Survival Mechanism In Cancer Cells Research Articles

Hsp Inhibitor is Affective Against Adenocarcinomic Human Alveolar Basal Epithelial Cells Through Modulating ERK/MAPK Signaling Pathway.

The extracellular signal-regulated kinase (ERK) - mitogen-activated protein kinase (MAPK) pathway regulates cell proliferation, differentiation, and apoptosis. Heat Shock Protein 90 (HSP90) is required to activate proto-oncogenic protein kinases and promotes tumor growth through anti-apoptotic effects on A549-non-small cell lung cancer (NSCLC). Therefore, deregulation of the ERK-MAPK pathway and abnormal expression of HSP90 are reasonably frequent events in NSCLC. In this study, novel perimidine-pyrazole compounds employed to block ERK-MAPK deregulation through inhibiting HSP dependent cancer cell survival mechanisms. A set of perimidine-pyrazole derivatives effects was monitored on NSCLC cell line. Array experiments performed to understand the effect of the compounds on signaling pathways and results were analyzed by gene enrichment analysis. Further, senescence and apoptosis experiments were performed to support the enrichment results along with in silico methods to determine perimidine-pyrazole/HSP interactions. Treatment of NSCLC cells with perimidine-pyrazole derivatives displayed cancer-inhibitory, pro-senescent and pro-apoptotic effects on NSCLC cells through ERK/MAPK pathway and these compounds are promising templates for designing anticancer drugs.

Immunohistochemical evaluation of cellular activities in canine osteoblastic osteosarcoma

Osteosarcoma is the most frequently occurring malignant bone tumor in animals and humans. A better understanding of the etiopathogenetic mechanisms of osteosarcoma, especially biomolecular ones, is fundamental to improving diagnosis and prognosis of the disease. Autophagy is a self-degrative process that removes dysfunctional cellular components, eliminates intracellular pathogens, and promotes cellular senescence. In neoplastic cells, autophagy suppresses tumorigenesis by inhibiting cancer cell survival mechanisms and promoting cell death. The aim of this study was to use immunohistochemistry to identify alterations in the expression of several apoptotic and proliferative biomarkers in multiple cases of canine osteosarcoma. Bcl-2, an intracellular membrane protein, inhibits cell death by blocking the p53-mediated pathway of apoptosis. While Bcl-2 overexpression has been described in many different premalignant and malignant lesions, it has yet to be analyzed in canine osteosarcoma. Our group investigated 10 primary canine osteoblastic osteosarcoma cases from the University of Perugia Department of Veterinary Medicine Teaching Hospital. Immunohistochemical analyses of proteins Bcl-2, Ki-67, and p53 have revealed interesting results as described in this paper. Expression of Bcl-2 was increased in all cases investigated while expression of p53 and Ki-67 was variable and no statistical association was observed between the expression patterns of p53, Bcl-2 and Ki-67.

Abstract 3101: Discovery of helical sulfonyl-γ-AApeptides targeting LC3 in cancer cells

Abstract Introduction: Autophagy pathway activation and upregulation is an important mechanism for cancer cell survival upon exposure to chemotherapeutic agents. LC3, a ubiquitin-like protein, plays a key role in the regulation of cargo selection and autophagosome formation. Given small molecule inhibitors have not been well developed to target LC3, we aim to design and synthesize an LC3 binder and evaluate its activity in the inhibition of autophagy pathway in cancer cells. Methods: The crystal structure of LC3 and p62 complex (PDB: 2ZJD) was used to assist our design of helical sulfonyl-γ-AApeptides to the LC3 interacting region of p62. The peptidomimetics were synthesized on the solid phase and purified by HPLC. We used fluorescent polarization assay to assess the binding affinity of the peptidomimetics to LC3. We then utilized an LC3 HiBiT reporter cell line (U2OS) from Promega, which was engineered to have a fused human LC3-HiBiT with a HaloTag in between to study the effect of the lead LC3 binder on autophagic flux. In addition, confocal microscopy enabled direction visualization of the colocalization of FITC-labeled LC3 binder with LC3 and lysosome, respectively. Cytotoxicity of LC3 binder was evaluated in colorectal cancer cell line HCT116. Results: We designed and synthesized 6 helical sulfonyl-γ-AApeptides (JC1-6) to target the binding interface of LC3 and p62. These peptidomimetic compounds were designed to retain essential LC3 and p62 interactions at the W and L regions and yet have different functional groups on the helical scaffold. Compounds JC1 and JC6 have binding affinity of Kd 280 nM and 150 nM with LC3, respectively. FITC-labelled JC6 was able to penetrate U2OS and HCT116 cells. It had intracytoplasmic colocalization with not only lysosomes but also LC3 aggregates. Direct visualization of intracytoplasmic LC3 aggregates showed that JC6 at 50 µM significantly inhibited the autophagic degradation of LC3 aggregates. This effect was augmented by the presence of 2 µM autophagy activator PP242. Lastly, JC6 demonstrated cytotoxicity with IC50 approximately 50 µM in HCT116 cells bearing baseline autophagy activation compared to lack of cytotoxicity in U2OS cells with no baseline autophagy activity. Conclusion: We designed and synthesized novel helical sulfonyl-γ-AApeptide LC3 binders. With the ability of cellular penetration, they demonstrated inhibition of autophagic flux and preliminary cytotoxicity in cancer cells. Future studies may focus on structural activity relationship and further evaluation of its mechanisms of action in cancer cells. Citation Format: Mentalla Mahmoud, Jianyu Chen, Jianfeng Cai, Hao Xie. Discovery of helical sulfonyl-γ-AApeptides targeting LC3 in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3101.

