Cell Level Research Articles

Dissecting the pleiotropic roles of reactive oxygen species (ROS) in lung cancer: From carcinogenesis towardtherapy.

Lung cancer is a major cause of morbidity and mortality. The specific pulmonary structure to directly connect with ambient air makes it more susceptible to damage from airborne toxins. External oxidative stimuli and endogenous reactive oxygen species (ROS) play a crucial role in promoting lung carcinogenesis and development. The biological properties of higher ROS levels in tumor cells than in normal cells make them more sensitive and vulnerable to ROS injury. Therefore, the strategy of targeting ROS has been proposed for cancer therapy for decades. However, it is embarrassing that countless attempts at ROS-based therapies have had very limited success, and no FDA approval in the anticancer list was mechanistically based on ROS manipulation. Even compared with the untargetable proteins, such as transcription factors, ROS are more difficult to be targeted due to their chemical properties. Thus, the pleiotropic roles of ROS provide therapeutic potential for anticancer drug discovery, while a better dissection of the mechanistic action and signaling pathways is a prerequisite for future breakthroughs. This review discusses the critical roles of ROS in cancer carcinogenesis, ROS-inspired signaling pathways, and ROS-based treatment, exemplified by lung cancer. In particular, an eight considerations rule is proposed for ROS-targeting strategies and drug design and development.

Heparin Oligosaccharides as Vasoactive Intestinal Peptide Inhibitors via their Binding Process Characterization.

It has been proven that vasoactive intestinal peptide (VIP) was involved in the pathogenesis of prostate cancer. Cardin et al. found that by an alanine scan, the heparin- binding site on VIP was exactly the same sequence in VIP and its receptor. Therefore, heparin could competitively block the binding of VIP and its receptor. However, the structure-activity relationship between heparin and VIP has not been reported, especially in terms of the sequence and sulfation patterns of heparin oligosaccharides upon binding to VIP. A variety of experiments were designed to study the binding process and structure-activity relationship between heparin oligosaccharides and VIP. Heparin was enzymatically digested and purified to produce heparin oligosaccharides, and the structures were characterized by NMR. The binding capacity between heparin oligosaccharides and VIP was analyzed by GMSA and ITC experiments. The binding between heparin oligosaccharides and VIP was simulated using a molecular docking program to show the complex. ELISA assay was used to investigate the effect of non-anticoagulant heparin oligosaccharides on the VIP-mediated cAMP/PKA signaling pathway in vitro. The results indicated that both the length and the sulfation pattern of heparin oligosaccharides affected its binding to VIP. VIP could induce the expression of cAMP at a higher level in PC3 cells, which could be regulated by the interaction of heparin oligosaccharides and VIP. The binding between heparin oligosaccharides and VIP could block the binding between VIP and its receptor on tumor cells. Downloading the regulation of the expression level of cAMP could possibly further affect the subsequent activation of PKA. These non-anticoagulant heparin oligosaccharides may block the VIP-mediated cAMP/PKA signaling pathway and thus exert their antitumor activity.

RNA polymerase III is involved in regulating Plasmodium falciparum virulence.

While often undetected and untreated, persistent seasonal asymptomatic malaria infections remain a global public health problem. Despite the presence of parasites in the peripheral blood, no symptoms develop. Disease severity is correlated with the levels of infected red blood cells (iRBCs) adhering within blood vessels. Changes in iRBC adhesion capacity have been linked to seasonal asymptomatic malaria infections, however how this is occurring is still unknown. Here, we present evidence that RNA polymerase III (RNA Pol III) transcription in Plasmodium falciparum is downregulated in field isolates obtained from asymptomatic individuals during the dry season. Through experiments with in vitro cultured parasites, we have uncovered an RNA Pol III-dependent mechanism that controls pathogen proliferation and expression of a major virulence factor in response to external stimuli. Our findings establish a connection between P. falciparum cytoadhesion and a non-coding RNA family transcribed by Pol III. Additionally, we have identified P. falciparum Maf1 as a pivotal regulator of Pol III transcription, both for maintaining cellular homeostasis and for responding adaptively to external signals. These results introduce a novel perspective that contributes to our understanding of P. falciparum virulence. Furthermore, they establish a connection between this regulatory process and the occurrence of seasonal asymptomatic malaria infections.

Prognostic value of long noncoding RNA LINC00924 in lung adenocarcinoma and its regulatory effect on tumor progression.

Long non-coding RNAs (lncRNAs) have been used in the study of tumor biomarkers in recent years. However, the prognostic role of lncRNA LINC00924 (LINC00924) in lung adenocarcinoma (LUAD) has not yet been concluded. Therefore, this study investigates the prognostic value of LINC00924 in LUAD and its regulatory effect on tumor progression. The LUAD tissues and adjacent normal tissues of 128 subjects were extracted, and the expressions of LINC00924 and miR-196a-5p in tissues and cells were detected by RT-qPCR. The prognostic value of LINC00924 in LUAD patients was obtained by Kaplan-Meier analysis and multivariate Cox regression test. The cell counting kit-8 (CCK-8) and Transwell assay were used to detect the effect of overexpression LINC00924 on LUAD cells. In LUAD tissues and cells, LINC00924 expression was down-regulated and miR-196a-5p expression was up-regulated compared with the normal control group. High expression of LINC00924 inhibited the proliferation level, migration ability and invasion situation of LUAD cells, which was more conducive to the survival and prognosis of LUAD patients. Bioinformatics studies indicated that overexpression of LINC00924 inhibited the development of LUAD by targeting miR-196a-5p, while miR-196a-5p mimic effectively weakened the inhibition. LINC00924 sponges of miR-196a-5p may be considered as a potential prognostic biomarker for LUAD.

