Response Of Cancer Cell Lines Research Articles

STAT3 Contributes a Favorable Response to Pembrolizumab Through IFN-γ-induced Apoptosis in Urothelial Cancer.

Pembrolizumab, a second-line therapy for platinum-refractory advanced urothelial carcinoma (UC), is needed to improve objective response rate. Hence, it is crucial to identify optimal predictive biomarkers of responses. This study aimed to clarify the predictive value and role of signal transducer and activator of transcription 3 (STAT3) in selecting patients with advanced UC who might benefit clinically from pembrolizumab therapy. We retrospectively analyzed 31 patients who received pembrolizumab therapy for UC. STAT3, phosphorylated STAT3 (p-STAT3), and PD-L1 expression were determined using tissue microarrays constructed from patient-derived specimens, and the association of these expression levels with overall survival was analyzed. We assessed the functional role of STAT3 in bladder cancer cell lines in response to interferon-gamma (IFN-γ). Patients with high STAT3 or p-STAT3 expression, and high platelet-to-lymphocyte ratio (PLR) (n=6) had a significantly shorter OS; in the other patients (n=25), high STAT3 or p-STAT3 expression was significantly associated with improved prognosis. IFN-γ-induced apoptosis was partially dependent on STAT3 in T24 cells but not in JMSU1 cells. In patients with advanced UC, STAT3 plays a key role in mediating the efficacy of pembrolizumab through apoptosis in response to IFN-γ.

Farnesoid X receptor activation inhibits pancreatic carcinogenesis

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily that controls bile acid (BA) homeostasis, has also been proposed as a tumor suppressor for breast and liver cancer. However, its role in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis remains controversial. We recently found that FXR attenuates acinar cell autophagy in chronic pancreatitis resulting in reduced autophagy and promotion of pancreatic carcinogenesis. Feeding Kras-p48-Cre (KC) mice with the BA chenodeoxycholic acid (CDCA), an FXR agonist, attenuated pancreatic intraepithelial neoplasia (PanIN) progression, reduced cell proliferation, neoplastic cells and autophagic activity, and increased acinar cells, elevated pro-inflammatory cytokines and chemokines, with a compensatory increase in the anti-inflammatory response. Surprisingly, FXR-deficient KC mice did not show any response to CDCA, suggesting that CDCA attenuates PanIN progression and decelerate tumorigenesis in KC mice through activating pancreatic FXR. FXR is activated in pancreatic cancer cell lines in response to CDCA in vitro. FXR levels were highly increased in adjuvant and neoadjuvant PDAC tissue compared to healthy pancreatic tissue, indicating that FXR is expressed and potentially activated in human PDAC. These results suggest that BA exposure activates inflammation and suppresses autophagy in KC mice, resulting in reduced PanIN lesion progression. These data suggest that activation of pancreatic FXR has a protective role by reducing the growth of pre-cancerous PDAC lesions in response to CDCA and possibly other FXR agonists.

Pharmacological activities of Artemisia absinthium and control of hepatic cancer by expression regulation of TGFβ1 and MYC genes.

Plant derived compounds have always been an important source of medicines and have received significant attention in recent years due to their diverse pharmacological properties. Millions of plant-based herbal or traditional medicines are used to cure various types of cancers especially due to activation of proliferative genes. The aim of the present study was to characterize the altered and attenuated gene expression of the selected growth factor namely Transforming growth factor Beta -1 (TGFβ1) and MYC in human hepatoma-derived (Huh7) liver cancer cell lines in response to extracts of Artemisia absinthium dissolved in selected organic solvents. Ethanolic, methanolic and acetone extract of different plant parts (leaf, stem and flowers) was used to access the antiproliferative activity by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Quantitative Real-Time RT-PCR revealed that the transcript levels of TGFβ1 are induced in the samples treated with methanolic extract of Artemisia absinthium. Furthermore, reduced expression levels of MYC gene was noticed in cancerous cells suggesting antiproliferative properties of the plant. This study further highlights the resistance profile of various microbes by antimicrobial susceptibility test with plant extracts. In addition, antidiabetic effect of Artemisia absinthium have also shown positive results. Our study elucidates the potentials of Artemisia absinthium as a medicinal plant, and highlights the differential expression of genes involved in its mitogenic and anti-proliferative activity with a brief account of its pharmacological action.

