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).
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.
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.
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.
Melanoma; BRAF(V600E); interleukin-1; tumor-associated fibroblasts (TAFs); cytotoxic T lymphocytes (CTL)
MET-signaling has been suggested a potential role in malignant peripheral nerve sheath tumors (MPNSTs). Here, MET function and blockade were preclinically assessed.
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.
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.
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.
Malignant peripheral nerve sheath tumor; MET; Biomarker; Therapeutic target; XL184
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.
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.
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.
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.
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.
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.
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.
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.
thyroid cancer; IQGAP1; gene copy gain; E-cadherin; BRAF mutation
This study was conducted to identify novel genes with importance to the biology of adult acute myelogenous leukemia (AML).
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.
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.
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.
AML; genomic microdeletions; NF1 mutations
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.
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.
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.
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.
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.
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.
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.
Given the urgent need for better systemic approaches to STS, clinical trials evaluating TRAIL in combination with low dose chemotherapy are potentially warranted.
Soft tissue sarcoma; TRAIL; doxorubicin; apoptosis; angiogenesis; Therapy
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.
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.
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.
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.
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.
linkage; haplotype; association; polymorphism; proliferation; tumor
The current research was undertaken to examine the association between genetic variations in DNA repair and pancreatic cancer risk.
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.
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).
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.
pancreatic cancer; DNA repair; oxidative stress; genetic polymorphisms; single nucleotide polymorphism (SNP)
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.
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).
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.
MORF pretargeting has now been shown to be a promising approach for tumor radiotherapy as well as diagnosis.
The Ewing Sarcoma Family of Tumors (ESFTs) comprises a group of aggressive, malignant bone and soft tissue tumors that predominantly affect children and young adults. These tumors frequently share expression of the EWS-FLI-1 translocation, which is central to tumor survival but not present in healthy cells. In this study, we examined EWS-FLI-1 antigens for their capacity to induce immunity against a range of ESFT types.
Computer prediction analysis of peptide binding, HLA-A2.1 stabilization assays, and induction of Cytotoxic T-Lymphocytes (CTL) in immunized HLA-A2.1 transgenic mice were used to assess the immunogenicity of native and modified peptides derived from the fusion region of EWS-FLI-1 type 1. CTL-killing of multiple ESFT family members in vitro, and control of established xenografts in vivo, was assessed. We also examined whether these peptides could induce human CTLs in vitro.
EWS-FLI-1 type 1 peptides were unable to stabilize cell surface HLA-A2.1 and induced weak CTL activity against Ewing Sarcoma cells. In contrast, peptides with modified anchor residues induced potent CTL killing of Ewing Sarcoma cells presenting endogenous (native) peptides. The adoptive transfer of CTL specific for the modified peptide YLNPSVDSV resulted in enhanced survival of mice with established Ewing Sarcoma xenografts. YLNPSVDSV-specific CTL displayed potent killing of multiple ESFT types in vitro: Ewing Sarcoma, pPNET, Askin’s Tumor, and Biphenotypic Sarcoma. Stimulation of human Peripheral Blood Mononuclear Cells with YLNPSVDSV peptide resulted in potent CTL-killing.
These data show that YLNPSVDSV peptide is a promising antigen for ESFT immunotherapy and warrants further clinical development.
Ewing Sarcoma Family of Tumors; Ewing Sarcoma; pPNET; Askin’s Tumor; Biphenotypic sarcoma; EWS-FLI-1; Immunotherapy; vaccine; cancer; HLA-A2.1; HLA-A*0201
The aim of this study was to show preclinical efficacy and clinical development potential of NVP-BKM120, a selective pan class I phosphatidylinositol-3 kinase (PI3K) inhibitor in human glioblastoma (GBM) cells in vitro and in vivo.
The effect of NVP-BKM120 on cellular growth was assessed by CellTiter-Blue assay. Flow cytometric analyses were carried out to measure the cell-cycle, apoptosis, and mitotic index. Mitotic catastrophe was detected by immunofluorescence. The efficacy of NVP-BKM120 was tested using intracranial U87 glioma model.