Ginsenoside Rg1 Suppresses Ferroptosis of Renal Tubular Epithelial Cells in Sepsis-induced Acute Kidney Injury via the FSP1-CoQ10- NAD(P)H Pathway.

Sepsis-induced acute kidney injury is related to an increased mortality rate by modulating ferroptosis through ginsenoside Rg1. In this study, we explored the specific mechanism of it. Human renal tubular epithelial cells (HK-2) were transfected with oe-ferroptosis suppressor protein 1 and treated with lipopolysaccharide for ferroptosis induction, and they were then treated with ginsenoside Rg1 and ferroptosis suppressor protein 1 inhibitor. Ferroptosis suppressor protein 1, CoQ10, CoQ10H2, and intracellular NADH levels in HK-2 cells were assessed by Western blot, ELISA kit, and NAD/NADH kit. NAD+/NADH ratio was also calculated, and 4-Hydroxynonal fluorescence intensity was assessed by immunofluorescence. HK-2 cell viability and death were assessed by CCK-8 and propidium iodide staining. Ferroptosis, lipid peroxidation, and reactive oxygen species accumulation were assessed by Western blot, kits, flow cytometry, and C11 BODIPY 581/591 molecular probe. Sepsis rat models were established by cecal ligation and perforation to investigate whether ginsenoside Rg1 regulated the ferroptosis suppressor protein 1-CoQ10-NAD(P)H pathway in vivo. LPS treatment diminished ferroptosis suppressor protein 1, CoQ10, CoQ10H2, and NADH contents in HK-2 cells, while facilitating NAD+/NADH ratio and relative 4- Hydroxynonal fluorescence intensity. FSP1 overexpression inhibited lipopolysaccharideinduced lipid peroxidation in HK-2 cells via the ferroptosis suppressor protein 1-CoQ10- NAD(P)H pathway. The ferroptosis suppressor protein 1-CoQ10-NAD(P)H pathway suppressed lipopolysaccharide-induced ferroptosis in HK-2 cells. Ginsenoside Rg1 alleviated ferroptosis in HK-2 cells by regulating the ferroptosis suppressor protein 1-CoQ10- NAD(P)H pathway. Moreover, ginsenoside Rg1 regulated the ferroptosis suppressor protein 1-CoQ10-NAD(P)H pathway in vivo. Ginsenoside Rg1 alleviated sepsis-induced acute kidney injury by blocking renal tubular epithelial cell ferroptosis via the ferroptosis suppressor protein 1-CoQ10- NAD(P)H pathway.

The prognostic and antitumor roles of key genes of ferroptosis in liver hepatocellular cancer and stomach adenocarcinoma.

Liver hepatocellular cancer (LIHC) and stomach adenocarcinoma (STAD) are common malignancies with high lethal ratios worldwide. Great progress has been achieved by using diverse therapeutic strategies; however, these diseases still have an unfavourable prognosis. Ferroptosis inducer drugs, unlike apoptosis-related drugs, can overcome the resistance to cancer therapy caused by traditional chemicals. However, the relationship between overall survival (OS) and ferroptosis-related genes, as well as the mechanisms involved, are largely unclear. The expression levels of AIFM2, GPX4, ACSL4, FTH1, NOS1, and PTGS2 in LIHC and STAD were obtained from UALCAN. The correlations of OS with these gene expression levels were obtained using the Kaplan-Meier Plotter database. The OS associated with genetic mutations of those genes compared to that of unchanged genes was analysed using the TIMER website. GO and KEGG enrichment analyses of ferroptosis-related genes and their coexpressed genes in LIHC and STAD were conducted using the STRING and DAVID databases. The relationship of PTGS2 and ACSL4 to immune cell infiltration was analysed using the TIMER website. The viability and GPX5 expression levels in LIHC cells treated with RSL3 and As2O3 were detected by MTT methods and western blotting, respectively. Our results showed that GPX4, FTH1 and AIFM2 were overexpressed in LIHC and STAD. High levels of GPX4, FTH1 and AIFM2 were prominently correlated with better prognosis in LIHC. However, GPX and FTH1 in STAD did not show significant correlations with OS. AIFM2 in STAD had the opposite trend with OS compared with that in LIHC. Moreover, a high mutation rate of these genes (35.74%) was also observed in LIHC patients, and genetic mutation of these genes was correlated with shorter OS. In contrast, the genetic mutation of these genes did not change OS in STAD. Enrichment analysis showed that the respiratory electron transport chain, cell chemotaxis and T-cell migration were related to ferroptosis. ASCL4 and PTGS2 coexpressed with cytokines associated with immune cell infiltration. Compared to RSL3 or As2O3 alone, As2O3 plus RSL3 significantly inhibited the growth of Huh7 cells. GPX4 was downregulated to an undetectable level when in combination with RSL3. Our results indicated that ferroptosis-related genes might play an important role in LIHC and STAD and might be risk factors for overall survival in LIHC and STAD.

