High expression of ALDH1A3 might independently influence poor progression‐free and overall survival in patients with glioma via maintaining glucose uptake and lactate production

Recent studies have found that the acetaldehyde dehydrogenase 1A3 (ALDH1A3) gene is a marker of glioma stem cells. A total of 115 brain glioma specimens were collected and classified into grade I–IV, while non‐tumor brain tissue specimens, taken from 12 patients of vascular malformation surgery, were used as control. ALDH1A3 gene promoter methylation in glioma tissues was detected by pyrosequencing, while immunohistochemistry and western blot were used to detect ALDH1A3 protein expressions in different grades of glioma tissues and normal brain tissues. The expression of ALDH1A3 in the glioma cell line U87 was detected by quantitative real‐time polymerase chain reaction and RNA‐Seq technology was applied to investigate differentially expressed genes before and after silencing the ALDH1A3 gene. Among the 115 glioma tissue specimens, 50 (43.48%) showed low and 65 (56.52%) high expression of ALDH1A3, but no expression was detected in the control. Univariate and multivariate COX regression analyses showed that the patient's tumor pathological grade, the methylation status of ALDH1A3 promoter, and the expression of ALDH1A3 protein were risk factors for progression‐free survival (PFS) and overall survival (OS) (all P < 0.05) and the OS of mice with silenced ALDH1A3 in a glioma nude mouse model was prolonged. U87 experiments revealed that ALDH1A3 expression had significant effects on apoptosis, proliferation, cell cycle, mitochondrial membrane potential, glucose consumption, lactate production, invasion ability, and expression of the pyruvate kinase M2 (PKM2) and hexokinase 2 (HK2) in glioma cells. ALDH1A3 protein expression is a marker for poor PFS and OS in glioma patients.     

Protein kinase D1 promotes anchorage‐independent growth,invasion, and angiogenesis by human pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases. Novel molecularly targeted therapies are urgently needed. Here, we extended our studies on the role of protein kinase D1 (PKD1) in PDAC cell lines. Given that Panc‐1 express moderate levels of PKD1, we used retroviral‐mediated gene transfer to create a Panc‐1 derivative that stably over‐expresses PKD1 (Panc‐1‐PKD1). Reciprocally, we used shRNA targeting PKD1 in Panc‐28 to produce a PKD1 under‐expressing Panc‐28 derivative (Panc‐28‐shPKD1). Our results demonstrate that Panc‐1‐PKD1 cells exhibit significantly increased anchorage‐independent growth in soft agar and increased in vitro invasion compared with Panc‐1‐mock. Reciprocally, Panc‐28‐shPKD1 cells show a significant decrease in anchorage‐independent growth and invasiveness, as compared with Panc‐28‐mock cells. The selective PKD family inhibitor CRT0066101 markedly decreased colony‐forming ability and invasiveness by either Panc‐1‐PKD1 or Panc‐28‐mock cells. Secretion of the pro‐angiogenic factors vascular endothelial growth factor (VEGF) and CXC chemokines (CXCL8) was significantly elevated by PKD1 over‐expression in Panc‐1 cells and reduced either by depletion of PKD1 via shRNA in Panc‐28 cells or by addition of CRT0066101 to either Panc‐1‐PKD1 or Panc‐28‐mock cells. Furthermore, human umbilical vein endothelial cell (HUVEC) tube formation was significantly enhanced by co‐culture with Panc‐1‐PKD1 compared with Panc‐1‐mock in an angiogenesis assay in vitro. Conversely, PKD1 depletion in Panc‐28 cells decreased their ability to induce endotube formation by HUVECs. PDAC‐induced angiogenesis in vitro and in vivo was markedly inhibited by CRT0066101. Our results lend further support to the hypothesis that PKD family members provide a novel target for PDAC therapy. J. Cell. Physiol. 226: 1074–1085, 2011. © 2010 Wiley‐Liss, Inc.     

