Impact of APE1/Ref-1 Redox Inhibition on Pancreatic Tumor Growth

Pancreatic cancer is especially a deadly form of cancer with a survival rate less than 2%. Pancreatic cancers respond poorly to existing chemotherapeutic agents and radiation, and progress for the treatment of pancreatic cancer remains elusive. To address this unmet medical need, a better understanding of critical pathways and molecular mechanisms involved in pancreatic tumor development, progression, and resistance to traditional therapy is therefore critical. Reduction–oxidation (redox) signaling systems are emerging as important targets in pancreatic cancer. AP endonuclease1/Redox effector factor 1 (APE1/Ref-1) is upregulated in human pancreatic cancer cells and modulation of its redox activity blocks the proliferation and migration of pancreatic cancer cells and pancreatic cancer-associated endothelial cells in vitro. Modulation of APE1/Ref-1 using a specific inhibitor of APE1/Ref-1′s redox function, E3330, leads to a decrease in transcription factor activity for NFκB, AP-1, and HIF1α in vitro. This study aims to further establish the redox signaling protein APE1/Ref-1 as a molecular target in pancreatic cancer. Here, we show that inhibition of APE1/Ref-1 via E3330 results in tumor growth inhibition in cell lines and pancreatic cancer xenograft models in mice. Pharmacokinetic studies also show that E3330 attains more than10 μmol/L blood concentrations and is detectable in tumor xenografts. Through inhibition of APE1/Ref-1, the activity of NFκB, AP-1, and HIF1α that are key transcriptional regulators involved in survival, invasion, and metastasis is blocked. These data indicate that E3330, inhibitor of APE1/Ref-1, has potential in pancreatic cancer and clinical investigation of APE1/Ref-1 molecular target is warranted. Mol Cancer Ther; 10(9); 1698–708. ©2011 AACR

Phyllanthus spp. Induces Selective Growth Inhibition of PC-3 and MeWo Human Cancer Cells through Modulation of Cell Cycle and Induction of Apoptosis

BACKGROUND: Phyllanthus is a traditional medicinal plant that has been used in the treatment of many diseases including hepatitis and diabetes. The main aim of the present work was to investigate the potential cytotoxic effects of aqueous and methanolic extracts of four Phyllanthus species (P.amarus, P.niruri, P.urinaria and P.watsonii) against skin melanoma and prostate cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: Phyllanthus plant appears to possess cytotoxic properties with half-maximal inhibitory concentration (IC(50)) values of 150-300 µg/ml for aqueous extract and 50-150 µg/ml for methanolic extract that were determined using the MTS reduction assay. In comparison, the plant extracts did not show any significant cytotoxicity on normal human skin (CCD-1127Sk) and prostate (RWPE-1) cells. The extracts appeared to act by causing the formation of a clear "ladder" fragmentation of apoptotic DNA on agarose gel, displayed TUNEL-positive cells with an elevation of caspase-3 and -7 activities. The Lactate Dehydrogenase (LDH) level was lower than 15% in Phyllanthus treated-cancer cells. These indicate that Phyllanthus extracts have the ability to induce apoptosis with minimal necrotic effects. Furthermore, cell cycle analysis revealed that Phyllanthus induced a Go/G1-phase arrest on PC-3 cells and a S-phase arrest on MeWo cells and these were accompanied by accumulation of cells in the Sub-G1 (apoptosis) phase. The cytotoxic properties may be due to the presence of polyphenol compounds such as ellagitannins, gallotannins, flavonoids and phenolic acids found both in the water and methanol extract of the plants. CONCLUSIONS/SIGNIFICANCE: Phyllanthus plant exerts its growth inhibition effect in a selective manner towards cancer cells through the modulation of cell cycle and induction of apoptosis via caspases activation in melanoma and prostate cancer cells. Hence, Phyllanthus may be sourced for the development of a potent apoptosis-inducing anticancer agent

A New Drug–Drug Interaction Between Hydroxychloroquine and Metformin? A Signal Detection Study