Incorporation of 53BP1 into phase-separated bodies in cancer cells during aberrant mitosis.

53BP1 (also known as TP53BP1) is a key mediator of the non-homologous end joining (NHEJ) DNA repair pathway, which is the primary repair pathway in interphase cells. However, the mitotic functions of 53BP1 are less well understood. Here, we describe 53BP1 mitotic stress bodies (MSBs) formed in cancer cell lines in response to delayed mitosis. These bodies displayed liquid-liquid phase separation characteristics, were close to centromeres, and included lamin A/C and the DNA repair protein RIF1. After release from mitotic arrest, 53BP1 MSBs decreased in number and moved away from the chromatin. Using GFP fusion constructs, we found that the 53BP1 oligomerization domain region was required for MSB formation, and that inclusion of the 53BP1 N terminus increased MSB size. Exogenous expression of 53BP1 did not increase MSB size or number but did increase levels of MSB-free 53BP1. This was associated with slower mitotic progression, elevated levels of DNA damage and increased apoptosis, which is consistent with MSBs suppressing a mitotic surveillance by 53BP1 through sequestration. The 53BP1 MSBs, which were also found spontaneously in a subset of normally dividing cancer cells but not in non-transformed cells (ARPE-19), might facilitate the survival of cancer cells following aberrant mitoses. This article has an associated First Person interview with the first author of the paper.

The modulatory effects of two bioflavonoids, quercetin and thymoquinone on the expression levels of DNA damage and repair genes in human breast, lung and prostate cancer cell lines.

The recent decade has witnessed the increasing potential of various flavonoids such as quercetin and thymoquinone in inhibiting cancer cells proliferation and growth and their therapeutic effects in various cancers. Therefore, in the current study, we aim to evaluate the expression levels of key factors of DNA damage response in human breast, lung and prostate cancer cell lines in response to treatment with quercetin and thymoquinone. MTT assay was applied to assess the effects of quercetin and thymoquinone on the viability of MCF-7, A549, and PC3 cancer cells. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to evaluate the expression levels of p53, RAD51, Ku70, XRCC1, and H2AX in treated cells. In addition, the expression rate of 8-hydroxy-deoxyguanosine (8-OH-dG) was assessed by ELISA kit. The quercetin and thymoquinone induce cytotoxicity in breast, lung, and prostate cancer cells effectively; MCF-7 cells were the most sensitive cells to quercetin with an IC50 value of 50μM and PC3 cells were more sensitive to thymoquinone with an IC50 value of 20μM. The expression levels of DNA damage markers, H2AX, and 8-OH-dG were significantly increased in all cancer cells treated with quercetin and thymoquinone (p<0.05). Moreover, both flavonoids significantly decreased the expression levels of DNA repair mediators, RAD51, Ku70, XRCC1, in cell lines. P53 was also increased in MCF-7 and A549 cells. We concluded that quercetin and thymoquinone may exert their effects through modulation of DNA damage response, increasing DNA damage, and suppressing DNA repair genes.

Circular dorsal ruffles disturb the growth factor-induced PI3K-AKT pathway in hepatocellular carcinoma Hep3B cells

