Hypoxic Pancreatic Cancer Cells Research Articles

Dual hypoxia-responsive supramolecular complex for cancer target therapy

The prognosis with pancreatic cancer is among the poorest of any human cancer. One of the important factors is the tumor hypoxia. Targeting tumor hypoxia is considered a desirable therapeutic option. However, it has not been translated into clinical success in the treatment of pancreatic cancer. With enhanced cytotoxicities against hypoxic pancreatic cancer cells, BE-43547A2 (BE) may serve as a promising template for hypoxia target strategy. Here, based on rational modification, a BE prodrug (NMP-BE) is encapsulated into sulfonated azocalix[5]arene (SAC5A) to generate a supramolecular dual hypoxia-responsive complex NMP-BE@SAC5A. Benefited from the selective load release within cancer cells, NMP-BE@SAC5A markedly suppresses tumor growth at low dose in pancreatic cancer cells xenograft murine model without developing systemic toxicity. This research presents a strategy for the modification of covalent compounds to achieve efficient delivery within tumors, a horizon for the realization of safe and reinforced hypoxia target therapy using a simple approach.

Effects of anoxic prognostic model on immune microenvironment in pancreatic cancer

Pancreatic cancer has one of the worst prognoses in the world, which suggests that the tumor microenvironment, which is characterized by hypoxia and immunosuppression, plays a significant role in the prognosis and progression of pancreatic cancer. We identified PLAU, LDHA, and PKM as key genes involved in pancreatic cancer hypoxia through GO/KEGG enrichment related hypoxia pathways and cox regression, established prognostic models, and studied their relationship to immune invasion through bioinformatics using R and related online databases. We verified the high expression of PLAU, LDHA, and PKM in pancreatic cancer cells using qPCR in vitro, and we also discovered that the expression of PLAU, LDHA, and PKM in hypoxic pancreatic cancer cells differed from that in normal cultured pancreatic cancer cells. Finally, we discovered that our prognostic model accurately predicted postrain in pancreatic cancer patients with hypoxia and immune infiltration.

M6A methylation regulates hypoxia-induced pancreatic cancer glycolytic metabolism through ALKBH5-HDAC4-HIF1α positive feedback loop

Pancreatic cancer (PC) is the most hypoxic cancer type among solid tumors. The dynamic changes of RNA N6-methyl-adenosine (m6A) contribute to tumor cells adaption to hypoxic microenvironmental. However, the regulatory mechanisms of hypoxia response in PC remains elusive. Here, we reported that the m6A demethylase ALKBH5 mediated a decrease of total mRNA m6A modification during hypoxia. Subsequently, methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with RNA sequencing (RNA-seq) revealed transcriptome-wide gene expression alteration and identified histone deacetylase type 4 (HDAC4) as a key target gene of m6A modification under hypoxic conditionds. Mechanistically, m6A methylation recognized by m6A reader-YTHDF2 enhanced the stability of HDAC4, and then promoted glycolytic metabolism and migration of PC cells. Our assays also demonstrated that hypoxia-induced HDAC4 enhanced HIF1a protein stability, and overexpressed HIF1a promoted transcription of ALKBH5 in hypoxic pancreatic cancer cells. Together, these results found a ALKBH5/HDAC4/HIF1α positive feedback loop for cellular response to hypoxia in pancreatic cancer. Our studies uncover the crosstalk between histone acetylation and RNA methylation modification on layer of epigenetic regulation.

Hypoxic exosomal HIF-1α-stabilizing circZNF91 promotes chemoresistance of normoxic pancreatic cancer cells via enhancing glycolysis.