Abstract 395: Characterizing the role of chaperone-mediated autophagy in prostate cancer treatment resistance

Abstract Androgen deprivation therapy (ADT) and androgen receptor pathway inhibition (ARPI) remains the standard of care for advanced prostate cancer (PCa). As part of its canonical activities, the androgen receptor (AR) plays an important role in PCa metabolism. AR inhibition with ARPI subjects PCa cells to acute metabolic stress caused by reduced biosynthesis and energy production. The upregulation of ARPI-induced stress response mechanisms is essential adaptive mechanism for cell proliferation and survival. This process requires PCa cells undergoing rapid phenotypic changes to adapt to their environment and circumvent lethal outcomes. By profiling the proteomic pathway alterations associated with the ARPI stress response in LNCaP cells, we noted a significant upregulation of chaperone mediated autophagy (CMA), a stress response mechanism yet to be defined in PCa. CMA, a selective protein degradation pathway, specifically targets protein substrates via a CMA recognition motif and is an essential survival mechanism in cancer cells during energy depleted metabolic stress. Within in-vitro and in-vivo PCa models, ARPI induced CMA is reflected through the increased activity of CMA lysosomes and specifically by the increased expression of the CMA marker, lysosomal associated membrane protein 2a (L2A). L2A knockdown using shRNA not only elicited a strong anti-proliferative effect in PCa cells, but also compromised PCa metabolism with decreases in ATP levels and mTORC1 signaling. Proteomic analysis by mass spectrometry further identified that CMA promotes chromatin silencing, and the suppression of pathways associated with transcription. Conversely, L2A overexpression not only promoted PCa cell proliferation during Enza treatment, but upregulated of hallmark target genes of AR-indifferent PCa growth. The upregulation of CMA facilitated proteome remodeling during ARPI, leading to increased mTORC1 signaling and biosynthesis. In summary, our data illustrates the importance of CMA in mediating the ARPI stress response in PCa, providing novel insights into the mechanisms of ARPI treatment resistance. Citation Format: Nicholas Nikesitch, Eliana Beraldi, Fan Zhang, Hans Adomat, Robert Bell, Ladan Fazli, Christopher Wells, Nicholas Pinette, Yuzhuo Wang, Martin Gleave. Characterizing the role of chaperone-mediated autophagy in prostate cancer treatment 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 395.

Role of autophagy in cholangiocarcinoma: Pathophysiology and implications for therapy.

Cholangiocarcinoma (CCA) is a malignant tumour of the biliary system that originates from the neoplastic transformation of cholangiocytes. CCA is characterized by late diagnosis and poor outcome, with surgery considered as the last option for management. Autophagy is a physiological lysosomal degradation process, essential for cellular homeostasis and ubiquitous in all eukaryotic cells. Several studies have reported a potential involvement of autophagy in cancer, but it remains unclear whether activation of this process represents a survival mechanism of cancer cells. In the present review, we examine the autophagic process and summarize the current knowledge about the involvement of autophagy in the progression of cancer. The link between autophagy and chemoresistance and the use of autophagic markers in diagnosis are also considered in detail. Preliminary evidence shows that the combination of autophagy modulators (activators or inhibitors) with conventional chemotherapeutic agents offers a possible treatment option against signalling pathways that are hyperactivated or altered in CCA. In vitro evidence suggests that combination of chemotherapy agents, such as cisplatin, under activation or inhibition of autophagic processes, in two different CCA cell lines, may improve chemosensitivity and reduce cell survival, respectively. A deeper understanding of these pathways, in both cancer and non-cancer cells, could unveil possible therapeutic targets to treat CCA patients.

