant to enzalutamide.
doi:10.1158/1078-0432.CCR-13-3296
PMCID: PMC4058390  PMID: 24740322
prostate cancer; enzalutamide; niclosamide; variants; drug resistance
21.  CS1, a Potential New Therapeutic Antibody Target for the Treatment of Multiple Myeloma 
Purpose
We generated a humanized antibody, HuLuc63, which specifically targets CS1 (CCND3 subset 1, CRACC, and SLAMF7), a cell surface glycoprotein not previously associated with multiple myeloma. To explore the therapeutic potential of HuLuc63 in multiple myeloma, we examined in detail the expression profile of CS1, the binding properties of HuLuc63 to normal and malignant cells, and the antimyeloma activity of HuLuc63 in preclinical models.
Experimental Design
CS1 was analyzed by gene expression profiling and immunohistochemistry of multiple myeloma samples and numerous normal tissues. HuLuc63-mediated anti-myeloma activity was tested in vitro in antibody-dependent cellular cytotoxicity (ADCC) assays and in vivo using the human OPM2 xenograft model in mice.
Results
CS1 mRNA was expressed in >90% of 532 multiple myeloma cases, regardless of cytogenetic abnormalities. Anti-CS1 antibody staining of tissues showed strong staining of myeloma cells in all plasmacytomas and bone marrow biopsies. Flow cytometric analysis of patient samples using HuLuc63 showed specific staining of CD138+ myeloma cells, natural killer (NK), NK-like Tcells, and CD8+ Tcells, with no binding detected on hematopoietic CD34+ stem cells. HuLuc63 exhibited significant in vitro ADCC using primary myeloma cells as targets and both allogeneic and autologous NK cells as effectors. HuLuc63 exerted significant in vivo antitumor activity, which depended on efficient Fc-CD16 interaction as well as the presence of NK cells in the mice.
Conclusions
These results suggest that HuLuc63 eliminates myeloma cells, at least in part, via NK-mediated ADCC and shows the therapeutic potential of targeting CS1 with HuLuc63 for the treatment of multiple myeloma.
doi:10.1158/1078-0432.CCR-07-4246
PMCID: PMC4433038  PMID: 18451245
22.  Recognition and killing of autologous, primary glioblastoma tumor cells by human cytomegalovirus pp65-specific cytotoxic T cells 
Purpose
Despite aggressive conventional therapy, glioblastoma multiforme (GBM) remains uniformly lethal. Immunotherapy, in which the immune system is harnessed to specifically attack malignant cells, offers a treatment option with less toxicity. The expression of cytomegalovirus (CMV) antigens in GBM presents a unique opportunity to target these viral proteins for tumor immunotherapy. Although the presence of CMV within malignant gliomas has been confirmed by several laboratories, its relevance as an immunological target in GBM has yet to be established. The objective of this study was to explore whether T cells stimulated by CMV pp65 RNA-transfected dendritic cells (DCs) target and eliminate autologous GBM tumor cells in an antigen-specific manner.
Experimental Design
T cells from patients with GBM were stimulated with autologous DCs pulsed with CMV pp65 RNA, and the function of the effector CMV pp65-specific T cells was measured.
Results
In this study, we demonstrate the ability to elicit CMV pp65-specific immune responses in vitro using RNA-pulsed autologous DCs generated from patients with newly diagnosed GBM. Importantly, CMV pp65-specific T cells lyse autologous, primary GBM tumor cells in an antigen-specific manner. Moreover, T cells expanded in vitro using DCs pulsed with total tumor RNA demonstrated a 10–20 fold expansion of CMV pp65-specific T cells as assessed by tetramer analysis and recognition and killing of CMV pp65-expressing target cells.
Conclusion
These data collectively demonstrate that CMV-specific T cells can effectively target glioblastoma tumor cells for immunologic killing and support the rationale for the development of CMV-directed immunotherapy in patients with GBM.
doi:10.1158/1078-0432.CCR-13-3268
PMCID: PMC4059212  PMID: 24658154
Cytomegalovirus; immunotherapy; dendritic cells; gliomas; T cells
23.  Preclinical and Early Clinical Evaluation of the Oral AKT Inhibitor, MK-2206, for the Treatment of Acute Myeloid Leukemia 
Purpose
Recent studies suggested that AKT activation might confer poor prognosis in acute myeloid leukemia (AML), providing the rationale for therapeutic targeting of this signaling pathway. We therefore explored the preclinical and clinical anti-AML activity of an oral AKT inhibitor, MK-2206.
Experimental Methods
We first studied the effects of MK-2206 in human AML cell lines and primary AML specimens in vitro. Subsequently, we conducted a phase 2 trial of MK-2206 (200 mg weekly) in adults requiring second salvage therapy for relapsed/refractory AML, and assessed target inhibition via reverse phase protein array (RPPA).
