Pancreatic Cancer Lines Research Articles

A Deimmunized Bispecific Ligand-Directed Toxin That Shows an Impressive Anti–Pancreatic Cancer Effect in a Systemic Nude Mouse Orthotopic Model

The objective was to test a bispecific ligand-directed toxin (BLT), with reduced immunogenicity for enhanced efficacy in targeting orthotopic pancreatic cancer in vivo. A new BLT was created in which both human epidermal growth factor (EGF) and interleukin 4 cytokines were cloned onto the same single chain molecule with deimmunized pseudomonas exotoxin (dEGF4KDEL). Key amino acids dictating B-cell generation of neutralizing antitoxin antibodies were mutated. Bioassays were used to determine whether mutation reduced potency, and enzyme-linked immunosorbent assay studies were performed to determine whether antitoxin antibodies were reduced. A genetically altered luciferase MIA PaCa-2 xenograft model was used to image in real time and determine effects on systemic malignant human cancer. Bispecific ligand-directed toxins targeting B cells were used as specificity controls. Deimmunized EGF4KDEL was significantly effective after systemic injection against established orthotopic MIA PaCa-2 pancreatic cancer and selectively prevented metastasis. Mutagenesis significantly reduced antitoxin levels in vivo with no apparent activity loss in vitro. The drug was effective against 3 human pancreatic cancer lines in vitro, MIA PaCa-2, SW1990, and S2VP10. Despite the metastatic nature of the MIA PaCa-2 orthotopic tumor xenografted in nude mice, high percentages of tumors responded to extended dEGFKDEL treatment resulting in significant anticancer effects and disease-free survivors.

Abstract 2186: Function and regulation of Polo-like Kinase 3 in human pancreatic cancer

Abstract Pancreatic cancer is currently the fourth leading cause of cancer death in the United States. Despite the recent advances in pancreatic cancer research, patients with this devastating disease still have a poor prognosis. Therefore, deciphering pancreatic cancer-related gene expression and defining the individual gene's specific functions will make a significant impact on the diagnosis and treatment of pancreatic cancer. Recently, our lab has demonstrated the tumorigenic transformation in an immortalized pancreatic cell line, HPNE/hTERT by sequential introduction of multiple genetic alterations. We found that Polo-like kinase 3 (Plk3) is significantly down-regulated in transformed tumor cell lines. Plk3 belongs to the Polo-like kinase family and is a multi-functional kinase. We previously reported that Plk3 played a pro-apoptotic role in pancreatic cancer lines when it was overexpressed. Plk3 down-regulation in transformed HPNE cells suggests that Plk3 may have tumor suppression function during pancreatic tumor development. In addition, we found Plk3 expression is regulated by Pten. In Pten-knockout MEF cells, Plk3 expression was significantly down-regulated. So Plk3 may be downstream of Pten in the tumor suppression pathway. These data suggest that Plk3 play an important role in pancreatic cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2186. doi:1538-7445.AM2012-2186

Abstract 4315: A novel role for the high affinity IL13 receptor in glioblastoma multiforme

Abstract IL13 Receptor alpha 2 (IL13Ra2) is one of two receptors for IL13 and is found to be highly expressed in a number of cancers including, ovarian, head and neck, pancreatic and glioblastoma multiforme (GBM). IL13Ra2 has been found to be expressed mostly in high grade GBMs but not in low grade gliomas or normal brain tissue. IL13Ra2 has a short intracellular domain that lacks a signaling domain and possesses a high affinity for IL13. It is thought that in GBMs IL13Ra2 acts primarily as a decoy receptor for IL13. However, recent evidence from pancreatic and ovarian cancer lines indicates that IL13Ra2 initiates signaling pathways that influence cell invasion and adhesion properties of cancer cells. We hypothesize the IL13Ra2 might play a role in GBMs distinct for its decoy receptor function. This study is designed to determine if IL13Ra2 possess functional properties and can modulate the behavior of glioma cells through its interaction with IL13. We examined the effect of IL13 interaction with IL13Ra2 in multiple glioma cell lines in vitro. A BrdU based proliferation assay was utilized to determine the effect of IL13 on the proliferation rate of glioma cells. Migration of glioma cells was also examined over a 24 hour period following exposure to IL13. A shRNA mediated knockdown of IL13Ra2 in glioma cells was also produced and the resultant phenotype analyzed by phalloidin staining for cytoskeletal actin and in the migration assay. Treatment of IL13Ra2 expressing glioma cell lines with IL13 did not influence their proliferation rates in vitro. However, cells that had been treated with IL13 demonstrated an increased migration response. Knockdowns were found to have decreased migration rates, both in the presence and absence of IL13. Knockdowns were also found to have dramatic changes in phenotype with increased cellular adhesion and altered actin structure. When the actin structure was examined following IL13 treatment, normal cells appeared to have an increase in peripheral actin and filopodia, while in knockdown cells treated cells did not appear different from untreated controls. Our data indicate that IL13 interaction with IL13Ra2 might contribute to changes in the phenotype of glioma cells that promote their invasive properties. Further research is needed to determine the underlying mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4315. doi:1538-7445.AM2012-4315

