Target For Pancreatic Cancer Therapy Research Articles

CXCL12–CXCR4 signalling axis confers gemcitabine resistance to pancreatic cancer cells: a novel target for therapy

Background:Pancreatic cancer cells are highly resistant to drug therapy; however, underlying causes remain largely unknown. We hypothesised that the activation of CXCL12–CXCR4 signalling confers drug resistance to pancreatic cancer cells by potentiating survival. CXCR4 is overexpressed in precancerous/malignant pancreatic lesions and cancer stem cells, and implicated in its pathogenesis.Methods:Effect of CXCR4 activation by CXCL12 on restricting the gemcitabine-induced cytotoxicity and stimulating the survival signalling was examined in pancreatic cancer cells by MTT, DNA laddering, caspase activity, immunoblot, and promoter-reporter assays. Subsequently, we examined the effect of CXCR4 antagonist, AMD3100, in abrogating the rescue effect of activated CXCL12–CXCR4 signalling.Results:The pancreatic cancer cells treated with gemcitabine exhibited reduced cytotoxicity in the presence of CXCL12 as compared with the cells treated with drug alone. CXCL12 induced the activation of FAK, ERK, and Akt signalling pathways, enhanced transcriptional activities of β-catenin and NF-κB, and expression of survival proteins. AMD3100 arrested the CXCL12-induced pancreatic cancer cell growth and drug resistance.Conclusion:Our findings demonstrate, for the first time, a role of CXCL12–CXCR4 signalling axis in conferring drug resistance to pancreatic cancer cells and suggest that it could serve as a novel therapeutic target for pancreatic cancer therapy, alone and in combination with the cytotoxic drug.

Amphiregulin regulates the activation of ERK and Akt through epidermal growth factor receptor and HER3 signals involved in the progression of pancreatic cancer

Pancreatic cancer is one of the most lethal malignancies. Epidermal growth factor receptor (EGFR), HER3, Akt, and amphiregulin have been recognized as targets for pancreatic cancer therapy. Although gemcitabine + erlotinib has been the recommended chemotherapy for pancreatic cancer, the prognosis is extremely poor. The development of molecularly targeted therapies has been required for patients with pancreatic cancer. To assess the validation of amphiregulin as a target for pancreatic cancer therapy, we examined its expression in pancreatic cancer using real-time PCR analyses and ELISA. We also measured the apoptotic cell rate using TUNEL assays. In addition, alterations in signaling pathways were detected by immunoblotting analyses. Treatment with gemcitabine, which reduced the cell viability and augmented the cell apoptotic rate, activated and subsequently attenuated ERK and EGFR signals. However, gemcitabine, paclitaxel, or cisplatin treatment enhanced the Akt activation, heterodimer formation of EGFR with HER3, and secretion of amphiregulin, indicating that the presence of gemcitabine promoted the activity of targeted molecules including amphiregulin, Akt, and HER3 for pancreatic cancer therapy. Combined treatment with an inhibitor for amphiregulin and gemcitabine, paclitaxel, or cisplatin induced synergistic antitumor effects, accompanied by the suppression of Akt and ERK activation. Blockade of amphiregulin suppressed the activities of EGFR, HER3, and Akt and the expression of amphiregulin itself. According to this evidence, combination chemotherapy of conventional anticancer drugs plus an inhibitor for amphiregulin would allow us to provide more favorable clinical outcomes for patients with pancreatic cancer.

Prognostic significance of xCT gene single nucleotide polymorphisms (SNPs) in patients (pts) with advanced pancreatic cancer (PC) treated with gemcitabine (GEM) plus platinum analogues (PLAT).