Autophagy inhibition in breast cancer cells induces ROS-mediated MIF expression and M1 macrophage polarization

Autophagy can function as a survival mechanism for cancer cells and therefore, its inhibition is currently being explored as a therapy for different cancer types. For breast cancer, triple negative breast cancer (TNBC) is the subtype most sensitive to the inhibition of autophagy; but its inhibition has also been shown to promote ROS-dependent secretion of macrophage migration inhibitory factor (MIF), a pro-tumorigenic cytokine. In this work, we explore the role of MIF in breast cancer, the mechanism by which autophagy inhibition promotes MIF secretion and its effects on neighboring cancer cell signaling and macrophage polarization. We analyzed MIF mRNA expression levels in tumors from breast cancer patients from different subtypes and found that Luminal B, HER2 and Basal subtypes, which are associated to high proliferation, displayed high MIF levels. However, MIF expression had no prognostic relevance in any breast cancer subtype. In addition, we found that autophagy inhibition in 66cl4 TNBC cells increased intracellular Reactive Oxygen Species (ROS) levels, which increased MIF expression and secretion. MIF secreted from 66cl4 TNBC cells induced the activation of MIF-regulated pathways in syngeneic cell lines, increasing Akt phosphorylation in 4T1 cells and ERK phosphorylation in 67NR cells. Regarding MIF/ chemokine receptors, higher levels of CD74 and CXCR2 were found in TNBC tumor cell lines when compared to non-tumorigenic cells and CXCR7 was elevated in the highly metastatic 4T1 cell line. Finally, secreted MIF from autophagy deficient 66cl4 cells induced macrophage polarization towards the M1 subtype. Together, our results indicate an important role for the inhibition of autophagy in the regulation of ROS-mediated MIF gene expression and secretion, with paracrine effects on cancer cell signaling and pro-inflammatory repercussions in macrophage M1 polarization. This data should be considered when considering the inhibition of autophagy as a therapy for different types of cancer.

Phenol-Boronic surface functionalization of gold nanoparticles; to induce ROS damage while inhibiting the survival mechanisms of cancer cells

The natural phenolic molecule caffeic acid, show promising effects on biological systems as an anti/pro-oxidant, anti-cancer, and anti-inflammatory agent. In nanoparticle functionalization designs, most organic nanoparticle coatings are utilized only for their ability to carry chemotherapeutics and targeting ligand. In this study, UV-light and auto-oxidation polymerization of caffeic acid on top of as-prepared gold nanoparticles was utilized to bring about a 5 nm multifunctional coating. The resulting polycaffeic acid (PCA) coating was used to conjugate both boronic acid containing compounds, chemotherapeutic bortezomib (BTZ) and cancer targeting ligand folate, while inducing mitochondrial reactive oxygen species that can damage intracellular proteins and DNA. This complements the drug payload, bortezomib’s cell survival inhibition properties. The drug, targeting ligand, and coating complexation are all pH cleavable under acidic pH condition (<5.0) which can be found in a tumor and endosomal microenvironment. The in vitro and in vivo experiments demonstrated cancer cytotoxicity and tumor inhibiting properties of the developed nanomedicine.

Expression of cell type incongruent alpha-cardiac actin 1 subunit in medulloblastoma reveals a novel mechanism for cancer cell survival and control of migration.

BackgroundAlterations in actin subunit expression have been reported in multiple cancers, but have not been investigated previously in medulloblastoma.MethodsBioinformatic analysis of multiple medulloblastoma tumor databases was performed to profile ACTC1 mRNA levels. Western blot was used to verify protein expression in established medulloblastoma cell lines. Immunofluorescence microscopy was performed to assess ACTC1 localization. Stable cell lines with ACTC1 overexpression were generated and shRNA knockdown of ACTC1 was accomplished. We used PARP1 cleavage by Western blot as a marker of apoptosis and cell survival was determined by FACS viability assay and colony formation. Cell migration with overexpression or knockdown of ACTC1 was determined by the scratch assay. Stress fiber length distribution was assessed by fluorescence microscopy.Results ACTC1 mRNA expression is highest in SHH and WNT medulloblastoma among all subgroups. ACTC1 protein was confirmed by Western blot in SHH subgroup and Group 3 subgroup cell lines with the lowest expression in Group 3 cells. Microscopy demonstrated ACTC1 co-localization with F-actin. Overexpression of ACTC1 in Group 3 cells abolished the apoptotic response to Aurora kinase B inhibition. Knockdown of ACTC1 in SHH cells and in Myc overexpressing SHH cells induced apoptosis, impaired colony formation, and inhibited migration. Changes in stress fiber length distribution in medulloblastoma cells are induced by alterations in ACTC1 abundance.ConclusionsAlpha-cardiac actin (ACTC1) is expressed in SHH medulloblastoma. Expression of this protein in medulloblastoma modifies stress fiber composition and functions in promoting resistance to apoptosis induced by mitotic inhibition, enhancing cell survival, and controlling migration.

