Human Pancreatic Adenocarcinoma Tissues Research Articles

HKDC1 enhances the proliferation, migration and glycolysis of pancreatic adenocarcinoma and is linked to immune infiltration.

Background: Understanding the molecular mechanisms of pancreatic adenocarcinoma (PAAD) development is vital for treating this disease, as the current prognosis and treatment options are highly discouraging. Objective: This study aimed to examine the involvement of Hexokinase Domain Containing 1 (HKDC1) in the progression of PAAD. Methods: The study utilized bioinformatics techniques to evaluate the relationship between the expression of HKDC1 and clinical characteristics. In vitro experiments were conducted to investigate the molecular mechanisms and biological functions of HKDC1 in PAAD. Results: The findings of this research indicate that the expression of HKDC1 was increased in various types of human cancers, and a significant correlation was observed between elevated HKDC1 expression in PAAD and unfavorable prognosis. According to the findings from univariate and multivariate Cox regression analyses, HKDC1 could potentially serve as a standalone prognostic indicator for individuals diagnosed with PAAD. After performing calculations, we determined that the HKDC1 high-expression group exhibited lower immunologic score and higher ESTIMATE score, indicating a difference in immune infiltration score. In order to validate the expression of HKDC1 in PAAD cell lines, we analyzed the PAAD cell lines through qPCR and protein blotting. The expression of HKDC1 in human PAAD tissues was also detected by western blotting. Additionally, we explored the involvement of HKDC1 in PAAD by conducting experiments such as colony formation, 5-ethynyl-2'-deoxyuridine (EdU), transwell, and wound healing assays. In our study, we discovered that disruption of HKDC1 expression in PAAD cell types resulted in a decrease in cell growth rate and inhibited cell movement and invasion. Conclusion: To conclude, our findings indicate that HKDC1 has a significant impact on the tumor microenvironment (TME) of PAAD and could potentially be a promising target for PAAD treatment, offering fresh perspectives on the management of PAAD.

BICC1 drives pancreatic cancer progression by inducing VEGF-independent angiogenesis

VEGF inhibitors are one of the most successful antiangiogenic drugs in the treatment of many solid tumors. Nevertheless, pancreatic adenocarcinoma (PAAD) cells can reinstate tumor angiogenesis via activation of VEGF-independent pathways, thereby conferring resistance to VEGF inhibitors. Bioinformatic analysis showed that BICC1 was one of the top genes involved in the specific angiogenesis process of PAAD. The analysis of our own cohort confirmed that BICC1 was overexpressed in human PAAD tissues and was correlated to increased microvessel density and tumor growth, and worse prognosis. In cells and mice with xenograft tumors, BICC1 facilitated angiogenesis in pancreatic cancer in a VEGF-independent manner. Mechanistically, as an RNA binding protein, BICC1 bounds to the 3’UTR of Lipocalin-2 (LCN2) mRNA and post-transcriptionally up-regulated LCN2 expression in PAAD cells. When its level is elevated, LCN2 binds to its receptor 24p3R, which directly phosphorylates JAK2 and activates JAK2/STAT3 signal, leading to increased production of an angiogenic factor CXCL1. Blocking of the BICC1/LCN2 signalling reduced the microvessel density and tumor volume of PAAD cell grafts in mice, and increased the tumor suppressive effect of gemcitabine. In conclusion, BICC1 plays a pivotal role in the process of VEGF-independent angiogenesis in pancreatic cancer, leading to resistance to VEGF inhibitors. BICC1/LCN2 signaling may serve as a promising anti-angiogenic therapeutic target for pancreatic cancer patients.

LACTB mRNA expression is increased in pancreatic adenocarcinoma and high expression indicates a poor prognosis.