Research has indicated that hypoxia profoundly contributes to chemoresistance of pancreatic cancer (PC), while the precise mechanism has not been fully elucidated. In this study, we report a hypoxic exosomal circular RNA (circRNA)-mediated mechanism of conferred chemoresistance in PC cells. Gemcitabine (GEM) resistance was enhanced in normoxic PC cells incubated with exosomes derived from hypoxic PC cells. CircRNA microarray displayed that circZNF91 was remarkably increased in hypoxic exosomes of PC cells compared with normoxic exosomes. Overexpression of circZNF91 obviously stimulated chemoresistance in PC cells, while knockdown of circZNF91 retarded the hypoxic exosome-transmitted chemoresistance. Mechanistically, the hypoxic-induced exosomal circZNF91 transmitted into normoxic PC cells could competitively bind to miR-23b-3p, which deprives the inhibition of miR-23b-3p on expression of deacetylase Sirtuin1 (SIRT1). Consequently, the upregulated SIRT1 enhanced deacetylation-dependent stability of HIF-1α protein, leading to glycolysis and GEM chemoresistance of recipient PC cells. In addition, we revealed that the increased circZNF91 in hypoxic exosome was attributed to the transcriptional regulation by HIF-1α. Coincidently, transmission of hypoxic exosomes into subcutaneous xenografts in nude mice obviously facilitated the chemoresistance of transplanted PC tumor, which could be reversed by depletion of circZNF91 or upregulation of miR-23b-3p. Furthermore, clinical data showed that circZNF91 was significantly upregulated in PC tissues and correlated with overexpression of glycolytic enzymes and short overall survival time. Collectively, exosomal circZNF91 can function as a cargo mediating the signal transmission between hypoxic and normoxic tumor cells to promote GEM chemoresistance of PC and may potentially serve as a therapeutic target.

Hypoxic Tumor-Derived Exosomal Long Noncoding RNA UCA1 Promotes Angiogenesis via miR-96-5p/AMOTL2 in Pancreatic Cancer

The hypoxic microenvironment, an important feature of solid tumors, promotes tumor cells to release exosomes and enhances tumor angiogenesis. However, the detailed functions of hypoxic exosomes and the mechanisms underlying their effects in pancreatic cancer (PC) remain mysterious. Here, we observed that hypoxic exosomes derived from PC cells promoted cell migration and tube formation of human umbilical vein endothelial cells (HUVECs). The long noncoding RNA (lncRNA) UCA1, a key factor, was highly expressed in exosomes derived from hypoxic PC cells and could be transferred to HUVECs through the exosomes. In addition, the expression levels of UCA1 in exosomes derived from PC patients’ serum were higher than in healthy controls and were associated with poor survival of PC patients. Moreover, hypoxic exosomal UCA1 could promote angiogenesis and tumor growth both in vitro and in vivo. With respect to the functional mechanism, UCA1 acted as a sponge of microRNA (miR)-96-5p, relieving the repressive effects of miR-96-5p on the expression of its target gene AMOTL2. Collectively, these results indicate that hypoxic exosomal UCA1 could promote angiogenesis and tumor growth through the miR-96-5p/AMOTL2/ERK1/2 axis and therefore, serve as a novel target for PC treatment.

Abstract 3082: Targeting hypoxic pancreatic cancer cells with glucose conjugated lactate dehydrogenase inhibitor NHI-Glc-2