Targeting ATR in vivo using the novel inhibitor VE-822 results in selective sensitization of pancreatic tumors to radiation

Combined radiochemotherapy is the currently used therapy for locally advanced pancreatic ductal adenocarcinoma (PDAC), but normal tissue toxicity limits its application. Here we test the hypothesis that inhibition of ATR (ATM-Rad3-related) could increase the sensitivity of the cancer cells to radiation or chemotherapy without affecting normal cells. We tested VE-822, an ATR inhibitor, for in vitro and in vivo radiosensitization. Chk1 phosphorylation was used to indicate ATR activity, γH2AX and 53BP1 foci as evidence of DNA damage and Rad51 foci for homologous recombination activity. Sensitivity to radiation (XRT) and gemcitabine was measured with clonogenic assays in vitro and tumor growth delay in vivo. Murine intestinal damage was evaluated after abdominal XRT. VE-822 inhibited ATR in vitro and in vivo. VE-822 decreased maintenance of cell-cycle checkpoints, increased persistent DNA damage and decreased homologous recombination in irradiated cancer cells. VE-822 decreased survival of pancreatic cancer cells but not normal cells in response to XRT or gemcitabine. VE-822 markedly prolonged growth delay of pancreatic cancer xenografts after XRT and gemcitabine-based chemoradiation without augmenting normal cell or tissue toxicity. These findings support ATR inhibition as a promising new approach to improve the therapeutic ration of radiochemotherapy for patients with PDAC.     

Reversing the Intractable Nature of Pancreatic Cancer by Selectively Targeting ALDH-High,Therapy-Resistant Cancer Cells

Human pancreatic ductal adenocarcinoma (PDAC) is a cancer with a dismal prognosis. The efficacy of PDAC anticancer therapies is often short-lived; however, there is little information on how this disease entity so frequently gains resistance to treatment. We adopted the concept of cancer stem cells (CSCs) to explain the mechanism of resistance and evaluated the efficacy of a candidate anticancer drug to target these therapy-resistant CSCs. We identified a subpopulation of cells in PDAC with CSC features that were enriched for aldehyde dehydrogenase (ALDH), a marker expressed in certain stem/progenitor cells. These cells were also highly resistant to, and were further enriched by, treatment with gemcitabine. Similarly, surgical specimens from PDAC patients showed that those who had undergone preoperative chemo-radiation therapy more frequently displayed cancers with ALDH strongly positive subpopulations compared with untreated patients. Importantly, these ALDH-high cancer cells were sensitive to disulfiram, an ALDH inhibitor, when tested in vitro. Furthermore, in vivo xenograft studies showed that the effect of disulfiram was additive to that of low-dose gemcitabine when applied in combination. In conclusion, human PDAC-derived cells that express high levels of ALDH show CSC features and have a key role in the development of resistance to anticancer therapies. Disulfiram can be used to suppress this therapy-resistant subpopulation.     

Retracted: Resistin effects on pancreatic cancer progression and chemoresistance are mediated through its receptors CAP1 and TLR4

Resistin, secreted by macrophages in tumor microenvironment, has never been investigated in pancreatic cancer models, despite a vibrant tumor microenvironment around pancreatic tumors. We evaluated serum resistin levels in healthy individuals versus pancreatic cancer patients representing different tumor grades. In vitro mechanistic analysis involved MiaPaCa-2 and SW1990 cells. Resistin signaling depends on binding of resistin to its cognitive receptors. Therefore, we silenced adenylyl cyclase-associated protein 1 (CAP1) and toll-like receptor 4 (TLR4), its two known receptors, individually as well as in combination, by short hairpin RNA (shRNA). Effect of resistin on cell proliferation, migration, invasion, cell cycle, and sensitivity to gemcitabine was studied without or with silencing of resistin receptors CAP1 and/or TLR4. The results were also confirmed in vivo in mice xenografted with MiaPaCa-2 cells without or with receptor silencing. We report high resistin levels in pancreatic cancer patients which correlate positively with tumor grades. We observed a marked reduction in the resistin-induced proliferation, migration, invasion, and cell cycle of pancreatic cancer cells MiaPaCa-2 and SW1990 when the receptors were silenced. The results were confirmed in vivo wherein resistin effects were significantly attenuated in MiaPaCa-2 xenografts with silenced receptors. The combined silencing of CAP1 and TLR4 was found to be most effective in vitro and in vivo. We found activation of STAT3 by resistin in vivo and in vitro which was dependent on the presence of CAP1 and TLR4. Further, resistin was found to induce resistance to gemcitabine through its receptors. Our results describe novel functional roles of resistin with implications toward a better understanding of pancreatic tumor microenvironment.     

The ARF tumor suppressor inhibits tumor cell colonization independent of p53 in a novel mouse model of pancreatic ductal adenocarcinoma metastasis

Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2, and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging. Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2-invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies.     