BackgroundCircular dorsal ruffles (CDRs) are rounded membrane ruffles induced on the dorsal surfaces of cells stimulated by growth factors (GF). They can serve as signal platforms to activate AKT protein kinase. After GF stimulation, phosphatidylinositol 3-kinase (PI3K) generates phosphatidylinositol (3,4,5)-triphosphate (PIP3) in the plasma membrane. PIP3 accumulates inside CDRs, recruits AKT into the structures, and phosphorylates them (pAKT). Given the importance of the PI3K-AKT pathway in GF signaling, CDRs are likely involved in cell growth. Interestingly, some cancer cell lines express CDRs. We hypothesized that CDRs contribute to carcinogenesis by modulating the AKT pathway. In the present study, we identified CDR-expressing cancer cell lines and investigated their cellular functions.MethodsCDR formation was examined in six cancer cell lines in response to epidermal growth factor (EGF) and insulin. The morphology of the CDRs was characterized, and the related signaling molecules were observed using confocal and scanning electron microscopy. The role of CDRs in the AKT pathway was studied using biochemical analysis. The actin inhibitor cytochalasin D (Cyto D) and the PI3K inhibitor TGX221 were used to block CDRs.ResultsGF treatment induced CDRs in the hepatocellular carcinoma (HCC) Hep3B cell line, but not in others, including HCC cell lines HepG2 and Huh7, and the LO2 hepatocyte cell line. Confocal microscopy and western blot analysis showed that the PI3K-PIP3-AKT pathway was activated at the CDRs and that receptor proteins were recruited to the structures. Cyto D and TGX221 completely blocked CDRs and partially attenuated GF-induced pAKT. These results indicate that CDRs regulate the receptor-mediated PI3K-AKT pathway in Hep3B cells and the existence of CDR-independent pAKT mechanisms.ConclusionsOur results showed that CDRs modulate the AKT pathway in Hep3B cells. Since CDRs were not observed in other HCC and hepatocyte cell lines, we propose that CDRs in Hep3B would determine the carcinoma characteristic of the cell by aberrantly triggering the AKT pathway. Signaling molecules involved in CDR formation are promising therapeutic targets for some types of HCC.9eb9aTWDk6mAQeLNgQGeJaVideo abstract

Abstract 2995: Autophagy as a key driver of waterpipe smoke-induced lung cancer cell plasticity and resistance

Abstract Smoking is the number one risk factor for lung cancer and tobacco smokers are at 20 to 40 times higher risk of developing lung cancer in comparison to non-smokers. We have previously shown that exposing lung cancer cell lines to tobacco smoke condensate generated from waterpipe (WP) interferes with cell proliferation, cell plasticity, DNA damage and tumor cell recognition and killing by natural killer (NK) cells. Global transcriptomic analysis identified an expression profile of genes that best distinguished treated and non-treated cells involving several pathways including those involved in epithelial to mesenchymal transition (EMT) and stemness. Because of the important role of autophagy in tumor resistance and progression, we investigated its relationship with WP smoking. We first showed an activation of autophagy as reflected by LC3 and P62 in lung cancer cell lines in response to WPS. Interestingly, our data revealed that WPS-induced EMT and cancer stemness involved autophagy induction. The autophagy response in smokers with lung adenocarcinoma as compared to non-smokers with lung adenocarcinoma was investigated using TCGA lung adenocarcinoma bulk RNA-seq dataset with the available patient metadata on smoking status. Preliminary results based on a machine learning classification model using Random Forest indicate that smokers have an increase in autophagy activating genes as compared to non-smokers. Comparative analysis of lung adenocarcinoma molecular signatures in affected patients with a long-term active exposure to smoke compared to patients who have not had any active exposure to smoke indicate a higher tumor mutational burden, CD8+ T-cell level and lower dysfunction level in smokers. Multivariate regression analyses confirm these results even after controlling for confounding factors like age, gender, and tumor stage. While the checkpoint genes tested PD1, PDL1, PDL2 and CTLA-4, had no difference between smokers and non-smokers, B7-1, B7-2, IDO1 and CD200R1 had higher expression in non-smokers than smokers. Because multiple factors in the tumor microenvironment dictate the success of immunotherapy, in addition to the expression of immune checkpoint genes, our analysis explains why patients who are smokers with lung adenocarcinoma respond better to immunotherapy, even though there are no relative differences in immune checkpoint genes in the two cohorts. Therefore, targeting autophagy in lung adenocarcinoma patients in combination with checkpoint inhibitors, targeted therapies or chemotherapy should be considered in smoker patients with lung adenocarcinoma. Citation Format: Rania F. Zaarour, Mohak Sharda, Bilal Azakir, Goutham Hassan Venkatesh, Raefa Abou Khouzam, Ayesha Rifath, Zohra Nizami, Fatima Abdullah, Fatin Mohammad, Husam Nawafleh, Yehia ElSayed, Salem Chouaib. Autophagy as a key driver of waterpipe smoke-induced lung cancer cell plasticity and resistance [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 2995.