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an abysmal disease with a 5-year survival rate of merely 8%. The tumor microenvironment of PDAC is one of the factors contributing to drug resistance. More specifically, the hypoxic tumor core and the metabolic switch to aerobic glycolysis (the Warburg effect), contribute to the lack of drug response. Therefore, we investigated the effect of several novel lactate dehydrogenase (LDH-A) inhibitors (N-Hydroxyindole-based LDH-A inhibitors, NHI-1 and NHI-2, and the glucose conjugate NHI-Glc-2) in PDAC cells in vitro and in vivo, in combination with the standard drug gemcitabine. For this purpose we used our primary PDAC cancer cell cultures, tested growth inhibition with the SRB chemosensitivity assay, used 3D cultures and established an in vivo orthotopic bioluminescent model. Additionally, LDH-A enzyme activity inhibition by NHI-Glc-2 was assessed by spectrophotometry. LDH-A is overexpressed in PDAC and its expression is correlated with the prognosis of metastatic PDAC. The glucose transporter 1 (GLUT-1) is also overexpressed in PDAC, which would enable an increased uptake of NHI-Glc-2 by the tumor cells. LDH-A mRNA expression and enzyme activity were about 2-fold higher under hypoxic conditions. NHI-1, NHI-2 and NHI-Glc-2 were 4-15-fold more effective under hypoxic conditions compared to normoxia, but gemcitabine was 10-20-fold less active under hypoxia. NHI-1 showed a synergistic effect with gemcitabine in hypoxic PANC-1 and LPC006 cells (combination index 0.14 ± 0.06 and 0.29 ± 0.53, respectively). NHI-Glc-2 inhibited PDAC cell growth in micromolar range under hypoxic conditions and also showed a synergistic effect with gemcitabine. In a 3D spheroid culture (with a hypoxic core), NHI-Glc-2 disrupted the spheroid integrity. Moreover, in an orthotopic PDAC model NHI-Glc-2 showed a more pronounced inhibition (almost complete) of tumor growth compared to gemcitabine. NHI-Glc-2 also showed a favorable pharmacokinetics with a peak plasma concentration of 26 µM at 4 hr, which is higher than the IC50. In conclusion, LDH-A is a viable target in PDAC, and novel LDH-A inhibitors offer an innovative therapeutic tool. Remarkably, the LDH-A inhibitors NHI-1 and NHI-2 increased the effect of gemcitabine under hypoxic conditions, while the glucose conjugated NHI-Glc-2 showed an improved uptake possibly because of the increased GLUT-1 expression, leading to a pronounced in vivo effect. Citation Format: Btissame El Hassouni, Rocco Sciarrillo, Valentina Edith Gómez, Mina Maftouh, Giulia Mantini, Christian M. Vonk, Carlotta Granchi, Niccola Funel, Filippo Minutolo, Godefridus J. Peters, Elisa Giovannetti. Targeting hypoxic pancreatic cancer cells with glucose conjugated lactate dehydrogenase inhibitor NHI-Glc-2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3082.

Hypoxic Tumor-Derived Exosomal miR-301a Mediates M2 Macrophage Polarization via PTEN/PI3Kγ to Promote Pancreatic Cancer Metastasis.

Exosomes are emerging as important mediators of the cross-talk between tumor cells and the microenvironment. However, the mechanisms by which exosomes modulate tumor development under hypoxia in pancreatic cancer remain largely unknown. Here, we found that hypoxic exosomes derived from pancreatic cancer cells activate macrophages to the M2 phenotype in a HIF1a or HIF2a-dependent manner, which then facilitates the migration, invasion, and epithelial-mesenchymal transition of pancreatic cancer cells. Given that exosomes have been shown to transport miRNAs to alter cellular functions, we discovered that miR-301a-3p was highly expressed in hypoxic pancreatic cancer cells and enriched in hypoxic pancreatic cancer cell-derived exosomes. Circulating exosomal miR-301a-3p levels positively associated with depth of invasion, lymph node metastasis, late TNM stage, and poor prognosis of pancreatic cancer. Hypoxic exosomal miR-301a-3p induced the M2 polarization of macrophages via activation of the PTEN/PI3Kγ signaling pathway. Coculturing of pancreatic cancer cells with macrophages in which miR-301a-3p was upregulated or treated with hypoxic exosomes enhanced their metastatic capacity. Collectively, these data indicate that pancreatic cancer cells generate miR-301a-3p-rich exosomes in a hypoxic microenvironment, which then polarize macrophages to promote malignant behaviors of pancreatic cancer cells. Targeting exosomal miR-301a-3p may provide a potential diagnosis and treatment strategy for pancreatic cancer.Significance: These findings identify an exosomal miRNA critical for microenvironmental cross-talk that may prove to be a potential target for diagnosis and treatment of pancreatic cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4586/F1.large.jpg Cancer Res; 78(16); 4586-98. ©2018 AACR.