Abstract 4866: The atypical kinase SgK223 is a novel mediator of cell invasion in pancreatic cancer

Abstract Pancreatic cancer still remains one of the most aggressive and lethal of human cancers, with a lack of effective treatments contributing to its poor prognosis and high mortality. Perturbations in tyrosine kinase signaling are characteristic of many human cancers. In order to detect aberrant protein tyrosine phosphorylation events in pancreatic cancer, and to subclassify pancreatic cancer cell lines based on their tyrosine phosphorylation patterns, tryptic digests from a wide panel of pancreatic cancer cell lines were subjected to anti-phosphotyrosine immunoprecipitation, then analysed by liquid chromatography coupled with tandem mass spectrometry (MS/MS). Proteins were ranked according to differences in median phosphorylation levels across the cell line panel, and those that exhibited significantly variable phosphorylation levels were selected as candidates for further evaluation as biomarkers and therapeutic targets. One candidate identified by this approach was SgK223, a 175 kDa atypical kinase with substitutions in the DFG motif of its kinase domain. Since this is one of the conserved motifs critical for full catalytic activity, SgK223 may be catalytically inactive. Western blotting revealed that SgK223 protein expression varied across the panel, with a subset of pancreatic cancer lines exhibiting overexpression relative to normal human pancreatic duct epithelial (HPDE) cells. To further interrogate the role of SgK223 in pancreatic cancer, SgK223 was overexpressed in HPDE cells. This resulted in an elongated cell morphology and increased cellular migration and invasion in transwell assays, with no effect on cellular proliferation or Erk and Akt activity. MS/MS analysis determined that SgK223 was phosphorylated on tyrosine residues 159 and 413. Since the former is a candidate Stat3 binding site, we characterised Stat3 signalling in this model. SgK223 overexpression led to enhanced Stat3 tyrosine phosphorylation and transcriptional activity. These data identify a role for SgK223 as a mediator of cell invasion in pancreatic cancer, and support the use of MS-based phosphoproteomic profiling as an effective discovery tool for cancer-associated signaling proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4866. doi:1538-7445.AM2012-4866

Abstract 3355: Signaling networks in pancreatic cancer cells with stem cell characteristics

Abstract Despite considerable efforts, the 5-year survival rate for pancreatic ductal adenocarcinoma (PDAC) has not changed in the last several decades. This poor prognosis is a function of the propensity of PDAC towards early metastatic spread and drug resistance. An ever-growing body of evidence attributes these characteristics to the presence of cancer stem cells (CSCs) within the tumor, the specific origin and nature of which is yet to be fully understood. Conserved pathways, e.g. TGF-β and Wnt, have been shown in general to regulate CSC hallmarks of self-renewal and differentiation, but these effects seem restricted to specialized cancer cells within the tumor mass. Notwithstanding, the function of Wnt signaling alone or in combination with other pathways in pancreatic CSCs remains elusive. We are elucidating whether microenvironmental cues are able to induce stemness in so-called facultative stem cells associated with the tumor mass. In light of mounting evidence, we are investigating canonical Wnt signaling and its interplay with other pathways as well as its function in regulating stemness-associated characteristics _ anoikis, epithelial-mesenchymal plasticity, drug-resistance, and tumorigenesis. To enrich for CSCs, we established sphere culture in serum-depleted media that yields cells exhibiting high levels of embryonic stem cell markers (Bmi1, Nanog, Oct3/4, Sox2), and increased colony-formation and drug-resistance relative to adherent cultures of the same line. Early immunofluorescence staining data reveal heterogeneous cellular β-Catenin distribution in the parental population of an established pancreatic cancer line (Panc1), increased nuclear translocation after Wnt3a stimulation, and higher nuclear β-Catenin staining in sphere cells. Known Wnt target genes (CyclinD1, Axin2) detected by qRT-PCR are significantly upregulated in spheroid cultures, further indicating that canonical Wnt-signaling could be intrinsically up regulated. In a sphere-forming, surrogate in vitro, assay for evaluation of stemness, constant stimulation with rhWnt3a increased the self-renewal capacity of floating pancreatic cancer cells. Together, these data support the hypothesis that activation of Wnt-signaling in an autocrine/paracrine manner might lead to stemness in susceptible CSC. We are deciphering patterns of multiple heterogeneously activated pathways, specifically the signaling sequence and chronology, that drives sphere-formation. Western Blot analysis shows enhanced activating phosphorylation of β-Catenin in sphere cells whereas SMAD3, a critical component of TGF-β-signaling, was activated in more differentiated pancreatic cancer cells. In conclusion, we show evidence for differential regulation of Wnt/TGF-β-signaling in pancreatic cancer cells, suggesting a reconsideration of current clinical strategies to include combining standard therapy with inhibitors of these signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3355. doi:1538-7445.AM2012-3355