TGF\u03b1-EGFR pathway in breast carcinogenesis, association with WWOX expression and estrogen activation

WWOX is a tumor-suppressive steroid dehydrogenase, which relationship with hormone receptors was shown both in animal models and breast cancer patients. Herein, through nAnT-iCAGE high-throughput gene expression profiling, we studied the interplay of estrogen receptors and the WWOX in breast cancer cell lines (MCF7, T47D, MDA-MB-231, BT20) under estrogen stimulation and either introduction of the WWOX gene by retroviral transfection (MDA-MB-231, T47D) or silenced with shRNA (MCF7, BT20). Additionally, we evaluated the consequent biological characteristics by proliferation, apoptosis, invasion, and adhesion assays. TGFα-EGFR signaling was found to be significantly affected in all examined breast cancer cell lines in response to estrogen and strongly associated with the level of WWOX expression, especially in ER-positive MCF7 cells. Under the influence of 17β-estradiol presence, biological characteristics of the cell lines were also delineated. The study revealed modulation of adhesion, invasion, and apoptosis. The obtained results point at a complex role of the WWOX gene in the carcinogenesis of the breast tissue, which seems to be closely related to the presence of estrogen α and/or β receptors.

Multiple G-quadruplex binding ligand induced transcriptomic map of cancer cell lines.

The G-quadruplexes (G4s) are a class of DNA secondary structureswith guanine rich DNA sequences that can fold into four stranded non-canonical structures. At the genomic level, their pivotal role is well established in DNA replication, telomerase functions, constitution of topologically associating domains, and the regulation of gene expression. Genome instability mediated by altered G4 formation and assembly has been associated with multiple disorders including cancers and neurodegenerative disorders. Multiple tools have also been developed to predict the potential G4 regions in genomes and the whole genome G4 maps are also being derived through sequencing approaches. Enrichment of G4s in the cis-regulatory elements of genes associated with tumorigenesis has accelerated the quest for identification of G4-DNAbinding ligands (G4DBLs) that can selectively bind and regulate the expression of such specific genes. In this context, the analysis of G4DBL responsive transcriptome in diverse cancer cell lines is inevitable for assessment of the specificity of novel G4DBLs. Towards this, we assembled the transcripts differentially regulated by different G4DBLs and have also identified a core set of genes regulated in diverse cancer cell lines in response to 3 or more of these ligands. With the mode of action of G4DBLs towards topology shifts, folding, or disruption of G4 structure being currently visualized, we believe that this dataset will serve as a platform for assembly of G4DBL responsive transcriptome for comparative analysis of G4DBLs in multiple cancer cells based on the expression of specific cis-regulatory G4 associated genes in the future.

A Potent, Selective CBX2 Chromodomain Ligand and Its Cellular Activity During Prostate Cancer Neuroendocrine Differentiation.

Polycomb group (PcG) proteins are epigenetic regulators that facilitate both embryonic development and cancer progression. PcG proteins form Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). PRC2 trimethylates histone H3 lysine 27 (H3K27me3), a histone mark recognized by the N-terminal chromodomain (ChD) of the CBX subunit of canonical PRC1. There are five PcG CBX paralogs in humans. CBX2 in particular is upregulated in a variety of cancers, particularly in advanced prostate cancers. Using CBX2 inhibitors to understand and target CBX2 in prostate cancer is highly desirable; however, high structural similarity among the CBX ChDs has been challenging for developing selective CBX ChD inhibitors. Here, we utilize selections of focused DNA encoded libraries (DELs) for the discovery of a selective CBX2 chromodomain probe, SW2_152F. SW2_152F binds to CBX2 ChD with a Kd of 80 nM and displays 24-1000-fold selectivity for CBX2 ChD over other CBX paralogs in vitro. SW2_152F is cell permeable, selectively inhibits CBX2 chromatin binding in cells, and blocks neuroendocrine differentiation of prostate cancer cell lines in response to androgen deprivation.

