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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.  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
3.  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
4.  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
5.  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)
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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)
15.  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
16.  Rb Loss is Characteristic of Prostatic Small Cell Neuroendocrine Carcinoma 
Purpose
Small cell neuroendocrine carcinoma of the prostate is likely to become increasingly common with recent advances in pharmacologic androgen suppression. Thus, developing molecular markers of small cell differentiation in prostate cancer will be important to guide diagnosis and therapy of this aggressive tumor.
Experimental Design
We examined the status of RB1, TP53 and PTEN in prostatic small cell and acinar carcinomas via immunohistochemistry (IHC), copy number alteration analysis and sequencing of formalin fixed paraffin-embedded specimens.
Results
We found Rb protein loss in 90% (26/29) of small cell carcinoma cases with RB1 allelic loss in 85% (11/13) of cases. Of acinar tumors occurring concurrently with prostatic small cell carcinoma, 43% (3/7) showed Rb protein loss. In contrast, only 7% (10/150) of primary high grade acinar carcinomas, 11% (4/35) of primary acinar carcinomas with neuroendocrine differentiation, and 15% (2/13) of metastatic castrate resistant acinar carcinomas showed Rb protein loss. Loss of PTEN protein was seen in 63% (17/27) of small cell carcinomas, with 38% (5/13) showing allelic loss. By IHC, accumulation of p53 was observed in 56% (14/25) of small cell carcinomas, with 60% (6/10) of cases showing TP53 mutation.
Conclusions
Loss of RB1 by deletion is a common event in prostatic small cell carcinoma and can be detected by validated IHC assay. As Rb protein loss rarely occurs in high grade acinar tumors, these data suggest that Rb loss is a critical event in the development of small cell carcinomas and may be a useful diagnostic and potential therapeutic target.
doi:10.1158/1078-0432.CCR-13-1982
PMCID: PMC3931005  PMID: 24323898
Prostatic adenocarcinoma; small cell carcinoma; tumor suppressor; RB1; TP53; PTEN
17.  Telomere Content and Risk of Second Malignant Neoplasm in Survivors of Childhood Cancer: A Report from the Childhood Cancer Survivor Study 
PURPOSE
Shorter constitutional telomere length has been associated with increased cancer incidence. Furthermore, telomere shortening is observed in response to intensive chemotherapy and/or ionizing radiation exposure. We aimed to determine if less telomere content was associated with treatment-related second malignant neoplasms (SMNs) in childhood cancer survivors.
EXPERIMENTAL DESIGN
Using a nested case-control design, 147 cancer survivors with breast cancer, thyroid cancer, or sarcoma developing after treatment for childhood cancer (cases) were matched (1:1) with childhood cancer survivors without a SMN (controls). Cases and controls were matched by primary cancer diagnosis, years since diagnosis, age at time of sample collection, years of follow up from childhood cancer diagnosis, exposure to specific chemotherapy agents, and to specific radiation fields. We performed conditional logistic regression using telomere content (TC) as a continuous variable to estimate odds ratios (ORs) with corresponding 95% confidence intervals (CI) for development of SMN. ORs were also estimated for specific SMN types, i.e., breast cancer, thyroid cancer, and sarcoma.
RESULTS
There was an inverse relationship between TC and SMN, with an adjusted OR of 0.3 per unit change in telomere length to single copy gene ratio (95% CI, 0.09–1.02, p=0.05). Patients with thyroid cancer SMN were less likely to have more telomere content (OR 0.04, 95% CI, 0.00–0.55, p=0.01), but statistically significant associations could not be demonstrated for breast cancer or sarcoma.
CONCLUSIONS
A relation between less telomere content and treatment-related thyroid cancer was observed, suggesting that shorter telomeres may contribute to certain SMNs in childhood cancer survivors.
doi:10.1158/1078-0432.CCR-13-2076
PMCID: PMC3944671  PMID: 24277454
telomere; second malignant neoplasm; risk; childhood cancer
18.  BET-bromodomain inhibition of MYC-amplified medulloblastoma 
Purpose
MYC-amplified medulloblastomas are highly lethal tumors. BET bromodomain inhibition has recently been shown to suppress MYC-associated transcriptional activity in other cancers. The compound JQ1 inhibits BET bromodomain-containing proteins, including BRD4. Here we investigate BET bromodomain targeting for the treatment of MYC-amplified medulloblastoma.
