Cancer Cell BxPC-3 Research Articles

Abstract 1613: BTK as a drug target for cancers that harbor inactivating mutations in the DPC4 gene

Abstract The Deleted in Pancreatic Cancer locus 4 (DPC4) is a tumor suppressor gene that is often inactivated in pancreatic and colorectal cancers. In pancreatic adenocarcinomas, the frequency of inactivation of DPC4 is approximately 55%, while in colorectal carcinomas has been reported to be 10% to 35%. The loss of DPC4 has been shown to be associated with the progression and malignancy of pancreatic cancer along with decreased patient survival. We employed a small interfering (siRNA) library based screening strategy to identify potential synthetic lethal partners of the DPC4 gene. We used a kinase focused siRNA library that consisted of two siRNA oligonucleotides for each of the 624 protein kinase genes. A DPC4 isogenic pair, BxPC3-Vector and BxPC3-DPC4 (Wang et al. 2006) were treated by the siRNA oligonucleotides in parallel and the effects of the siRNA oligonucleotides on the growth of the cell lines were then compared. siRNA oligonucleotides that selectively inhibited the cell growth of the BxPC3-Vector cell line were selected as potential positive hits. These genes, once validated, represented potential drug targets that are very specific to cancer cells harboring mutations in the DPC4 gene. One of the top-ranked hits we identified from this screening is the Bruton agammaglobulinemia tyrosine kinase (BTK). With BTK siRNA treatment, BxPC3-Vector cells, which are DPC4 null, showed ∼30% more cell growth inhibition than the BxPC3-DPC4 cells, which are DPC4 wildtype. This selectivity of BTK siRNA against DPC4 null cells was further confirmed in a confirmation screen using two pancreatic cancer cell lines, BxPC3 and PANC-1, which are DPC4 null and DPC4 wildtype, respectively. BTK siRNA also showed significant selectivity against DPC4 deficiency in another pair of DPC4 isogenic colon cancer cell lines, HCT-116 and HCT-116-DPC4-knockout. To validate the BTK siRNA selectivity findings, we evaluated the BTK inhibitor PCI-32765 in our cancer cell line models and observed the same selectively against DPC4 null pancreatic and colon cancer cell lines. These results indicate that BTK is a potential molecular target for pancreas, colon, and other cancers harboring inactivating mutations in the DPC4 gene. (The work was supported by a NCI grant P01CA109552.) 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 1613.

Abstract 1609: Functional analysis of SFRS10: A novel molecular target for anti-cancer drug

Abstract [Background] Development of novel molecular-target drugs is actively conducted: A large number of such drugs are under clinical trials. However, an ideal molecular-target drug with high-specificity and low-toxicity is yet to be developed. For epoch-making development of cancer drugs, it is necessary to further identify target candidates by unprecedented approach. [Purpose] Last year, we reported 13 candidate genes that contributed to an immortalization through comprehensive gene expression analysis of human mortal- and immortal-cells and tissues. Under the same procedure, we here added SFRS10 gene to the list. To evaluate its possibility as drug target, we attempted here a functional analysis of SFRS10. [Materials and Methods] Human colon cancer cell lines DLD-1 and HCC48 and pancreatic cancer cells MIAPaCa-2, KLM-1, and BxPC-3 were used in each experiment. Quantitative analyses of gene expression levels were conducted by real-time RT-PCR method using gene specific-primers and Universal Probe Library probes (Roche). Microarray analysis was conducted with 4×44K Whole Human Genome Microarray (Agilent). Pathway analysis was performed through the use of Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, www.ingenuity.com). Knockdown experiments were performed using Stealth RNAi and RNAiMax reagent (Invitrogen). Cell proliferation was measured with MTT assay. Caspase activities were analyzed using ApoAlert Caspase Profiling Plate (Clontech). Cell cycle profile was obtained by measuring PI-stained cells with FACS “Calibur” (BD Biosciences). [Results] SFRS10 knockdown induced inhibition of cell proliferation of all the tested cells. Because knockdown efficiencies and cytostatic activities showed a good correlation in the experiment, we speculated that SFRS10 may have an important role for the cell proliferation. Treatment with SFRS10 siRNA induced cell cycle arrest in the S and G2/M phases and apoptosis in DLD-1 and KLM-1 cells. Moreover, we observed that some cell cycle-related genes, such as MAD2L1 and RFC4, altered their expression levels in response to SFRS10 knockdown as assessed by microarray analysis; cell cycle checkpoint pathways were significantly changed with network analyses. The alteration of gene expression levels were confirmed by real-time RT-PCR analyses. [Conclusion] These results suggested that SFRS10 may have an important role in regulation of cell proliferation and may control cell cycle checkpoints. Taken together, SFRS10 may be one molecule responsible for immortalization of cancer cells and the useful molecular target for cancer therapy. 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 1609.