Identifying a Novel Eight-NK Cell-related Gene Signature for Ovarian Cancer Prognosis Prediction.

Ovarian cancer (OVC) is the most common and costly tumor in the world with unfavorable overall survival and prognosis. This study is aimed to explore the prognostic value of natural killer cells related genes for OVC treatment. RNA-seq and clinical information were acquired from the TCGA-OVC dataset (training dataset) and the GSE51800 dataset (validation dataset). Genes linked to NK cells were obtained from the immPort dataset. Moreover, ConsensusClusterPlus facilitated the screening of molecular subtypes. Following this, the risk model was established by LASSO analysis, and immune infiltration and immunotherapy were then detected by CIBERSORT, ssGSEA, ESTIMATE, and TIDE algorithms. Based on 23 NK cell-related genes with prognosis, TCGA-OVC samples were classified into two clusters, namely C1 and C2. Of these, C1 had better survival outcomes as well as enhanced immune infiltration and tumor stem cells. Additionally, it was more suitable for immunotherapy and was also sensitive to traditional chemotherapy drugs. The eight-gene prognosis model was constructed and verified via the GSE51800 dataset. Additionally, a high infiltration level of immune cells was observed in low-risk patients. Low-risk samples also benefited from immunotherapy and chemotherapy drugs. Finally, a nomogram and ROC curves were applied to validate model accuracy. The present study identified a RiskScore signature, which could stratify patients with different infiltration levels, immunotherapy, and chemotherapy drugs. Our study provided a basis for precisely evaluating OVC therapy and prognosis.

Abstract 2021: The RXR agonist MSU-42011 and the MEK inhibitor selumetinib reduce pERK levels in NF1-deficient cells and inhibit cytokine production in macrophages

Abstract Neurofibromatosis type 1 (NF1) is a common genetic disease that predisposes approximately 50% of affected individuals to develop plexiform neurofibromas (PNFs), which can progress to highly aggressive malignant peripheral nerve sheath tumors (MPNSTs) in approximately 10% of patients. NF1 is caused by mutations in the tumor suppressor gene NF1, which encodes for neurofibromin, a negative regulator of RAS activity. Selumetinib, a specific inhibitor of MEK1/2, is the only FDA-approved drug for NF1-associated PNFs. However, the anti-tumor effects of selumetinib are limited in MPNSTs and have dose-limiting side effects. Deficiency of the NF1 gene not only promotes tumorigenesis but also has broad effects on the immune cells and cytokine signaling driven by hyperactive RAS signaling. Because macrophages account for almost half of cells in NF1 lesions and their infiltration correlates with disease progression, we hypothesized that targeting tumor-promoting immune cells could be an alternative approach for treating NF1. The novel retinoid X receptor (RXR) agonist MSU-42011 reduces tumor growth in experimental Kras-driven cancers by decreasing pERK expression, reducing tumor-promoting immune cells like CD206+ macrophages and regulatory T cells, and increasing activated cytotoxic T cells. Here, we treated NF1-deficient cells and macrophages with MSU-42011 and selumetinib, either alone or in combination, using monoculture and conditioned media (CM) conditions. In human PNF cells and mouse MPNSTs, treatment with 200 nM MSU-40211 or 50 nM selumetinib for 3 hours reduced pERK protein levels compared with untreated controls, and the combination treatment enhanced this reduction in pERK protein levels. Additionally, there was a trend toward reduction in cell viability with increasing drug concentrations after 72 hours of the combination treatment. Moreover, CM from human and mouse PNF cells increased the mRNA expression of monocyte chemoattractant CCL2 (C-C motif chemokine ligand 2) and the secretion of IL-6 and TNFα in human THP1 monocytes/macrophages and bone marrow derived macrophages (BMDM). Notably, MSU-42011 and selumetinib alone inhibited CCL2 mRNA expression in THP1 macrophages and BMDM stimulated with CM from human and mouse PNF cells, respectively, and the inhibition of CCL2 mRNA expression was greatest with combination treatment. The combination of MSU42011 and selumetinib also significantly reduced tumor burden in a LL2 model of lung cancer driven by an activating Kras mutation. Based on the similarities in RAS activation and immune cell infiltration in NF1 and lung cancer, our next step is to confirm the immunomodulatory and anti-tumor effects of MSU-42011 and selumetinib in a syngeneic model of PNF and MPNST. Citation Format: Pei-Yu Hung, Jessica A. Moerland, Karen T. Liby. The RXR agonist MSU-42011 and the MEK inhibitor selumetinib reduce pERK levels in NF1-deficient cells and inhibit cytokine production in macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2021.