Paclitaxel-induced stress granules increase LINE-1 mRNA stability to promote drug resistance in breast cancer cells.

Abnormal expression of long interspersed element-1 (LINE-1) has been implicated in drug resistance, while our previous study showed that chemotherapy drug paclitaxel (PTX) increased LINE-1 level with unknown mechanism. Bioinformatics analysis suggested the regulation of LINE-1 mRNA by drug-induced stress granules (SGs). This study aimed to explore whether and how SGs are involved in drug-induced LINE-1 increase and thereby promotes drug resistance of triple negative breast cancer (TNBC) cells. We demonstrated that SGs increased LINE-1 expression by recruiting and stabilizing LINE-1 mRNA under drug stress, thereby adapting TNBC cells to chemotherapy drugs. Moreover, LINE-1 inhibitor efavirenz (EFV) could inhibit drug-induced SG to destabilize LINE-1. Our study provides the first evidence of the regulation of LINE-1 by SGs that could be an important survival mechanism for cancer cells exposed to chemotherapy drugs. The findings provide a useful clue for developing new chemotherapeutic strategies against TNBCs.

AGE-RAGEsynergy influences programmed cell death signaling to promote cancer.

Advanced glycation end products (AGEs) are formed as a result of non-enzymatic reaction between the free reducing sugars and proteins, lipids, or nucleic acids. AGEs are predominantly synthesized during chronic hyperglycemic conditions or aging. AGEs interact with their receptor RAGE and activate various sets of genes and proteins of the signal transduction pathway. Accumulation of AGEs and upregulated expression of RAGE is associated with various pathological conditions including diabetes, cardiovascular diseases, neurodegenerative disorders, and cancer. The role of AGE-RAGE signaling has been demonstrated in the progression of various types of cancer and other pathological disorders. The expression of RAGE increases manifold during cancer progression. The activation of AGE-RAGE signaling also perturbs the cellular redox balance and modulates various cell death pathways. The programmed cell death signaling often altered during the progression of malignancies. The cellular reprogramming of AGE-RAGE signaling with cell death machinery during tumorigenesis is interesting to understand the complex signaling mechanism of cancer cells. The present review focus on multiple molecular paradigms relevant to cell death particularly Apoptosis, Autophagy, and Necroptosis that are considerably influenced by the AGE-RAGE signaling in the cancer cells. Furthermore, the review also attempts to shed light on the provenienceof AGE-RAGE signaling on oxidative stress and consequences of cell survival mechanism of cancer cells.