Fibulin-3 negatively regulates ALDH1 via c-MET suppression and increases γ-radiation-induced sensitivity in some pancreatic cancer cell lines

Fibulin-3 (FBLN-3) has been postulated to be either a tumor suppressor or promoter depending on the cell type, and hypermethylation of the FBLN-3 promoter is often associated with human disease, especially cancer. We report that the promoter region of the FBLN-3 was significantly methylated (>95%) in some pancreatic cancer cell lines and thus FBLN-3 was poorly expressed in pancreatic cancer cell lines such as AsPC-1 and MiaPaCa-2. FBLN-3 overexpression significantly down-regulated the cellular level of c-MET and inhibited hepatocyte growth factor-induced c-MET activation, which were closely associated with γ-radiation resistance of cancer cells. Moreover, we also showed that c-MET suppression or inactivation decreased the cellular level of ALDH1 isozymes (ALDH1A1 or ALDH1A3), which serve as cancer stem cell markers, and subsequently induced inhibition of cell growth in pancreatic cancer cells. Therefore, forced overexpression of FBLN-3 sensitized cells to cytotoxic agents such as γ-radiation and strongly inhibited the stemness and epithelial to mesenchymal transition (EMT) property of pancreatic cancer cells. On the other hand, if FBLN3 was suppressed in FBLN-3-expressing BxPC3 cells, the results were opposite. This study provides the first demonstration that the FBLN-3/c-MET/ALDH1 axis in pancreatic cancer cells partially modulates stemness and EMT as well as sensitization of cells to the detrimental effects of γ-radiation.     

Relevance of biopsy-derived pancreatic organoids in the development of efficient transcriptomic signatures to predict adjuvant chemosensitivity in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) patients are frequently treated by chemotherapy. Even if personalized therapy based on molecular analysis can be performed for some tumors, PDAC regimens selection is still mainly based on patients' performance status and expected efficacy. Therefore, the establishment of molecular predictors of chemotherapeutic efficacy could potentially improve prognosis by tailoring treatments. We have recently developed an RNA-based signature that predicts the efficacy of adjuvant gemcitabine using 38 PDAC primary cell cultures. While demonstrated its efficiency, a significant association with the classical/basal-like PDAC spectrum was observed. We hypothesized that this flaw was due to the basal-like biased phenotype of cellular models used in our strategy. To overcome this limitation, we generated a prospective cohort of 27 consecutive biopsied derived pancreatic organoids (BDPO) and include them in the signature identification strategy. As BDPO's do not have the same biased phenotype as primary cell cultures we expect they can compensate one with each other and cover a broader range of molecular phenotypes. We then obtained an improved signature predicting gemcitabine sensibility that was validated in a cohort of 300 resected PDAC patients that have or have not received adjuvant gemcitabine. We demonstrated a significant association between the improved signature and the overall and disease-free survival in patients predicted as sensitive and treated with adjuvant gemcitabine. We propose then that including BDPO along primary cell cultures represent a powerful strategy that helps to overcome primary cell cultures limitations producing unbiased RNA-based signatures predictive of adjuvant treatments in PDAC.     

Omeprazole inhibits proliferation and modulates autophagy in pancreatic cancer cells

Background

Omeprazole has recently been described as a modulator of tumour chemoresistance, although its underlying molecular mechanisms remain controversial. Since pancreatic tumours are highly chemoresistant, a logical step would be to investigate the pharmacodynamic, morphological and biochemical effects of omeprazole on pancreatic cancer cell lines.

Methodology/Principal Findings

Dose-effect curves of omeprazole, pantoprazole, gemcitabine, 5-fluorouracil and the combinations of omeprazole and 5-fluorouracil or gemcitabine were generated for the pancreatic cancer cell lines MiaPaCa-2, ASPC-1, Colo357, PancTu-1, Panc1 and Panc89. They revealed that omeprazole inhibited proliferation at probably non-toxic concentrations and reversed the hormesis phenomena of 5-fluorouracil. Electron microscopy showed that omeprazole led to accumulation of phagophores and early autophagosomes in ASPC-1 and MiaPaCa-2 cells. Signal changes indicating inhibited proliferation and programmed cell death were found by proton NMR spectroscopy of both cell lines when treated with omeprazole which was identified intracellularly. Omeprazole modulates the lysosomal transport pathway as shown by Western blot analysis of the expression of LAMP-1, Cathepsin-D and β-COP in lysosome- and Golgi complex containing cell fractions. Acridine orange staining revealed that the pump function of the vATPase was not specifically inhibited by omeprazole. Gene expression of the autophagy-related LC3 gene as well as of Bad, Mdr-1, Atg12 and the vATPase was analysed after treatment of cells with 5-fluorouracil and omeprazole and confirmed the above mentioned results.