Changes in Density and Localization of GLUT1 and MCT1 Transporters of Cancer Cells Induced by Ketones

Ketone supplementation has been investigated as a potential therapy and/or as adjuvant with traditional cancer treatments. Previous studies have indicated that ketone supplementation decreases viability and proliferation of cultured tumor cells, as well as prolongs survival of mice with metastatic cancer. The primary fuel for cancer cells is glucose that enters the cells through glucose transporters (GLUT), while lactate, another key energy source for cancer cells that has been shown to enhance tumor angiogenesis, is exported through monocarboxylate transmembrane transporters (MCT). The inhibition of these transporters could lead to promising anti-cancer therapies, therefore, we investigated the changes in the density and localization of GLUT1 and MCT1 transporters in various cancer cell lines in response to R,S-sodium-3-hydroxybutyrate (βHB) in the culture media, in order to identify underlying mechanisms of the previously observed anti-cancer effects. We studied mice metastatic cancer (VM-M3), human brain cancer (U87MG) and human lung cancer (A549) cells using indirect immunofluorescence assay with microplate photometer and confocal microscopy. Cells were cultured in 12 or 96-well plates, and after 2 days they were treated with graded concentrations of βHB for 24 hrs, followed by immunofluorescent reaction. In VM-M3 cells only 1 nM βHB treatment decreased GLUT1 density, but not higher concentrations. A statistically significant decrease in GLUT1 density was detected with increasing βHB concentration starting at 2 nM in U87 cells and at 5nM in A549 cells. The density of MCT1 transporter decreased in all cell types with βHB treatment, and less protein was localized in the cell nucleus. These results suggest a possible mechanism for ketone bodies to decrease cancer cell viability via decreased ability to take up glucose and export lactate. However, the lowest effective concentration was different in each cell type and it is possible that higher concentrations might induce compensatory mechanisms. Further exploration of the effects of ketones on the expression of cell surface proteins and transporters relevant to cancer cell metabolism may improve our understanding of the clinical relevance of ketone supplementation in the treatment of cancer.

Tumor heterogeneity in autophagy-dependent ferroptosis

ABSTRACT Macroautophagy (hereafter referred to as “autophagy”) is a lysosome-mediated degradation process that plays a complex role in cellular stress, either promoting survival or triggering death. Early studies suggest that ferroptosis, an iron-dependent form of regulated cell death, is not related to autophagy. Conversely, recent evidence indicates that the molecular machinery of autophagy facilitates ferroptosis through the selective degradation of anti-ferroptosis regulators. However, the mechanism of autophagy-dependent ferroptosis remains incompletely understood. Here, we examine the early dynamic change in protein expression of autophagic (e.g., MAP1LC3B and SQSTM1) or ferroptotic (e.g., SLC7A11 and GPX4) regulators in 60 human cancer cell lines in response to two classical ferroptosis activators (erastin and RSL3) in the absence or presence of the lysosomal inhibitor chloroquine. Compared to erastin, RSL3 exhibits wider and stronger activity in the upregulation of MAP1LC3B-II or downregulation of SQSTM1 in 80% (48/60) or 63% (38/60) of cell lines, respectively. Both RSL3 and erastin failed to affect SLC7A11 expression, but they led to GPX4 downregulation in 12% (7/60) and 3% (2/60) of cell lines, respectively. Additionally, the intracellular iron exporter SLC40A1/ferroportin-1 was identified as a new substrate for autophagic elimination, and its degradation by SQSTM1 promoted ferroptosis in vitro and in xenograft tumor mouse models. Together, these findings show tumor heterogeneity in autophagy-dependent ferroptosis, which might have different biological behaviors with regard to the dynamic characteristics of cell death. Abbreviations: ATG: Autophagy-related; CQ: Chloroquine; GPX4: Glutathione peroxidase 4; MAP1LC3B/LC3: Microtubule-associated protein 1 light chain 3 beta: NCOA4: Nuclear Receptor Coactivator 4; ROS: Reactive Oxygen Species; SLC40A1/ferroportin-1: Solute Carrier family 40 Member 1; SLC7A11: Solute Carrier Family 7 Member 11; SQSTM1/p62: Sequestosome 1

Differential metabolic responses in breast cancer cell lines to acidosis and lactic acidosis revealed by stable isotope assisted metabolomics

