3D imaging analysis on an organoid-based platform guides personalized treatment in pancreatic ductal adenocarcinoma.

BACKGROUNDPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with unpredictable responses to chemotherapy. Approaches to assay patient tumors before treatment and identify effective treatment regimens based on tumor sensitivities are lacking. We developed an organoid-based platform (OBP) to visually quantify patient-derived organoid (PDO) responses to drug treatments and associated tumor-stroma modulation for personalized PDAC therapy.METHODSWe retrospectively quantified apoptotic responses and tumor-stroma cell proportions in PDOs via 3D immunofluorescence imaging through annexin A5, α-smooth muscle actin (α-SMA), and cytokeratin 19 (CK-19) levels. Simultaneously, an ex vivo organoid drug sensitivity assay (ODSA) was used to measure responses to standard-of-care regimens. Differences between ODSA results and patient tumor responses were assessed by exact McNemar's test.RESULTSImmunofluorescence signals, organoid growth curves, and Ki-67 levels were measured and authenticated through the OBP for up to 14 days. ODSA drug responses were not different from patient tumor responses, as reflected by CA19-9 reductions following neoadjuvant chemotherapy (P = 0.99). PDOs demonstrated unique apoptotic and tumor-stroma modulation profiles (P < 0.0001). α-SMA/CK-19 ratio levels of more than 1.0 were associated with improved outcomes (P = 0.0179) and longer parental patient survival by Kaplan-Meier analysis (P = 0.0046).CONCLUSIONHeterogenous apoptotic drug responses and tumor-stroma modulation are present in PDOs after standard-of-care chemotherapy. Ratios of α-SMA and CK-19 levels in PDOs are associated with patient survival, and the OBP could aid in the selection of personalized therapies to improve the efficacy of systemic therapy in patients with PDAC.FUNDINGNIH/National Cancer Institute grants (K08CA218690, P01 CA117969, R50 CA243707-01A1, U54CA224065), the Skip Viragh Foundation, the Bettie Willerson Driver Cancer Research Fund, and a Cancer Center Support Grant for the Flow Cytometry and Cellular Imaging Core Facility (P30CA16672).

Compound NSC84167 selectively targets NRF2-activated pancreatic cancer by inhibiting asparagine synthesis pathway.

Nuclear factor erythroid 2-related factor 2 (NRF2) is aberrantly activated in about 93% of pancreatic cancers. Activated NRF2 regulates multiple downstream molecules involved in cancer cell metabolic reprogramming, translational control, and treatment resistance; however, targeting NRF2 for pancreatic cancer therapy remains largely unexplored. In this study, we used the online computational tool CellMinerTM to explore the NCI-60 drug databases for compounds with anticancer activities correlating most closely with the mRNA expression of NQO1, a marker for NRF2 pathway activity. Among the >100,000 compounds analyzed, NSC84167, termed herein as NRF2 synthetic lethality compound-01 (NSLC01), was one of the top hits (r = 0.71, P < 0.001) and selected for functional characterization. NSLC01 selectively inhibited the viabilities of four out of seven conventional pancreatic cancer cell lines and induced dramatic apoptosis in the cells with high NRF2 activation. The selective anticancer activity of NSLC01 was further validated with a panel of nine low-passage pancreatic patient-derived cell lines, and a significant reverse correlation between log(IC50) of NSLC01 and NQO1 expression was confirmed (r = -0.5563, P = 0.024). Notably, screening of a panel of nine patient-derived xenografts (PDXs) revealed six PDXs with high NQO1/NRF2 activation, and NSLC01 dramatically inhibited the viabilities and induced apoptosis in ex vivo cultures of PDX tumors. Consistent with the ex vivo results, NSLC01 inhibited the tumor growth of two NRF2-activated PDX models in vivo (P < 0.01, n = 7-8) but had no effects on the NRF2-low counterpart. To characterize the mechanism of action, we employed a metabolomic isotope tracer assay that demonstrated that NSLC01-mediated inhibition of de novo synthesis of multiple amino acids, including asparagine and methionine. Importantly, we further found that NSLC01 suppresses the eEF2K/eEF2 translation elongation cascade and protein translation of asparagine synthetase. In summary, this study identified a novel compound that selectively targets protein translation and induces synthetic lethal effects in NRF2-activated pancreatic cancers.