SMURF1 Amplification Promotes Invasiveness in Pancreatic Cancer

Pancreatic cancer is a deadly disease, and new therapeutic targets are urgently needed. We previously identified DNA amplification at 7q21-q22 in pancreatic cancer cell lines. Now, by high-resolution genomic profiling of human pancreatic cancer cell lines and human tumors (engrafted in immunodeficient mice to enrich the cancer epithelial fraction), we define a 325 Kb minimal amplicon spanning SMURF1, an E3 ubiquitin ligase and known negative regulator of transforming growth factor β (TGFβ) growth inhibitory signaling. SMURF1 amplification was confirmed in primary human pancreatic cancers by fluorescence in situ hybridization (FISH), where 4 of 95 cases (4.2%) exhibited amplification. By RNA interference (RNAi), knockdown of SMURF1 in a human pancreatic cancer line with focal amplification (AsPC-1) did not alter cell growth, but led to reduced cell invasion and anchorage-independent growth. Interestingly, this effect was not mediated through altered TGFβ signaling, assayed by transcriptional reporter. Finally, overexpression of SMURF1 (but not a catalytic mutant) led to loss of contact inhibition in NIH-3T3 mouse embryo fibroblast cells. Together, these findings identify SMURF1 as an amplified oncogene driving multiple tumorigenic phenotypes in pancreatic cancer, and provide a new druggable target for molecularly directed therapy.

Abstract 2466: RON receptor tyrosine kinase as a targeting moiety for pancreatic cancer stem-like cells by antibody directed immunoliposomes for enhanced cytotoxicity

Abstract Pancreatic adenocarcinoma (PAC) is a malignant disease characterized by metastasis and chemo-resistance. Accumulated evidence has suggested that PAC malignancies arise due to functional presence of cancer stem cells that have high potential for self renewal, differentiation, and resistance to cytotoxic agents. Currently, therapeutics targeting PAC stem cells are not available. The study in this project describes a novel strategy and potential therapeutics targeting PAC stem cells for enhanced cytotoxicity. PAC stem like cells (PSLC) were obtained from human L3.6pl pancreatic cancer line under stem cell culture conditions. These cells show a typical sphere-like morphology in suspension and re-differentiate into epithelial appearance when subjected to serum inclusive adherent conditions. PSLC express transcription factor Bmi-1, a unique self-renewal marker, Aldh-1a, a metabolic enzyme expressed in stem-like cells, and cell-surface markers including CD24, CD44, and ESA as evidenced by Western blot and flow cytometry analyses. PSLC also displayed a phenotype characterized as epithelial to mesenchymal transition. Analysis of a panel of receptor tyrosine kinases has revealed that expression of EGFR, VEGFR, and MET was significantly reduced in PSLC. In contrast, RON receptor tyrosine kinase expression was sustained, which provides the molecular basis for antibody-directed approach for targeting PSLC. To achieve this goal, Zt/c9 monoclonal antibody was post-inserted onto doxorubicin liposomes to form anti-RON immunoliposomes (Zt/c9-Dox-IL). In vitro analysis indicated that; a) Zt/c9 was highly effective in inducing receptor internalization, which facilitates intracellular delivery of chemoagents; b) Zt/c9-Dox-IL was effective in killing Panc 0327 and L3.6pl PAC cells. An average of 7 fold decrease in IC50 value was obtained compared to control Dox-IL inserted with mouse IgG (mIgG-Dox-IL); and c) Zt/c9-Dox-IL displayed increased cytotoxicity against PSLC with the IC50 at 8 µM compared to 69 µM for mIgG-Dox-IL. In conclusion, PSLC enriched from L3.6pl cells display a phenotype similar to cancer stem cells with properties of increased self renewal and EMT. RON expression was maintained in PSLC, which was used as the drug delivery moiety for enhanced cytotoxicity. Increased PSLC cytotoxicity by Zt/c9-Dox-IL suggests that antibody-directed RON targeted delivery of cytotoxic agents is a rational approach for targeting PSLC to achieve enhanced cytotoxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2466. doi:10.1158/1538-7445.AM2011-2466

X‐ray irradiation and Rho‐kinase inhibitor additively induce invasiveness of the cells of the pancreatic cancer line, MIAPaCa‐2, which exhibits mesenchymal and amoeboid motility