Abstract B170: Downregulation of Yes-associated protein 1 (YAP1) expression reduces cell proliferation and clonogenicity of pancreatic cancer cells

Abstract Recent genetic studies in Drosophila have shown the Hippo pathway to be a potential tumor suppressor pathway that could play vital role(s) in mammalian cancer development. The Hippo pathway regulates organ size by inhibiting cell proliferation and promotes cell apoptosis upon its activation. The Yes Associated Protein 1 (YAP1) is a nuclear effector that promotes cell growth as a transcription co-activator. In human cancer, YAP1 has been shown to be a candidate oncogene as the chromosomal region containing YAP1 (11q22) has been reported as amplified and over-expressed in several tumor types, such as colon, liver, and lung (Overholtzer et al. 2006 PNAS, 103: 12405–12410). To determine the role of YAP1 in the tumorigenesis and progression of pancreatic cancer, here we examine YAP1 protein expression in pancreatic tumors and investigate the phenotypic effects of YAP1 down-regulation in cultured pancreatic cancer cells. Initially, we evaluated YAP1 expression in pancreatic tumors through immunohistochemistry (IHC) using tissue microarrays (TMA) to determine relative protein abundance and localization at the tissue and cellular level. YAP1 staining intensities were moderate to strong in the nucleus and cytoplasm of the cells, whereas the companion normal pancreas tissue showed negative to weak staining. In cultured cells, YAP1 expression and localization was modulated by cell densities. YAP1 total protein expression and Ser127 phosphorylation increased concurrently with cell density in whole cell lysates and cytosolic fractions, but does not in nuclear fractions. Additionally, transfection of YAP1-targeting siRNA oligonucleotides within pancreatic cancer cell lines, BxPC-3 and PANC-1, significantly decreased cell viability and reduced cell proliferation in vitro. An additional observation was the diminished anchorage-independent growth on soft agar of BxPC-3 and PANC-1 cells upon transfection with YAP1 siRNA oligonucleotides. The number of colonies formed were approximately 5% in BxPC-3 (p<0.007) and 20% (p<0.04) in PANC-1 compared to the negative siRNA control. Furthermore, the reduction of YAP1 protein expression by siRNA oligonucleotides correlates with the reduced growth on soft agar. The silencing of YAP1 abated clonogenicity of pancreatic cancer cells, suggesting a role of YAP1 in tumorigenesis. In summary, we demonstrate that YAP1 is overexpressed in pancreatic cancer and potentially plays an important role in the tumorigenesis and growth of pancreatic cancer. (This work was supported by a grant from the National Foundation for Cancer Research.) Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B170.