Abstract 4316: Investigation the role of DDI2 in autophagy

Abstract Background: The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are critical intracellular protein degradation pathways. In response to proteasome dysfunction, cells initiate compensatory autophagy to manage proteotoxic stress, which impacts cancer therapy efficacy. Despite current insights, the exact molecular mechanisms underlying the induction of autophagy due to impaired proteasome function remain unclear. The transcription factor nuclear factor erythroid-2-related factor 1 (NRF1) is the master regulator of proteasome subunit genes, which is activated by the protease DNA damage inducible 1 homolog 2 (DDI2) upon proteasome inhibition. DDI2 also acts as an ubiquitin shuttling factor, and its depletion causes the accumulation of highly ubiquitinated proteins, resulting in increased sensitivity to proteasome inhibition in cancer cells. However, cancer cells might trigger compensatory autophagy in response to DDI2 depletion to degrade ubiquitinated protein aggregates. Here, we demonstrated that DDI2 depletion plays a role in autophagy induction. Methods: A panel of cell lines of different origins (EW16 and ES1 human Ewing sarcoma, MIA PaCa-2 human pancreatic cancer, and NIH-3T3 murine fibroblasts) either control or DDI2-deficient were examined for autophagy activity following treatment with the autophagy inhibitor chloroquine (CQ). Through total proteome analysis, we identified an increase in cellular communication network factor 1 (CCN1) protein levels in DDI2-depleted cells, which plays a role in autophagy induction. To determine whether CCN1 is required and sufficient to induce autophagy, we applied small interfering RNA (siRNA), and viral constructs to knockdown and overexpress CCN1. Results: The autophagic flux measurements in four different DDI2-deficient cell lines revealed elevated LC3B-II protein levels, a known autophagy marker, following CQ treatment compared to their control. Western blotting analysis confirmed increased CCN1 protein levels in the same cell lines. The autophagy induction observed in DDI2-deficient cells was diminished by CCN1-siRNA, demonstrating that CCN1 may be required for autophagy induction. Furthermore, overexpressing CCN1 in wild-type cells resulted in increased LC3B-II protein levels, indicating that CCN1 alone is sufficient to induce autophagy. Conclusion: Targeting autophagy is a promising therapeutic strategy to improve the effects of anti-cancer therapies. We demonstrated that upon depletion of ubiquitin shuttle factor DDI2, secretory protein CCN1 accumulates leading to the subsequent induction of autophagy. These findings improve our understanding of the molecular basis governing DDI2’s role in crosstalk between protein degradation pathways. Targeting DDI2 in combination with autophagy inhibition could further potentiate these drugs as anti-cancer therapies. Citation Format: Nayyerehalsadat Hosseini, Holly A. Byers, Senthil K. Radhakrishnan. Investigation the role of DDI2 in autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4316.

Abstract 3005: Low-density lipoprotein receptor-related protein 1B (LRP1B) depletion promotes tumorigenicity by modulating fibroblasts in the tumor microenvironment in esophageal squamous cell carcinoma

Abstract Background: Esophageal squamous cell carcinoma (ESCC) is widespread worldwide with a poor survival rate. Tumor cells shedding from tumor tissues traveling to other organs through the blood circulatory system are called circulating tumor cells (CTCs). The CTCs in the blood of ESCC patients were enriched and then subjected to CTC mutational profiling by target sequencing. Our results suggested that CTC mutations in low-density lipoprotein receptor-related protein 1B (LRP1B) may have a potential prognostic role for ESCC. LRP1B belongs to the low-density receptor (LDLR) family with diverse roles in cellular functions and development, but the function of LRP1B in ESCC remains unclear. Therefore, this study aims to do a functional characterization of LRP1B. Methods: The CRISPR cas9 system was used to functionally knock down the candidate gene in ESCC cell lines with the highest endogenous level of LRP1B. The in vitro cell proliferation rate and viability were measured by MTT assay, colony formation assay, and wound healing assay. The in vivo tumorigenicity assay was performed to evaluate the tumor suppressive effect by subcutaneous injection. To examine the clinical relevance of LRP1B, we also performed immunohistochemistry (IHC) in tumor tissue microarray (TMA). Results: The reduction or loss of LRP1B mRNA was demonstrated in 60% of a panel of 15 ESCC cell lines. The depletion of LRP1B did not influence cell proliferation, cell clonogenic ability, or cell migration in ECSS cell lines. However, LRP1B depletion significantly promoted the growth of tumors in mouse xenograft models. Therefore, we hypothesize that LRP1B may be involved in modulating the tumor microenvironment (TME) by impacting various stromal components leading to an immuno-suppressive TME. We utilized RT-qPCR to investigate markers of various stromal components and observed that fibroblast markers (acta2, pdgfrb, and vimentin) had significantly higher expression levels after LRP1B downregulation in ESCC cell-derived mouse xenografts. The higher level of fibroblast cells was then verified by IHC staining. Further molecular mechanistic studies are underway and are expected to provide novel therapeutic insights by targeting the LRP1B genes. The LRP1B ESCC TMA data analysis is underway and statistical analysis will be presented. Conclusions: LRP1B plays a tumor-suppressive role in ESCC by modulating the tumor-associated fibroblasts in the TME. Acknowledgements: TBRS grant T12-701/17R and HMRF grant 05160926 to MLL Citation Format: KaiYan Xu, Josephine Mun-yee KO, Valen Zhuo you Yu, Maria Li LUNG. Low-density lipoprotein receptor-related protein 1B (LRP1B) depletion promotes tumorigenicity by modulating fibroblasts in the tumor microenvironment in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3005.