Tumor cells can migrate and invade tissue by two modes of motility: mesenchymal and amoeboid. X-ray or γ-ray irradiation increases the invasiveness of tumor cells with mesenchymal motility through the induction of matrix metalloproteinases (MMP), and this increase is suppressed by MMP inhibitors (MMPI). However, the effects of X-ray or γ-ray irradiation on the invasiveness of tumor cells with amoeboid motility remain unclear. We investigated the effect of irradiation on amoeboid motility by using cells of the human pancreatic cancer line, MIAPaCa-2, which exhibits both modes of motility. The X-ray-induced invasiveness of MIAPaCa-2 cells was associated with the upregulation of MMP2 at both the RNA and protein levels and was inhibited by MMPI treatment. Amoeboid-mesenchymal transition was slightly induced after irradiation. The MMPI treatment caused mesenchymal-amoeboid transition without significant increase in invasiveness, while the ROCK inhibitor (ROCKI) stimulated amoeboid-mesenchymal transition and enhanced invasiveness under both non-irradiated and irradiated conditions. This ROCKI-induced transition was accompanied by the upregulation of MMP2 mRNA and protein. Exposure to both irradiation and ROCKI further enhanced MMP2 expression and had an additive effect on the invasiveness of MIAPaCa-2 cells. Additionally, exposure to MMPI led to significant suppression of both radiation-induced and the basal invasiveness of MIAPaCa-2 cells. This suggests that ROCKI treatment, especially with concomitant X-ray irradiation, can induce invasion of cancer cells and should be used only for certain types of cancer cells. Simultaneous use of inhibitors, ROCKI and MMPI may be effective in suppressing invasiveness under both X-ray-irradiated and non-irradiated conditions.

Proteome-based biomarkers in pancreatic cancer

Pancreatic cancer, as a highly malignant cancer and the fourth cause of cancer-related death in world, is characterized by dismal prognosis, due to rapid disease progression, highly invasive tumour phenotype, and resistance to chemotherapy. Despite significant advances in treatment of the disease during the past decade, the survival rate is little improved. A contributory factor to the poor outcome is the lack of appropriate sensitive and specific biomarkers for early diagnosis. Furthermore, biomarkers for targeting, directing and assessing therapeutic intervention, as well as for detection of residual or recurrent cancer are also needed. Thus, the identification of adequate biomarkers in pancreatic cancer is of extreme importance. Recently, accompanying the development of proteomic technology and devices, more and more potential biomarkers have appeared and are being reported. In this review, we provide an overview of the role of proteome-based biomarkers in pancreatic cancer, including tissue, serum, juice, urine and cell lines. We also discuss the possible mechanism and prospects in the future. That information hopefully might be helpful for further research in the field.

Proteome analysis of human pancreatic cancer cell lines with highly liver metastatic potential by antibody microarray

Antibody microarrays have been successfully used to determine relative abundance of key proteins in various cancers and other diseases. We have previously showed liver metastatic-related genes between the metastatic pancreatic cancer line (SW1990HM) and its parental line (SW1990). In this study, we searched for potential markers for metastatic progression using antibody microarrays. The SpringBio Antibody Microarrays were used to analysis the different proteomes between SW1990HM and SW1990 cells. A standard ≥2.0-fold cutoff value was used to determine differentially expressed proteins and Western blotting analysis further confirmed the results. Antibody microarrays revealed that 40 proteins were reproducibly altered more than 2-fold between the selected variant and its parental counterpart; 14 of the proteins were up-regulated, and 26 were down-regulated. Most of the up-regulated proteins (7/14) play a role in tumor signal transduction, while a number of down-regulated proteins (10/26) function in cell differentiation; this might be crucial for pancreatic cancer metastasis. Four dysregulated proteins were validated by western blotting in the cell lines. Interestingly, the up-regulation of Glucagon and down-regulation of Prolactin were further confirmed in the culture supernatants by western blotting. These proteomic data are valuable for understanding pancreatic cancer metastasis and searching for potential markers of metastatic progression.

Abstract A22: Hypoxia-dependent antitumor activity of TH-302, a hypoxia-activated prodrug, in preclinical pancreatic xenograft models