This study aimed to find the prognostic value of Beta-lactamase-like (LACTB) in pancreatic adenocarcinoma (PAAD) patients. The mRNA expression of LACTB was upregulated in PAAD and was correlated with vital status (P = 0.0199). The immunoreactive scores of LACTB protein in human PAAD tissues were significantly higher than those in adjacent noncancerous pancreatic tissues. Receiver operating characteristic (ROC) curve assessment showed that LACTB mRNA expression has high diagnostic value in PAAD. Kaplan-Meier curve and Cox analyses suggested that patients with high LACTB mRNA expression have a poor prognosis, indicating that LACTB mRNA is an independent prognostic factor for overall survival [hazard ratio (HR) = 1.72, P = 0.015, 95% confidence interval (CI) = 1.106-2.253] and disease-specific survival (HR = 1.97, P = 0.004, 95% CI = 1.238-3.152) of PAAD patients. Gene set enrichment analysis (GSEA) revealed that hallmark_g2m_checkpoint, hallmark_myc_targets_v1, hallmark_e2f_targets, and kegg_cell_cycle were differentially enriched in phenotypes with high LACTB expression. In addition, CDC20, CDK4, MCM6, MAD2L1, MCM2 and MCM5 were leading genes intersecting in these four pathways, and a positive correlation between mRNA expression and LACTB was observed in most normal and cancer tissues. Finally, elevated LACTB mRNA expression was significantly related to multiple immune marker sets. Our results elucidate that LACTB is involved in the development of cancer, and that high LACTB expression in patients with PAAD can predict a poor prognosis. High LACTB expression was significantly correlated with cell cycle-related genes and multiple immune marker sets.

UPLC‑MS/MS‑based metabolomic characterization and comparison of pancreatic adenocarcinoma tissues using formalin‑fixed, paraffin‑embedded and optimal cutting temperature‑embedded materials

The purpose of the present study was to compare metabolites from formalin-fixed and paraffin-embedded (FFPE) pancreatic tissue blocks with those identified in optimal cutting temperature (OCT)-embedded pancreatic tissue blocks. Thus, ultra-performance liquid chromatograph-mass spectrometry/mass spectrometry-based metabolic profiling was performed in paired frozen (n=13) and FFPE (n=13) human pancreatic adenocarcinoma tissue samples, in addition to their benign counterparts. A total of 206 metabolites were identified in both OCT-embedded and FFPE tissue samples. The method feasibility was confirmed through reproducibility and a consistency assessment. Partial least-squares discriminant analysis and heatmap analysis reliably distinguished tumor and normal tissue phenotypes. The expression of 10 compounds, including N-acetylaspartate and creatinine, was significantly different in both OCT-embedded and FFPE tumor samples. These ten compounds may be viable candidate biomarkers of malignant pancreatic tissues. The super-categories to which they belonged exhibited no significant differences between FFPE and OCT-embedded samples. Furthermore, purine, arginine and proline, and pyrimidine metabolism used a shared pathway found in both OCT-embedded and FFPE tissue samples. These results supported the notion that metabolomic data acquired from FFPE pancreatic cancer specimens are reliable for use in retrospective and clinical studies.

Overexpression of CBX3 in Pancreatic Adenocarcinoma Promotes Cell Cycle Transition-Associated Tumor Progression.

Background: Previous studies showed that Chromobox protein homolog 3 (CBX3) was overexpressed in several types of human cancers, however its pattern and role in pancreatic adenocarcinoma (PAAD) has not yet been understood. The aim of this study was to identify the expression and function of CBX3 in PAAD. Methods: Data of transcriptomic and protein expression of CBX3 in PAAD were collected from different databases and analyzed. The in vitro and in vivo role of CBX3 in PAAD was examined. Results: CBX3 was overexpressed in human PAAD tissues, which was associated with poor prognosis of overall and disease-free survival of the patients. Overexpression of CBX3 induced the in vitro proliferation, anchorage-free growth, migration and invasion of the PAAD cells, and led to in vivo growth of orthotoptic PAAD tumors in mice. GO and KEGG pathway analysis, as well as experimental observation showed that CBX3 may be associated with cell cycle transition of PAAD cells, and cyclin-dependent kinase 1 (CDK1) and proliferating cell nuclear antigen (PCNA) may mediate the tumor-promoting action of CBX3. CDK1 knockdown attenuated the cell cycle transition, proliferation and invasion of CBX3-overexpressing PAAD cells. Conclusion: Our findings suggest the tumor-promoting role of CBX3 in PAAD to be targeted by novel therapeutic strategies.

MicroRNA-217 inhibits cell proliferation, invasion and migration by targeting Tpd52l2 in human pancreatic adenocarcinoma.