Abstract 2941: APE1/Ref-1 redox signaling regulates HIF1a-mediated CA9 expression in hypoxic pancreatic cancer cells: Combination treatment in patient-derived pancreatic tumor models

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by aggressive metastasis and therapeutic resistance. Reactive stroma in PDAC tumors leads to fibrosis, inflammation, and hypoxia. Hypoxia signaling creates a more aggressive phenotype with increased potential for metastasis and decreased therapeutic efficacy. Carbonic anhydrase IX (CA9) functions as part of the cellular response to hypoxia by regulating intracellular pH to promote cell survival. Apurinic/Apyrimidinic Endonuclease-1-Reduction/oxidation Effector Factor 1 (APE1/Ref-1) is a multifunctional protein with endonuclease activity in DNA base excision repair and redox signaling activity. This redox activity is responsible for reducing oxidized cysteines on specific transcription factors, including hypoxia inducible factor 1 alpha (HIF1α), enabling them to bind target sequences in DNA. We evaluated the mechanisms underlying PDAC cell responses to hypoxia and APE1/Ref-1 redox signaling control of HIF1α, a critical factor in hypoxia-induced CA9 transcription. We hypothesized that obstructing the HIF-CA9 axis at two points via APE1/Ref-1 inhibition (which results in a decrease in CA9 expression) and direct CA9 inhibition results in enhanced PDAC cell killing under hypoxic conditions. In our studies, HIF1α-mediated induction of CA9 is significantly attenuated following APE1/Ref-1 knock down or redox signaling inhibition in patient-derived PDAC cells and pancreatic cancer-associated fibroblast cells using the APE1/Ref-1 redox signaling inhibitor APX3330 (currently in clinical trials). Additionally, dual-targeting of APE1/Ref-1 redox signaling activity and CA9 activity results in additive-to-synergistic enhancement of acidification and cytotoxicity of PDAC cells under hypoxic conditions as well as decreased tumor growth in an ex vivo 3-dimensional tumor co-culture model. These studies are clinically relevant as we used the CA9 inhibitor SLC-0111 (phase I clinical trial completed), as well as APX3330 (Apexian Pharmaceuticals: IND 125360), for which a phase I clinical trial has opened. Further experiments characterized novel analogs of APX3330: APX2009 and APX2014, which demonstrated up to 50-fold improved potency as measured by pH reduction, cytotoxicity, and inhibition of hypoxia-induced CA9 expression. An SLC-0111 analog, FC12-531A, demonstrated up to 75-fold improved potency as measured by cytotoxicity. In combination, these analogs resulted in synergistic inhibition of 3D tumor spheroid growth at nanomolar-to-low-micromolar concentrations. These results underscore the concept that proper combination therapy has significant clinical utility of blocking APE1/Ref-1 and CA9 function for novel PDAC therapeutic treatment. Citation Format: Derek Logsdon, Fenil Shah, Fabrizio Carta, Claudiu Supuran, Melissa Fishel, Mark R. Kelley. APE1/Ref-1 redox signaling regulates HIF1a-mediated CA9 expression in hypoxic pancreatic cancer cells: Combination treatment in patient-derived pancreatic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2941.

PO-042 Targeting hypoxic pancreatic cancer cells with glucose conjugated lactate dehydrogenase inhibitor NHI-Glc-2

IntroductionPancreatic ductal adenocarcinoma (PDAC) is an abysmal disease with a 5 year survival rate of merely 8%. The tumour microenvironment is one of the factors contributing to PDAC chemoresistance. More specifically, the hypoxic tumour cores and the metabolic switch to aerobic glycolysis (e.g. the Warburg effect), contribute to the lack of drug response. Interestingly, two glycolysis components glucose transporter 1 (GLUT-1) and lactate dehydrogenase A (LDH-A) are overexpressed in PDAC. The latter, LDH-A, is also correlated with prognosis in metastatic PDAC.N-Hydroxyindole-based LDH-A inhibitors (NHI-1 and NHI-2) have shown a synergistic effect in hypoxic PDAC cells when combined with gemcitabine. A glucose conjugated NHI-Glc-2 was designed to exploit the GLUT-1 overexpression in PDAC cells and in the present study we evaluated whether this novel compound further improved the pharmacological effect of LDH-A inhibitors.Material and methodsThe effect of NHI-Glc-2 on cell growth is tested in our primary PDAC cancer cell cultures, characterised for their hypoxic signature and LDH-A/GLUT-1 expression levels by next-generation sequencing. Inhibition of cell and tumour growth was evaluated by the SRB assay, 3D spheroid-cultures and with an orthotopic bioluminescent in vivo model. Additionally, LDH-A enzyme activity inhibition and the effect on the glycolytic rate by NHI-Glc-2 were assessed by spectrophotometry and with the Seahorse XF analyzer, respectively.Results and discussionsNHI-Glc-2 is capable of inhibiting PDAC cell growth in, especially in hypoxia, in nanomolar range and shows a synergistic effect with gemcitabine. In 3D cultures NHI-Glc-2 disrupts spheroid integrity, and preliminary in vivo studies show promising results.ConclusionLactate dehydrogenase A is a viable target in PDAC, and the novel LDH-A inhibitor showed improved pharmacological effect in normoxic and hypoxic PDAC cells compared to NHI-1 and NHI-2. Moreover, this compound displays a synergistic cytotoxic activity with gemcitabine, offering an innovative tool in hypoxic tumours.