Abstract TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP). In hypoxic cells the DNA cross-linker bromo-isophosphoramide mustard (Br-IPM) is released. TH-302 demonstrates hypoxia-dependent cytotoxicty in cell-based in vitro assays with human tumor cell lines, efficacy both as monotherapy and combination therapy in human tumor xenografts in nude mice, and is currently in multiple clinical trials for the treatment of cancer, including pancreatic cancer. In the present study, we investigated the antitumor activity and mechanism of action of TH-302 in two pancreatic xenograft models. The Hs766t and SU.86.86 human pancreatic cancer lines reflect two different pancreatic adenocarcinoma cell phenotypes. The Hs766t cells are more mesenchymal, having passed through the epithelial- mesenchymal transition (EMT), while the SU.86.86 cell line retains a more epithelial differentiative state. By employing a histological and immunohistochemical approach, we show that the two cell types form xenograft tumors with distinct microenvironmental features. As characterized by CD31 and Hoechst 33342 staining, SU.86.86 is more highly vascularized and well-perfused than Hs766t. With the exogenous hypoxia marker pimonidazole, a much larger hypoxic volume is present in Hs766t tumors compared with that in SU.86.86 (11.5% ± 1.4% vs. 4.5% ± 0.9%, p<0.001, tumors ~250 mm3). We tested TH-302 and gemcitabine, both as single agents and in combination, in these two pancreatic cancer models with in vitro cell-based models and in vivo tumor xenograft models. In the ectopic xenograft models, TH-302 (75mg/kg, Q3Dx5, ip), gemcitabine (60mg/kg, Q3Dx5, ip) or the combination of TH-302 and gemcitabine was administered to the animals. The TH-302 was given 4 hours before the gemcitabine. The schedule of TH-302 and gemcitabine mirrors the clinical trial schedule where TH-302 and gemcitabine are given on the same day weekly (3 weeks on, 1 week off per cycle). In the Hs766t model, tumor growth inhibition (TGI) of TH-302 alone and gemcitabine alone was 75%, and 45%, respectively. When TH-302 was combined with gemcitabine, the antitumor activity was significantly enhanced, and TGI was 89% (p<0.001 vs. Vehicle and p<0.05 vs. gemcitabine alone). In contrast, gemcitabine alone was highly efficacious in the SU.86.86 xenograft model and showed TGI of 104%, while the antitumor activity of TH-302 monotherapy was modest and TGI was only 28% (the combination TGI was 105%). To characterize specific pharmacodynamic effects of TH-302 treatment on the tumors, we examined Hs766t tumors immunohistochemically 48 hours after a single dose of TH-302 treatment (150mg/kg, ip). The hypoxic volume was significantly reduced from 11.5% (n=9) to 4.6% (n=5) (p<0.01) after TH-302 treatment. The gemcitabine and TH-302 showed no synergy when tested together in vitro. The results support the hypothesis that the two agents (TH-302 and gemcitabine) work in a complementary manner by targeting two distinct tumor compartments and add support for the hypothesis of hypoxic compartment selectivity of TH-302's mechanism of action. Recently published studies have implicated the mesenchymal phenotype of the Hs766t model being associated with gemcitabine resistance. Thus the enhanced anti-tumor efficacy observed with the addition of TH-302 to the gemcitabine regimen in the xenografts may model inter-patient response differences to therapy and lead to biomarker strategies for patient stratification. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A22.

Pancreatic Stellate Cells Increase the Invasion of Human Pancreatic Cancer Cells through the Stromal Cell-Derived Factor-1/CXCR4 Axis

Aim: Both pancreatic stellate cells (PSCs) and the stromal cell-derived factor-1(SDF-1)/CXCR4 receptor ligand system have important roles in pancreatic cancer progression. This study set out to detect if PSCs express SDF-1 and promote the invasion of pancreatic cancer through the SDF-1/CXCR4 receptor ligand axis. Methods: RT-PCR was performed to detect the expression of SDF-1 and CXCR4 in PSCs, pancreatic cancer lines and cancer tissue samples. ELISA was used to investigate the concentration of SDF-1 in PSC supernatants. An MTT assay was applied to detect the proliferation of pancreatic cancer cells. A transwell chamber migration assay was employed to detect the migration of AsPC-1 cells. An in vitro invasion assay was used to detect the invasion of AsPC-1 cells. Results: CXCR4 expression was detected in PSCs; AsPC-1, SW1990 and BxPC-3 cancer cells; and cancer tissues. SDF-1 was detected in PSCs and cancer tissues, but not in AsPC-1, SW1990 and BxPC-3 cells. SDF-1α protein was found in PSC supernatants. PSC-conditioned media can promote the proliferation, migration and invasion of pancreatic cancer cells. SDF-1 neutralizing antibody or AMD3100 can significantly inhibit these promotive effects. Conclusion: PSCs can secrete SDF-1 and increase the invasion of pancreatic cancer cells through the SDF-1/CXCR4 axis.

Abstract 5341: Sensitivity of NHL to killing by anti-HLA-DR and anti-CD74 mAbs is increased by interferon-gamma