Extracellular acidosis is considered as a hallmark of most human tumors, which plays an important role in promoting tumor malignant and aggressive phenotype in tumorigenesis. Acidosis and lactic acidosis can induce different responses in tumors. Previous studies have associated the response to lactic acidosis of tumors with good survival outcomes. In this study, we investigated the metabolomic changes in triple negative and luminal subtype breast cancer cell lines in response to acidosis and lactic acidosis. Our results showed that acidosis results in the reduction of cell viability and glycolysis in breast cancer cells, which is reversely correlated with the malignancy of cell lines. Under lactic acidosis, this reduction is reversed slightly. Untargeted metabolomic profiling revealed that glutaminolysis and fatty acid synthesis in cancer cells under acidosis are increased, while TCA cycle and glycolysis are decreased. Under lactic acidosis, the pentose phosphate pathway and acetate release are increased in MDA-MB-231 cells. The current results uncovered the different metabolic responses of breast cancer cells to acidosis and lactic acidosis, demonstrating the power of combined untargeted and stable isotope assisted metabolomics in comprehensive metabolomic analysis.

Collagen and fibronectin promote an aggressive cancer phenotype in breast cancer cells but drive autonomous gene expression patterns.

Understanding how various pathologies of breast cancer respond to their environment may be imperative in the creation of novel therapeutic targets. Central to the organisation and behaviour of cells within the tumour microenvironment is the extracellular matrix (ECM), a meshwork of fibrous proteins and glycoproteins that directly influences cell behaviour and the bioavailability of signalling molecules. Our appreciation on how the composition of the ECM can influence cancer behaviour has evolved significantly and although we are highly cognisant of the dramatic impact the ECM can have on cancer cell behaviour, we continue to neglect this during diagnosis and treatment. In the following study, we aimed to identify how three breast cancer cell lines respond functionally and genetically to common components of the ECM. Using real time and end point assays we have identified similar patterns of behaviour among the three breast cancer cell lines in response to commonly found ECM components of the breast. Using a selected gene panel, we have been able to identify cell line specific changes in gene differentiation when breast cancer cells are in contact with these elements. Although the response of our cells to these elements differ at the genetic level, their functional responses are consistent. This work adds to the growing arguments that highlight a need for histologically assessing ECM composition of breast tumours. In particular monitoring of fibrous protein deposition at the site of malignancy could provide critical information during clinical assessment influencing disease prognosis and treatment decisions for breast cancer patients.

A mass spectrometry-based proteome map of drug action in lung cancer cell lines.

Mass spectrometry-based discovery proteomics is an essential tool for the proximal readout of cellular drug action. Here, we apply a robust proteomic workflow to rapidly profile the proteomes of five lung cancer cell lines in response to more than 50 drugs. Integration of millions of quantitative protein-drug associations substantially improved the mechanism of action (MoA) deconvolution of single compounds. For example, MoA specificity increased after removal of proteins that frequently responded to drugs and the aggregation of proteome changes across cell lines resolved compound effects on proteostasis. We leveraged these findings to demonstrate efficient target identification of chemical protein degraders. Aggregating drug response across cell lines also revealed that one-quarter of compounds modulated the abundance of one of their known protein targets. Finally, the proteomic data led us to discover that inhibition of mitochondrial function is an off-target mechanism of the MAP2K1/2 inhibitor PD184352 and that the ALK inhibitor ceritinib modulates autophagy.

Inhibition of β-glucosidase overcomes gastric cancer chemoresistance through inducing lysosomal dysfunction

The clinical management of gastric cancer still remains challenge due to its poor response to chemotherapy. Better understanding the underlying mechanisms is required for the identification of more comprehensive therapies to overcome chemoresistance in gastric cancer. GBA1 level was systematically analyzed in gastric cancer patients before and after chemotherapy, and gastric cancer cells exposed to long-term chemo agent's treatment. The roles of GBA1 and its downstream mechanisms were investigated using pharmacological and genetic approaches. We observed the time-dependent upregulation of GBA1 expression and enzyme activity in multiple gastric cancer cell lines in response to prolonged exposure of 5-FU. It is noted that this phenomenon was also observed in gastric cancer patients after chemotherapy. Interestingly, no significant differences on GBA1 expression were detected between normal and malignant gastric tissues. These suggest that the predominant role of GBA1 is in the development of gastric cancer chemoresistance rather than tumorigenesis. Functional analysis demonstrated that GBA1 inhibition suppressed gastric cancer growth and survival without affecting migration, and augmented 5-FU's efficacy. Consistently, GBA1 inhibition was active against 5-FU-resistant gastric cancer cells. Mechanism studies showed that GBA1 inhibition led to loss of lysosomal integrity and function in 5-FU-resistant gastric cancer cells. We are the first to show that inhibition of β-glucosidase (encoded by GBA1) sensitizes gastric cancer to chemotherapy. Our findings demonstrate the therapeutic value of inhibiting GBA1 in gastric cancer, particularly in those who develop chemoresistance.