4065 Background: The plasma membrane xc- cystine/glutamate transporter mediates cellular uptake of cystine in exchange for intracellular glutamate and is highly expressed by PC cells. The xCT gene, encoding the cystine-specific xCT protein subunit of xc-, is important in regulating intracellular glutathione levels, critical for cancer cell protection against oxidative stress, tumor growth and resistance to chemotherapeutic agents including PLAT. Methods: We have investigated the relationship between xCT gene SNPs and overall survival (OS) of pts with PC treated with GEM ± PLAT. We examined 4 SNPs of the xCT gene in 269 advanced PC pts who received first line GEM with or without cisplatin or oxaliplatin. Genomic DNA was isolated from peripheral blood mononuclear cells. Genotyping was performed using Taqman real-time PCR assays. The association between SNPs and OS was analyzed by Kaplan-Meier plot, log-rank test and Cox proportional hazard regression models and corrected for previously identified prognostic factors, including CA 19-9, stage, and performance status. Results: A statistically significant correlation was noted between the 3’ untranslated region (UTR) xCT SNP rs7674870 and OS: Median survival time (MST) was 10.9 and 13.6 months, respectively, for the TT and TC/CC genotypes (p = 0.027). Stratified analysis showed the genotype effect was significant in pts receiving GEM+PLAT therapy (n = 145): MST was 10.5 vs. 14.1 months for the TT and TC/CC genotypes, respectively (p = 0.013). This effect remained statistically significant after adjusting for clinical factors (hazard ratio: 0.60, 95% CI, 0.40-0.89 and p = 0.011). The genotype effect was not significant in pts receiving GEM alone (n = 124): MST was 10.9 months for TT and 12.0 months for TC/CC genotypes (p = 0.47). None of the 3 synonymous SNPs examined were associated with outcome. Conclusions: The 3’ UTR xCT SNP rs7674870 may correlate with OS in PC pts receiving GEM+PLAT therapy. These data suggest that polymorphic variants of xCT may have predictive value, and that the xc- transporter may represent an important target for therapy in PC. No significant financial relationships to disclose.

PAR-4 as a possible new target for pancreatic cancer therapy

Importance of the field: Pancreatic cancer (PC) is a deadly disease that is intractable to currently available treatment regimens. Although well described in different tumors types, the importance of apoptosis inducer prostate apoptosis response-4 (Par-4) in PC has not been appreciated. PC is an oncogenic kras driven disease, which is known to downregulate Par-4. Therefore, this review highlights its significance and builds a strong case supporting the role of Par-4 as a possible therapeutic target in PC.Areas covered in this review: Literature-based evidence spanning the last 15 years on Par-4 and its significance in PC.What the reader will gain: This review provides comprehensive knowledge of the significance of Par-4 and its association with kras status in PC, along with the crosstalk with crucial resistance and survival molecules NF-κB and Bcl-2 that ultimately are responsible for the overall poor outcome of different therapeutic approaches in this disease.Take home message: Par-4 holds promise as a potential therapeutic target that can be induced by chemopreventive agents and small-molecule inhibitors either alone or in combination with standard chemotherapeutics leading to selective apoptosis in PC cells. It also acts as a chemosensitizer and therefore warrants further clinical investigations in this disease.

Crosstalk between Insulin/Insulin-like Growth Factor-1 Receptors and G Protein-Coupled Receptor Signaling Systems: A Novel Target for the Antidiabetic Drug Metformin in Pancreatic Cancer

Insulin/insulin-like growth factor 1(IGF-1) receptors and G protein-coupled receptors (GPCR) signaling systems are implicated in autocrine-paracrine stimulation of a variety of malignancies, including ductal adenocarcinoma of the pancreas, one of the most lethal human diseases. Novel targets for pancreatic cancer therapy are urgently needed. We identified a crosstalk between insulin/IGF-1 receptors and GPCR signaling systems in pancreatic cancer cells, leading to enhanced signaling, DNA synthesis, and proliferation. Crosstalk between these signaling systems depends on mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Metformin, the most widely used drug in the treatment of type 2 diabetes, activates AMP kinase (AMPK), which negatively regulates mTORC1. Recent results show that metformin-induced activation of AMPK disrupts crosstalk between insulin/IGF-1 receptor and GPCR signaling in pancreatic cancer cells and inhibits the growth of these cells in xenograft models. Given that insulin/IGF-1 and GPCRs are implicated in other malignancies, a similar crosstalk mechanism may be operative in other cancer cell types. Recent epidemiological studies linked administration of metformin with a reduced risk of pancreatic, breast, and prostate cancer in diabetic patients. We posit that crosstalk between insulin/IGF-1 receptor and GPCR signaling is a mechanism for promoting the development of certain types of cancer and a target for the prevention and therapy of these diseases via metformin administration.