Introduction Hydroxychloroquine was recently promoted in patients infected with COVID-19 infection. A recent experimental study has suggested an increased toxicity of hydroxychloroquine in association with metformin in mice. Objective The present study was undertaken to investigate the reality of this putative drug–drug interaction between hydroxychloroquine and metformin using pharmacovigilance data. Methods Using VigiBase®, the WHO pharmacovigilance database, we performed a disproportionality analysis (case/non-case study). Cases were reports of fatal outcomes with the drugs of interest and non-cases were all other reports for these drugs registered between 1 January 2000 and 31 December 2019. Data with hydroxychloroquine (or metformin) alone were compared with the association hydroxychloroquine + metformin. Results are reported as ROR (reporting odds ratio) with their 95% confidence interval. Results Of the 10,771 Individual Case Safety Reports (ICSR) involving hydroxychloroquine, 52 were recorded as ‘fatal outcomes’. In comparison with hydroxychloroquine alone, hydroxychloroquine + metformin was associated with an ROR value of 57.7 (23.9–139.3). In comparison with metformin alone, hydroxychloroquine + metformin was associated with an ROR value of 6.0 (2.6–13.8). Conclusion Our study identified a signal for the association hydroxychloroquine + metformin that appears to be more at risk of fatal outcomes (particularly by completed suicides) than one of the two drugs when given alone

High Expression of Wee1 Is Associated with Poor Disease-Free Survival in Malignant Melanoma: Potential for Targeted Therapy

Notoriously resistant malignant melanoma is one of the most increasing forms of cancer worldwide; there is thus a precarious need for new treatment options. The Wee1 kinase is a major regulator of the G2/M checkpoint, and halts the cell cycle by adding a negative phosphorylation on CDK1 (Tyr15). Additionally, Wee1 has a function in safeguarding the genome integrity during DNA synthesis. To assess the role of Wee1 in development and progression of malignant melanoma we examined its expression in a panel of paraffin-embedded patient derived tissue of benign nevi and primary- and metastatic melanomas, as well as in agarose-embedded cultured melanocytes. We found that Wee1 expression increased in the direction of malignancy, and showed a strong, positive correlation with known biomarkers involved in cell cycle regulation: Cyclin A (p<0.0001), Ki67 (p<0.0001), Cyclin D3 (p = 0.001), p21Cip1/WAF1 (p = 0.003), p53 (p = 0.025). Furthermore, high Wee1 expression was associated with thicker primary tumors (p = 0.001), ulceration (p = 0.005) and poor disease-free survival (p = 0.008). Transfections using siWee1 in metastatic melanoma cell lines; WM239WTp53, WM45.1MUTp53 and LOXWTp53, further support our hypothesis of a tumor promoting role of Wee1 in melanomas. Whereas no effect was observed in LOX cells, transfection with siWee1 led to accumulation of cells in G1/S and S phase of the cell cycle in WM239 and WM45.1 cells, respectively. Both latter cell lines displayed DNA damage and induction of apoptosis, in the absence of Wee1, indicating that the effect of silencing Wee1 may not be solely dependent of the p53 status of the cells. Together these results reveal the importance of Wee1 as a prognostic biomarker in melanomas, and indicate a potential role for targeted therapy, alone or in combination with other agents