Biomarker Profile for Prediction of Patient Response to Smac Mimetic Monotherapy in Pediatric Precursor B-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. While improved multi-agent chemotherapy regimens with individualized risk stratification have led to increased survival rates of approximately 80 percent, 20 percent of patients respond poorly to therapy or relapse. Therefore, novel therapeutic avenues are urgently needed to improve treatment outcome, overcome resistance and reduce side effects. Failure to undergo cell death represents a key survival mechanism of cancer cells and results in drug resistance and clonal escape. Since inhibitor of apoptosis proteins (IAPs) are often overexpressed in malignant cells and their overexpression correlates with inferior survival rates, they provide an attractive molecular target for therapeutic intervention. Small molecule inhibitors have been developed that act as SMAC mimetics (SMs) to counteract the cell death inhibitory function of IAPs. SMs can activate and/or modulate cell death pathways, and are currently being evaluated in clinical trials. Their successful therapeutic implementation requires identification of patients who could benefit from a SM-based treatment regimen ideally before start of therapy. Here, we analyzed the intrinsic activity of two monovalent (AT406 and LCL161) and two bivalent (Birinapant or BV6) SMs on 29 unselected patient-derived pediatric precursor B-cell (BCP)-ALL samples and identified a subset of BCP-ALL primografts to be sensitive to SM treatment (n=8). When we compared gene expression of SM-sensitive (n=8) and SM-insensitive (n=6) patient-derived BCP-ALL samples, we identified a characteristic gene expression signature with 127 differentially regulated genes, amongst them upregulation of TNFRSF1A (TNFR1) in the SM-sensitive subset. In line with previous reports, we confirmed a critical role of the TNF/TNFR1-axis for SM-induced cell death in BCP-ALL by functional analysis. Expression of TNFRSF1A alone, however, did not correlate with sensitivity to SM-induced cell death indicating that TNFR1 is not the only factor regulating cell fate decisions in response to SM treatment. To identify potential biomarker genes for prediction of patient response to SM monotherapy in BCP-ALL, we compared differentially regulated genes of SM responders and non-responders from our cohort with data from a published cohort. Interestingly, we found 4 genes to overlap between these two cohorts. Of these 4 genes TSPAN7, FAM69C, and TNFRSF1A were upregulated whereas MTX2 was downregulated in SM-sensitive samples. The signature identified may reflect a particular TNF network. Analysis of expression levels of these 4 genes in BCP-ALL cell lines (Nalm6, Reh, UoCB6 and RS4;11) revealed that Reh cells, sensitive to SM-induced cell death, exhibited the biomarker profile of primograft sensitivity, i.e. upregulation of TSPAN7, FAM69C, TNFRSF1A and downregulation of MTX2. Nalm6 cells resembled the expression pattern of SM-insensitive samples with a downregulation of TSPAN7, FAM69C, TNFRSF1A and an upregulation of MTX2 and were resistant to SM-induced cell death. RS4;11 and UoCB6 cells showed no pattern. Based on these findings we hypothesized that the respective expression patterns of TSPAN7, FAM69C, TNFRSF1A and MTX2 could predict sensitivity to SMs. An extended screen of additional primary BCP-ALL samples for their expression levels of TSPAN7, FAM69C, TNFRSF1A and MTX2 and response to SMs substantiated this hypothesis. In summary, the subset of primary BCP-ALL samples with sensitivity to SMs is characterized by a gene signature with MTX2 low and TSPAN7, FAM69C and TNFRSF1A high. By using this expression profile, sensitivity to SMs in BCP-ALL could be identified in cell lines and additional primografts. Based on these results, we suggest the identified gene expression pattern as a biomarker for selecting patients to be treated by SM monotherapy in clinical trials. Disclosures No relevant conflicts of interest to declare.

Acid-induced autophagy protects human gastric cancer cells from apoptosis by activating Erk1/2 pathway

Background: Acidic microenvironments exist widely in tumors. However, the specific mechanism of cancer cell survival under an acidic microenvironment remains unknown. This study aims to investigate whether acid can induce autophagy and examine the mechanism of autophagy in gastric cancer cells. Methods: Human gastric adenocarcinoma (AGS) cells were cultured in media with different pH values in vitro and then subjected to autophagy detection under different conditions. To determine the effect of an acidic microenvironment on autophagy, we employed real-time quantitative polymerase chain reaction (PCR), Western blot, mRFP-GFP-LC3 immunofluorescence, and transmission electron microscopy (TEM) to detect the expression of various autophagy indicators. We also performed cell counting kit 8 (CCK8) and cell invasion and migration assays to examine cell viability and invasion, respectively. Results: We found that the protein expression of autophagy markers such as LC3II/I and Beclin1 was higher in AGS cells treated with an acidic microenvironment than in control cells. The protein expression level of P62 was obviously decreased in acid-treated cells compared to that in control cells. Furthermore, the expression of Erk1/2 pathway markers, including p-Erk1/2, was also increased in response to acidic pH. Dense LC3 puncta were observed in cells cultured under acidic conditions, whereas untreated cells exhibited diffuse and weak LC3 puncta; an increased autophagy flux could also be observed. The presence of autophagosomes was observed by TEM in AGS cells subjected to low pH. Additionally, autophagy was inhibited by the autophagy inhibitor Bafilomycin A1 (Baf) and apoptosis was obviously increased. Moreover, cells exposed to an acidic microenvironment displayed facilitated growth compared with that in control cells. Conclusions: Taken together, these results indicate that the acidic microenvironment promotes AGS cell growth by upregulating autophagy through the Erk1/2 pathway, which acts as a survival adaptation.

Acid-induced autophagy protects human gastric cancer cells from apoptosis by activating Erk1/2 pathway