We tested the biologic effects of a selective PI3K inhibitor NVP-BKM120 in a set of glioma cell lines. NVP-BKM120 treatment for 72 hours resulted in a dose-dependent growth inhibition and effectively blocked the PI3K/Akt signaling cascade. Although we found no obvious relationship between the cell line's sensitivity to NVP-BKM120 and the phosphatase and tensin homolog (PTEN) and epidermal growth factor receptor (EGFR) statuses, we did observe a differential sensitivity pattern with respect to p53 status, with glioma cells containing wild-type p53 more sensitive than cells with mutated or deleted p53. NVP-BKM120 showed differential forms of cell death on the basis of p53 status of the cells with p53 wild-type cells undergoing apoptotic cell death and p53 mutant/deleted cells having a mitotic catastrophe cell death. NVP-BKM120 mediates mitotic catastrophe mainly through Aurora B kinase. Knockdown of p53 in p53 wild-type U87 glioma cells displayed microtubule misalignment, multiple centrosomes, and mitotic catastrophe cell death. Parallel to the assessment of the compound in in vitro settings, in vivo efficacy studies using an intracranial U87 tumor model showed an increased median survival from 26 days (control cohort) to 38 and 48 days (treated cohorts).
Our present findings establish that NVP-BKM120 inhibits the PI3K signaling pathways, leading to different forms of cell death on the basis of p53 statuses. Further studies are warranted to determine if NVP-BKM120 has potential as a glioma treatment.
MicroRNA (miRNA) expression profiles improve classification, diagnosis, and prognostic information of malignancies, including lung cancer. This study uncovered unique growthsuppressive miRNAs in lung cancer.
miRNA arrays were done on normal lung tissues and adenocarcinomas from wild-type and proteasome degradation-resistant cyclin E transgenic mice to reveal repressed miRNAs in lung cancer. Real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays validated these findings. Lung cancer cell lines were derived from each transgenic line (designated as ED-1 and ED-2 cells, respectively). Each highlighted miRNA was independently transfected into these cells. Growth-suppressive mechanisms were explored. Expression of a computationally predictedmiRNA target was examined.ThesemiRNAs were studied in a paired normal-malignant human lung tissue bank.
miR-34c, miR-145, and miR-142-5p were repressed in transgenic lung cancers. Findings were confirmed by real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays. Similar miRNA profiles occurred in human normal versus malignant lung tissues. Individual overexpression of miR-34c, miR-145, and miR-142-5p in ED-1and ED-2 cells markedly repressed cell growth. Anti-miR cotransfections antagonized this inhibition. The miR-34c target, cyclin E, was repressed by miR-34c transfection and provided amechanism for observed growth suppression.
miR-34c, miR-145, and miR-142-5p were repressed in murine and human lung cancers. Transfection of each miRNA significantly repressed lung cancer cell growth. Thus, these miRNAs were growth suppressive and are proposed to exert antineoplastic effects in the lung.
We evaluated X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) protein in head and neck squamous cell carcinoma (HNSCC) patients in association with outcome.
XRCC1 protein expression was assessed by immunohistochemical (IHC) staining of pretreatment tissue samples in 138 consecutive HNSCC patients treated with surgery (n = 31), radiation (15), surgery and radiation (23), surgery and adjuvant chemoradiation (17), primary chemoradiation (51), and palliative measures (1).
Patients with high XRCC1 expression by IHC (n = 77) compared with patients with low XRCC1 expression (n = 60) had poorer median overall survival (OS; 41.0 months vs. OS not reached, P = 0.009) and poorer progression-free survival (28.0 months vs. 73.0 months, P = 0.031). This association was primarily due to patients who received chemoradiation (median OS of high- and low-XRCC1 expression patients, 35.5 months and not reached respectively, HR 3.48; 95% CI: 1.44–8.38; P = 0.006). In patients treated with nonchemoradiation modalities, there was no survival difference by XRCC1 expression. In multivariable analysis, high XRCC1 expression and p16INK4a-positive status were independently associated with survival in the overall study population (HR = 2.62; 95% CI: 1.52–4.52; P < 0.001 and HR = 0.21; 95% CI: 0.06–0.71; P = 0.012, respectively) and among chemoradiation patients (HR = 6.02; 95% CI: 2.36–15.37; P < 0.001 and HR = 0.26; 95% CI: 0.08–0.92, respectively; P = 0.037).
In HNSCC, high XRCC1 protein expression is associated with poorer survival, particularly in patients receiving chemoradiation. Future validation of these findings may enable identification of HNSCC expressing patients who benefit from chemoradiation treatment.
The purpose of this preclinical study was to determine the effectiveness of RAF265, a multi-kinase inhibitor, for treatment of human metastatic melanoma and to characterize traits associated with drug response.
Advanced metastatic melanoma tumors from 34 patients were orthotopically implanted to nude mice. Tumors that grew in mice (17 of 34) were evaluated for response to RAF265 (40 mg/kg, every day) over 30 days. The relation between patient characteristics, gene mutation profile, global gene expression profile, and RAF265 effects on tumor growth, mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation, proliferation, and apoptosis markers was evaluated.