Abstract Background: HLA-DR and CD74 are similarly, but not identically, expressed and induced by interferons on a variety of cells. Expression of both antigens on hematological malignancies led to their development as targets for antibody-based therapy. The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab (Immunomedics Inc, Morris Plains, NJ), is in clinical evaluation for therapy of NHL, multiple myeloma (MM), and CLL after preclinical evidence of activity in these tumor types. A humanized anti-HLA-DR mAb, hL243γ4P (IMMU-114, Immunomedics) has demonstrated anti-tumor activity in vitro and in vivo, and clinical evaluation is planned. In addition to expression in hematologic cancers, these antigens are expressed on the surface of other tumor types, including melanoma and renal cell carcinoma, and in the cytoplasm of others, including pancreatic and colonic carcinomas, and glioblastomas (GBM). Methods: We examined whether the ability of anti-HLA-DR and anti-CD74 mAbs to kill cancer cells can be increased by using IFNγ as an inducer of antigen expression. Using a panel of diverse cancer cell lines (including NHL, MM, GBM, and pancreatic and colonic carcinomas), we examined IFNγ-induced changes in surface and cytoplasmic HLA-DR and CD74 expression. Sensitivity of malignant cells to milatuzumab and hL243γ4P was assessed with and without INFγ by cytotoxicity assays. Results: Without IFNγ surface expression of HLA-DR and CD74 were present on 2/2 NHL, 2/2 MM, and only weakly positive on 2/2 GBM cell lines. Surface CD74 and HLA-DR were weak or undetectable on 4/4 colon and 4/4 pancreatic carcinomas. Cytoplasmic CD74 and HLA-DR were seen in NHL, MM, GBM, and 1/4 colon and 1/4 pancreatic (CD74 only) carcinomas. Two-day incubation with IFNγ increased surface and cytoplasmic expression of both HLA-DR and CD74 in all the NHL and GBM, and 3/4 pancreatic cancer lines, but not MM cell lines. In all 4 colon lines, IFNγ increased cytoplasmic expression of both antigens, and surface expression of HLA-DR in 3/4 and CD74 in 2/4. Upregulation of HLA-DR and CD74 ranged from 23-3700%. Increased killing by both hL243γ4P (58%) and milatuzumab (33%) was seen in vitro after INFγ exposure in WSU-FSCCL NHL cells. No cell killing was observed using these mAbs in vitro on U118 (GBM), Capan-1 (pancreatic carcinoma), or LoVo (colon carcinoma), despite upregulation of the antigens in these cell lines. A CD74-transfected version of the U118 GBM cell line has been prepared for comparison of milatuzumab sensitivity based on antigen density only. Conclusions: Cell surface and cytoplasmic expression of CD74 and HLA-DR are increased on cell lines from a variety of cancer types after INFγ exposure. This increased expression correlates with increased toxicity of anti-HLA-DR and anti-CD74 mAbs in a NHL cell line, and is under evaluation in other cancer types. These studies could prove useful in predicting the potential benefit of combined INFγ and mAb therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5341.

Nanomonitor: A Miniature Electronic Biosensor for Glycan Biomarker Detection

The goal of our research is to develop an ultrasensitive diagnostic platform called 'NanoMonitor' to enable rapid label-free analysis of a highly promising class of biomarkers called glycans (oligosaccharide chains attached to proteins) with high sensitivity and selectivity. The glycosylation of fetuin - a serum protein - and extracts from a human pancreatic cancer line was analyzed to demonstrate the capabilities of the NanoMonitor. The NanoMonitor device consists of a silicon chip with an array of gold electrodes forming multiple sensor sites and works on the principle of electrochemical impedance spectroscopy. Each sensor site is overlaid with a nanoporous alumina membrane that forms a high density of nanowells on top of each electrode. Lectins (proteins that bind to and recognize specific glycan structures) are conjugated to the surface of the electrode. When specific glycans from a test sample bind to lectins at the base of each nanowell, a perturbation of electrical double-layer occurs, which results in a change in the impedance. Using the lectins Sambucs nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA), subtle variations to the glycan chains of fetuin were investigated. Protein extracts from BXPC-3, a cultured human pancreatic cancer cell line were also analyzed for binding to SNA and MAA lectins. The performance of the NanoMonitor was compared to a conventional laboratory technique: lectin-based enzyme linked immunosorbent assay (ELISA). The NanoMonitor was used to identify glycoform variants of fetuin and global differences in glycosylation of protein extracts from cultured human pancreatic cancerous versus normal cells. While results from NanoMonitor correlate very well with results from lectin-based ELISA, the NanoMonitor is rapid, completely label free, requires just 10 microl of sample, is approximately five orders of magnitude more sensitive and highly selective over a broad dynamic range of glycoprotein concentrations. Based on its performance metrics, the NanoMonitor has excellent potential for development as a point-of-care handheld electronic biosensor device for routine detection of glycan biomarkers from clinical samples.