Conclusions

We hypothesise that omeprazole interacts with the regulatory functions of the vATPase without inhibiting its pump function. A modulation of the lysosomal transport pathway and autophagy is caused in pancreatic cancer cells leading to programmed cell death. This may circumvent common resistance mechanisms of pancreatic cancer. Since omeprazole use has already been established in clinical practice these results could lead to new clinical applications.     

ENO1-targeted superparamagnetic iron oxide nanoparticles for detecting pancreatic cancer by magnetic resonance imaging

The aim of this study was to investigate in vitro magnetic resonance imaging (MRI) of PDAC using ENO1-targeted superparamagnetic iron oxide nanoparticles and xenograft models. Expression level and location of ENO1 protein in pancreatic cancer cell lines of CFPAC-1 and MiaPaCa-2 were detected by Western blotting, flow cytometry and confocal microscopy. Dex-g-PCL/SPIO nanoparticles targeting ENO1 were constructed with ENO1 antibody and characterized by MRI. In addition, ENO1-Dex-g-PCL/SPIO nanoparticles were tested to assess their efficacy on the detection of PDAC using in vitro and in vivo MRI. The results showed that ENO1 was expressed in both human PDAC cell lines of CFPAC-1 and MiaPaCa-2, demonstrating that the localization of cytoplasm and membrane was dominant. It was confirmed that ENO1 antibody was connected to the SPIO surface in ENO1-Dex-g-PCL/SPIO nanoparticles. The nanoparticles had satisfactory superparamagnetism and significantly enhance the detection of PDAC by in vivo and in vitro MRI. In conclusion, ENO1 can serve as a membrane protein expressed on human PDAC cell lines. ENO1-targeted SPIO nanoparticles using ENO1 antibody can increase the efficiency of detection of PDAC by in vitro and in vivo MRI.     

In Vitro Selective Suppression of Tumor Cells by an Oncolytic Peptide in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a typically violent sort of malignancy and a major source of morbidity and mortality worldwide. Most of the radiation as well as chemotherapeutic agents used for the treatment of PDAC exhibit strong adverse effects with low specificity. Henceforth, a need for an alternative demanded that prompted us to design a novel anticancer peptide (KOR-19) in such a way that it interacts predominantly with cancer cells but exhibits low toxicity with normal mammalian cells. Cell proliferation (XTT) assay and crystal violet assay revealed that the novel designed peptide exhibited vital toxic activities against four different PDAC cell lines (BxPC3, Colo-357, Panc89, and Panc1). On the opposite, this peptide depicted negligible or very low toxicity towards a non-cancerous primary mouse pancreatic stellate cells (MPSC). In the LDH-release assay, we have detected potential membrane damaging properties of KOR-19 as its major mode of action. Further, the hemolytic assay revealed very low or negligible toxic activities towards both mouse- and human RBCs. The flow cytometric analysis demonstrated that the KOR-19 expanded both apoptotic and necrotic cell death in all PDAC cell lines in a dose-dependent manner. Morphological assessments also supported the notion of necrosis assassination of cancer cells as there were elevated swollen vesicle-like structure and cell aggregation noticed in a cell type-dependent manner. In summary, KOR-19 exhibits excellent “druggable” properties due to its promising oncolytic and growth inhibitory activities against PDAC cells, making it a promising agent for the future remedy choice for PDAC.

     

Lipid Peroxidation Plays an Important Role in Chemotherapeutic Effects of Temozolomide and the Development of Therapy Resistance in Human Glioblastoma