The expression of β-adrenoceptors in human pancreatic cancer cell lines and their roles in cell invasiveness

Objective To investigate the effect and mechanism of β-adrenoceptors on norepi-nephrine-induced invasiveness of pancreatic cancer cell lines. Methods The expression of β-adrenocep-tors mRNA in human pancreatic cancer cell lines MiaPaCa-2 and BxPC3 was detected by using RT-PCR. The cells were randomly divided into control group.10 mol/L NE intervention group, 1 mol/L propranolol intervention group and NE + propranolol intervention group. After 48 h , transwell invasiveness test was used to examine the changes in invasive ability of MiaPaCa-2. The expression of MMP-2, MMP-9 and VEGF mRNA was measured by semi-quantitative RT-PCR. The levels of MMP-2 , MMP-9 and VECF proteins were assayed by immunocytochemistry. Results Both MiaPaCa-2 and BxPC3 expressed β1-and β2-adrenocep-tors. The absorbance ( A) values of invasive cells in NE, NE + propranolol, propranolol and control groups were 0.78±0.02 ,0.32±0.03 ,0.26±0.01 and 0.28±0.02 , respectively, and those in NE intervention group were significantly higher than in control and NE + propranolol groups ( P 0. 05) . In NE group , the expression index of MMP-2 , MMP-9 and VEGF mRNA was 0. 87±0.02 , 1.04±0.02 and 0. 92±0. 01 , and the gray value of the protein expression was 131.20±2.34,105.32±7.21 and 115.60 ±5. 03 , respectively, which were higher than those in control and NE + propranolol groups ( P 0.05 ) . Conclusion β-adrenoceptors play an important role in the process of norepinephrine-induced invasiveness of pancreatic cancer cells. NE can promote the invasiveness of MiaPaCa-2 through up-regulating the expression of MMP-2 , MMP-9 and VEGF via β-adre-noceptors. Key words: Pancreatic carcinoma; Invasiveness; β-adrenoceptors; Norepinephrine

DCE-MRI using small-molecular and albumin-binding contrast agents in experimental carcinomas with different stromal content

Objectives To compare DCE-MRI experiments performed using a standard small-molecular (Gd-DTPA) and an albumin-binding (MS-325) contrast agent in two carcinoma models with different stromal content. Materials and methods DU-145 or BXPC-3 cancer cells were subcutaneously injected into nude mice. DCE-MRI was performed by a bolus injection of Gd-DTPA or MS-325 about 2 weeks after inoculation. For quantitative analysis a volume of interest was manually drawn over each tumor. To address the heterogeneous enhancement, each tumor volume was then divided into the 20% most-enhancing and the remaining 80% least-enhancing fractions. Mean tumor enhancement was calculated over these selected tumor volumes and compared between tumor groups and contrast agents. Maps of differential enhancement, peak enhancement and time-to-peak were used for visual evaluation. CD31 and VEGF immunohistochemistry were performed in excised tumors. Results In the 80% least-enhancing volume, at late time points of the dynamic scan, the mean enhancement elicited by MS-325 was higher in BXPC-3 than in DU-145 tumors. In the 20% most-enhancing volume, using either contrast agents, significant difference between the two tumors types were observed only early, while at later time points of the dynamic scan the difference were obscured by the faster washout observed in the BXPC-3 tumors. Enhancement maps confirmed that BXPC-3 tumors were characterized by marked washout rate using either contrast agent, particularly in the higher enhancing peripheral rim. With MS-325 this washout pattern appeared to be specific to the BXPC-3 carcinomas, since it was not observed in the DU-145 tumors. Finally, in both tumor types, MS-325 produced significantly higher enhancement than Gd-DTPA in the late phase of the dynamic scan. Ex vivo analysis confirmed the marked presence of aberrant infiltrative stroma in BXPC-3 tumors, in which tumor vessels were embedded. In all tumors the central portion was less viable and less infiltrated by stromal tissue then the peripheral areas. Conclusions Contrast distribution proved to be related to stromal content, which presumably produced the higher enhancement and faster washout observed in the BXPC-3 tumors. In particular, ‘early’ contrast-enhanced MRI, appeared as the most sensitive technique to detect the tumor portions characterized by a high stromal content, i.e. the peripheral rim of the BXPC-3 tumors. Since the same tumor models were recently investigated using FDG-PET imaging, showing inverse relationship between FDG uptake and stromal content, contrast-enhanced MRI and FDG-PET could provide complementary and comprehensive sensitivity in the assessment of carcinomas.