Survivin: A Potential Target of Adoptive Immunotherapy for Pediatric Sarcomas (101.19)

Abstract Cell based immunity is driven by CD8+ cytotoxic T cells bearing TCR that recognize specific tumor-associated peptides bound to class I MHC molecules. Survivin, an inhibitor of apoptosis protein (IAP), is a tumor-associated antigen that is widely expressed in nearly all human cancer cells and undetectable in most normal tissues. Although there is evidence for cytotoxic T cell responses against survivin in patients, tolerance to self antigens can be a limiting factor in generating highly avid T cells capable of killing tumor cells. An alternate approach involves peptide immunization of HLA-A2 transgenic mice that express the human class I HLA-A2 molecule, which could in principle generate more highly avid TCRs to human antigens. In this study, we vaccinated A2 transgenic mice with a modified survivin epitope, Surv96M (LMLGEFLKL) and successfully generated CD8+ CTL clones that react against Survivin96-104 and a human pancreatic cancer line. The α- and β- chains for the TCR of reactive CTLs were cloned and inserted into an retrovirus vector incorporating 2A linker peptides between coding sequences of α- and β- chains of TCRs against survivin. Transduction of survivin specific TCR retrovirus constructs into human PBMCs yielded up to 70% TCR redirected lymphocytes and specific effector function as measured by IFNgamma release following co-culture with peptide-pulsed targets and one A2+ pediatric osteosarcoma line. Ongoing work is underway to increase the TCR affinity to potency.

Imexon enhances gemcitabine cytotoxicity by inhibition of ribonucleotide reductase

Gemcitabine (GEM) is currently the standard first line treatment for pancreatic cancer; however, the overall survival of patients with this disease remains poor. Imexon is a pro-oxidant small molecule which produced a high response rate in combination with GEM in a phase I trial in pancreatic cancer. In this study, we investigate the combination of GEM with a novel redox-active agent, imexon, in vitro and in vivo. Median effect analysis was used for in vitro combination cytotoxicity. The effect of imexon on GEM metabolism and uptake into cells and into DNA and effects on ribonucleotide reductase (RNR) were examined in vitro. The pharmacokinetics and antitumor efficacy of the imexon/GEM combination was evaluated in mouse models. In three human pancreatic cancer lines, there was additivity for the imexon/GEM combination. There was significantly greater efficacy for the drug combination in Panc-1 xenograft tumors. A pharmacokinetic study in mice showed a near doubling in the AUC of imexon when GEM was co-administered, with no effect of imexon on GEM's pharmacokinetic disposition. In vitro, imexon did not alter GEM's metabolism or uptake into DNA, but significantly inhibited RNR, and this effect was greater when combined with GEM. These results suggest that the interaction between imexon and GEM may be due to complimentary inhibition of RNR plus an enhanced exposure to imexon when the GEM is administered in vivo. This combination is currently being tested in a randomized phase II trial in pancreatic cancer.

MUC4 down-regulation reverses chemoresistance of pancreatic cancer stem/progenitor cells and their progenies

The present study was undertaken to estimate the therapeutic benefit to down-regulate the MUC4 mucin for reversing chemoresistance of pancreatic cancer (PC) stem/progenitor cells and their progenies. The results have revealed that MUC4 mucin is overexpressed in CD133 + and CD133 − pancreatic cells (PCs) detected in patient’s adenocarcinoma tissues while no significant expression was seen in normal pancreatic tissues. The gain- and loss-of-function analyses have indicated that the overexpression of MUC4 in PC lines is associated with a higher resistance to the anti-proliferative, anti-invasive and apoptotic effects induced by gemcitabine. Importantly, the treatment of the MUC4-overexpressing CD18/HPAF-Src cells with gemcitabine resulted in an enrichment of the side population (SP) cells expressing CD133 while the total PC cells including non-SP cells detected in MUC4 knockdown CD18/HPAF-shMUC4 cells were responsive to the cytotoxic effects induced by gemcitabine. These data suggest that the MUC4 down-regulation may constitute a potential therapeutic strategy for improving the efficacy of gemcitabine to eradicate the total PC cell mass, and thereby preventing disease relapse.

Radiosensitization of Epidermal Growth Factor Receptor/HER2–Positive Pancreatic Cancer Is Mediated by Inhibition of Akt Independent of Ras Mutational Status

Epidermal growth factor receptor (EGFR) family members (e.g., EGFR, HER2, HER3, and HER4) are commonly overexpressed in pancreatic cancer. We investigated the effects of inhibition of EGFR/HER2 signaling on pancreatic cancer to elucidate the role(s) of EGFR/HER2 in radiosensitization and to provide evidence in support of further clinical investigations. Expression of EGFR family members in pancreatic cancer lines was assessed by quantitative reverse transcription-PCR. Cell growth inhibition was determined by MTS assay. The effects of inhibition of EGFR family receptors and downstream signaling pathways on in vitro radiosensitivity were evaluated using clonogenic assays. Growth delay was used to evaluate the effects of nelfinavir on in vivo tumor radiosensitivity. Lapatinib inhibited cell growth in four pancreatic cancer cell lines, but radiosensitized only wild-type K-ras-expressing T3M4 cells. Akt activation was blocked in a wild-type K-ras cell line, whereas constitutive phosphorylation of Akt and extracellular signal-regulated kinase (ERK) was seen in lines expressing mutant K-ras. Overexpression of constitutively active K-ras (G12V) abrogated lapatinib-mediated inhibition of both Akt phosphorylation and radiosensitization. Inhibition of MAP/ERK kinase/ERK signaling with U0126 had no effect on radiosensitization, whereas inhibition of activated Akt with LY294002 (enhancement ratio, 1.2-1.8) or nelfinavir (enhancement ratio, 1.2-1.4) radiosensitized cells regardless of K-ras mutation status. Oral nelfinavir administration to mice bearing mutant K-ras-containing Capan-2 xenografts resulted in a greater than additive increase in radiation-mediated tumor growth delay (synergy assessment ratio of 1.5). Inhibition of EGFR/HER2 enhances radiosensitivity in wild-type K-ras pancreatic cancer. Nelfinavir, and other phosphoinositide 3-kinase/Akt inhibitors, are effective pancreatic radiosensitizers regardless of K-ras mutation status.