Experimental Design
We evaluated the effects of genetic and pharmacological inhibition of BET bromodomains on proliferation, cell cycle, and apoptosis in established and newly generated patient- and GEMM-derived medulloblastoma cell lines and xenografts that harbored amplifications of MYC or MYCN. We also assessed the effect of JQ1 on MYC expression and global MYC-associated transcriptional activity. We assessed in vivo efficacy of JQ1 in orthotopic xenografts established in immunocompromised mice.
Results
Treatment of MYC-amplified medulloblastoma cells with JQ1 decreased cell viability associated with arrest at G1 and apoptosis. We observed down-regulation of MYC expression and confirmed inhibition of MYC-associated transcriptional targets. Exogenous expression of MYC from a retroviral promoter reduced the effect of JQ1 on cell viability, suggesting that attenuated levels of MYC contribute to the functional effects of JQ1. JQ1 significantly prolonged survival of orthotopic xenograft models of MYC-amplified medulloblastoma (p<0.001). Xenografts harvested from mice after five doses of JQ1 had reduced expression of MYC mRNA and a reduced proliferative index.
Conclusion
JQ1 suppresses MYC expression and MYC-associated transcriptional activity in medulloblastomas, resulting in an overall decrease in medulloblastoma cell viability. These preclinical findings highlight the promise of BET bromodomain inhibitors as novel agents for MYC-amplified medulloblastoma.
doi:10.1158/1078-0432.CCR-13-2281
PMCID: PMC4198154  PMID: 24297863
BET-bromodomain; JQ1; MYC; MYCN; MYCL1; medulloblastoma
19.  Direct inhibition of Retinoblastoma phosphorylation by Nimbolide causes cell cycle arrest and suppresses glioblastoma growth 
Purpose
Classical pharmacology allows the use and development of conventional phytomedicine faster and more economically than conventional drugs. This approach should be tested for their efficacy in terms of complementarity and disease control. The purpose of this study was to determine the molecular mechanisms by which nimbolide, a triterpenoid found in the well-known medicinal plant Azadirachta indica controls glioblastoma (GBM) growth.
Experimental Design
Using in vitro signaling, anchorage-independent growth, kinase assays, and xenograft models, we investigated the mechanisms of its growth inhibition in glioblastoma.
Results
We show that nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains nimbolide as the principal cytotoxic agent is highly cytotoxic against GBM in vitro and in vivo. Azt caused cell cycle arrest, most prominently at the G1-S stage in GBM cells expressing EGFRvIII, an oncogene present in about 20-25% of GBMs. Azt/nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retinoblastoma (RB) protein, cell cycle arrest at G1-S and cell death. Independent of RB hypophosphorylation, Azt also significantly reduced proliferative and survival advantage of GBM cells in vitro and in tumor xenografts by downregulating Bcl2 and blocking growth factor induced phosphorylation of Akt, Erk1/2 and STAT3. These effects were specific since Azt did not affect mTOR or other cell cycle regulators. In vivo, Azt completely prevented initiation and inhibited progression of GBM growth.
Conclusions
Our preclinical findings demonstrate Nimbolide as a potent anti-glioma agent that blocks cell cycle and inhibits glioma growth in vitro and in vivo.
doi:10.1158/1078-0432.CCR-13-0762
PMCID: PMC3947304  PMID: 24170547
20.  Transient activation of Hedgehog pathway rescued irradiation-induced hyposalivation by preserving salivary stem/progenitor cells and parasympathetic innervation 
Purpose
To examine effects and mechanisms of transient activation of Hedgehog pathway on rescuing radiotherapy-induced hyposalivation in head and neck cancer survivors.
Experimental Design
Mouse salivary glands and cultured human salivary epithelial cells were irradiated by single 15Gy dose. Hedgehog pathway was transiently activated in mouse salivary glands by shortly over-expressing Sonic hedgehog (Shh) transgene or administrating Smoothened Agonist and in human salivary epithelial cells by infecting with adenovirus encoding Gli1. Activity of Hedgehog signaling was examined by expression of Ptch1-lacZ reporter and endogenous Hedgehog target genes. Salivary flow rate was measured following pilocarpine stimulation. Salivary stem/progenitor cells (SSPCs), parasympathetic innervation and expression of related genes were examined by flow cytometry, salisphere assay, IHC, quantitative RT-PCR, Western blot and ELISA.
Results
Irradiation does not activate Hedgehog signaling in mouse salivary glands. Transient Shh over-expression activated Hedgehog pathway in ductal epithelia and that after irradiation rescued salivary function in male mice, which is related with preservation of functional SSPCs and parasympathetic innervation. The preservation of SSPCs was likely mediated by rescue of signaling activities of Bmi1 and Chrm1/HB-EGF pathways. The preservation of parasympathetic innervation was related with rescue of expression of neurotrophic factors such as Bdnf and Nrtn. The expression of genes related with maintenance of salivary stem/progenitor cells and parasympathetic innervation in female salivary glands and cultured human salivary epithelial cells was similarly affected by irradiation and transient Hedgehog activation.