Results
In preclinical studies, MK-2206 dose-dependently inhibited growth and induced apoptosis in AML cell lines and primary AML blasts. We then treated 19 patients with MK-2206 but, among 18 evaluable participants, observed only 1 (95% CI: 0–17%) response (complete remission with incomplete platelet count recovery), leading to early study termination. The most common grade 3/4 drug-related toxicity was a pruritic rash in 6/18 patients. Nevertheless, despite the use of MK-2206 at maximum tolerated doses, RPPA analyses indicated only modest decreases in Ser473 AKT (median 28%; range, 12–45%) and limited inhibition of downstream targets.
Conclusions
While preclinical activity of MK-2206 can be demonstrated, this inhibitor has insufficient clinical anti-leukemia activity when given alone at tolerated doses, and alternative approaches to block AKT signaling should be explored.
doi:10.1158/1078-0432.CCR-13-1978
PMCID: PMC3989412  PMID: 24583795
Acute myeloid leukemia; MK-2206; AKT pathway
24.  Ultra Low-Dose IL-2 for GVHD prophylaxis after Allogeneic Hematopoietic Stem Cell Transplantation mediates Expansion of Regulatory T Cells Without Diminishing Anti-viral and Anti-leukemic Activity 
Purpose
Graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with low numbers of circulating CD4+CD25+FoxP3+ regulatory T-cells(Tregs). Because Tregs express high levels of the IL-2 receptor, they may selectively expand in vivo in response to doses of IL-2 insufficient to stimulate T-effector T-cell populations, thereby preventing GvHD.
Experimental Design
We prospectively evaluated the effects of ultra low-dose (ULD) IL-2 injections on Treg recovery in pediatric patients after alloSCT and compared this recovery with Treg reconstitution post alloSCT in patients without IL-2. Sixteen recipients of related(n=12) or unrelated(n=4) donor grafts received ULD-IL-2 post HSCT (100,000–200,000 IU/m2 3×weekly), starting
Results
No grade 3/4 toxicities were associated with ULD-IL-2. CD4+CD25+FoxP3+ Tregs increased from a mean of 4.8%(range, 0–11.0%) pre IL-2 to 11.1%(range,1.2–31.1%) following therapy, with the greatest change occurring in the recipients of MRD transplants. No IL-2 patients developed grade II-IV aGvHD, compared to 4/33(12%) of the comparator group who did not receive IL-2. IL-2 recipients retained T-cells reactive to viral and leukemia antigens, and in the MRD recipients, only 2/13(15%) of the IL-2 patients developed viral infections versus 63% of the comparator group (p=0.022).
Conclusions
Hence, ULD-IL-2 is well-tolerated, expands a Treg population in vivo, and may be associated with a lower incidence of viral infections and GvHD.
doi:10.1158/1078-0432.CCR-13-3205
PMCID: PMC3989436  PMID: 24573552
allogeneic hematopoietic stem cell transplantation; Graft versus host disease; Regulatory T cells; Interleukin-2
Purpose
EphA2 is an attractive therapeutic target due to its diverse roles in cancer growth and progression. Dasatinib is a multi-kinase inhibitor that targets EphA2 and other kinases. However, reliable predictive markers and a better understanding of the mechanisms of response to this agent are needed.
Experimental design
The effects of dasatinib on human uterine cancer cell lines were examined using a series of in vitro experiments, including MTT, Western blot, and plasmid transfection. In vivo, an orthotopic mouse model of uterine cancer was utilized to identify the biological effects of dasatinib. Molecular markers for response prediction and the mechanisms relevant to response to dasatinib were identified by using RPPA, immunoprecipitation, and double immunofluorescence staining.
Results
We show that high levels of CAV-1, EphA2 phosphorylation at S897 and the status of PTEN are key determinants of dasatinib response in uterine carcinoma. A set of markers essential for dasatinib response was also identified and includes CRaf, pCRafS338, pMAPKT202/Y204 (MAPK pathway), pS6S240/244, p70S6kT389 (mTOR pathway) and pAKTS473. A novel mechanism for response was discovered whereby high expression level of CAV-1 at the plasma membrane disrupts the BRaf/CRaf heterodimer and thus inhibits the activation of MAPK pathway during dasatinib treatment.
Conclusions
Our in vitro and in vivo results provide a new understanding of EphA2 targeting by dasatinib and identify key predictors of therapeutic response. These findings have implications for ongoing dasatinib-based clinical trials.
doi:10.1158/1078-0432.CCR-13-2141
PMCID: PMC3975695  PMID: 24486585
Dasatinib; EphA2; Caveolin-1 (CAV-1); Uterine Cancer

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