Nine of the 17 tumors that successfully implanted (53%) were mutant BRAF (BRAFV600E/K), whereas eight of 17 (47%) tumors were BRAF wild type (BRAFWT). Tumor implants from 7 of 17 patients (41%) responded to RAF265 treatment with more than 50% reduction in tumor growth. Five of the 7 (71%) responders were BRAFWT, of which 1 carried c-KITL576P and another N-RASQ61R mutation, while only 2 (29%) of the responding tumors were BRAFV600E/K. Gene expression microarray data from nonimplanted tumors revealed that responders exhibited enriched expression of genes involved in cell growth, proliferation, development, cell signaling, gene expression, and cancer pathways. Although response to RAF265 did not correlate with pERK1/2 reduction, RAF265 responders did exhibit reduced pMEK1, reduced proliferation based upon reduced Ki-67, cyclin D1 and polo-like kinase1 levels, and induction of the apoptosis mediator BCL2-like 11.
Orthotopic implants of patient tumors in mice may predict prognosis and treatment response for melanoma patients. A subpopulation of human melanoma tumors responds to RAF265 and can be characterized by gene mutation and gene expression profiles.
To establish feasibility, maximum tolerated dose, and potential efficacy of ablative dose total marrow irradiation (TMI) delivered by helical tomotherapy, in patients with multiple myeloma (MM).
Patients with responding or stable MM received tandem autologous transplants (TASCT), first with melphalan 200 mg/m2, and ≥ 60 days later, with TMI. TMI doses were to be escalated from 1000 cGy, by increments of 200 cGy. All patients received thalidomide and dexamethasone maintenance.
Twenty two of 25 enrolled patients (79%) received TASCT: TMI was administered a median of 63.5 days (44–119) after melphalan. Dose limiting toxicities at level 5 (1800 cGy) included reversible grade 3 pneumonitis, congestive heart failure, and enteritis (1), and grade 3 hypotension (1). The estimated median radiation dose to normal organs was 11–81% of the prescribed marrow dose. Late toxicities included reversible enteritis (1), and lower extremity deep venous thrombosis during maintenance therapy (2). The complete and very good partial response rates were 55% and 27%, following TASCT and maintenance therapy. At a median of 35 months of follow-up (21-50+ months) progression-free and overall survival for all patients are 49 % (95% CI 0.27-0.71) and 82% (0.67-1.00).
Ablative dose TMI as part of TASCT is feasible, and the complete response rate is encouraging. Careful monitoring of late toxicities is needed. Further assessment of this modality is justified at the 1600 cGy maximum tolerated dose level in MM patients who are candidates for ASCT.
multiple myeloma; total marrow irradiation; tandem autologous transplantation
Hypoxia is a cause for resistance to cancer therapies. Molecularly targeted recombinant cytotoxins have shown clinical efficacy in the treatment of patients with primary brain tumors, glioblastoma multiforme, but it is not known whether hypoxia influences their antitumor effect.
We have exposed glioblastoma multiforme cells, such as U-251 MG, U-373 MG, SNB-19, and A-172 MG, to either anoxia or hypoxia and then reoxygenated them while treating with an interleukin (IL)-13-based diphtheria toxin (DT)-containing cytotoxin, DT-IL13QM. We measured the levels of immunoreactive IL-13Rα2, a receptor that mediates IL-13-cytotoxin cell killing, and the levels of active form of furin, a protease that activates the bacterial toxin portion in a cytotoxin.
We found that anoxia/hypoxia significantly alters the responsiveness of glioblastoma multiforme cells to DT-IL13QM. Interestingly, bringing these cells back to normoxia caused them to become even more susceptible to the cytotoxin than the cells maintained under normoxia. Anoxia/hypoxia caused a highly prominent decrease in the immunoreactive levels of both IL-13R and active forms of furin, and reoxygenation not only restored their levels but also became higher than that in normoxic glioblastoma multiforme cells.
Our results show that a recombinant cytotoxin directed against glioblastoma multiforme cells kills these cells much less efficiently under anoxic/hypoxic conditions. The reoxygenation brings unexpected additional benefit of making glioblastoma multiforme cells even more responsive to the killing effect of a cytotoxin.
This study evaluates the proapoptotic function of integrin β3 in human hepatocellular carcinoma (HCC).
The expression of integrin β3 in 67 HCC specimens paired with corresponding neighboring nontumorous tissue was studied by quantitative real-time PCR and Western blot. The proapoptotic function of integrin β3 in SMMC-7721 human hepatoma cells overexpressing ITGB3 (gene coding integrin β3) was determined through colony formation, serum starvation, and anoikis assay.