MicroRNAs (miRNAs) play important roles in the regulation of various tumor biological processes including proliferation and apoptosis. miR-217 has been implicated in many types of cancer, whereas its expression and potential biological function in human pancreatic adenocarcinoma (HPAC) remain unclear. We aimed to investigate the clinical significance of miR-217 in patients with pancreatic carcinoma and its role and underlying molecular mechanism in HPAC. We collected 15pairs of pancreatic cancer and normal pancreas tissues to evaluate the expression of miR-217 and tumor protein D52-like2 (Tpd52l2). Then, we transfected AsPC-1 cells with miR-217 mimics or Tpd52l2 siRNA to detect the effect on cell proliferation, apoptosis, invasion, migration and the cell cycle. In addition, miR-217 mimics and Tpd52l2 expression plasmids were co-transfected into AsPC-1 cells to further investigate the mechanism of miR-217 and Tpd52l2 in HPAC tumorigenesis. Finally, exploration of related signaling pathways was carried out. Herein, we found that the expression of miR-217 was significantly downregulated in HPAC tissues as compared with that observed in adjacent normal tissues. Further functional assays showed that restoration of the expression of miR-217 inhibited cell proliferation, invasion and migration, induced apoptosis, and caused cell cycle arrest of HPAC cells. Notably, Tpd52l2 was identified as a functional target of miR-217 in HPAC. Furthermore, an inverse correlation between miR-217 and Tpd52l2 expression was observed in the HPAC tissues. Downregulation of Tpd52l2 had an effect similar to that following overexpression of miR-217, and upregulation of Tpd52l2 reversed the effects of the overexpression of miR-217. Finally, we found that overexpression of miR-217 or knockdown of Tpd52l2 suppressed the PIK3CA/AKT signaling pathways. In addition, this may explain the effect of miR-217/Tpd52l2 on HPAC development. Taken together, these results suggest a critical role of miR-217 in suppressing proliferation, migration and invasion of HPAC cells by targeting Tpd52l2. Targeting the miR-217/Tpd52l2 axis may be a new therapeutic application with which to treat patients with HPAC in the future.

Downregulation of ULK1 by microRNA-372 inhibits the survival of human pancreatic adenocarcinoma cells.

Dysregulation of microRNA (miRNA) expression in various cancers and their role in cancer progression is well documented. The purpose of this study was to investigate the biological role of miR‐372 in human pancreatic adenocarcinoma (HPAC). We collected 20 pairs of HPAC tissues and adjacent non‐cancerous tissues to detect miR‐372 expression levels. We transfected BXPC‐3 and PANC‐1 cells with miR‐372 inhibitor/mimics to study their effect on cell proliferation, apoptosis, invasion, migration and autophagy. In addition, miR‐372 mimics and a tumor protein UNC51‐like kinase 1 (ULK1) siRNA were co‐transfected into BXPC‐3 and PANC‐1 cells to explore the mechanism of miR‐372 and ULK1 on HPAC tumorigenesis. We found that the expression of miR‐372 was markedly downregulated in HPAC cells compared to adjacent normal tissues. Furthermore, functional assays showed that miR‐372 inhibited cell proliferation, invasion, migration and autophagy in BXPC‐3 and PANC‐1 cells. An inverse correlation between miR‐372 expression and ULK1 expression was observed in HPAC tissues. Downregulation of ULK1 inhibited the overexpression effects of miR‐372, and upregulation of ULK1 reversed the effects of overexpressed miR‐372. Finally, we found that silencing ULK1 or inhibiting autophagy partly rescued the effects of miR‐372 knockdown in HPAC cells, which may explain the influence of miR‐372/ULK1 in HPAC development. Taken together, these results revealed a significant role of the miR‐372/ULK1 axis in suppressing HPAC cell proliferation, migration, invasion and autophagy.

P300 inhibition enhances gemcitabine-induced apoptosis of pancreatic cancer.

The transcriptional cofactor p300 has histone acetyltransferase activity (HAT) and has been reported to participate in chromatin remodeling and DNA repair. We hypothesized that targeting p300 can enhance the cytotoxicity of gemcitabine, which induces pancreatic cancer cell apoptosis by damaging DNA. Expression of p300 was confirmed in pancreatic cancer cell lines and human pancreatic adenocarcinoma tissues by western blotting and immunohistochemistry. When pancreatic cancer cells were treated with gemcitabine, p300 was recruited to chromatin within 24 hours, indicating the role in response to DNA damage. When p300 was gene-silenced with siRNA, histone acetylation was substantially reduced and pancreatic cancer cells were sensitized to gemcitabine. The selective p300 HAT inhibitor C646 similarly decreased histone acetylation, increased gemcitabine-induced apoptosis and thus enhanced the cytotoxicity of gemcitabine on pancreatic cancer cells. These findings indicate that p300 contributes to chemo-resistance of pancreatic cancer against gemcitabine and suggest that p300 and its HAT activity may be a potential therapeutic target to improve outcomes in patients with pancreatic cancer.