Enhanced Sensitivity of Pancreatic Cancer Cells to Concurrent Inhibition of Aberrant Signal Transducer and Activator of Transcription 3 and Epidermal Growth Factor Receptor or Src

Many molecular aberrations occur in pancreatic cancer. Although aberrant epidermal growth factor receptor (EGFR), Src, and signal transducer and activator of transcription 3 (Stat3) are implicated in pancreatic cancer, therapies that target only one of these entities are undermined by signaling cross-talk. In the human pancreatic cancer lines, Panc-1 and Colo-357, pY845EGFR, pY1068EGFR, pY1086EGFR, and pY1173EGFR levels and pY416c-Src are concurrently elevated with aberrantly active Stat3 in a complex signaling cross-talk. Thus, understanding the signaling integration would facilitate the design of effective multiple-targeted therapeutic modalities. In Panc-1 and Colo-357 lines, pY845EGFR, pY1068EGFR, and pY1086EGFR levels are responsive to c-Src inhibition in contrast to pY1173EGFR, which is EGFR kinase-dependent. Constitutively active Stat3 is sensitive to both EGFR and Src inhibition, but the early suppression of aberrantly active Stat3 in response to the inhibition of EGFR and Src is countered by a Janus kinase (Jaks)-dependent reactivation, suggesting that Jaks activity is a compensatory mechanism for Stat3 induction. The inhibition of EGFR, Src, or Stat3 alone induced weak biological responses. By contrast, the concurrent inhibition of Stat3 and EGFR or Src induced greater viability loss and apoptosis and decreased the migration/invasion of pancreatic cancer cells in vitro. Significantly, the concurrent inhibition, compared with monotargeting modality, induced stronger human pancreatic tumor growth inhibition in xenografts. We infer that the tumor growth inhibition in vivo is caused by the simultaneous suppression of the abnormal functions of Stat3 and EGFR or Src. These studies strongly suggest that the concurrent targeting of Stat3 and EGFR or Src could be a beneficial therapeutic approach for pancreatic cancer.

A novel small-molecule disrupts Stat3 SH2 domain–phosphotyrosine interactions and Stat3-dependent tumor processes

The molecular modeling of the phosphotyrosine (pTyr)–SH2 domain interaction in the Stat3:Stat3 dimerization, combined with in silico structural analysis of the Stat3 dimerization disruptor, S3I-201, has furnished a diverse set of analogs. We present evidence from in vitro biochemical and biophysical studies that the structural analog, S3I-201.1066 directly interacts with Stat3 or the SH2 domain, with an affinity ( K D) of 2.74 μM, and disrupts the binding of Stat3 to the cognate pTyr–peptide, GpYLPQTV–NH 2, with an IC 50 of 23 μM. Moreover, S3I-201.1066 selectively blocks the association of Stat3 with the epidermal growth factor receptor (EGFR), and inhibits Stat3 tyrosine phosphorylation and nuclear translocation in EGF-stimulated mouse fibroblasts. In cancer cells that harbor aberrant Stat3 activity, S3I-201.1066 inhibits constitutive Stat3 DNA-binding and transcriptional activities. By contrast, S3I-201.1066 has no effect on Src activation or the EGFR-mediated activation of the Erk1/2 MAPK pathway. S3I-201.1066 selectively suppresses the viability, survival, and malignant transformation of the human breast and pancreatic cancer lines and the v-Src-transformed mouse fibroblasts harboring persistently active Stat3. Treatment with S3I-201.1066 of malignant cells harboring aberrantly active Stat3 down-regulated the expression of c-Myc, Bcl-xL, Survivin, the matrix metalloproteinase 9, and VEGF. The in vivo administration of S3I-201.1066-induced significant antitumor response in mouse models of human breast cancer, which correlates with the inhibition of constitutively active Stat3 and the suppression of known Stat3-regulated genes. Our studies identify a novel small-molecule that binds with a high affinity to Stat3, blocks Stat3 activation and function, and thereby induces antitumor response in human breast tumor xenografts harboring persistently active Stat3.