Compared with neighboring pathologically normal liver tissue, ~60% of the HCC specimens showed a significantdown-regulated level of integrin β3 expression. Transient expression of integrin β3 in SMMC-7721resulted in an enhanced level of apoptosis and suppression of colony formation. Cell growth inhibition on serum/ligand deprivation and incidences of anoikis were remarkably increased in SMMC-7721with stable expression of integrin β3 in comparison with vector control transfectants. In addition, expression of fibrinogen and vitronectin, two native ligands for integrin αvβ3 in liver, was inhibited, which was correlated with the decreased integrin β3 expression. Replenishing these ligands to the starved SMMC-7721 stable transfectants effectively restored the proapoptotic function of integrin β3.
Down-regulation of integrin β3 and its ligands in liver is related to the aggressive growth of HCC. Thus, reconstitution of integrin β3 in HCC may be a potential therapeutic approach to inhibit aggressive growth of liver cancer.
There is growing evidence implicating that neutrophil gelatinase–associated lipocalin (NGAL) plays a role in the development and progression of cancers. However, the effect of NGAL in colorectal carcinoma (CRC) has not been clearly elucidated. In this study, we investigated the role of NGAL in the tumorigenesis and progression of CRC and evaluated the clinical value of NGAL expression.
We examined NGAL expression in 526 colorectal tissue samples, including 53 sets of matched specimens (histologically normal mucosa, adenomas, and carcinomas) using immunohistochemical analysis. In CRCs, correlations between NGAL expression and clinicopathologic parameters were analyzed, and survival analysis was conducted. The role of NGAL was further tested using mouse xenograft models.
NGAL expression was elevated during the colorectal adenoma–carcinoma sequence both among the 526 cases (rs = 0.66, P < 0.001) and in the 53 sets of matched specimens (rs = 0.60, P < 0.001). In CRCs, NGAL expression was associated with cancer stage (P = 0.041) and tumor recurrence in stage II patients (P = 0.037). Survival analysis revealed that NGAL expression was an independent prognostic factor for overall survival (HR = 1.84, P = 0.004) and for disease-free survival of stage II patients (HR = 5.88, P = 0.021). In mouse models, the xenografts in cecum and spleen were heavier and more numerous in the group injected with NGAL-overexpressing CRC cells (P < 0.05).
NGAL overexpression may promote the tumorigenesis and progression of CRC. Detecting NGAL expression in tumor tissues may be useful for evaluating prognosis of patients with CRC.
To characterize proliferative changes in tumors during the sunitinib malate exposure/withdrawal using 3′-Deoxy-3′-[18F]fluorothymidine (FLT) PET/CT imaging.
Patients and Methods
Patients with advanced solid malignancies and no prior anti-VEGF exposure were enrolled. All patients had metastatic lesions amenable to FLT PET/CT imaging. Sunitinib was initiated at the standard dose of 50 mg PO daily either on a 4/2 or 2/1 schedule. FLT PET/CT scans were obtained at baseline, during sunitinib exposure, and after sunitinib withdrawal within cycle #1 of therapy. VEGF levels and sunitinib pharmacokinetic data were assessed at the same time points.
16 patients (8 pts on 4/2 schedule; 8 pts on 2/1 schedule) completed all three planned FLT PET/CT scans, and were evaluable for pharmacodynamic imaging evaluation. During sunitinib withdrawal (change from scan 2 to 3), median FLT PET SUVmean increased +15% (range −14% to +277%) (p=0.047) for the 4/2 schedule and +19% (range −5.3% to +200%) (p=0.047) for the 2/1 schedule. Sunitinib PK and VEGF ligand levels increased during sunitinib exposure, and returned towards baseline during the treatment withdrawal.
The increase of cellular proliferation during sunitinib withdrawal in patients with renal cell carcinoma and other solid malignancies is consistent with a VEGFR TKI withdrawal flare. Univariate and multivariate analysis suggest that plasma VEGF is associated with this flare, with an exploratory analysis implying that patients who experience less clinical benefit have a larger withdrawal flare. This might suggest that patients with a robust compensatory response to VEGFR TKI therapy experience early “angiogenic escape”.
Angiogenesis; molecular imaging; VEGFR TKI
The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM).
Both cell line–derived OCI-Ly10 and primary human lymphoma–derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMycCα/Bcl-XL GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc–disseminated model of iMycCα/ Bcl-XL was used to determine antitumor activity and effects on osteolytic bone disease.
MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMycCα/Bcl-XL GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model.
Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials.
We have previously shown that within tumors, recombinant interleukin-2 (rIL-2, Aldesleukin) consistently activates tumor-associated macrophages and up-regulates interferon stimulated genes (ISGs) while inducing minimal migration, activation or proliferation of T-cells. These effects are independent of tumor response to treatment. Here, we prospectively evaluated transcriptional alterations induced by rIL-2 in peripheral blood mononuclear cells (PBMCs) and within melanoma metastases.