Aldehyde Dehydrogenase 1B1 as a Modulator of Pancreatic Adenocarcinoma.

The aim of the current study was to examine expression and the role, if any, of aldehyde dehydrogenase (ALDH)1B1 in pancreatic adenocarcinoma. A tissue microarray of 61 pancreatic cancer patients were evaluated for protein expression of ALDH1B1 by immunohistochemistry. The ALDH1B1 small interfering (RNA) was used to assess the contribution of ALDH1B1 on proliferation of pancreatic cancer cells. In normal human pancreas, ALDH1B1 is abundantly expressed in glandular cells, but sparsely in the ducts (ALDH1B1 immunopositivity = 16.7 ± 1.7). In pancreatic ductal carcinoma, we found high ALDH1B1 expression in ductal cancerous tissues (ALDH1B1 immunopositivity = 197.2 ± 29.4). Analysis of ALDH1B1 expression in a human pancreatic adenocarcinoma tissue microarray showed the greatest expression in tumors that were more invasive. A variation in ALDH1B1 expression was also observed in 16 human pancreatic cancer cell lines. Knockdown of ALDH1B1 caused a 35% reduction in cell growth in the high ALDH1B1-expressing cell lines. Our data show for the first time that ALDH1B1 is expressed at very high levels in human pancreatic cancer, and it contributes to proliferation in these tumor cells. These data suggest a potential modulatory role for ALDH1B1 in pancreatic cancer.

Abstract 3613: P300 inhibition enhances cytotoxic effect of Gemcitabine through E2F1 activation in pancreatic cancer

Abstract Background: Transcriptional cofactor, P300 has been reported to regulate cell cycle on G1/S transition. Previously, we shown that prolonged S-phase is associated with increased apoptosis induced by Gemcitabine (GEM) in pancreatic cancer cells. Thus, we hypothesized that targeting P300 enhances the cytotoxic effect of GEM on pancreatic cancer. Methods: We studied 2 human pancreatic cancer cell lines, Panc1 and MIAPaCa2. P300 was gene-silenced using siRNA, and P300 histone acetyltransferase (HAT) activity was inhibited by specific inhibitor C646. Cell viability was measured by WST-8 assay and GEM-induced apoptosis was measured using Caspase3 and PARP by Western blotting, Tunel assay by immunofluorescence staining, and Annexin V-PI staining by flow cytometry. CDK4/6 inhibitor Palbociclib was used to block S-phase entry. Results: Robust P300 expression was observed in pancreatic cancer cell lines and human pancreatic adenocarcinoma tissues. When P300 expression was depleted by specific P300 siRNA, cell viability after GEM treatment was decreased significantly compared with the control pancreatic cancer cells (43% decrease in MIAPaCa2 and 39% decrease in Panc1, respectively, p<0.05). Increased GEM-induced apoptosis was confirmed by increased cleaved forms of Caspase3 and PARP, increased tunel positive cells, and increased Annexin positive cells in P300-depleted cells compared to the control cells. When S phase entry was blocked by Palbociclib, the effect of gene silencing of P300 on GEM-induced apoptosis was abolished. As Rb-E2F1 complex is known as the key regulator of the S phase specific gene transcriptions, we evaluated the effect of P300 gene silencing on Rb and E2F1. While P300 gene-silencing did not affect Rb phosphorylation after GEM treatment, it increased E2F1 expression significantly (56% increase in MIAPaCa2 and 119% increase in Panc1, respectively, p<0.05). When pancreatic cancer cells were treated with combination of GEM and C646, P300 specific HAT inhibitor, the cell viability was reduced significantly compared with when treated with GEM alone (48% decrease for Panc1, and 58% for MIAPaCa2, respectively, p<0.05). Conclusion: Targeting P300 by siRNA or HAT inhibitor enhances the efficacy of GEM on pancreatic cancer cells. Our observation suggests the presence of Rb-independent E2F1 regulatory mechanism in pancreatic cancer cells. This pathway may be an important therapeutic target to potentiate GEM chemotherapy in the treatment of pancreatic cancer. Citation Format: Hiroaki Ono, Marc D. Basson, Hiromichi Ito. P300 inhibition enhances cytotoxic effect of Gemcitabine through E2F1 activation in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3613. doi:10.1158/1538-7445.AM2015-3613