Medicinal and ethnoveterinary remedies of hunters in Trinidad

BACKGROUND: Ethnomedicines are used by hunters for themselves and their hunting dogs in Trinidad. Plants are used for snakebites, scorpion stings, for injuries and mange of dogs and to facilitate hunting success. RESULTS: Plants used include Piper hispidum, Pithecelobium unguis-cati, Bauhinia excisa, Bauhinia cumanensis, Cecropia peltata, Aframomum melegueta, Aristolochia rugosa, Aristolochia trilobata, Jatropha curcas, Jatropha gossypifolia, Nicotiana tabacum, Vernonia scorpioides, Petiveria alliacea, Renealmia alpinia, Justicia secunda, Phyllanthus urinaria,Phyllanthus niruri,Momordica charantia, Xiphidium caeruleum, Ottonia ovata, Lepianthes peltata, Capsicum frutescens, Costus scaber, Dendropanax arboreus, Siparuma guianensis, Syngonium podophyllum, Monstera dubia, Solanum species, Eclipta prostrata, Spiranthes acaulis, Croton gossypifolius, Barleria lupulina, Cola nitida, Acrocomia ierensis (tentative ID). CONCLUSION: Plant use is based on odour, and plant morphological characteristics and is embedded in a complex cultural context based on indigenous Amerindian beliefs. It is suggested that the medicinal plants exerted a physiological action on the hunter or his dog. Some of the plants mentioned contain chemicals that may explain the ethnomedicinal and ethnoveterinary use. For instance some of the plants influence the immune system or are effective against internal and external parasites. Plant baths may contribute to the health and well being of the hunting dogs

Abstract 3247: Functional CRISPR-Cas9 screens identify master regulators of resistance to chemical targeting of RNA polymerase I

Abstract Increased ribosome biogenesis is a hallmark of cancer and targeting this process with RNA polymerase I (Pol I) inhibitors is a promising strategy for cancer therapy. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and induces degradation of the enzyme. This approach is effective across many cancer types. However, a heterogeneous response was observed in cancer cell lines emphasizing the need to increase knowledge of factors that underlie the response to this therapeutic strategy. To identify genes that modulate the response of cancer cells to BMH-21, we performed genome-wide CRISPR-Cas9-based positive selection screens in human colorectal carcinoma cells. These screens identified high-confidence hits accounting for BMH-21 drug resistance that included all key positive regulators of the mTORC1 complex. Given that p53 has been identified as a downstream effector of Pol I transcription stress, we conducted the screens in TP53 isogenic cells. Notably, the mTORC1 pathway hits were identified in all screens indicating that the resistance was p53 independent. These findings are particularly striking given that mTOR is a major driver of ribosome biogenesis and cellular translational programs. The findings were validated using chemical and genetic approaches. Torin-1, a catalytic mTOR inhibitor, was found to cause resistance to BMH-21. mTOR signaling pathway knockout and rescue cell lines were generated and tested for changes in the drug responses by growth, viability, colony formation and GFP competition assays. In each case, compromised mTOR activity led to resistance to BMH-21. However, compromised mTOR activity did not abrogate Pol I transcription inhibition by BMH-21. To assess impact on protein translation, we used polysome profiling. BMH-21 treatment was found to cause a severe ribosome biogenesis defect. Surprisingly, mTOR inactivation partially rescued the translation ability under the drug treatment suggesting that this translation is pivotal for cell survival. To uncover factors critical for the survival, we performed Ribo-seq and RNA-seq in the drug-treated BMH-21 sensitive and resistant cells. The profiling results revealed that mTOR inactivation led to elevated translation efficiency of mRNAs encoding ribosomal proteins. These findings suggest that mTOR inactivation evokes compensatory selective translation of ribosomal proteins under severe ribosome biogenesis defect caused by the Pol I inhibition. The findings indicate that mTOR inactivation regulates selective translation as means to bypass Pol I inhibition, and more generally, that maintenance of translational capacity strongly contributes to treatment resistance. These findings reveal an unexpected complication by mTOR inhibitory strategies as well as have implications on exploring drug combinations in cancer. Citation Format: Wenjun Fan, Hester Liu, Stephanie Pitts, Brittany Ford, Rajeshkumar NV, Marikki Laiho. Functional CRISPR-Cas9 screens identify master regulators of resistance to chemical targeting of RNA polymerase I [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3247.</jats:p