Abstract 2799: Exosomes from hypoxic pancreatic cancer cells confer resistance to subsequent hypoxia insult

Abstract Pancreatic cancer (PC) remains one of the leading causes of cancer-associated deaths in the US due to its highly aggressive nature and lack of effective treatments. Pancreatic tumors are characterized by a dense, desmoplastic reaction and poor vasculature leading to reduced oxygen levels within the tumor microenvironment. It is believed that this encounter of pancreatic tumor cells to intratumoral hypoxic condition is a major cause of their persistent survival under a variety of stresses, increased aggressiveness and therapeutic-resistance. However, underlying molecular mechanisms responsible for PC's adaptation to hypoxia (along with enhanced growth and aggressiveness) remain elusive. In this study, we examined the role of exosomes, which are nanoscale biological vesicles with a lipid bilayer membrane, in imparting survival benefit to PC cells. For this, we first examined the effect of conditioned media (CM) from PC cells cultured under hypoxic (0.1% O2, H-CM) or normoxic (21% O2, N-CM) conditions on the growth of fresh PC cells cultured under hypoxic (0.1% O2) conditions. We found that H-CM conferred significant survival benefit to cancer cells under hypoxia as compared to N-CM. Thereafter, we isolated exosomes from N-CM (N-exo) or H-CM (H-exo) and used them to treat pancreatic cancer cells under hypoxic culture conditions. We observed that H-exo imparted a significantly higher survival advantage to cancer cells under hypoxia as compared to N-exo. Interestingly, protein quantification indicated the presence of higher levels of exosomes from cells cultured under hypoxic conditions as compared to those grown in normoxic environment. Western blot analysis with anti-CD9 and anti-CD63 antibodies confirmed enrichment of exosomes. Moreover, Dynamic Light Scattering-based size distribution suggested that H-exo were smaller in size compared to N-exo. Further studies are underway to identify the molecular determinants in exosomes responsible for inducing growth-promoting effects under hypoxia. Overall, our data provides the first evidence that hypoxic PC cells secrete exosomes that enhance survivability under hypoxic conditions. Citation Format: Mary C. Patton, Haseeb Zubair, Girijesh K. Patel, Seema Singh, Ajay P. Singh. Exosomes from hypoxic pancreatic cancer cells confer resistance to subsequent hypoxia insult. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2799.

Hypoxia-Responsive Polymersomes for Drug Delivery to Hypoxic Pancreatic Cancer Cells

Hypoxia in tumors contributes to overall tumor progression by assisting in epithelial-to-mesenchymal transition, angiogenesis, and metastasis of cancer. In this study, we have synthesized a hypoxia-responsive, diblock copolymer poly(lactic acid)-azobenzene-poly(ethylene glycol), which self-assembles to form polymersomes in an aqueous medium. The polymersomes did not release any encapsulated contents for 50 min under normoxic conditions. However, under hypoxia, 90% of the encapsulated dye was released in 50 min. The polymersomes encapsulated the combination of anticancer drugs gemcitabine and erlotinib with entrapment efficiency of 40% and 28%, respectively. We used three-dimensional spheroid cultures of pancreatic cancer cells BxPC-3 to demonstrate hypoxia-mediated release of the drugs from the polymersomes. The vesicles were nontoxic. However, a significant decrease in cell viability was observed in hypoxic spheroidal cultures of BxPC-3 cells in the presence of drug encapsulated polymersomes. These polymersomes have potential for future applications in imaging and treatment of hypoxic tumors.