Dexmedetomidine enhances hypoxia-induced cancer cell progression.

Dexmedetomidine (DEX) is widely used in perioperative settings for analgesia and sedation; however, little is known about its effects on the hypoxia-induced progression of tumor cells. In the present study, the effects of DEX on hypoxia-induced growth and metastasis of lung cancer cells and colorectal cancer cells was examined. A549 cells and HCT116 cells were treated with normoxia, hypoxia, co-treatment of hypoxia and DEX, and atipamezole (an α2 adrenoceptor antagonist) for 4 h. The proliferation rate of cells was determined by MTT assays. Cell metastatic potential was evaluated by Transwell assays. Survivin and hypoxia inducible factor (HIF)-1α were detected by western blotting. Matrix metalloproteinase (MMP)-2 and MMP-9 were measured using reverse transcription-quantitative PCR. It was demonstrated that hypoxia treatment promoted the proliferation and may promote the metastasis of the two cancer cell lines. DEX substantially contributed to the survival and aggressiveness of the two cancer cell lines following hypoxia. Furthermore, DEX upregulated the expression of survivin, MMP-2, MMP-9 and HIF-1α in the two cancer cell lines in response to hypoxia. Finally, the effects of DEX on the hypoxia-induced growth and metastatic potential of cancer cells were reversed by atipamezole. Collectively, DEX enhances the hypoxia-induced progression of lung cancer cells and colorectal cancer cells by regulating HIF-1α signaling, which may be associated with the α2 adrenoceptor pathway.

PPARα agonist alleviates tumor growth and chemo-resistance associated with the inhibition of glucose metabolic pathway

As a nuclear receptor, peroxisome-proliferator-activated receptor α (PPARα) plays a critical role in regulation of metabolism and cancer, while the effect of PPARα agonist on cancer cell glucose metabolism-linked tumor growth is still unclear. Here we found that PPARα agonist (Wy14,643) decreased Glut1 (Glucose transporter 1) gene and protein expressions of colorectal cancer cell lines in response to normoxia or hypoxia. Dual-luciferase analysis showed that Wy14,643 inhibited Glut1 transcription activity. Importantly, ChIP-qPCR analysis showed that Wy14,643 increased the binding of PPARα to Glut1 promoter region. Wy14,643 suppressed Glut1 transcription activity resulting in reduced influx of glucose in cancer cells in response to normoxia or hypoxia. Further analysis showed that Wy14,643-mediated inhibition of tumor growth and chemo-resistance was associated with inhibition of mTOR pathway. Taken together, PPARα agonist Wy14,643 suppressed Glut1 transcription activity, glucose uptake and mTOR pathway in colorectal cancer cells, which was involved in reduced tumor growth and chemo-resistance. These findings provided a novel therapy strategy for cancer progression.

Abstract 4596: Synthesis and biological evaluation of small molecules inhibitors targeting heterotrimeric Gαi2 protein