Molecular targeting of IGF-I receptor for human pancreatic cancer

14051 Background: Pancreatic cancer is one of the worst prognostic diseases. Continuous tumor growth depends on the altered regulation of the cell cycle that is modulated by signals from growth factors and their receptors, including insulin-like growth factors (IGF) and IGF-I receptor (IGF-Ir). It have been reported that blockade of IGF-Ir can suppress tumorigenicity and induce regression of established tumors. Recently several approaches of targeting IGF-Ir have been reported, including monoclonal antibodies and tyrosine kinase inhibitors. Methods: To study the mechanism of this effect and develop potential targeted therapeutics, we have constructed truncated IGF-Irs that function as dominant negative inhibitor (IGF-Ir/dn) and short hairpin RNA for IGF-Ir (shIGF-Ir). Those were cloned into recombinant adenoviruses (ad-IGF-Ir/dn and ad-shIGF-Ir). The effects of these adenoviruses were studied in human pancreatic cancer cell lines, BxPC3, Panc1, and AsPC1. We assessed efficiency of IGF-Ir signaling blockade, the effect of the adenoviruses on cell growth, steady-state, radiation, and chemotherapy induced apoptosis, and in vivo therapeutic efficacy in xenografts. Results: The blockade for IGF-Ir suppressed tumorigenicity of cancer cells both in vitro and in vivo and increased the amount of steady-state apoptosis in cancer cells. IGF-Ir/dn effectively blocked both IGF-I and IGF-II signaling through Akt-1 and shIGF-Ir reduced both IGF-Ir expression and ligands inducing phosphorylaion. The blockade for IGF-Ir increased chemotherapy and radiation induced apoptosis in vitro and the combination therapy with chemotherapy was very effective against established tumors in mice. Conclusions: These data suggest that the blocking of IGF-Ir signals, by ad-IGF-Ir/dn or ad-shIGF-Ir, may be a useful therapeutic strategy for pancreatic cancers. No significant financial relationships to disclose.

Treatment with rAD001 improves the efficacy of siRNA mediated down-regulation of survivin/BIRC5 in pancreatic carcinoma cell lines

15146 The rapamycin derived mTOR inhibitor RAD001 (everolimus) is cytotoxic to a number of cell lines. Because survivin, an important mediator of apoptosis and cell survival, is one of the targets down-regulated by RAD001, we tested the ability of a combination of RAD001 and siRNA mediated inhibition of survivin to enhance the cytotoxic effects. We have used four pancreatic cancer cell lines, MiaPaCa-2, BxPC-3, AsPC-1 and Panc-1. We found that the cytotoxic effect was enhanced in all cell lines after treatment with RAD001 and survivin siRNA. An efficient way of treatment was the initial down-regulation of survivin with RAD001 and a subsequent incubation with survivin siRNA. We conclude, that the transformation efficiency of siRNA, still a limiting factor in using this method, is more sufficiently combining both ways to down-regulate survivin. Our data indicate that a combination of mTOR inhibitors like RAD001 and survivin-targeted down-regulation with siRNA may improve the efficacy of therapy in pancreatic cancer. No significant financial relationships to disclose.