Smad4 Decreases the Population of Pancreatic Cancer–Initiating Cells through Transcriptional Repression of ALDH1A1

Cancer progression involves a rare population of undifferentiated cancer-initiating cells that have stem cell-like properties for self-renewal capacity and high tumorigenicity. We investigated how maintenance of pancreatic cancer-initiating cells is influenced by Smad4, which is frequently deleted or mutated in pancreatic cancers cells. Smad4 silencing up-regulated the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA, whereas forced expression of Smad4 in pancreatic cancer cells down-regulated it. Smad4 and ALDH1 expression inversely correlated in some human clinical pancreatic adenocarcinoma tissues, suggesting that ALDH1 in pancreatic cancer cells was associated with decreased Smad4 expression. We then examined whether ALDH1 served as a marker of pancreatic cancer-initiating cells. Pancreatic cancer cells contained ALDH1(hi) cells in 3% to 10% of total cells, with high tumorigenic potential. Because Smad4 is a major mediator of transforming growth factor (TGF)-β family signaling, we investigated the regulatory mechanism of ALDH activity by TGF-β and bone morphogenetic proteins. Treatment with TGF-β attenuated ALDH1(hi) cells in several pancreatic cancer cells, whereas bone morphogenetic protein-4 was not as potent. Biochemical experiments revealed that TGF-β regulated ALDH1A1 mRNA transcription through binding ofSmad4 to its regulatory sequence. It appears that TGF-β negatively regulates ALDH1 expression in pancreatic cancer cells in a Smad-dependent manner and in turn impairs the activity of pancreatic cancer-initiating cells.

Neurotrophic Factor Artemin Promotes Invasiveness and Neurotrophic Function of Pancreatic Adenocarcinoma In Vivo and In Vitro

ObjectivesThe aim of this study was to investigate the effect of the neurotrophic factor Artemin on neuroplasticity and perineural invasion of pancreatic adenocarcinoma.MethodsArtemin expressions were detected in human pancreatic adenocarcinoma tissues by Western blot and immunohistochemistry. Artemin overexpression and RNA interference in the pancreatic cancer cell lines were performed to evaluate the effects of Artemin on cell proliferation, invasion, and neurotrophic activity in vitro and in nude orthotopic transplantation tumor models.ResultsArtemin expression in pancreatic cancer tissues was related to the incidence of lymphatic metastasis and perineural invasion as well as the mean density and total area of nerve fibers. Overexpression of Artemin in pancreatic cancer cell lines improved colony formation, cell migration, matrigel invasion, and neurotrophic activity in vitro. This overexpression also increased the volume of nude orthotopic transplantation tumors; promoted cancer cell invasion of the peripheral organs, nerves, vessels, and lymph nodes; and stimulated the proliferation of peritumoral nerve fibers. Artemin depletion by RNA interference had an inhibitory effect mentioned previously.ConclusionsArtemin could promote invasiveness and neurotrophic function of pancreatic adenocarcinoma in vivo and in vitro. Therefore, Artemin could be used as a new therapeutic target of pancreatic carcinoma.

Antibody Against CD44s Inhibits Pancreatic Tumor Initiation and Postradiation Recurrence in Mice