DDR1-induced neutrophil extracellular traps drive pancreatic cancer metastasis.

Pancreatic ductal adenocarcinoma (PDAC) tumors are characterized by a desmoplastic reaction resulting in dense deposition of collagen that is known to promote cancer progression. A central mediator of protumorigenic collagen signaling is the receptor tyrosine kinase discoid domain receptor 1 (DDR1). DDR1 is a critical driver of a mesenchymal and invasive cancer cell PDAC phenotype. Previous studies have demonstrated that genetic or pharmacologic inhibition of DDR1 reduces PDAC tumorigenesis and metastasis. Here, we investigated whether DDR1 signaling has cancer cell nonautonomous effects that promote PDAC progression and metastasis. We demonstrate that collagen-induced DDR1 activation in cancer cells is a major stimulus for CXCL5 production, resulting in the recruitment of tumor-associated neutrophils (TANs), the formation of neutrophil extracellular traps (NETs), and subsequent cancer cell invasion and metastasis. Moreover, we have identified that collagen-induced CXCL5 production was mediated by a DDR1/PKCθ/SYK/NF-κB signaling cascade. Together, these results highlight the critical contribution of the collagen I-DDR1 interaction in the formation of an immune microenvironment that promotes PDAC metastasis.

Oncogenic KRAS Recruits an Expansive Transcriptional Network through Mutant p53 to Drive Pancreatic Cancer Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most TP53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis, yet the extent of direct cooperation between KRAS effectors and mutant p53 remains largely undefined. We show that oncogenic KRAS effectors activate CREB1 to allow physical interactions with mutant p53 that hyperactivate multiple prometastatic transcriptional networks. Specifically, mutant p53 and CREB1 upregulate the prometastatic, pioneer transcription factor FOXA1, activating its transcriptional network while promoting WNT/ß-catenin signaling, together driving PDAC metastasis. Pharmacologic CREB1 inhibition dramatically reduced FOXA1 and ß-catenin expression and dampened PDAC metastasis, identifying a new therapeutic strategy to disrupt cooperation between oncogenic KRAS and mutant p53 to mitigate metastasis. SIGNIFICANCE: Oncogenic KRAS and mutant p53 are the most commonly mutated oncogene and tumor suppressor gene in human cancers, yet direct interactions between these genetic drivers remain undefined. We identified a cooperative node between oncogenic KRAS effectors and mutant p53 that can be therapeutically targeted to undermine cooperation and mitigate metastasis.This article is highlighted in the In This Issue feature, p. 1861.

Rational combination with PDK1 inhibition overcomes cetuximab resistance in head and neck squamous cell carcinoma.

Cetuximab, an EGFR-blocking antibody, is currently approved for treatment of metastatic head and neck squamous cell carcinoma (HNSCC), but its response rate is limited. In addition to blocking EGFR-stimulated cell signaling, cetuximab can induce endocytosis of ASCT2, a glutamine transporter associated with EGFR in a complex, leading to glutathione biosynthesis inhibition and cellular sensitization to ROS. Pyruvate dehydrogenase kinase-1 (PDK1), a key mitochondrial enzyme overexpressed in cancer cells, redirects glucose metabolism from oxidative phosphorylation toward aerobic glycolysis. In this study, we tested the hypothesis that targeting PDK1 is a rational approach to synergize with cetuximab through ROS overproduction. We found that combination of PDK1 knockdown or inhibition by dichloroacetic acid (DCA) with ASCT2 knockdown or with cetuximab treatment induced ROS overproduction and apoptosis in HNSCC cells, and this effect was independent of effective inhibition of EGFR downstream pathways but could be lessened by N-acetyl cysteine, an anti-oxidative agent. In several cetuximab-resistant HNSCC xenograft models, DCA plus cetuximab induced marked tumor regression, whereas either agent alone failed to induce tumor regression. Our findings call for potentially novel clinical trials of combining cetuximab and DCA in patients with cetuximab-sensitive EGFR-overexpressing tumors and patients with cetuximab-resistant EGFR-overexpressing tumors.