BACKGROUND: Glioblastoma (GBM) is the most malignant primary brain tumor. Relapse occurs regularly, and the clinical behavior seems to be due to a therapy-resistant subpopulation of glioma-initiating cells that belong to the group of cancer stem cells. Aldehyde dehydrogenase (ALDH) has been identified as a marker for this cell population, and we have shown previously that ALDH1A3-positive GBM cells are more resistant against temozolomide (TMZ) treatment. However, it is still unclear how ALDH expression mediates chemoresistance. MATERIALS AND METHODS: ALDH1A3 expression was analyzed in 112 specimens from primary and secondary surgical resections of 56 patients with GBM (WHO grade IV). All patients received combined adjuvant radiochemotherapy. For experimental analysis, CRISPR-Cas9–induced knockout cells from three established GBM cell lines (LN229, U87MG, T98G) and two glioma stem-like cell lines were investigated after TMZ treatment. RESULTS: ALDH1A3 knockout cells were more sensitive to TMZ, and oxidative stress seemed to be the molecular process where ALDH1A3 exerts its role in resistance against TMZ. Oxidative stress led to lipid peroxidation, yielding active aldehydes that were detoxified by ALDH enzymatic activity. During the metabolic process, autophagy was induced leading to downregulation of the enzyme, but ALDH1A3 is upregulated to even higher expression levels after finishing the TMZ therapy in vitro. Recurrent GBMs show significantly higher ALDH1A3 expression than the respective samples from the primary tumor, and patients suffering from GBM with high ALDH1A3 expression showed a shorter median survival time (12 months vs 21 months, P < .05). CONCLUSION: Oxidative stress is an important and clinically relevant component of TMZ-induced therapeutic effects. Cytotoxicity seems to be mediated by aldehydes resulting from lipid peroxidation, and ALDH1A3 is able to reduce the number of toxic aldehydes. Therefore, we present a molecular explanation of the role of ALDH1A3 in therapeutic resistance of human GBM cells.     

Plk1 inhibition enhances the efficacy of gemcitabine in human pancreatic cancer

Gemcitabine is the standard-of-care for chemotherapy in patients with pancreatic adenocarcinoma and it can directly incorporate into DNA or inhibit ribonucleotide reductase to prevent DNA replication and, thus, tumor cell growth. Most pancreatic tumors, however, develop resistance to gemcitabine. Polo-like kinase 1 (Plk1), a critical regulator in many cell cycle events, is significantly elevated in human pancreatic cancer. In this study, we show that Plk1 is required for the G1/S transition and that inhibition of Plk1 significantly reduces the DNA synthesis rate in human pancreatic cancer cells. Furthermore, the combined effect of a specific Plk1 inhibitor GSK461364A with gemcitabine was examined. We show that inhibition of Plk1 significantly potentiates the anti-neoplastic activity of gemcitabine in both cultured pancreatic cancer cells and Panc1-derived orthotopic pancreatic cancer xenograft tumors. Overall, our study demonstrates that co-targeting Plk1 can significantly enhance the efficacy of gemcitabine, offering a promising new therapeutic option for the treatment of gemcitabine-resistant human pancreatic cancer.     

The anti-myeloma activity of a novel purine scaffold HSP90 inhibitor PU-H71 is via inhibition of both HSP90A and HSP90B1

Background

The ribonucleotide reductase M1 (RRM1) gene encodes the regulatory subunit of ribonucleotide reductase, the molecular target of gemcitabine. The overexpression of RRM1 mRNA in tumor tissues is reported to be associated with gemcitabine resistance. Thus, single nucleotide polymorphisms (SNPs) of the RRM1 gene are potential biomarkers of the response to gemcitabine chemotherapy. We investigated whether RRM1 expression in peripheral blood mononuclear cells (PBMCs) or SNPs were associated with clinical outcome after gemcitabine-based chemotherapy in advanced non-small cell lung cancer (NSCLC) patients.

Methods

PBMC samples were obtained from 62 stage IIIB and IV patients treated with gemcitabine-based chemotherapy. RRM1 mRNA expression levels were assessed by real-time PCR. Three RRM1 SNPs, -37C→A, 2455A→G and 2464G→A, were assessed by direct sequencing.

Results

RRM1 expression was detectable in 57 PBMC samples, and SNPs were sequenced in 56 samples. The overall response rate to gemcitabine was 18%; there was no significant association between RRM1 mRNA expression and response rate (P = 0.560). The median progression-free survival (PFS) was 23.3 weeks in the lower expression group and 26.9 weeks in the higher expression group (P = 0.659). For the -37C→A polymorphism, the median PFS was 30.7 weeks in the C(-)37A group, 24.7 weeks in the A(-)37A group, and 23.3 weeks in the C(-)37C group (P = 0.043). No significant difference in PFS was observed for the SNP 2455A→G or 2464G→A.

Conclusions

The RRM1 polymorphism -37C→A correlated with PFS in NSCLC patients treated with gemcitabine-based chemotherapy. No significant correlation was found between PBMC RRM1 mRNA expression and the efficacy of gemcitabine.     

Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK,DNA Synthesis and Proliferation in Pancreatic Cancer Cells

Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2) with the isoquinoline alkaloid berberine (0.3–6 µM) inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC) at Ser⁷⁹. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr³⁸⁹ and S6 at Ser^240/244) and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α₁ and α₂ catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK) and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.