We evaluated gene expression changes by serially comparing pre- to post-treatment samples in 14 patients and also compared transcriptional differences among lesions displaying different responsiveness to therapy, focusing on 2 lesions decreasing in size and 2 remaining stable (responding lesions) compared to non-responding ones.
As previously described, the effects of rIL-2 were dramatic within PBMC, while effects within the tumor microenvironment were lesion-specific and limited. However, distinct signatures specific to response could be observed in responding lesions pre-treatment that were amplified following rIL-2 administration. These signatures match the functional profile observed in other human or experimental models in which immune-mediated tissue-specific destruction (TSD) occurs underlying a common pathways leading to rejection. Moreover, the signatures observed in pre-treatment lesions were qualitatively similar to those associated with TSD underlining a determinism to immune responsiveness that depends upon the genetic background of the host or the intrinsic genetic makeup of individual tumors.
This is the first prospectively collected insight on global transcriptional events occurring during high-dose rIL-2 therapy in melanoma metastases responding to treatment.
One of the most important rate-limiting steps in adoptive cell transfer (ACT) is the inefficient migration of T cells to tumors. Since melanomas specifically express the chemokines CXCL1 and CXCL8 that are known to facilitate the CXCR2-dependent migration by monocytes, our aim is to evaluate whether introduction of the CXCR2 gene into tumor-specific T cells could further improve the effectiveness of ACT, by enhancing T-cell migration to tumor.
In this study, we utilized transgenic pmel-1 T cells which recognize gp100 in the context of H-2Db, that were transduced with luciferase gene to monitor the migration of transferred T cells in vivo. In order to visualize luciferase-expressing T cells within a tumor, a non-pigmented tumor is required. Therefore, we utilized the MC38 tumor model which naturally expresses CXCL1.
Mice bearing MC38/gp100 tumor cells treated with CXCR2/luciferase-transduced pmel-1 T cells showed enhanced tumor regression and survival compared to mice receiving control luciferase transduced pmel-1 T cells. We also observed preferential accumulation of CXCR2-expressing pmel-1 T cells in the tumor sites of these mice using bioluminescence imaging. A similar enhancement in tumor regression and survival was observed when CXCR2-transduced pmel-1 T cells were transferred into mice bearing CXCL1-transduced B16 tumors compared to mice treated with control pmel-1 T cells.
These results implicate that the introduction of the CXCR2 gene into tumor-specific T cells can enhance their localization to tumors and improve antitumor immune responses. This strategy may ultimately enable personalization of cancer therapies based on chemokine expression by tumors.
CXCR2; T cell-mediated antitumor immune response; Adoptive cell transfer (ACT); melanoma immunotherapy
Molecular deregulations underlying epithelioid sarcoma (ES) progression are poorly understood yet critically needed to develop new therapies. EGFR is overexpressed in ES; using preclinical models, we examined the ES EGFR role and assessed anti-ES EGFR blockade effects, alone and with mTOR inhibition.
EGFR and mTOR expression/activation was examined via tissue microarray (n=27 human ES specimens; IHC) and in human ES cell lines (WB and qRTPCR). Cell proliferation, survival, migration, and invasion effects of EGFR and mTOR activation and erlotinib (anti-EGFR small molecule inhibitor) alone and combined with rapamycin were assessed in cell culture assays. In vivo growth effects of erlotinib alone or with rapamycin, were evaluated using SCID mouse ES xenograft models.
EGFR was expressed and activated in ES specimens and cell lines. EGFR activation increased ES cell proliferation, motility, and invasion, and induced cyclin D1, MMP2, and MMP9 expression. EGFR blockade inhibited these processes and caused significant cytostatic ES growth inhibition in vivo. mTOR pathway activation at varying levels was identified in all TMA-evaluable ES tissues; 88% of samples had no or reduced PTEN expression. Similarly, both ES cell lines demonstrated enhanced mTOR activity; VAESBJ cells exhibited constitutive mTOR activation uncoupled from EGFR signaling. Most importantly, combined erlotinib/rapamycin resulted in synergistic anti-ES effects in vitro and induced superior tumor growth inhibition in vivo versus single agent administration.
EGFR- and mTOR-signaling pathways are deregulated in ES. Preclinical ES model-derived insights suggest that combined inhibition of these targets might be beneficial, supporting evaluations in clinical trials.
Epitheioid sarcoma; EGFR; mTOR; PTEN; Targeted Therapy; preclinical models