Hypoxia-Induced Autophagy Degrades Stromal Lumican into Tumor Microenvironment of Pancreatic Ductal Adenocarcinoma: A Mini-Review.

The extracellular matrix (ECM) in the tumor microenvironment (TME) has gained considerable interest in recent years as a crucial component in fundamental cellular processes and provides novel therapeutic targets. Lumican is a class II small leucine-rich proteoglycan with a key role in ECM organization and modulation of biological functions dependent on tumor type, abundance, and stage of disease. The presence of stromal lumican in the ECM surrounding pancreatic ductal adenocarcinoma (PDAC) inhibits cancer cell replication and is associated with improved patient outcomes after multimodal therapies. In this mini-review, were-present our novel findings describing how hypoxia (1% O2) within the TME influences stromal lumican expression and secretion. We observed that hypoxia specifically inhibited lumican expression and secretion post-transcriptionally only from pancreatic stellate cells. Hypoxia-induced increased lactate production did not influence lumican expression. Notably, autophagy was induced by hypoxia in ex vivo cultures of patient-derived primary PDAC xenograft and pancreatic stellate cells; however, the cancer cells remain unaffected. Moreover, hypoxia-inducible factor (HIF)-1α expression or inhibition of AMP-regulated protein kinase (AMPK) activation within hypoxic stellate cells restored lumican expression levels. Interestingly, AMPK inhibition attenuated hypoxia-reduced phosphorylation of the mTOR/p70S6K/4EBP signaling pathway. The aim of this mini-review is to summarize our recent publication that hypoxia reduces stromal lumican in PDAC through autophagy-mediated degradation and reduction in protein synthesis within pancreatic cancer stellate cells. This may provide another plausible mechanism by which hypoxia-induced stromal autophagy leads to cancer growth.

Antineoplastic effects of auranofin in human pancreatic adenocarcinoma preclinical models.

BACKGROUND: Auranofin, a Food and Drug Administration-approved anti-rheumatic agent with anticancer properties for lung and ovarian cancer, has never been studied for pancreatic cancer. We hypothesize that auranofin may prevent pancreatic ductal adenocarcinoma progression by inhibition of Txnrd1 and HIF-1α. METHODS: In vitro sensitivity of human pancreatic ductal adenocarcinoma cell lines was determined based on IC50. Western blot assays were used to interrogate mechanisms of apoptosis and resistance. Ex vivo live tissue slice assays of xenografts allowed for testing of a larger number of PDX samples with high efficiency. In vivo pancreatic ductal adenocarcinoma orthotopic mouse models using MiaPaCa-2 Luc + cells were designed to determine optimal dose and antitumor effect. RESULTS: We found that 10 of 15 tested pancreatic ductal adenocarcinoma cell lines were sensitive to auranofin based on IC50s below 5 µmol/L. Ex vivo tissue growth inhibition greater than 44% was observed for 13 PDX tissue cases treated with 10 µmol/L auranofin. High Txnrd1 expression was observed for resistant cell lines. In vivo studies showed 15 mg/kg IP as the optimal dose with absence of gross solid organ metastasis up to 13 weeks post-treatment (median survival 8 and 12 weeks, respectively; P = .0953). CONCLUSIONS: We have demonstrated that auranofin prevents pancreatic ductal adenocarcinoma progression using multiple models. Our study suggests inhibition of Txnrd1 and HIF-1α as possible mechanisms of action, and Txnrd1 as a biomarker of resistance. Based on these data, an off-label Phase 0 clinical trial with this FDA-approved drug should be considered for patients with pancreatic cancer.