Abstract 5325: Cytokine CCL9, new downstream target of oncogene Kras to modulate acinar-to-ductal metaplasia during pancreatic cancer initiation

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer with a current five-year survival rate at 12% in the US, and is dominated by oncogenic Kras mutations. Accumulating evidence showed that PDAC can be originated from acinar-to-ductal metaplasia (ADM). In this process, pancreatic acini harboring oncogenic Kras mutations are transdifferentiated to a duct-like phenotype that further progresses to become pancreatic intraepithelial neoplasia (PanIN) lesions, giving rise to PDAC. Although ADM formation is commonly observed in KrasG12D transgenic mouse models of PDAC, the exact mechanisms of how oncogenic KrasG12D modulates this process remain unclear. Here, we unveil a new downstream target of oncogenic Kras, cytokine CCL9 during ADM formation. Either exogenously treating primary pancreatic acini with recombinant CCL9 or ectopically expressing CCL9 in primary pancreatic acini resulted in ADM formation in a 3D organoid culture system. Furthermore, knockdown of CCL9 in KrasG12D-expressed acini reduced KrasG12D-induced ADM. Higher levels of reactive oxygen species (ROS) were detected in the KrasG12D-expressed pancreatic acini, and knockdown of CCL9 in these acini decreased ROS levels. Overexpression of CCL9 in primary pancreatic acini elevated cellular ROS of pancreatic acinar cells. Furthermore, depletion of ROS in 3D organoid culture of CCL9-expressed acini reduced CCL9-induced ADM formation. In transgenic mice of p48cre:KrasG12D, blockade of CCL9 through its specific neutralizing antibody attenuated ADM formation as well as PanIN structures. Altogether, our results indicated oncogenic KrasG12D initiates PDAC through a new downstream target molecule, CCL9, to enhance ROS levels of pancreatic acinar cells. Citation Format: Geou-Yarh (Stancy) Liou, Justin K. Messex, Crystal J. Byrd. Cytokine CCL9, new downstream target of oncogene Kras to modulate acinar-to-ductal metaplasia during pancreatic cancer initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5325.

Abstract 4756: Targeting DNA topoisomerase II to manage acquired resistance to third generation EGFR tyrosine kinase inhibitors in the treatment of EGFR mutant NSCLC

Abstract Development of effective strategies to manage the inevitable acquired resistance to osimertinib, a 3rd generation EGFR inhibitor with proven clinical efficacy in prolonging survival of patients with EGFR mutant (EGFRm) non-small cell lung cancer (NSCLC), is an urgent and critical area of unmet need in the clinic. Effort toward this direction led us to find that the DNA topoisomerase II (Topo II) inhibitors, doxorubicin and etoposide (VP-16), but not other chemotherapeutic agents, synergistically decreased the survival of osimertinib-resistant cell lines when combined with osimertinib. The combination of osimertinib with either doxorubcin or VP-16 effectively induced DNA damage and apoptosis in osimertinib-resistant cells, suppressed the growth of osimertinib-resistant tumors, and delayed the emergence of acquired resistance to osimertinib. Mechanistic studies found that osimertinib as well other EGFR-TKIs effectively decreased Topo IIα levels in EGFRm NSCLC cells by facilitating its proteasomal degradation and induced DNA damage; these effects were lost in cell lines with osimertinib acquired resistance that possessed elevated basal levels of Topo IIα. In the majority of EGFRm NSCLC tissues relapsed from EGFR-TKI treatment, Topo IIα levels were substantially increased. Enforced expression of ectopic TOP2A genes in sensitive EGFRm NSCLC cells conferred resistance to osimertinib in inducing DNA damage and apoptosis, whereas knockdown of TOP2A in osimertinib-resistant cell lines restored their responses to undergo osimertinib-induced DNA damage and apoptosis. Together, these results reveal a previously undiscovered essential role of Topo IIα modulation in regulating the responses of EGFRm NSCLC cells to osimertinib, providing scientific rationale for targeting Topo II to manage acquired resistance to osimertinib and possibly other third generation EGFR-TKIs, thus warranting clinical validation of this strategy. (This study was supported by NIH/NCI R01 CA223220 and UG1 CA233259 and Emory Winship Cancer Institute lung cancer research pilot funds). Citation Format: Zhen Chen, Karin A. Vallega, Dongsheng Wang, Zihan Quan, Songqing Fan, Qiming Wang, Ticiana A. Leal, Suresh S. Ramalingam, Shi-Yong Sun. Targeting DNA topoisomerase II to manage acquired resistance to third generation EGFR tyrosine kinase inhibitors in the treatment of EGFR mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4756.

Multiple receptor tyrosine kinases regulate dengue infection of hepatocytes.