Targeting Notch signaling in pancreatic cancer

Importance of the field: With some 220,000 new cases/year in the world, pancreatic adenocarcinoma is the fourth highest cause of death by cancers. Among newly diagnosed patients about 210,000 will die within 9 months following diagnosis. Therefore, effective adjuncts to current treatment strategies are necessary. Because embryological signaling pathways are upregulated in pancreatic adenocarcinoma, they represent potential targets for future therapies.Areas covered in this review: Our aim is to present the Notch pathway, and to describe its involvement in pancreatic pathophysiology/carcinogenesis. This pathway appeared as a prime target for pancreatic cancer therapy. In the light of the crosstalk of Notch with other survival/embryologic pathways, drugs affecting more than one pathway may have to be combined.What the reader will gain: Drugs against γ-secretases could thus serve in cancer treatment and can be combined with drugs targeting survival pathways interplaying with Notch such as Hedgehog.Take home message: Downregulation of Notch contributes to the inhibition and apoptosis of pancreatic cancer cells whereas Hedgehog inhibition will allow for enhanced delivery of drugs to the tumor. Both pathway inhibitors appear to have synergistic effects for future therapeutics for pancreatic adenocarcinoma, once safety issues of compounds are overcome.

Masitinib Combined with Standard Gemcitabine Chemotherapy: In Vitro and In Vivo Studies in Human Pancreatic Tumour Cell Lines and Ectopic Mouse Model

BackgroundTyrosine kinases are attractive targets for pancreatic cancer therapy because several are over-expressed, including PDGFRα/β, FAK, Src and Lyn. A critical role of mast cells in the development of pancreatic cancer has also been reported. Masitinib is a tyrosine kinase inhibitor that selectively targets c-Kit, PDGFRα/β, Lyn, and to a lesser extent the FAK pathway, without inhibiting kinases of known toxicities. Masitinib is particularly efficient in controlling the proliferation, differentiation and degranulation of mast cells. This study evaluates the therapeutic potential of masitinib in pancreatic cancer, as a single agent and in combination with gemcitabine.Methodology/FindingsProof-of-concept studies were performed in vitro on human pancreatic tumour cell lines and then in vivo using a mouse model of human pancreatic cancer. Molecular mechanisms were investigated via gene expression profiling. Masitinib as a single agent had no significant antiproliferative activity while the masitinib/gemcitabine combination showed synergy in vitro on proliferation of gemcitabine-refractory cell lines Mia Paca2 and Panc1, and to a lesser extent in vivo on Mia Paca2 cell tumour growth. Specifically, masitinib at 10 µM strongly sensitised Mia Paca2 cells to gemcitabine (>400-fold reduction in IC50); and moderately sensitised Panc1 cells (10-fold reduction). Transcriptional analysis identified the Wnt/β-catenin signalling pathway as down-regulated in the cell lines resensitised by the masitinib/gemcitabine combination.ConclusionsThese data establish proof-of-concept that masitinib can sensitise gemcitabine-refractory pancreatic cancer cell lines and warrant further in vivo investigation. Indeed, such an effect has been recently observed in a phase 2 clinical study of patients with pancreatic cancer who received a masitinib/gemcitabine combination.

Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Pancreatic cancer is the fourth leading cause of cancer deaths in the United States, with an overall 5-year survival rate of <5%. Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is highly resistant to conventional chemotherapies, underscoring the critical need for new molecular targets for pancreatic cancer chemotherapy. The KRAS proto-oncogene is mutated in >90% of PDAC. Protein kinase Ciota (PKCiota) is required for the oncogenic Ras-mediated transformed growth of lung cancer and intestinal epithelial cells. However, little is known about the role of PKCiota in pancreatic cancer. In this study, we evaluated the expression of PKCiota in human pancreatic cancer and the requirement for PKCiota for the transformed growth and tumorigenicity of PDAC cells. We find that PKCiota is significantly overexpressed in human pancreatic cancer, and high PKCiota expression correlates with poor patient survival. Inhibition of PKCiota expression blocks PDAC cell transformed growth in vitro and tumorigenicity in vivo. Inhibition of PKCiota expression in pancreatic tumors also significantly reduces tumor angiogenesis and metastasis. Analysis of downstream PKCiota effectors implicates the Rac1-MEK/ERK1/2 signaling axis in PKCiota-mediated transformed growth and cellular invasion. Taken together, our data show a required role for PKCiota in the transformed growth of pancreatic cancer cells and reveal a novel role for PKCiota in pancreatic cancer cell metastasis and angiogenesis in vivo. Our results strongly indicate that PKCiota will be an effective target for pancreatic cancer therapy.

Validation of TPX2 as a Potential Therapeutic Target in Pancreatic Cancer Cells

The targeting protein for Xklp2 (TPX2) has recently gained attention as a putative oncogene possibly amplified in several human malignancies, including pancreatic adenocarcinoma. In this work, we sought to evaluate the copy number and expression of TPX2 in pancreatic cancer cell lines and tumor tissues and to further explore the potential of TPX2 as a therapeutic target. The DNA copy number and expression of the TPX2 gene were surveyed in pancreatic cancer cell lines and tumor tissues and compared with those of immortalized normal pancreatic ductal cells and normal pancreatic tissues. The cellular effects of TPX2 knockdown using small interfering RNA oligonucleotides in pancreatic cancer cells, such as growth in tissue culture, in soft agar, and in nude mice; apoptosis; and sensitivity to paclitaxel, were also investigated using various assays. Low-copy-number TPX2 amplification was found in pancreatic cancer cell lines and low-passage pancreatic cancer tumor xenografts. TPX2 expression was upregulated in pancreatic cancer cell lines at both the mRNA and protein levels relative to the immortalized pancreatic ductal epithelial cell line HPDE6. Immunohistochemical staining of a tissue microarray showed that TPX2 expression was higher in pancreatic tumors compared with their normal counterparts. Treatment with TPX2 targeting small interfering RNAs effectively reduced pancreatic cancer cell growth in tissue culture, induced apoptosis, and inhibited growth in soft agar and in nude mice. Knockdown of TPX2 also sensitized pancreatic cancer cells to paclitaxel treatment. Our results suggest that TPX2 might be an attractive target for pancreatic cancer therapy.

Endocrine glands-derived vascular endothelial growth factor protects pancreatic cancer cells from apoptosis via upregulation of the myeloid cell leukemia-1 protein

Endocrine glands-derived vascular endothelial growth factor (EG-VEGF, also termed as Prok1)—a novel cytokine that selectively acts on the endothelial cells of endocrine glands—was recently reported to be involved in the regulation of tumor cell growth and survival. However, its roles in the regulation of pancreatic cancer progression remain unclear. In this report, we investigated the suppressive effects of EG-VEGF on pancreatic cancer cell apoptosis and the relevant mechanisms. By using reverse-transcriptase polymerase chain reaction (RT-PCR) we found that the Mia PaCa II cells of the pancreatic cancer cell line express the mRNAs of both EG-VEGF (Prok1) and its receptors. EG-VEGF protects pancreatic cancer cells from apoptosis through upregulation of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein of the bcl-2 family. Treatment of pancreatic cancer cells with EG-VEGF results in the rapid phosphorylation of mitogen-activated protein kinase (MAPK), STAT3, and AKT, which are involved in the upregulation of Mcl-1 expression. EG-VEGF (Prok1) protects Mia PaCa II cells from apoptosis through G protein-coupled receptor (GPR)-induced activation of multiple signal pathways, and hence can be a novel target for pancreatic cancer therapy.