BackgroundAcidic microenvironments exist widely in tumors. However, the specific mechanism of cancer cell survival under an acidic microenvironment remains unknown. This study aims to investigate whether acid can induce autophagy and examine the mechanism of autophagy in gastric cancer cells.MethodsHuman gastric adenocarcinoma (AGS) cells were cultured in media with different pH values in vitro and then subjected to autophagy detection under different conditions. To determine the effect of an acidic microenvironment on autophagy, we employed real-time quantitative polymerase chain reaction (PCR), Western blot, mRFP-GFP-LC3 immunofluorescence, and transmission electron microscopy (TEM) to detect the expression of various autophagy indicators. We also performed cell counting kit 8 (CCK8) and cell invasion and migration assays to examine cell viability and invasion, respectively.ResultsWe found that the protein expression of autophagy markers such as LC3II/I and Beclin1 was higher in AGS cells treated with an acidic microenvironment than in control cells. The protein expression level of P62 was obviously decreased in acid-treated cells compared to that in control cells. Furthermore, the expression of Erk1/2 pathway markers, including p-Erk1/2, was also increased in response to acidic pH. Dense LC3 puncta were observed in cells cultured under acidic conditions, whereas untreated cells exhibited diffuse and weak LC3 puncta; an increased autophagy flux could also be observed. The presence of autophagosomes was observed by TEM in AGS cells subjected to low pH. Additionally, autophagy was inhibited by the autophagy inhibitor Bafilomycin A1 (Baf) and apoptosis was obviously increased. Moreover, cells exposed to an acidic microenvironment displayed facilitated growth compared with that in control cells.ConclusionsTaken together, these results indicate that the acidic microenvironment promotes AGS cell growth by upregulating autophagy through the Erk1/2 pathway, which acts as a survival adaptation.

Crystal structures and snapshots along the reaction pathway of human phosphoserine phosphatase.

The equilibrium between phosphorylation and dephosphorylation is one of the most important processes that takes place in living cells. Human phosphoserine phosphatase (hPSP) is a key enzyme in the production of serine by the dephosphorylation of phospho-L-serine. It is directly involved in the biosynthesis of other important metabolites such as glycine and D-serine (a neuromodulator). hPSP is involved in the survival mechanism of cancer cells and has recently been found to be an essential biomarker. Here, three new high-resolution crystal structures of hPSP (1.5-2.0 Å) in complexes with phosphoserine and with serine, which are the substrate and the product of the reaction, respectively, and in complex with a noncleavable substrate analogue (homocysteic acid) are presented. New types of interactions take place between the enzyme and its ligands. Moreover, the loop involved in the open/closed state of the enzyme is fully refined in a totally unfolded conformation. This loop is further studied through molecular-dynamics simulations. Finally, all of these analyses allow a more complete reaction mechanism for this enzyme to be proposed which is consistent with previous publications on the subject.

O-GlcNAcylation Is Involved in the Regulation of Stem Cell Markers Expression in Colon Cancer Cells.

The dynamic O-linked-N-acetylglucosamine posttranslational modification of nucleocytoplasmic proteins has emerged as a key regulator of diverse cellular processes including several hallmarks of cancer. However, the role played by this modification in the establishment of CSC phenotype has been poorly studied so far and remains unclear. In this study we confirmed the previous reports showing that colon cancer cells exhibit higher O-GlcNAc basal levels than non-malignant cells, and investigated the role played by O-GlcNAcylation in the regulation of CSC phenotype. We found that the modification of O-GlcNAcylation levels by pharmacological inhibition of the O-GlcNAc-transferase enzyme that adds O-GlcNAc (OGT), but not of the enzyme that removes it (OGA), increased the expression of all stem cell markers tested in our colon malignant cell lines, and induced the appearance of a double positive (CD44+/CD133+) small stem cell-like subpopulation (which corresponded to 1–10%) that displayed very aggressive malignant phenotype such as increased clonogenicity and spheroid formation abilities in 3D culture. We reasoned that OGT inhibition would mimic in the tumor the presence of severe nutritional stress, and indeed, we demonstrated that nutritional stress reproduced in colon cancer cells the effects obtained with OGT inhibition. Thus, our data strongly suggests that stemness is regulated by HBP/O-GlcNAcylation nutrient sensing pathway, and that O-GlcNAc nutrient sensor represents an important survival mechanism in cancer cells under nutritional stressful conditions.

The HDAC6 inhibitor C1A modulates autophagy substrates in diverse cancer cells and induces cell death

BackgroundCytosolic deacetylase histone deacetylase 6 (HDAC6) is involved in the autophagy degradation pathway of malformed proteins, an important survival mechanism in cancer cells. We evaluated modulation of autophagy-related proteins and cell death by the HDAC6-selective inhibitor C1A.MethodsAutophagy substrates (light chain-3 (LC-3) and p62 proteins) and endoplasmic reticulum (ER) stress phenotype were determined. Caspase-3/7 activation and cellular proliferation assays were used to assess consequences of autophagy modulation.ResultsC1A potently resolved autophagy substrates induced by 3-methyladenine and chloroquine. The mechanism of autophagy inhibition by HDAC6 genetic knockout or C1A treatment was consistent with abrogation of autophagosome–lysosome fusion, and decrease of Myc protein. C1A alone or combined with the proteasome inhibitor, bortezomib, enhanced cell death in malignant cells, demonstrating the complementary roles of the proteasome and autophagy pathways for clearing malformed proteins. Myc-positive neuroblastoma, KRAS-positive colorectal cancer and multiple myeloma cells showed marked cell growth inhibition in response to HDAC6 inhibitors. Finally, growth of neuroblastoma xenografts was arrested in vivo by single agent C1A, while combination with bortezomib slowed the growth of colorectal cancer xenografts.ConclusionsC1A resolves autophagy substrates in malignant cells and induces cell death, warranting its use for in vivo pre-clinical autophagy research.