Dengue is an arboviral disease causing severe illness in over 500,000 people each year. Currently, there is no way to constrain dengue in the clinic. Host kinase regulators of dengue virus (DENV) infection have the potential to be disrupted by existing therapeutics to prevent infection and/or disease progression. To evaluate kinase regulation of DENV infection, we performed kinase regression (KiR), a machine learning approach that predicts kinase regulators of infection using existing drug-target information and a small drug screen. We infected hepatocytes with DENV in vitro in the presence of a panel of 38 kinase inhibitors then quantified the effect of each inhibitor on infection rate. We employed elastic net regularization on these data to obtain predictions of which of 291 kinases are regulating DENV infection. Thirty-six kinases were predicted to have a functional role. Intriguingly, seven of the predicted kinases - EPH receptor A4 (EPHA4), EPH receptor B3 (EPHB3), EPH receptor B4 (EPHB4), erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 2 (FGFR2), Insulin like growth factor 1 receptor (IGF1R), and ret proto-oncogene (RET) - belong to the receptor tyrosine kinase (RTK) family, which are already therapeutic targets in the clinic. We demonstrate that predicted RTKs are expressed at higher levels in DENV infected cells. Knockdown of EPHB4, ERBB2, FGFR2, or IGF1R reduces DENV infection in hepatocytes. Finally, we observe differential temporal induction of ERBB2 and IGF1R following DENV infection, highlighting their unique roles in regulating DENV. Collectively, our findings underscore the significance of multiple RTKs in DENV infection and advocate further exploration of RTK-oriented interventions against dengue.

Abstract 5411: Effect of different fatty acids on ccRCC behavior and metabolism

Abstract Introduction: Clear Cell Renal Cell Carcinoma (ccRCC) is the most common type of kidney cancer, presenting with defining characteristics of increased lipid droplets formation, loss of the VHL tumor suppressor, and low levels of fatty acid oxidation (FAO). Obesity is associated with an increase in ccRCC incidence, although obese patients respond better to different therapies, a phenomenon called as obesity paradox. The consumption of diets rich in lipids and leading to obesity has been increasing, but how the dietary composition affects ccRCC progression, or could be modulated in combination with other therapies for better responses is still unclear. In the present study, we aimed to evaluate how different fatty acids (FAs) affect ccRCC behavior and metabolism. Methods: We used 786-O and 769-P ccRCC cell lines. Palmitic acid (PA), oleic acid (OA), and linoleic acid (LA), which are saturated, monounsaturated, and polyunsaturated fatty acids, respectively were used as stimuli for the cells at 50 or 200µM. All stimuli were diluted in culture media with 5% FBS. Lipid droplets were evaluated by Oil Red O staining; viability was assessed by MTT; protein expression was tested by Western Blotting; Oxygen Consumption rate (OCR) by Seahorse; survival was quantified by clonogenic assay; and mRNA was measured by RT-qPCR. Results: All FAs increased the amount of lipid droplets compared to the control, but OA was the most potent. PA impaired the growth rate for cells compared to control, and other FAs in both cell lines, and did not increase the expression of cleaved caspase-3. PA, even in a lower concentration [50µM], impaired the formation of colonies for both cell lines compared to control and other FAs. LA impaired colony formation only in the higher concentration [200µM], while OA only induced a difference for 769-P cells at the smaller concentration, increasing the number of colonies. OA, and mainly PA, decreased 769-P cells migration, at the time of 6 and 12 h, while only PA decreased migration for 786-O cells, at the time of 12 h. LA, and mainly PA, increased CPT1a mRNA levels in 786-O cells but did not induce a significant difference to 769-P, and all FAs decreased FASN in both cell lines. Conclusion: It was demonstrated that OA was a potent inducer of lipid droplet formation, which can be protective in avoiding lipid peroxidation. On the other hand, PA and LA induced fewer lipid droplets probably because they are going to FAO, as we observed increased mRNA CPT1a levels, which is the rate-limiting enzyme in FAO. PA impaired potentially the formation of colonies and growth rate, and it is important to highlight that it was without inducing caspase 3-dependent programmed cell death. Taken together our data suggest that PA could be a target therapy for ccRCC, but it is still necessary for more studies in vitro and in vivo to evaluate how PA and enriched lipid diet can impact a better outcome for patients. Citation Format: Isadora Ramos de Andrade, Dazhi Wang, Scott Welford. Effect of different fatty acids on ccRCC behavior and metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5411.

Abstract 7418: LongFuse: Detecting gene fusion transcripts from high throughput long-read single cell RNA sequencing data