Identification of C2orf18, termed ANT2BP (ANT2‐binding protein), as one of the key molecules involved in pancreatic carcinogenesis

Pancreatic ductal adenocarcinoma (PDAC) shows one of the worst mortality rates among the common malignancies, and the great majority of PDAC patients are diagnosed at an advanced stage where no effective therapy is presently available. Hence, identification of novel molecular targets and development of molecular therapy for PDAC are urgently required. Through our genome-wide gene expression profiles of microdissected PDAC cells, we here identified a novel gene C2orf18 as a molecular target for PDAC treatment. Transcriptional and immunohistochemical analysis validated its overexpression in PDAC cells and limited expression in normal adult organs. Knockdown of C2orf18 by small-interfering RNA in PDAC cell lines resulted in induction of apoptosis and suppression of cancer cell growth, suggesting its essential role in maintaining viability of PDAC cells. We showed that C2orf18 was localized in the mitochondria and it could interact with adenine nucleotide translocase 2 (ANT2), which is involved in maintenance of the mitochondrial membrane potential and energy homeostasis, and was indicated some roles in apoptosis. These findings implicated that C2orf18, termed ANT2-binding protein (ANT2BP), might serve as a candidate molecular target for pancreatic cancer therapy.

Activated Epidermal Growth Factor Receptor as a Novel Target in Pancreatic Cancer Therapy

Pancreatic cancer is one of the most fatal among all solid malignancies. Targeted therapeutic approaches have the potential to transform cancer therapy as exemplified by the success of several tyrosine kinase inhibitors. Prompted by this, comprehensive profiling of tyrosine kinases and their substrates was carried out using a panel of low passage pancreatic cancer cell lines. One of the pancreatic cancer cell lines, P196, which showed dramatic upregulation of tyrosine kinase activity as compared to non-neoplastic cells, was systematically studied using a quantitative proteomic approach called stable isotope labeling with amino acids in cell culture (SILAC). A careful analysis of activated tyrosine kinase pathways revealed aberrant activation of epidermal growth factor receptor pathway in this cell line. Mouse xenograft based studies using EGFR inhibitor erlotinib confirmed EGFR pathway to be responsible for proliferation in these tumors. By a systematic study across low passage pancreatic cancer cell lines and mice carrying pancreatic cancer xenografts, we have demonstrated activated epidermal growth factor receptor as an attractive candidate for targeted therapy in a subset of pancreatic cancers. Further, we propose immunohistochemical labeling of activated EGFR (pEGFR (1068)) as an efficient screening tool to select patients who are more likely to respond to EGFR inhibitors.

ADAM8 expression is associated with increased invasiveness and reduced patient survival in pancreatic cancer

ADAM8 belongs to a family of transmembrane proteins implicated in cell–cell interactions, proteolysis of membrane proteins, and various aspects of carcinogenesis. In the present study, we aimed to evaluate the expression and function of ADAM8 in pancreatic cancer. ADAM8 mRNA levels were analysed by quantitative RT-PCR and correlated to patient survival. Immunohistochemistry was performed to localize ADAM8 in pancreatic tis-sues. Silencing of ADAM8 expression was carried out by transfection with specific siRNA oligonucleotides. Cell growth and invasion assays were used to assess the functional consequences of ADAM8 silencing. SELDI-TOF-MS was performed to detect the proteolytic activity of ADAM8 in pancreatic cancer cells. ADAM8 mRNA was significantly overexpressed in pancreatic ductal adenocarcinoma (PDAC) compared with normal pancreatic tissues (5.3-fold increase; P= 0.0008), and high ADAM8 mRNA and protein expression levels correlated with reduced survival time of PDAC patients (P= 0.048 and P= 0.065, respectively). Silencing of ADAM8 expression did not significantly influence pancreatic cancer cell growth but suppressed invasiveness. In addition, decreased proteolytic activity was measured in cell culture supernatants following silencing of ADAM8. In conclusion, ADAM8 is overexpressed in PDAC, influences cancer cell invasiveness and correlates with reduced survival, suggesting that ADAM8 might be a potential target in pancreatic cancer therapy.