Hypoxia-induced autophagy of stellate cells inhibits expression and secretion of lumican into microenvironment of pancreatic ductal adenocarcinoma.

Lumican is secreted by pancreatic stellate cells and inhibits cancer progression. Extracellular lumican inhibits cancer cell replication and restrains growth of early-stage pancreatic adenocarcinoma (PDAC) such that patients with tumors containing stromal lumican experience a three-fold longer survival after treatment. In the present study, patient tumor tissues, ex-vivo cultures of patient-derived xenografts (PDX), PDAC stellate and tumor cells were used to investigate whether hypoxia (1% O2) within the tumor microenvironment influences stromal lumican expression and secretion. We observed that hypoxia significantly reduced lumican expression and secretion from pancreatic stellate cells, but not cancer cells. Although hypoxia enhanced lactate dehydrogenase A (LDHA) expression and lactate secretion from all cells, neither hypoxia-induced nor exogenous lactate influenced lumican expression. Autophagy was induced by hypoxia in ex vivo cultures of PDX and pancreatic stellate cells, but not cancer cells cultured in 2D. Autophagic flux inhibitors, bafilomycin A1, chloroquine diphosphate salt, and ammonium chloride prevented hypoxia-mediated reduction in lumican expression in stellate cells. Furthermore, inhibition of AMP-regulated protein kinase (AMPK) phosphorylation or hypoxia-inducible factor (HIF)-1α expression within hypoxic stellate cells restored lumican expression levels. Hypoxia did not affect lumican mRNA expression, indicating that hypoxia-induced reduction of lumican occurs post-transcriptionally; in addition, AMPK inhibition prevented hypoxia-reduced phosphorylation of the mTOR/p70S6K/4EBP signaling pathway, a key contributor to protein synthesis. Taken together, these findings demonstrate that hypoxia reduces stromal lumican in PDAC through autophagy-mediated degradation and reduction in protein synthesis within pancreatic cancer stellate cells.

Preclinical Evaluation of Sequential Combination of Oncolytic Adenovirus Delta-24-RGD and Phosphatidylserine-Targeting Antibody in Pancreatic Ductal Adenocarcinoma.

Delta-24-RGD (DNX-2401) is a conditional replication-competent oncolytic virus engineered to preferentially replicate in and lyse tumor cells with abnormality of p16/RB/E2F pathway. In a phase I clinical trial, Delta-24-RGD has shown favorable safety profile and promising clinical efficacy in brain tumor, which prompted us to evaluate its anticancer activity in pancreatic ductal adenocarcinoma (PDAC), which also has high frequency of homozygous deletion and promoter methylation of CDKN2A encoding the p16 protein. Our results demonstrate that Delta-24-RGD can induce dramatic cytotoxicity in a subset of PDAC cell lines with high cyclin D1 expression. Induction of autophagy and apoptosis by Delta-24-RGD in sensitive PDAC cells was confirmed with LC3B-GFP autophagy reporter and acridine orange staining as well as Western blotting analysis of LC3B-II expression. Notably, we found that Delta-24-RGD induced phosphatidylserine exposure in infected cells independent of cells' sensitivity to Delta-24-RGD, which renders a rationale for combination of Delta-24-RGD viral therapy and phosphatidylserine targeting antibody for PDAC. In a mouse PDAC model derived from a liver metastatic pancreatic cancer cell line, Delta-24-RGD significantly inhibited tumor growth compared with control (P < 0.001), and combination of phosphatidylserine targeting antibody 1N11 further enhanced its anticancer activity (P < 0.01) possibly through inducing synergistic anticancer immune responses. Given that these 2 agents are currently in clinical evaluation, our study warrants further clinical evaluation of this novel combination strategy in pancreatic cancer therapy. Mol Cancer Ther; 16(4); 662-70. ©2016 AACR.