Abstract Gene fusion resulted from genomic translocation plays important oncogenic roles including but not limited to gene activation and changing protein coding sequences. Historically, fusion gene analysis has been limited to bulk levels resulting in mixed oncogene activation models. Single cell Nanopore RNA sequencing (scNanoRNAseq) has the unique strength in detecting full-length chimeric transcripts in thousands of single cells in parallel. However, robust computational tools for detecting fusion genes in single cells from scNanoRNAseq data are missing. Here, we developed LongFuse, a fast and accurate tool, to detect fusion genes and their splicing isoforms in single cells from scNanoRNAseq data. LongFuse implements an XOR logic gate algorithm to speed up fusion candidate searching and stringent criteria to eliminate false discoveries due to trans-splicing events and randomly ligated chimeras. We applied LongFUSE onto scNanoRNAseq data of both human prostate cancer cell line, VCaP, and tumor tissues with known TMPRSS2-ERG fusion. Our results showed that both cancer cell line and prostate tumors harbored a mixture of fusion positive and fusion negative tumor cells. Gene expression analysis revealed transcriptional differences between fusion positive and negative cells including genes involved in cancer cell proliferation and invasion programs such as ELL2, ZBTB16, IGF1R, ANK3, MYPOP and PLPP1 genes. LongFUSE also calculates the splicing isoforms of fusion gene transcripts, allowing prediction of protein sequences of fusion gene transcripts. Taken together, our data demonstrated that LongFUSE is a robust computational tool for detecting gene fusions and their splicing isoforms at single cell level from scNanoRNAseq data. Citation Format: Cheng-Kai Shiau, Yueying He, Hsiao-Yun Lin, Lina Lu, Xiao-Dong Lu, Jindan Yu, Ruli Gao. LongFuse: Detecting gene fusion transcripts from high throughput long-read single cell RNA sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7418.

Abstract 5485: Spatial characterization of the immunosuppressive tumor microenvironment in estrogen receptor positive breast cancer at single cell level

Abstract We characterized immune cell exclusion in estrogen receptor positive (ER+) breast cancer through the study of spatial relationships among various subsets of cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), and other immune cells using a novel in-situ spatial transcriptomic technology. ER+ breast cancer is the most common breast cancer subtype. While endocrine therapy and immune checkpoint inhibitors have considerably reduced relapse and mortality for breast cancer patients, tumor resistance to treatment remains a major challenge. The tumor microenvironment (TME) plays an important role in this mechanism of resistance. Our previous spatial proteomic analysis of ER+ breast FFPE samples demonstrated the intricate relationship between tumor and immunosuppressive cells such as CAFs and TAMs via the CXCR4-CXCL12 axis associated with the exclusion of CD8 T cells from the TME. However, the limited number of antibodies in the panel made it challenging to investigate the diversity of those CAF and TAM populations and better understand their contribution to T cell exclusion. For this purpose, we further characterized the TME of 4 human ER+ breast cancer FFPE cores using the Xenium in-situ transcriptomic platform with a breast cancer-focused panel of 280 genes from 10x Genomics plus 100 additional genes selected from literature and publicly available single cell datasets to delineate CAF and TAM sub-populations. Image analysis enabled the quantification and spatial mapping of the target genes within each cell boundary. Cells were manually annotated through unsupervised clustering of over 1 million cells. The cell phenotyping analysis allowed us to annotate major cell types including tumor cells, macrophages, T cells and fibroblasts. With our curated panel, we further characterized CAF and TAM sub-populations by identifying myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen-presenting CAFs (apCAFs) in addition to M1 and M2-like macrophages as reported in the literature. We then performed spatial neighborhood analysis based on the similarity of the cell type composition in the vicinity of each cell. The results showed that myCAFs and M2-like macrophages are found in proximity to tumor cells and may play a role in T-cell exclusion. In addition, iCAFs mainly express CXCL12, a chemokine known to attract immunosuppressive cells such as regulatory T cells and M2-like macrophages as well as repel cytotoxic T cells from infiltrating the tumor via the CXCR4-CXCL12 axis. Overall, our work shows the potential of spatial transcriptomics to elucidate the spatial distribution of cells and their contribution to the immunosuppressive TME, which may enable researchers to improve understanding of tumor biology and drug target discovery efforts. Citation Format: Julien Tessier, Sandra Curras-Alonso, Joon Sang Lee, Fabien Delahaye, Hong Ma, Katie Malley, Dinesh Bangari, Donald Jackson, Angela Hadjipanayis. Spatial characterization of the immunosuppressive tumor microenvironment in estrogen receptor positive breast cancer at single cell level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5485.

RACGAP1 drives proliferation, migration and invasion and suppresses autophagy of gastric cancer cells via inhibiting SIRT1/Mfn2.

Gastric cancer is the most frequent gastrointestinal malignancy with a poor prognosis. Rac GTPase activation protein 1 (RACGAP1) is a novel tumor promotor, whose detailed effect on gastric cancer remains to be further elucidated. Hence, this study identifies the action of RACGAP1 on gastric cancer and investigates the potential mechanism. RACGAP1 expression in gastric cancer was analyzed based on the data of The Cancer Genome Atlas (TCGA) database. Cell proliferation was measured by CCK-8 and colony formation assay. Cell migration and invasion were evaluated by transwell assay. Cell apoptosis was assessed by flow cytometry. Cell autophagy was evaluated via determining LC3. RACGAP1 presented at high level in gastric cancer cells. Overexpressed RACGAP1 potentiated gastric cancer cell proliferation, migration, and invasion. Besides, silenced RACGAP1 induced cell apoptosis and autophagy. Furthermore, RACGAP1 suppressed the expression of SIRT1 and Mfn2. RACGAP1 was overexpressed in gastric cancer. RACGAP1 potentiated aggressive behaviors of gastric cancer, and suppressed cell apoptosis and autophagy via modulating SIRT1/Mfn2. RACGAP1 may be a valuable target in the treatment of gastric cancer.