CD44s is a surface marker of tumor-initiating cells (TICs); high tumor levels correlate with metastasis and recurrence, as well as poor outcomes for patients. Monoclonal antibodies against CD44s might eliminate TICs with minimal toxicity. This strategy is unclear for treatment of pancreatic cancer, and little is known about how anti-CD44s affect pancreatic cancer initiation or recurrence after radiotherapy. One hundred ninety-two pairs of human pancreatic adenocarcinoma and adjacent nontumor pancreatic tissues were collected from patients undergoing surgery. We measured CD44s levels in tissue samples and pancreatic cancer cell lines by immunohistochemistry, real-time polymerase chain reaction, and immunoblot; levels were correlated with patient survival times. We studied the effects of anti-CD44s in mice with human pancreatic tumor xenografts and used flow cytometry to determine the effects on TICs. Changes in CD44s signaling were examined by real-time polymerase chain reaction, immunoblot, reporter assay, and invitro tumorsphere formation assays. Levels of CD44s were significantly higher in pancreatic cancer than adjacent nontumor tissues. Patients whose tumors expressed high levels of CD44s had a median survival of 10 months compared with >43 months for those with low levels. Anti-CD44s reduced growth, metastasis, and postradiation recurrence of pancreatic xenograft tumors in mice. The antibody reduced the number of TICs in cultured pancreatic cancer cells and xenograft tumors, as well as their tumorigenicity. In cultured pancreatic cancer cell lines, anti-CD44s down-regulated the stem cell self-renewal genes Nanog, Sox-2, and Rex-1 and inhibited signal transducer and activator of transcription 3-mediated cell proliferation and survival signaling. The TIC marker CD44s is up-regulated in human pancreatic tumors and associated with patient survival time. CD44s is required for initiation, growth, metastasis, and postradiation recurrence of xenograft tumors in mice. Anti-CD44s eliminated bulk tumor cells as well as TICs from the tumors. Strategies to target CD44s cab be developed to block pancreatic tumor formation and post-radiotherapy recurrence in patients.

Covering the Cover

Covering the Cover

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8. Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12. In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1). Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity. Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas. In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues, but staining on paraffin-embedded slides had been a challenge until recently. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Hypermethylation of HIC1 Promoter and Aberrant Expression of HIC1/SIRT1 Might Contribute to the Carcinogenesis of Pancreatic Cancer

DNA hypermethylation is proved to be involved in carcinogenesis. Because chronic pancreatitis (CP) is a consistent risk factor for pancreatic cancer, the possible alteration and tumor contribute effects of hypermethylated in cancer-1 (HIC1) promoter methylation in CP was investigated. Methylation of HIC1 promoter HIC1 and SIRT1 expression were detected in human normal pancreas (NP), CP and pancreatic adenocarcinoma tissues. Furthermore, HIC1/SIRT1 pathway was regulated by demethylating reagent and exogenous expression in PANC-1, BxPC-3 and AsPC-1 cell lines, cell biology behavior including proliferation, apoptosis, cell cycle and senescence were detected. The methylation of HIC1 promoter was demonstrated in 70.3 % pancreatic carcinoma (45 of 64), 47.5 % CP (19 of 40) and 11.4 % NP tissues (4 of 35). Moreover, hypermethylation of HIC1 promoter and deregulation of HIC1 expression in pancreatic cancer were significantly related to high-stage tumor and older patient age. HIC1 promoter hypermethylation was also observed in pancreatic cancer cell lines including PANC-1, BxPC-3 and AsPC-1. Restoration of HIC1 function with 5-aza-dC treatment or pCDNA3FlagHIC1 plasmid transfection leaded to a reduction in cell proliferation, obvious cell senescence, cell cycle arrest and apoptosis, accompanied with acetylated p53 and p21(WAF1 of Cip1) upregulation. While after further transfected with pCDNA3FlagSIRT1 plasmid, the growth inhibition, senescence and cycle arrest without apoptosis were partially rescued with deregulated acetylated p53 and p21(WAF1 of Cip1). Our results indicate that hypermethylation of HIC1 promoter in CP may contribute to the aberrant expression of HIC1/SIRT1 pathway and then involve in the pancreatic carcinogenesis.

Abstract 1854: Lysophosphatidic Acid Acyltransferase-Beta contributes to proliferation and anchorage-independent growth in pancreatic adenocarcinoma