ASCT2 (SLC1A5) is an EGFR-associated protein that can be co-targeted by cetuximab to sensitize cancer cells to ROS-induced apoptosis.

Therapeutic targeting of ASCT2, a glutamine transporter that plays a major role in glutamine uptake in cancer cells, is challenging because ASCT2 also has a biological role in normal tissues. In this study, we report our novel finding that ASCT2 is physically associated in a molecular complex with epidermal growth factor receptor (EGFR), which is often overexpressed in human head and neck squamous cell carcinoma (HNSCC). Furthermore, we found that ASCT2 can be co-targeted by cetuximab, an EGFR antibody approved for treating metastatic HNSCC. We demonstrated that cetuximab downregulated ASCT2 in an EGFR expression-dependent manner via cetuximab-mediated EGFR endocytosis. Downregulation of ASCT2 by cetuximab led to decreased intracellular uptake of glutamine and subsequently a decreased glutathione level. Cetuximab thereby sensitized HNSCC cells to reactive oxygen species (ROS)-induced apoptosis and, importantly, it is independent of effective inhibition of EGFR downstream signaling by cetuximab. In contrast, knockdown of EGFR by siRNA or inhibition of EGFR kinase with gefitinib, an EGFR kinase inhibitor, failed to sensitize HNSCC cells to ROS-induced apoptosis. Our findings support a novel therapeutic strategy for EGFR-overexpressing and cetuximab-resistant cancers by combining cetuximab with an oxidative therapy.

Ex Vivo Testing of Patient-Derived Xenografts Mirrors the Clinical Outcome of Patients with Pancreatic Ductal Adenocarcinoma.

PURPOSE: Translation of the patient-derived xenograft (PDX) model into a method for practical personalized cancer treatment is prevented by the intense resources and time necessary to generate and test each tumorgraft. We aimed to develop a high-throughput ex vivo drug testing approach that can be used for personalized cancer treatment design. EXPERIMENTAL DESIGN: We developed a unique ex vivo live tissue sensitivity assay (LTSA), in which precision-cut and uniform small tissue slices derived from pancreatic ductal adenocarcinoma PDX tumors were arrayed in a 96-well plate and screened against clinically relevant regimens within 3 to 5 days. The correlation between the sensitivities of tissue slices to the regimens and patients' clinical responses and outcome were statistically analyzed. The results of LTSA assay were further confirmed with biochemical methods in vitro and animal PDX model in vivo RESULTS: The ex vivo tissue slices remain viable for at least 5 days, and the tumor parenchyma, including stroma, vascular structures, and signaling pathways, are all retained. The sensitivities of the ex vivo tissue slices to gemcitabine and irinotecan was consistent with the clinical responses and outcomes of the patients from whom the tumorgrafts were derived (r = 0.77; P = 0.0002). Retrospective analysis showed that the patients who received LTSA-sensitive regimens had remarkably longer progression-free survival than patients who received LTSA-resistant regimens (16.33 vs. 3.8 months; n = 18, P = 0.011). CONCLUSIONS: The results from these PDX and LTSA methods reflect clinical patients' responses and could be used as a personalized strategy for improving systemic therapy effectiveness in patients with pancreatic cancer. Clin Cancer Res; 22(24); 6021-30. ©2016 AACR.