Inhibition of protein synthesis by imexon reduces HIF-1α expression in normoxic and hypoxic pancreatic cancer cells

Hypoxia-inducing factor-1 alpha (HIF-1alpha), is a major survival factor for tumor cells growing in a low oxygen environment. The anti-cancer agent imexon binds thiols and causes accumulation of reactive oxygen species (ROS) in pancreatic cancer cells. Unlike many cytotoxic agents, imexon is equi-cytotoxic in human MiaPaCa-2 and Panc-1 cells grown in normoxic (21% O(2)) and hypoxic (1% O(2)) conditions. Western blot analyses of imexon-treated cells demonstrated that imexon reduces HIF-1alpha protein levels in both normoxic and hypoxic conditions in a time- and concentration-dependant fashion. Gemcitabine did not similarly affect HIF-1alpha levels. Imexon did not reduce transcription of new HIF-1alpha mRNA, but did reduce the synthesis of new proteins, including HIF-1alpha, measured by (35)S methionine/cysteine (Met/Cys) incorporation. Concurrently, the half-life of existing HIF-1alpha protein was increased by imexon, in association with a marked inhibition of chymotryptic activity in the 20S proteasome. The inhibition of HIF-1alpha translation was not specific, rather it was part of a general decrease in protein translation caused by imexon. This inhibitory effect on translation did not involve phosphorylation of eukaryotic initiation factor-2alpha (eIF-2alpha) and was not closely correlated to cell growth inhibition by imexon, suggesting that mechanisms other than protein synthesis inhibition contribute to the drug's cytotoxic effects. In summary, imexon blocks the translation of new proteins, including HIF-1alpha, and this effect overcomes an increase in the stability of preformed HIF-1alpha due to proteasome inhibition by imexon. Because net HIF-1alpha levels are reduced by imexon, combination studies with other drugs affected by HIF-1alpha survival signaling are warranted.

Effects of YC-1 on Hypoxia-Inducible Factor 1-Driven Transcription Activity, Cell Proliferative Vitality, and Apoptosis in Hypoxic Human Pancreatic Cancer Cells

To investigate the effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) on HIF-1-driven transcription activity, cell proliferative vitality, and apoptosis in hypoxic human pancreatic cancer cells. Human pancreatic cancer PC-3 cells were incubated under normoxic or hypoxic conditions. YC-1 was added to the media with different concentrations. The HIF-1alpha protein expression was detected by means of immunocytochemical staining and Western blotting. Semiquantitative reverse transcriptase polymerase chain reaction was used to determine the mRNA expression of HIF-1alpha, vascular endothelial growth factor (VEGF), and glucose phosphate isomerase (GPI). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry were used to detect the cells' proliferative vitality and apoptosis. Hypoxic PC-3 cells expressed a higher level of HIF-alpha protein in nucleus compared with the normoxic controls. When the dose of YC-1 was at 100 micromol/L, the expression location of HIF-alpha shifted from nucleus to cytoplasm. Western blotting revealed that YC-1 reduced the level of HIF-1alpha protein expression, and the inhibitory effect was dose dependent. Moreover, YC-1 dose dependently inhibited mRNA expression levels of VEGF and GPI in hypoxic cells. YC-1 inhibited proliferative vitality and induced apoptosis of hypoxic PC-3 cells in a dose-dependent manner. YC-1 inhibits HIF-1alpha expression in hypoxic pancreatic cancer cells, which is accompanied by the translocation of HIF-1alpha from nucleus to cytoplasm, decreased mRNA expression of VEGF and GPI, reduced cell proliferative vitality, and increased apoptosis. These results suggest that HIF-1 is a potential therapeutic target for pancreatic cancer.