Abstract 5837: Acquired resistance to anticancer drug of high glucose-exposed HepG2

Abstract Reports show that diabetic patients with cancer tend to show resistance to anticancer drug treatment and have poor prognosis compared to non-diabetic patients. We observed that HepG2-HG, which is a derivative of the hepatocellular carcinoma cell line HepG2 exposed continuously to high glucose, shows high resistance to etoposide. In this study, we aimed to understand the involvement of excretion transporter and apoptosis regulatory factor in anti-cancer drug resistance in HepG2-HG. HepG2 was cultured in minimum essential medium (MEM) (with 5.5 mM glucose), and HepG2-HG was cultured in MEM supplemented with up to 25 mM glucose. Sensitivity to anticancer drugs was evaluated by the SRB assay. mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on Rotor-Gene Q using the KAPA SYBR FAST qPCR kit. Proteins extracted using the Ultra-RIPA kit were electrophoresed, transferred to a membrane by the iBlot 2 gel transfer device, reacted with antibodies, and evaluated for expression using enhanced chemiluminescence (ECL) prime reagent. IC50 values of etoposide in HepG2 and HepG2-HG were 0.59 µM and 17.5 µM, respectively. Comparison of the expression of the excretion transporter showed that the expression of p-glycoprotein (p-gp) was markedly enhanced in HepG2-HG compared to that in HepG2. Continuous contact of HepG2-HG with high glucose induced the expression of the glucose transporter GLUT1, which may provide a survival mechanism for cancer cells via escape from hypoxia-induced apoptosis. Furthermore, the expression of phospho-p70S6K was high in HepG2-HG, suggesting an increase in the activity of the mammalian target of rapamycin (mTOR). Therefore, we suggest that increased p-gp expression and mTOR activity in HepG2-HG provided resistance to etoposide by decreasing intracellular drug levels and suppressing the apoptotic mechanism. Estimation of the contribution of both mechanisms to etoposide resistance of HepG2-HG requires further examination. Citation Format: Shuichi Kishimoto, Shoji Fukushima. Acquired resistance to anticancer drug of high glucose-exposed HepG2 [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 5837.

Abstract LB-052: Stress response through theCaMK2A/NRF2/GSTP1 axis strengthens cancer cell resilience and mediates stemness phenotype through ROS attenuation

Abstract Lung cancer is the leading cause of cancer mortality. Adaptive responses to microenvironmental stresses are important survival mechanisms of cancer cells that lead to long term treatment failure, with accentuated resilience in cell subsets identified by cancer stem cell markers such as CD44 and ALDH. Reactive oxidative species (ROS) are harmful by-products of both cancer cell proliferation and treatment-induced cell death, the elimination of which is critical for maintaining cellular homeostasis and survival. We previously observed the calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) could enhance cancer stem cell (CSC) properties and attenuate ROS in lung cancer cells. To understand the molecular mechanisms, we focused on its relation to the master transcription factor for ROS regulation, NRF2, and the downstream pathways. To test if NRF2 could be a substrate activated by CaMK2A, total NRF2 proteins were measured in nuclear protein extracted from lung cancer cells. CaMK2A-overexpression (OE) increased, while shRNA-mediated CaMK2A-knockdown (shCaMK2A) reduced nuclear NRF2, indicating NRF2 is a mediator of CaMK2A. To identify downstream molecular pathways, screening for known NRF2 target genes was performed using qPCR. The results identified Glutathione S-transferase P1 (GSTP1), an anti-oxidant and detoxifying enzyme, was a target observed consistently in CaMK2A-OE and shCaMK2A cell lines. This finding was confirmed at the protein levels by western blot, supporting CaMK2A/NRF2/GSTP1 might be a candidate axis playing a supportive role for lung CSC. To obtain further support, we studied the expression levels of GSTP1 in 44 clinical lung cancers from our cohort and the TCGA database, respectively, which showed significant up-regulation in cancers compared to corresponding normal lung samples. Further investigations showed GSTP1 protein levels were higher in tumor-spheres than monolayers of five established lung cancer cell lines. Stable knockdown of GSTP1 led to significant reduction of tumor sphere formation, expression of CD44 and ALDH1, as well as mouse xenograft tumorigenicity. On the other hand, sensitivities to chemotherapy and mutant EGFR targeting drugs were increased. Thus, the in vitro and in vivo data supported that GSTP1 played a role in the maintenance of the stem cell-like phenotypic state in lung cancer. To assess if GSTP1 maintained the CSC phenotype through regulating oxidative stress, its effects on ROS levels was measured using cellROX deep red probes. In shGSTP1 cells, the ROS level was significantly raised while it was reciprocally reduced on GSTP1- OE. Furthermore, ROS challenge experiments using N-acetyl-L-cysteine (NAC) in shGSTP1 cell lines resulted in increased cell sphere formation. Together, the results support the CaMK2A/NRF2/GSTP1 axis increases cancer cell resilience through ROS elimination and enhancement of stem cell-like properties of lung carcinoma, indicating its potential role as a long-term treatment target. Citation Format: SIQI WANG, Zhijie Xiao, Pui Chi Tin, Xuyuan Gao, Judy Wai Ping Yam, Maria Pik Wong. Stress response through theCaMK2A/NRF2/GSTP1 axis strengthens cancer cell resilience and mediates stemness phenotype through ROS attenuation [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 LB-052.