Abstract Abstract: Lysophosphatidic acid acyltransferase-beta (LPAAT-α) catalyzes the production of phosphatidic acid (PA) from lysophosphatidic acid (LPA). PA is a lipid cofactor that contributes to the activation of c-Raf, BRAF, mTOR and PKC-α. LPAAT-α expression is a prognostic factor in gynecologic malignancies and is being investigated as a therapeutic target in a variety of tumor types. We previously reported that LPAAT-α is expressed in pancreatic cancer cell lines and in human pancreatic adenocarcinoma tissues. We employed siRNA knockdown of LPAAT-α expression in pancreatic cancer cells in vitro. We find that reduction in the expression of LPAAT-α protein is associated with decreased proliferation and impairment in anchorage dependent growth of pancreatic cancer cells. Methods: MiaPaCa and Panc-1 cells were transfected with two different siRNAs targeted to LPAAT-α at 10 nM and 25 nM for 48 hrs and 72 hrs. A non-targeting siRNA was used as a control. The cells were then plated on tissue culture dishes or in soft agar and allowed to proliferate. Proliferation on plastic dishes was measured using Alamar Blue. Colony formation in soft agar was determined by directly counting macroscopic colonies. Results: Transfection of MiaPaCa and Panc-1 cells with siRNA at 25 nM for 72 hours achieved almost 100% inhibition of LPAAT-α protein expression by Western blotting. Under these conditions there was a 50% reduction in proliferation and colony formation. Studies to determine the effects of depleting LPAAT-α on the levels of p-PKC-α, p-MEK, p-4E-BP1 and p-S6Kinase are ongoing and will be presented. Conclusions: Suppression of expression of LPAAT-α protein in pancreatic cancer cell lines reduces proliferation and anchorage independent growth. This contributes to the validation of LPAAT-α as a therapeutic target in pancreatic cancer. 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 1854. doi:1538-7445.AM2012-1854

High expression of S100A11 in pancreatic adenocarcinoma is an unfavorable prognostic marker

S100A11 is a member of S100 protein family, and our previous study showed that S100A11 is one of the up-regulated proteins that have not been reported to be associated with pancreatic carcinoma. The purpose of this study was to investigate the relation between S100A11 expression and the clinicopathological variables and clinical outcome in patients with pancreatic adenocarcinoma. Immunohistochemistry analysis was performed for S100A11 in 78 pairs of specimens of human pancreatic adenocarcinoma tissues and adjacent nontumorous tissues. The univariate and multivariate survival analyses were also performed to determine its prognostic significance. S100A11 expression in pancreatic adenocarcinoma (62/78) was significantly higher than that in the adjacent nontumorous tissues (19/78) (P = 0.000). High expression of S100A11 was associated with the lymph node metastasis and histological differentiation (P = 0.003 and 0.004, respectively). Univariate analysis showed that S100A11 expression was associated with poor prognosis (P = 0.0000). Multivariate analysis using the Cox regression model indicated that age ≥ 65 years, CA19-9 ≥ 1,000 U/ml and positive S100A11 were independent prognostic indicators of pancreatic adenocarcinoma (P = 0.002, 0.004 and 0.001, respectively). These results suggested that S100A11 might be a significant tumor marker for pancreatic adenocarcinoma and an unfavorable predictor for prognosis of patients who have undergone surgical resection.

Abstract 1658: STAT5b is expressed in human pancreatic cancer and serves as a target for anti-neoplastic therapy

Abstract The transcription factor STAT5b (signal transducer and activator of transcription 5b) is implicated in tumor growth and metastases in various cancer entities including breast and prostate cancer. Activation of STAT5 signaling can be mediated via oncogene mutation (e.g. src kinase), cytokines (e.g. IL-6) and growth factors (e.g. EGF). So far, the role of STAT5b in human pancreatic adenocarcinoma (HPAC) has not been elucidated. Hence, HPAC tissue samples were investigated for nuclear STAT5b expression by immunohistochemical staining. Results show that nuclear expression of STAT5b can be detected in 42/80 HPAC tissue samples. Since specific inhibitors of STAT5b are currently not available, a shRNA approach was chosen for stable knock-down in human pancreatic cancer cells (HPAF-II, BxPC-3, L3.6pl). In vitro, no differences regarding tumor growth were detected by MTT assays and cell counts. However, knock-down of STAT5b led to significant reduction of tumor cell motility as determined in Boyden Chamber assays (P<0.05). Furthermore, upon stimulation with interleukin-6 (IL-6) and epidermal growth factor (EGF) as significant reduction in tumor cell migration was observed (P<0.05). In addition, expression of prometastatic factors Cav-1, MMP-2 and uPAR were impaired upon STAT5b blockade as determined by RT-PCR. In vivo, blockade of STAT5b significantly reduced tumor growth in subcutaneous tumor models (P<0.05). Immunohistochemical analyses of these tumors revealed no differences in tumor cell proliferation (BrdU) whereas vessel area (CD31) was significantly reduced (P<0.05). In conclusion, STAT5b is expressed in almost 50% of HPACs investigated in our study. Since blockade of STAT5b impairs tumor growth and cancer cell motility, this might be an interesting novel target for therapy of human pancreatic adenocarcinoma. 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 1658. doi:10.1158/1538-7445.AM2011-1658