Transforming Growth Factor-ß Limits Secretion of Lumican by Activated Stellate Cells within Primary Pancreatic Adenocarcinoma Tumors.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is lethal cancer whose primary tumor is characterized by dense composition of cancer cells, stromal cells, and extracellular matrix (ECM) composed largely of collagen. Within the PDAC tumor microenvironment, activated pancreatic stellate cells (PSC) are the dominant stromal cell type and responsible for collagen deposition. Lumican is a secreted proteoglycan that regulates collagen fibril assembly. We have previously identified that the presence of lumican in the ECM surrounding PDAC cells is associated with improved patient outcome after multimodal therapy and surgical removal of localized PDAC. EXPERIMENTAL DESIGN: Lumican expression in PDAC from 27 patients was determined by IHC and quantitatively analyzed for colocalization with PSCs. In vitro studies examined the molecular mechanisms of lumican transcription and secretion from PSCs (HPSCs and HPaSteC), and cell adhesion and migration assays examined the effect of lumican on PSCs in a collagen-rich environment. RESULTS: Here we identify PSCs as a significant source of extracellular lumican production through quantitative IHC analysis. We demonstrate that the cytokine, TGF-ß, negatively regulates lumican gene transcription within HPSCs through its canonical signaling pathway and binding of SMAD4 to novel SBEs identified within the promoter region. In addition, we found that the ability of HPSCs to produce and secrete extracellular lumican significantly enhances HPSCs adhesion and mobility on collagen. CONCLUSIONS: Our results demonstrate that activated pancreatic stellate cells within PDAC secrete lumican under the negative control of TGF-ß; once secreted, the extracellular lumican enhances stellate cell adhesion and mobility in a collagen-rich environment. Clin Cancer Res; 22(19); 4934-46. ©2016 AACR.

AMPK-mediated energy homeostasis and associated metabolic effects on cancer cell response and resistance to cetuximab.

We previously reported that cetuximab, an EGFR-blocking antibody, inhibits cancer metabolism via downregulation of HIF-1α and reverses the Warburg effect in cancer cells. Here, we report that inhibition of HIF-1 transcriptional activity by cetuximab does not necessarily lead to successful inhibition of cell proliferation. In several head and neck squamous cell carcinoma (HNSCC) cell lines, we observed a pattern of oscillating decrease and increase of intracellular ATP level after cetuximab treatment, and the magnitude and kinetics of which varied by cell line and appeared to be linked to the extent of cellular response to cetuximab. In HNSCC cells with low basal level of AMPK activity and that responded to cetuximab-induced growth inhibition, there was a transient, LKB1-dependent activation of AMPK. In contrast, HNSCC cells that had a high basal level of AMPK activity were less sensitive to cetuximab-induced growth inhibition despite effective inhibition of EGFR downstream signaling by cetuximab. Knockdown or inhibition of AMPK markedly enhanced response to cetuximab via induction of apoptosis. These findings indicate that a transient activation of AMPK is an early metabolic marker of cellular response to cetuximab and that high and sustained AMPK activity is an important mechanism by which cancer cells survive cetuximab treatment.

Extracellular lumican inhibits pancreatic cancer cell growth and is associated with prolonged survival after surgery.

PURPOSE: To evaluate the relevance between lumican expression patterns and the clinical course of patients with pancreatic ductal adenocarcinoma (PDAC), and to investigate the role of lumican in PDAC progression. EXPERIMENTAL DESIGN: One hundred thirty-one patient tumors were chosen for tissue microarray staining, and Cox regression analysis was used to test the associations between lumican expression and clinical, pathologic, and oncologic outcomes in all patients. Primary PDAC cells and recombinant human lumican protein were used to establish a working model to mimic the in vivo interactions between stromal lumican and PDAC cells. Using this model, we tested the effects of lumican on EGFR signaling via Akt and hypoxia-inducible factor-1α (HIF1α) and its subsequent influence on glucose consumption, lactate production, intracellular ATP, and apoptotic cell death. RESULTS: Lumican was present in the stroma surrounding PDAC cells in roughly one-half of primary tumors and the direct xenografts. Patients with stromal lumican were associated with a profound reduction in metastatic recurrence after surgery and 3-fold longer survival than patients without stromal lumican. In PDAC cells, extracellular lumican reduced EGFR expression and phosphorylation through enhanced dimerization and internalization of EGFR and the resultant inhibition of Akt kinase activity. Lumican also reduced HIF1α expression and activity via Akt. PDAC cells with enhanced HIF1α activity were resistant to lumican-induced inhibition of glucose consumption, lactate production, intracellular ATP, and apoptosis. CONCLUSIONS: There is a positive association between stromal lumican in primary PDAC tumors and prolonged survival after tumor resection. Lumican plays a restrictive role in EGFR-expressing pancreatic cancer progression.