Conclusions
These findings suggest that transient activation of Hedgehog pathway has the potential to restore irradiation-induced salivary gland dysfunction.
doi:10.1158/1078-0432.CCR-13-1434
PMCID: PMC3951215  PMID: 24150232
hyposalivation; Hedgehog signaling; radiotherapy; stem cells; parasympathetic innervation
21.  Interleukin-7 mediates selective expansion of tumor-redirected cytotoxic T lymphocytes without enhancement of regulatory T-cell inhibition 
Purpose
The antitumor activity of chimeric antigen receptor (CAR)-redirected cytotoxic T lymphocytes (CTLs) should be enhanced if it were possible to increase their proliferation and function after adoptive transfer without concomitantly increasing the proliferation and function of regulatory T cells (Tregs). Here we explored whether the lack of IL-7Rα in Tregs can be exploited by the targeted manipulation of the interleukin-7 (IL-7) cytokine-cytokine receptor axis in CAR-engrafted Epstein Barr Virus-specific CTLs (EBV-CTLs) to selectively augment their growth and anti-tumor activity even in the presence of Tregs.
Experimental Design
We generated a bicistronic retroviral vector encoding a GD2-specific CAR and the IL-7Rα subunit, expressed the genes in EBV-CTLs and assessed their capacity to control tumor growth in the presence of Tregs in vitro and in vivo when exposed to either interleukin-2 (IL-2) or IL-7 in a neuroblastoma xenograft.
Results
We found that IL-7, in sharp contrast to IL-2, supports the proliferation and antitumor activity of IL-7Rα.CAR-GD2+ EBV-CTLs both in vitro and in vivo even in the presence of fully functional Tregs.
Conclusions
IL-7 selectively favors the survival, proliferation, and effector function of IL-7Rα-transgenic/CAR-redirected EBV-CTLs in the presence of Tregs both in vitro and in vivo. Thus, IL-7 can have a significant impact in sustaining expansion and persistence of adoptively CAR-redirected CTLs.
doi:10.1158/1078-0432.CCR-13-1016
PMCID: PMC4113428  PMID: 24097874
22.  Comprehensive biomarker analysis and final efficacy results of sorafenib in the BATTLE (Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination) trial 
Purpose
To report the clinical efficacy of sorafenib and to evaluate biomarkers associated with sorafenib clinical benefit in the BATTLE program.
Patients and Methods
Patients with previously treated non-small–cell lung cancer (NSCLC) received sorafenib until progression or unacceptable toxicity. Eight-week disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were assessed. Prespecified biomarkers included K-RAS, EGFR, and B-RAF mutations, and EGFR gene copy number. Gene expression profiles from NSCLC cell lines and patient tumor biopsies with wild-type EGFR were used to develop a sorafenib sensitivity signature (SSS).
Results
105 patients were eligible and randomized to receive sorafenib. Among 98 patients evaluable for 8-week DCR, the observed DCR was 58.2%. The median PFS and OS were 2.83 (95% confidence interval [CI], 2.04-3.58) and 8.48 months (95% CI, 5.78-10.97), respectively. Eight-week DCR was higher in patients with wt-EGFR than patients with EGFR mutation (P=0.012), and in patients with EGFR gene copy number gain (FISH positive) versus patients FISH negative (P=0.048). In wt-EGFR tumors, the SSS was associated with improved PFS (median PFS 3.61 months in high SSS versus 1.84 months in low SSS, P=0.026) but not with 8-week DCR. Increased expression of fibroblast growth factor-1, NF-kB and hypoxia pathways were identified potential drivers of sorafenib resistance.
Conclusion
Sorafenib demonstrates clinical activity in NSCLC, especially with wt-EGFR. SSS was associated with improved PFS. These data identify subgroups that may derive clinical benefit from sorafenib and merit investigation in future trials. ClinicalTrials.gov: NCT00411671.
doi:10.1158/1078-0432.CCR-12-1818
PMCID: PMC3905243  PMID: 24166906
multikinase inhibitor; non–small cell lung cancer; sorafenib; biomarkers; targeted treatment
23.  Targeting Src and tubulin in mucinous ovarian carcinoma 
Purpose
To investigate the antitumor effects of targeting Src and tubulin in mucinous ovarian carcinoma.
Experimental design
The in vitro and in vivo effects and molecular mechanisms of KX-01, which inhibits Src pathway and tubulin polymerization, were examined in mucinous ovarian cancer models.