206 POSTER EGFR and PDGFR crosstalk may dictate the resistance to EGFR therapy in bladder cancer

The aim of this study was to improve fluorescence laparoscopy of pancreatic cancer in an orthotopic mouse model with the use of a light-emitting diode (LED) light source and optimal fluorophore combinations.Human pancreatic cancer models were established with fluorescent FG-RFP, MiaPaca2-GFP, BxPC-3-RFP, and BxPC-3 cancer cells implanted in 6-week-old female athymic mice. Two weeks postimplantation, diagnostic laparoscopy was performed with a Stryker L9000 LED light source or a Stryker X8000 xenon light source 24 hours after tail-vein injection of CEA antibodies conjugated with Alexa 488 or Alexa 555. Cancer lesions were detected and localized under each light mode. Intravital images were also obtained with the OV-100 Olympus and Maestro CRI Small Animal Imaging Systems, serving as a positive control. Tumors were collected for histologic analysis.Fluorescence laparoscopy with a 495-nm emission filter and an LED light source enabled real-time visualization of the fluorescence-labeled tumor deposits in the peritoneal cavity. The simultaneous use of different fluorophores (Alexa 488 and Alexa 555), conjugated to antibodies, brightened the fluorescence signal, enhancing detection of submillimeter lesions without compromising background illumination. Adjustments to the LED light source permitted simultaneous detection of tumor lesions of different fluorescent colors and surrounding structures with minimal autofluorescence.Using an LED light source with adjustments to the red, blue, and green wavelengths, it is possible to simultaneously identify tumor metastases expressing fluorescent proteins of different wavelengths, which greatly enhanced the signal without compromising background illumination. Development of this fluorescence laparoscopy technology for clinical use can improve staging and resection of pancreatic cancer.

CURCUMIN SENSITIZES PANCREATIC CANCER CELLS TO APOG2 INDUCED KILLING

Objectives: In Pancreatic cancer, several important survival molecules such as NF-κB, a key transcription factor playing important roles in tumorigenesis, metastasis and inflammation is highly upregulated along with NF-κB downstream proteins like Bcl-2 and Bcl-xL. Thus novel agents that have the capacity of inactivating such crucial target proteins could enhance the antitumor activity. ApoG2, new derivative of natural product gossypol, is a potent small molecule inhibitor of Bcl-2 and Bcl-xL proteins. Curcumin, an active ingredient from the rhizome of the plant, Curcuma longa, has anti oxidant, anti-inflammatory and anti-cancer activities and works by inactivation of NF-κB activity. In this study we have tested our hypothesis whether inactivation of NF-κB by curcumin could enhance anti-tumor activity of ApoG2. Methods: The BxPC-3 human pancreatic cell line was used in this study. For cell viability, apoptosis and NF-κB studies, MTT assay, ELISA and electrophoretic mobility shift assay were used respectively. Western blot assays was done to study Bcl-2 and Bcl-xL. Results: Treatment of human pancreatic cell line BxPC-3 with curcumin sensitized ApoG2 induced cell killing. In the dose escalation study with ApoG2 at 72 hrs, 1μM showed 60% cell viability, while 5μM of curcumin at 72 hrs showed 78 % cell viability. Combination treatment with 1μM of ApoG2 and 5μM of curcumin at 72 hrs showed 45 % cell viability. (P = <0.0001). Apoptosis by Histone/DNA ELISA showed 7-fold induction with ApoG2 and 6-fold induction with curcumin at 72 hrs. However, combination treatment with curcumin and ApoG2 at 72 hrs showed 11-fold induction in apoptosis compared to the control. Bcl-xL and Bcl-2 expression levels were down regulated along with the inactivation of NF-κB in the combination treatment group compared with curcumin and ApoG2 alone. Conclusion: From these results, we conclude that curcumin enhanced ApoG2 induced cell growth inhibition and apoptosis of Pancreatic BxPC-3 cancer cells. This effect is partly due to inactivation of NF-κB activation by curcumin and inhibition of Bcl-2 and Bcl-xL.

Regulatory Effects of X-linked Inhibitor of Apoptosis Protein and Pro-apoptotic Protein Smac on Apoptosis Resistance to Chemotherapy in Pancreatic Cancer Cells