Genistein potentiates the antitumor effect of 5-Fluorouracil by inducing apoptosis and autophagy in human pancreatic cancer cells.

BACKGROUND: Although 5-fluorouracil (5-FU)-based combination chemotherapy (i.e. FOLFIRINOX) has demonstrated effectiveness against pancreatic cancer, novel therapeutic strategies must be developed to increase the therapeutic window of these cytotoxic agents. Genistein is a soy-derived isoflavone with pleiotropic biological effects that can enhance the antitumor effect of chemotherapeutic agents. MATERIALS AND METHODS: To understand how genistein potentiates the antitumor effects of 5-FU, we examined apoptosis and autophagy in MIA PaCa-2 human pancreatic cancer cells and their derived xenografts. Apoptosis was evaluated using DNA fragmentation assays, and western blots of poly(ADP ribose)polymerase and caspase-3. Meanwhile, autophagy was evaluated using western blots of microtubule-associated protein light chain 3 (LC3)-I/II, fluorescent microscopy observation of green fluorescent protein-LC3B puncta formation, and acidic vesicular organelle formation using acridine orange staining. Tumors from animal treatment studies were examined for apoptosis and autophagy using the TdT-mediated dUTP nick-end labeling assay and immunohistochemical staining of LC3B, respectively. RESULTS: We observed that genistein increased 5-FU-induced cell death through increased apoptosis, as well as autophagy. The increased autophagy was accompanied by decreased B-cell lymphoma 2 (Bcl2) and increased beclin-1 protein levels. Animal treatment studies supported these observations. The combination of 5-FU and genistein significantly reduced final xenograft tumor volume when compared to 5-FU-alone by inducing apoptosis as well as autophagy. CONCLUSION: Genistein can potentiate the antitumor effect of 5-FU by inducing apoptotic as well as autophagic cell death. These results demonstrate the potential of genistein as an adjuvant therapeutic agent against pancreatic cancer.

Neuroprotection by Orexin-A via HIF-1α induction in a cellular model of Parkinson's disease.

Orexin-A, a neuropeptide secreted by hypothalamic neurons, may be neuroprotective in many neurological conditions such as cerebral ischaemia. One mechanism postulated to be involved in the neuroprotection by Orexin-A is the induction of hypoxia inducible factor 1 alpha (HIF-1α). Parkinson's disease (PD) is a progressive neurodegenerative disorder and mitochondrial dysfunction has been demonstrated to play a role in its pathogenesis. Mitochondrial dysfunction may cause reduction of O2 consumption and subsequently activate prolyl hydroxylase, which leads to decreased level of HIF-1α. In this study, we used MPP(+)-treated SH-SY5Y cells as an in vitro cellular model of PD to test the role of Orexin-A as an inducer of HIF-1α. Our results showed that Orexin-A not only induced HIF-1α but also activated downstream targets of HIF-1α, such as vascular endothelial growth factor and erythropoietin. Thus, Orexin-A treatment attenuated MPP(+)-induced cell injury and this effect was blocked when HIF-1α was suppressed. Hence, we conclude that induction of HIF-1α is one of the mechanisms involved in the neuroprotection by Orexin-A.

Cetuximab reverses the Warburg effect by inhibiting HIF-1-regulated LDH-A.