Abstract Heterotrimeric G-proteins are ubiquitously expressed in cells, and they transduce signals from activated G-protein coupled receptors. It is well known that these proteins are differentially expressed in cancer, thereby activating signals that lead to different biological functions such as proliferation and cell motility. Previously, we have shown that Gαi2 protein is essential for cell migration and invasion in prostate cancer cell lines, and its action is downstream of PI3-kinase/Rac1 activation. We have also shown that in PC3 prostate cancer cells, pre-treated with Pertussis toxin (PTX), a well know inhibitor of Gαi/0, resulted in the attenuation of TGFβ1- and oxytocin-induced migration and PI3-kinase activation, without affecting EGF-induced PI3-kinase activation and cell migration. Here, we synthesized new small molecules inhibitors, GDIs, capable of preventing nucleotide exchange and subunit activation. Compounds #29 and #46 are analogs of the lead compound, lacking the thiophene OH-group and with a thiol- to N-methyl amino-group substitution, respectively. Compound #35, a methyl ether derivative of #46, is designed to test the effect of the modification to the phenolic group on Gαi2 inhibition activity. We pretreated PC3 cells with the small molecules and then stimulated with EGF and oxytocin to activate Gαi2 subunit. Immunoprecipitation of the active Gαi and western blot for Gαi2 showed a decrease in the amount of active Gαi2 that was pulled down compared with the controls. Using cell migration assay, we observed that #29 and #46, both at 10 μM, caused inhibition of migration in different prostate cancer cell lines in response to EGF treatments. In contrast, #35 is inactive in this assay at the same concentration. To determine if the effects of Gαi2 are the same in other cancer cells, we first knockdown Gαi2 protein expression in breast and ovarian cancer cells and performed migration assays. The knock-down of endogenous Gαi2 attenuated cell migration, compared to the control cells. We also performed migration assays in the same cells using compound #29 at 10 μM and observed significant inhibition of migratory behavior in response to EGF- and FBS stimuli compared to the controls. We conclude that the small molecules we used in this study may be considered as potential therapeutic tools, inhibiting the migratory capability of metastatic cells. Citation Format: Silvia Caggia, Autumn S. Garrett, Aditi Kumar, Subhasish Tapadar, Adegboyega K. Oyelere, Smrruthi Vaidegi Venugopal, Shafiq A. Khan. Synthesis and biological evaluation of small molecules inhibitors targeting heterotrimeric Gαi2 protein [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 4596.

Abstract 270: Temozolomide resistant MGMT negative/MMR proficient cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival

Abstract MGMT loss is used to predict response to temozolomide (TMZ), however, some patients do not benefit, suggesting that additional criteria for selection remain to be identified. Using patient derived and well-established cancer lines cancer lines we observed a 100% association between mismatched repair (MMR) defects and TMZ resistance; however, only 16% of MGMT-/MMR+ lines were sensitive to TMZ at clinically relevant concentrations. Activation of DNA damage and repair signaling was noted in both sensitive and resistant bladder cancer cell lines in response to TMZ but protracted DNA damage, accompanied by persistent G2/M arrest, was observed only in the sensitive models. ATR inhibition sensitized MGMT-/MMR proficient resistant models to TMZ resulting in measurable DNA damage, prolonged G2/M arrest and persistent growth inhibition: an effect that was mediated by Chk1. Homologous recombination and repair has been shown to play an important role in repairing TMZ-induced DNA damage. We demonstrate that ATR inhibition abrogated homologous recombination (HR) activity in a MGMT-/MMR proficient TMZ resistant bladder model and that a TMZ sensitive bladder model had minimal HR (assessed by pDR-GFP/SceI assay). Moreover, HR -deficiency (mediated by BRCA1 defects or RAD51 inhibition) conferred sensitivity to TMZ in combination with an ATR inhibitor. Our in vitro data suggest that a MGMT-/MMR+/HR-Deficiency profile identifies models that respond to TMZ/ATR inhibitor combination. Indeed, one Patient-derived Xenograft (PDX) model (available in the Patient Derived Models Repository), selected by this criterion (MGMT-/MMR+ and LOH&amp;gt;40%) showed delayed tumor growth and increased survival in the TMZ-VX970 combination arm, compared to single agents. Overall, our data suggest that MGMT-/MMR+ cells rely on ATR-dependent signaling for repair of TMZ-induced DSBs and that HR defects should be evaluated as criteria for selecting patients that will benefit from an ATR inhibitor/TMZ combination regimen. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Lara El Touny, Erik Harris, Curtis Hose, John Connelly, Diana Vengsarkar, Anne Monks, Ralph E. Parchment, James H. Doroshow, Beverly A. Teicher, Annamaria Rapisarda. Temozolomide resistant MGMT negative/MMR proficient cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival [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 270.