Widespread germline genetic heterogeneity of human ribosomal RNA genes

AbstractPolymorphism drives survival under stress and provides adaptability. Genetic polymorphism of ribosomal RNA (rRNA) genes derives from internal repeat variation of this multicopy gene, and from interindividual variation. A considerable amount of rRNA sequence heterogeneity has been proposed but has been challenging to estimate given the scarcity of accurate reference sequences. We identified four rDNA copies on chromosome 21 (GRCh38) with 99% similarity to recently introduced reference sequence KY962518.1. Pairwise alignment of the rRNA coding sequences of these copies showed differences in sequence and length. We customized a GATK bioinformatics pipeline using the four rDNA loci, spanning a total 145 kb, for variant calling. We employed whole genome sequencing (WGS) data from the 1000 Genomes Project phase 3 and analyzed variants in 2,504 individuals from 26 populations. Using the pipeline, we identified a total of 3,790 variant positions. The variants positioned non-randomly on the rRNA gene. Invariant regions included the promoter, early 5’ ETS, 5.8S, ITS1 and certain regions of the 28S rRNA, and large areas of the intragenic spacer. 18S rRNA coding region had very few variants, while a total of 470 variant positions were observed on 28S rRNA. The majority of the 28S rRNA variants located on highly flexible human-expanded rRNA helical folds ES7L and ES27L, suggesting that these represent positions of diversity and are potentially under continuous evolution. These findings provide a genetic view for rRNA heterogeneity and raise the need to functional assess how the 28S rRNA variants affect ribosome functions.</jats:p

Abstract 2446: Targeting pancreatic cancer stem cells via the tumor microenvironment

Abstract Pancreatic adenocarcinoma is a highly aggressive disease with a propensity for metastasis early in its course. Pancreatic cancer stem cells (CSCs) have been thought to play a key role in this process. We previously found that pancreatic CSCs express increased levels of aldehyde dehydrogenase (ALDH) and at the time of diagnosis the presence of ALDH+ cells in primary tumors is associated with worse overall survival. The development of CSC-targeting strategies have largely focused on intrinsic signaling pathways, but normal stem cells are primarily regulated by the extracellular stem cell niche that consists of non-malignant cells and specific extracellular matrix (ECM) proteins. The densely fibrotic desmoplastic reaction (DR) is a common feature of pancreatic tumors, thus we examined the role of the ECM on pancreatic CSCs. We initially studied the distribution of ALDH+ cells in primary pancreatic tumors by immunohistochemistry and found that they were more frequently located at the invasive edge of tumors adjacent to the DR compared to the rest of the tumor (&amp;lt;5% of all cells). These findings suggested that the DR influences CSC properties and we cultured pancreatic cancer cell lines and primary tumors in the presence of type I collagen since it is the major ECM component of the DR and has been shown to increase the proliferation and survival of pancreatic tumor cells. Compared to cells cultured under standard conditions, type I collagen increased the proportion of ALDH+ cells, clonogenic growth and migratory potential in vitro. To determine if collagen provides a selective advantage to ALDH+ cells or induces ALDHneg cells to acquire a CSC phenotype, we studied isolated ALDH+ cells and found that they maintained their CSC phenotype to a higher degree (2-3 fold) in the presence of type I collagen compared to those cultured on plastic or matrigel. In contrast, ALDHneg cells cultured on collagen remained phenotypically similar to those cultured on plastic or matrigel. We also found that type I collagen induced further mesenchymal properties in ALDH+ cells as evidenced by decreased expression of E-cadherin and greater migratory potential. Furthermore, we found that expression of α2β1 integrin, a specific receptor for type I collagen, was restricted to ALDH+ cells. In order to target the interaction between type I collagen and pancreatic CSCs we studied focal adhesion kinase (FAK), a major component of integrin signal transduction, and found that ALDH+ cells expressed higher levels of activated, phosphorylated FAK compared to the bulk tumor cell population. Finally, we found that pharmacologic inhibition of FAK resulted in the loss of ALDH+ cells, clonogenic growth, and cellular migration. Our data suggest that the interaction between pancreatic CSCs and the DR results in enhanced tumorigenicity and metastatic potential. Furthermore, the disruption of FAK signaling can inhibit pancreatic CSC function and may serve as a novel therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2446. doi:10.1158/1538-7445.AM2011-2446</jats:p