Objective: To investigate the relation of X-linked inhibitor of apoptosis (XIAP) and second mitochondria-derived activator of caspase (Smac) signaling pathway to chemoresistance in human pancreatic cancer Panc-1 and BXPC-3 cells. Methods: Apoptosis and the changes of XIAP expression in permeabilized cells induced by cisplatin and 5-fluorouracil (FU) were measured by flow cytometry. The cytosolic expression of XIAP, Smac and caspase-3 was detected by Western blot. A recombinant plasmid vector pEGFP-N1/Smac was constructed and transfected into of Panc-1 cells. The effect of cytosolic overexpression of Smac on apoptosis of Panc-1 cells was evaluated by flow cytometry. Results: Panc-1 was more resistant to cisplatin or 5-FU induced apoptosis than BXPC-3. Western blot revealed that chemoresistant Panc-1 highly expressed XIAP, and increased cytosolic expression of Smac might be responsible for the marked down-regulation of XIAP in chemo-sensitive BXPC-3 cells after exposure to cisplatin or 5-FU. Furthermore, cytosolic overexpression of Smac could significantly down-regulate the levels of XIAP and promote the activity of caspase-3, as well as sensitize Panc-1 cells to anticancer drug-induced apoptosis. Conclusion: Anticancer drug-induced apoptosis requires mitochondrial release of Smac and downregulation of XIAP, which may be an important determinant of chemo-sensitivity in pancreatic cancer cells. Up-regulation of cytosolic expression of Smac may act as an effective modifying signal to overcome apoptosis resistance to chemotherapy in pancreatic cancer cells.

Molecular cloning and characterization of OSR1 on human chromosome 2p24

During Drosophila hindgut development, bowl, caudal/CDX, brachyenteron/Brachyury/TBX, fork head/FOX, drumstick, lines, and wingless/WNT play important roles. Drosophila bowl gene is homologous to Drosophila odd-skipped (odd) gene and odd-skipped related gene (sob). Here, human OSR1, related to Drosophila odd, was isolated using bioinformatics and cDNA-PCR. OSR1 was found to encode 266 amino-acid protein with three C2H2-type zinc fingers, a tyrosine phosphorylation site (Tyr 203), and several putative PXXP SH3 binding motifs. Three zinc fingers and a tyrosine phosphorylation site were conserved among human OSR1, OSR2, Drosophila odd, sob, and bowl. OSR1 showed 63.6% total amino-acid identity with OSR2. OSR1 gene consisting of three exons was located on human chromosome 2p24. OSR1 mRNA of 2.3-kb in size was detected in adult colon, small intestine, prostate, testis, and fetal lung. OSR1 mRNA was significantly up-regulated in a pancreatic cancer cell line MIA PaCa-2, and was weakly expressed in gastric cancer cell lines OKAJIMA, MKN45, pancreatic cancer cell lines PANC-1, BxPC-3, AsPC-1, PSN-1, Hs766T, and esophageal cancer cell line TE10. Among 10 cases of primary gastric cancer, OSR1 mRNA was up-regulated in 5 cases, and was down-regulated in 2 cases. This is the first report on molecular cloning and characterization of human OSR1.

Gastrin regulates growth of human pancreatic cancer in a tonic and autocrine fashion.

The gastrointestinal peptides gastrin and cholecystokinin (CCK) stimulate growth of human pancreatic cancer through a CCK-B/gastrin- like receptor. In the present study we evaluated whether growth of human pancreatic cancer is endogenously regulated by gastrin. Immunohistomical examination of BxPC-3 cells and tumor xenografts revealed specifc gastrin immunoreactivity. Gastrin was detected by radioimmunoassay in pancreatic cancer cell extracts and in pancreatic cancer cell extracts and in the growth media. With use of reverse-transcriptase polymerase chain reaction gastrin gene expression was detected in both cultured BxPC-3 cancer cells and transplanted tumors, as well as seven addition human pancreatic cancer cell lines. Growth of BxPC-3 human pancreatic cancer cell in serum-free medium was inhibited by the addition of the CCK-B/gastrin receptor antagonist L-365,260, and gastrin treatment reversed the inhibitory effect of the antagonist. A selective gastrin antibody (Ab repressed growth of BxPC-3 cells. Gastrin immunoreactivity was detected in fresh human pancreatic cancer specimens but not in normal human pancreatic tissue. These data provide the first evidence that growth of a human pancreatic cancer is tonically stimulated by the autocrine production of gastrin. Evidence for the ubiquity of this system was provided by the detection of gastrin gene expression in multiple human pancreatic cancer cell lines and detection of gastrin in cell lines and fresh pancreatic tumors.