Targeting of cathepsin C induces autophagic dysregulation that directs ER stress mediated cellular cytotoxicity in colorectal cancer cells

As Autophagy is a pivotal mechanism of cancer cell survival and the development of chemotherapeutic resistance; therefore, new approaches are warranted for its targeting which may be fulfilled by cathepsins regulation. Amongst cathepsins, cathepsin C (CTSC) is highly expressed in various cancers and possesses significant therapeutic potential in autoimmune disorders; however, its role in colorectal cancer has not been explored. Herein, we aimed to investigate the role of CTSC in autophagy regulation mediated colorectal carcinoma cell proliferation. Cathepsin C targeting through inhibitors/siRNA leads to the accumulation of light chain 3 II and p62 without affecting the lysosomal integrity, revealed dysfunctional autolysosomal degradation which is also substantiated by proteolytic studies. Cathepsin C inhibition showed comparable autophagy blockade with E64d and augmented the autophagy blockade mediated by bafilomycin. Loss of CTSC function also induced ER stress-mediated JNK phosphorylation accompanied by the translocation of mitochondrial cyt c followed by apoptotic cell death in colorectal carcinoma cells. Taken together, the study reveals that CTSC targeting plays a key role in the regulation of autophagy mediated colorectal cancer cell proliferation. Further investigations are required to determine the functional role of CTSC in other tumors also which may have implications for the therapeutic prevention of cancer in the future.

Novel Targeting of Transcription and Metabolism in Glioblastoma.

Purpose: Glioblastoma (GBM) is highly resistant to treatment, largely due to disease heterogeneity and resistance mechanisms. We sought to investigate a promising drug that can inhibit multiple aspects of cancer cell survival mechanisms and become an effective therapeutic for GBM patients.Experimental Design: To investigate TG02, an agent with known penetration of the blood-brain barrier, we examined the effects as single agent and in combination with temozolomide, a commonly used chemotherapy in GBM. We used human GBM cells and a syngeneic mouse orthotopic GBM model, evaluating survival and the pharmacodynamics of TG02. Mechanistic studies included TG02-induced transcriptional regulation, apoptosis, and RNA sequencing in treated GBM cells as well as the investigation of mitochondrial and glycolytic function assays.Results: We demonstrated that TG02 inhibited cell proliferation, induced cell death, and synergized with temozolomide in GBM cells with different genetic background but not in astrocytes. TG02-induced cytotoxicity was blocked by the overexpression of phosphorylated CDK9, suggesting a CDK9-dependent cell killing. TG02 suppressed transcriptional progression of antiapoptotic proteins and induced apoptosis in GBM cells. We further demonstrated that TG02 caused mitochondrial dysfunction and glycolytic suppression and ultimately ATP depletion in GBM. A prolonged survival was observed in GBM mice receiving combined treatment of TG02 and temozolomide. The TG02-induced decrease of CDK9 phosphorylation was confirmed in the brain tumor tissue.Conclusions: TG02 inhibits multiple survival mechanisms and synergistically decreases energy production with temozolomide, representing a promising therapeutic strategy in GBM, currently under investigation in an ongoing clinical trial. Clin Cancer Res; 24(5); 1124-37. ©2017 AACR.