PAK1 Promotes Inflammation Induced by Sepsis through the Snail/CXCL2 Signaling Pathway.

Sepsis is a severe syndrome characterized by organ dysfunction, resulting from a systemic imbalance in response to infection. PAK1 plays a critical role in various diseases. The present study aimed to explore and delineate the mechanism of PAK1 in inflammation induced by sepsis. Bioinformatics analysis was performed to assess PAK1, snail, and CXCL2 expression in the whole blood of septic patients and the pathways enriched with PAK1. To simulate the sepsis model, THP-1 cells were stimulated with lipopolysaccharide. Gene expression was evaluated using qRT-PCR, while cell viability was assessed using CCK-8 assay. Cell apoptosis was tested with flow cytometry. Expression of inflammatory factors in cells following different treatments was analyzed using the enzyme linked immunosorbent assay (ELISA). Dual-luciferase and chromatin immunoprecipitation assays were conducted to verify the binding relationship between PAK1 and the snail. Mouse models of cecal ligation and puncture were established, and hematoxylin and eosin staining and ELISA were employed to detect the infiltration levels of inflammatory cells and the expression of related protective factors in lung, liver, and kidney tissues. The results demonstrated upregulation of PAK1, snail, and CXCL2 in the whole blood of septic patients, with PAK1 being enriched in the chemokine-related pathway. Knockdown of PAK1 significantly promoted the apoptosis of LPS-stimulated THP-1 cells and inhibited the expression of inflammatory factors. PAK1 upregulated the expression of the snail, which in turn promoted the expression of CXCL2. Thus, PAK1 mediated the sepsis-induced inflammatory response through the snail/CXCL2 pathway. In conclusion, PAK1 played a role in promoting inflammation induced by sepsis through the snail/CXCL2 axis, thereby providing a potential therapeutic target for the management of sepsis.

GSH-related enzyme activity and tumor relation: glutathione peroxidase and glutathione reductase status under hypoxia in HepG2 cells

Abstract Objectives In hepatocellular carcinoma (HCC), tumorigenesis, hypoxia and reactive oxygen species (ROS) play a crucial role in altering the tumor microenvironment (TME). Until now, the time-dependent alteration of hypoxia-inducible factor-1α (HIF-1α), the antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR) under hypoxic conditions in HCC were not clear. Consequently, our main target was to investigate the role of GPx and GR status in HCC cell line (HepG2) under hypoxic conditions. Methods HIF-1α protein levels in cell lysates were determined by ELISA assay and protein expressions were identified using western blot. GPx and GR activity levels of the cell lysates were measured spectrophotometrically. Results HIF-1α protein levels were determined under normoxic and hypoxic conditions (p<0.001). Also, HIF-1α protein levels and expressions were observed under time-dependent hypoxic conditions, the HIF-1α protein level is found to be reached its peak point at 4 h in the HepG2 cell line. We also have detected decreased activity levels of GPx and increased GR activity levels under hypoxia for 4 h (p<0.001). Conclusions More than 4 h of exposure to hypoxic environment reducted the HIF-1α levels in HCC cells. According to the results, we suggest the ideal exposure time to hypoxic conditions as 4 h for the HepG2 cell line. In addition, hypoxia also stimulated the activity levels of GPx and GR. Our results suggest that the activity levels of GPx and/or GR enzymes may be therapeutic targets in the hypoxia-dependent HCC tumorigenesis process.

ITPR2 Mediated Calcium Homeostasis in Oligodendrocytes is Essential for Myelination and Involved in Depressive-Like Behavior in Adolescent Mice.

Ca2+ signaling is essential for oligodendrocyte (OL) development and myelin formation. Inositol 1,4,5-trisphosphate receptor type 2 (ITPR2) is an endoplasmic reticulum calcium channel and shows stage-dependent high levels in postmitotic oligodendrocyte precursor cells (OPCs). The role and potential mechanism of ITPR2 in OLs remain unclear. In this study, it is revealed that loss of Itpr2 in OLs disturbs Ca2+ homeostasis and inhibits myelination in adolescent mice. Animals with OL-specific deletion of Itpr2 exhibit anxiety/depressive-like behaviors and manifest with interrupted OPC proliferation, leading to fewer mature OLs in the brain. Detailed transcriptome profiling and signal pathway analysis suggest that MAPK/ERK-CDK6/cyclin D1 axis underlies the interfered cell cycle progression in Itpr2 ablated OPCs. Besides, blocking MAPK/ERK pathway significantly improves the delayed OPC differentiation and myelination in Itpr2 mutant. Notably, the resting [Ca2+]i is increased in Itpr2 ablated OPCs, with the elevation of several plasma calcium channels. Antagonists against these plasma calcium channels can normalize the resting [Ca2+]i level and enhance lineage progression in Itpr2-ablated OPCs. Together, the findings reveal novel insights for calcium homeostasis in manipulating developmental transition from OPCs to pre-OLs; additionally, the involvement of OLs-originated ITPR2 in depressive behaviors provides new therapeutic strategies to alleviate myelin-associated psychiatric disorders.