Results
In vitro studies using RMUG-S and RMUG-L cell lines showed that KX-01 inhibited cell proliferation, induced apoptosis, arrested the cell cycle at the G2/M phase, and enhanced the cytotoxicity of oxaliplatin in the KX-01-sensitive cell line, RMUG-S. In vivo studies showed that KX-01 significantly decreased tumor burden in RMUG-S and RMUG-L mouse models relative to untreated controls, and the effects were greater when KX-01 was combined with oxaliplatin. KX-01 alone and in combination with oxaliplatin significantly inhibited tumor growth by reducing cell proliferation and inducing apoptosis in vivo. PTEN knock-in experiments in RMUG-L cells showed improved response to KX-01. Reverse phase protein array analysis showed that in addition to blocking downstream molecules of Src family kinases, KX-01 also activated acute stress-inducing molecules.
Conclusion
Our results showed that targeting both the Src pathway and tubulin with KX-01 significantly inhibited tumor growth in preclinical mucinous ovarian cancer models, suggesting that this may be a promising therapeutic approach for patients with mucinous ovarian carcinoma.
doi:10.1158/1078-0432.CCR-13-1305
PMCID: PMC3852199  PMID: 24100628
Ovarian carcinoma; Mucinous; Src kinase; Tubulin; KX-01
24.  Molecular chaperone gp96 is a novel therapeutic target of multiple myeloma 
Purpose
gp96 (grp94) is a key downstream chaperone in the ER to mediate unfolded protein response (UPR) and the pathogenesis of multiple myeloma (MM) is closely linked to dysregulated UPR. In this study, we aimed to determine the roles of gp96 in the initiation and progression of MM in vivo and in vitro.
Experimental design
We generated a mouse model with over-expression of XBP1s and conditional deletion of gp96 in B cell compartment simultaneously to identify the roles of gp96 in the development of MM in vivo. Using a shRNA system, we silenced gp96 in multiple human MM cells and examined the effect of gp96 knockdown on MM cells by cell proliferation, cell cycle analysis, apoptosis assay, immunohistochemistry and human myeloma xenograft model. The anti-cancer activity of gp96 selective inhibitor, WS13 was evaluated by apoptosis assay and MTT assay.
Results
Genetic deletion of gp96 in XBP1s-Tg mice attenuates multiple myeloma. Silencing of gp96 causes severe compromise in human MM cell growth through inhibiting Wnt-LRP-survivin pathway. We also confirmed that knockdown of gp96 decreased human MM growth in a murine xenograft model. The targeted gp96 inhibitor induced apoptosis and blocked MM cell growth, but did not induce apoptosis in pre-B leukemic cells. We have demonstrated that myeloma growth is dependent on gp96 both genetically and pharmacologically.
Conclusions
gp96 is essential for MM cell proliferation and survival, suggesting that gp96 is a novel therapeutic target for multiple myeloma.
doi:10.1158/1078-0432.CCR-13-2083
PMCID: PMC3851294  PMID: 24077352
Multiple myeloma; gp96; unfolded protein response; Wnt signaling; survivin
25.  Lessons Learned from Radiation Oncology Clinical Trials 
A Workshop entitled “Lessons Learned from Radiation Oncology Trials” was held on December 7–8th, 2011 in Bethesda, MD, to present and discuss some of the recently conducted Radiation Oncology clinical trials with a focus on those that failed to refute the null hypothesis. The objectives of this Workshop were to summarize and examine the questions that these trials provoked, to assess the quality and limitations of the pre-clinical data that supported the hypotheses underlying these trials, and to consider possible solutions to these challenges for the design of future clinical trials.
Several themes emerged from the discussions, including the: a) opportunities to learn from null-hypothesis trials through tissue and imaging studies; b) value of pre-clinical data supporting the design of combinatorial therapies; c) significance of validated biomarkers; d) necessity of quality assurance in radiotherapy delivery; e) conduct of sufficiently-powered studies to address the central hypothesis; and f) importance of publishing results of the trials regardless of the outcome.
The fact that well-designed hypothesis-driven clinical trials produce null or negative results is expected given the limitations of trial design, and complexities of cancer biology. It is important to understand the reasons underlying such null results however, in order to effectively merge the technological innovations with the rapidly evolving biology for maximal patient benefit, through the design of future clinical trials.
doi:10.1158/1078-0432.CCR-13-1116
PMCID: PMC3965328  PMID: 24043463
Cancer; Null hypothesis; Radiation oncology; Radiation therapy; Randomized clinical trial

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