Hypoxia-inducible factor-1 (HIF-1) plays a critical role in reprogramming cancer metabolism toward aerobic glycolysis (i.e., the Warburg effect), which is critical to supplying cancer cells with the biomass needed for proliferation. Previous studies have shown that cetuximab, an EGF receptor-blocking monoclonal antibody, downregulates the alpha subunit of HIF-1 (HIF-1α) through the inhibition of EGF receptor downstream cell signaling and that downregulation of HIF-1α is required for cetuximab-induced antiproliferative effects. However, the mechanism underlying these actions has yet to be identified. In this study, we used the Seahorse XF96 extracellular flux analyzer to assess the effect of cetuximab treatment on changes in glycolysis and mitochondrial respiration, the two major energy-producing pathways, in live cells. We found that cetuximab downregulated lactate dehydrogenase A (LDH-A) and inhibited glycolysis in cetuximab-sensitive head and neck squamous cell carcinoma (HNSCC) cells in an HIF-1α downregulation-dependent manner. HNSCC cells with acquired cetuximab resistance expressed a high level of HIF-1α and were highly glycolytic. Overexpression of a HIF-1α mutant (HIF-1α/ΔODD) conferred resistance to cetuximab-induced G1 phase cell-cycle arrest, which could be overcome by knockdown of LDH-A expression. Inhibition of LDH-A activity with oxamate enhanced the response of cetuximab-resistant cells to cetuximab. Cetuximab had no noticeable inhibitory effect on glycolysis in nontransformed cells. These findings provide novel mechanistic insights into cetuximab-induced cell-cycle arrest from the perspective of cancer metabolism and suggest novel strategies for enhancing cetuximab response.

Immunoproteomic analysis of the antibody response obtained in mouse following vaccination with a T-cell vaccine.

T-cell vaccination (TCV), the application of irradiated activated T cells, has been shown to prevent effectively and treat experimental autoimmune diseases. It has been reported that anti-lymphocytic antibodies induced by TCV were capable of strongly inhibiting T-cell proliferation and of ameliorating experimental autoimmune disease. The present study was undertaken to characterize the antigen specificity of these Abs. We used activated mouse ovalbumin (OVA)-specific T cells (OVA-T) as vaccine immunized mice. By combination of 2-DE, 2-D Western blot and Q-TOF mass spectrometry we have identified 11 antigens in activated T cells that were recognized by the anti-T-cell Abs. The resulting antigenic molecules included calreticulin (CRT), ERp57, Vimentin, HSP70-4, tubulin ß5 chain, coronin-1A, pyruvate kinase, ATP synthase ß chain and transketolase most of which belong to so-called damage-associated molecular pattern molecules (DAMPs). CRT, ERp57 and vementin were further examined by Western blot and cellular ELISA to identify molecular targets which may be involved in the TCV immunotherapy. On the basis of our results, γ-radiation induced the activated T cells "immunogenic apoptosis" and exposed/secreted DAMPs (CRT, ERp57 and Vementin) played an important role in TCV therapy.

Resveratrol-activated AMPK/SIRT1/autophagy in cellular models of Parkinson's disease.

Excessive misfolded proteins and/or dysfunctional mitochondria, which may cause energy deficiency, have been implicated in the etiopathogenesis of Parkinson's disease (PD). Enhanced clearance of misfolded proteins or injured mitochondria via autophagy has been reported to have neuroprotective roles in PD models. The fact that resveratrol is a known compound with multiple beneficial effects similar to those associated with energy metabolism led us to explore whether neuroprotective effects of resveratrol are related to its role in autophagy regulation. We tested whether modulation of mammalian silent information regulator 2 (SIRT1) and/or metabolic energy sensor AMP-activated protein kinase (AMPK) are involved in autophagy induction by resveratrol, leading to neuronal survival. Our results showed that resveratrol protected against rotenone-induced apoptosis in SH-SY5Y cells and enhanced degradation of α-synucleins in α-synuclein-expressing PC12 cell lines via autophagy induction. We found that suppression of AMPK and/or SIRT1 caused decrease of protein level of LC3-II, indicating that AMPK and/or SIRT1 are required in resveratrol-mediated autophagy induction. Moreover, suppression of AMPK caused inhibition of SIRT1 activity and attenuated protective effects of resveratrol on rotenone-induced apoptosis, further suggesting that AMPK-SIRT1-autophagy pathway plays an important role in the neuroprotection by resveratrol on PD cellular models.