Activated epidermal growth factor receptor as a novel target in pancreatic cancer therapy

Pancreatic cancer is one of the most devastating and rapidly fatal of all solid malignancies. The main reasons for such high mortality rates have been the late presentation of the disease and lack of effective therapies. Targeted therapeutic approaches have the potential to transform cancer therapy as exemplified by the success of several tyrosine kinase inhibitors. Prompted by this, comprehensive profiling of tyrosine kinase pathways was carried out in a subset of pancreatic cancers using a quantitative proteomic approach called stable isotope labeling with amino acids in cell culture (SILAC). By a systematic study across low passage pancreatic cancer cell lines and mice bearing pancreatic cancer xenografts, we demonstrate activated epidermal growth factor receptor (EGFR) as a candidate for targeted therapy in a subset of pancreatic cancers. Further, we establish immunohistochemical labeling of activated epidermal growth factor receptor (phosphoEGFR) as an efficient screening tool to select patients who are more likely to respond to EGFR inhibitors.

EphA4 receptor, overexpressed in pancreatic ductal adenocarcinoma, promotes cancer cell growth

To isolate novel diagnostic markers and drug targets for pancreatic ductal adenocarcinoma (PDAC), we previously performed expression profile analysis of PDAC cells using a genome-wide cDNA microarray combined with laser microdissection. Among dozens of up-regulated genes identified in PDAC cells, we herein focused on one tyrosine kinase receptor, Eph receptor A4 (EphA4), as a molecular target for PDAC therapy. Immunohistochemical analysis validated EphA4 overexpression in approximately half of the PDAC tissues. To investigate its biological function in PDAC cells, we knocked down EphA4 expression by siRNA, which drastically attenuated PDAC cell viability. In concordance with the siRNA experiment, PDAC-derivative cells that were designed to constitutively express exogenous EphA4 showed a more rapid growth rate than cells transfected with mock vector, suggesting a growth-promoting effect of EphA4 on PDAC cells. Furthermore, the expression analysis for ephrin ligand family members indicated the coexistence of ephrinA3 ligand in PDAC cells with EphA4 receptor, and knockdown of ephrinA3 by siRNA also attenuated PDAC cell viability. These results suggest that the EphA4-ephrinA3 pathway is likely to be a promising molecular target for pancreatic cancer therapy.

TIMP-1 antisense gene transfection attenuates the invasive potential of pancreatic cancer cells in vitro and inhibits tumor growth in vivo

Background TIMP-1 overexpression decreases the invasive potential of pancreatic cancer cells. By tissue inhibitors of metalloproteinase (TIMP)-1 antisense gene transfection, we expected to produce aggressive pancreatic cancer cells with increased in vitro and in vivo invasive potential. Methods PANC-1 cells were transfected with either TIMP-1 gene (CD-1), antisense TIMP-1 gene (AS-3), or empty vector (MB-3). The in vitro cell growth kinetics and invasive potential of each cell line were compared. Total and active matrix metalloproteinase (MMP)-2 levels were determined. Each cell line was then implanted in athymic mice and the resultant tumors were compared for size, weight, MMP activity, and TIMP-1 expression. Results TIMP-1 modulation did not affect cell proliferation in vitro, but its underexpression and, to a lesser extent, overexpression resulted in attenuated tumor growth in vivo. AS-3 cells showed marked decreases in cell invasion and MMP-2 activity in vitro and in vivo. Conclusion TIMP-1 manipulation, particularly underexpression, greatly reduces the invasive potential of pancreatic cancer by limiting MMP-2 activity without affecting in vitro cell growth. TIMP-1 is a reasonable molecular target in pancreatic cancer therapy.

Complications, continence and quality of life after an intensive preoperative radio-chemotherapy regimen for locally advanced rectal cancer: analysis of a phase I–II study

Complications, continence and quality of life after an intensive preoperative radio-chemotherapy regimen for locally advanced rectal cancer: analysis of a phase I–II study

MTOR signaling is a possible new biochemical target for pancreatic cancer therapy

mTOR signaling is a possible new biochemical target for pancreatic cancer therapy