Proliferation Of Pancreatic Cancer Cells Research Articles

MSLN induced EMT, cancer stem cell traits and chemotherapy resistance of pancreatic cancer cells

Chemoresistance is one of the main reasons for poor prognosis of pancreatic cancer. The effects of mesothelin (MSLN) on chemoresistance in pancreatic cancer are still unclear. We aim to investigate potential roles of MSLN in chemoresistance and its relationship with proliferation, epithelial-mesenchymal transition (EMT) and cancer stemness of pancreatic cancer cells. Human pancreatic cancer cell lines ASPC-1 and Mia PaCa-2 with high and low expression of MSLN, respectively, were selected. The ASPC-1 with MSLN knockout (KO) and Mia PaCa-2 of MSLN overexpression (OE) were generated. The effects of MSLN on cell phenotypes, expression of EMT-related markers, clone formation, tumor sphere formation, and pathologic role of MSLN in tumorigenesis were detected. Sensitivity of tumor cells to gemcitabine was evaluated. The results showed that adhesion, proliferation, migration and invasion were decreased significantly in ASPC-1 with MSLN KO, whereas increased significantly in Mia PaCa-2 with MSLN OE. The size and the number of clones and tumor spheres were decreased in ASPC-1 with MSLN KO, and increased in Mia PaCa-2 with MSLN OE. In xenograft model, tumor volume was decreased (tumor grew slower) in MSLN KO group compared to control group, while increased in MSLN OE group. Mia PaCa-2 with MSLN OE had a higher IC50 of gemcitabine, while ASPC-1 with MSLN KO had a lower IC50. We concluded that MSLN could induce chemoresistance by enhancing migration, invasion, EMT and cancer stem cell traits of pancreatic cancer cells. Targeting MSLN could represent a promising therapeutic strategy for reversing EMT and chemoresistance in pancreatic cancer cells.

β-hydroxybutyrate inhibits malignant phenotypes of prostate cancer cells through β-hydroxybutyrylation of indoleacetamide-N-methyltransferase

BackgroundProstate cancer (PCa) is one of the most prevalent cancers in men and is associated with high mortality and disability rates. β-hydroxybutyrate (BHB), a ketone body, has received increasing attention for its role in cancer. However, its role in PCa remains unclear. This study aimed to explore the mechanism and feasibility of BHB as a treatment alternative for PCa.MethodsColony formation assay, flow cytometry, western blot assay, and transwell assays were performed to determine the effect of BHB on the proliferation and metastasis of PCa cells. Tumor sphere formation and aldehyde dehydrogenase assays were used to identify the impact of BHB or indoleacetamide-N-methyltransferase (INMT) on the stemness of PCa cells. N6-methyladenosine (m6A)–meRIP real-time reverse transcription polymerase chain reaction and dual luciferase assays were conducted to confirm INMT upregulation via the METTL3–m6A pathway. Co-IP assay was used to detect the epigenetic modification of INMT by BHB-mediated β-hydroxybutyrylation (kbhb) and screen enzymes that regulate INMT kbhb. Mouse xenograft experiments demonstrated the antitumor effects of BHB in vivo.ResultsBHB can inhibit the proliferation, migration, and invasion of PCa cells by suppressing their stemness. Mechanistically, INMT, whose expression is upregulated by the METTL3–m6A pathway, was demonstrated to be an oncogenic gene that promotes the stem-like characteristics of PCa cells. BHB can suppress the malignant phenotypes of PCa by kbhb of INMT, which in turn inhibits INMT expression.ConclusionsOur findings indicate a role of BHB in PCa metabolic therapy, thereby suggesting an epigenetic therapeutic strategy to target INMT in aggressive PCa.Trial registrationNot applicable.

The DDX6/KIFC1 signaling axis, as regulated by YY1, contributes to the malignant behavior of pancreatic cancer.

Human DEAD/H box RNA helicase DDX6 acts as an oncogene in several different types of cancer, where it participates in RNA processing. Nevertheless, the role of DDX6 in pancreatic cancer (PC), together with the underlying mechanism, has yet to be fully elucidated. In the present study, compared with adjacent tissues, the level of DDX6 was abnormally increased in human PC tissues, and this increased level of expression was associated with poor prognosis. Furthermore, the role of DDX6 in PC was investigated by overexpressing or silencing the DDX6 in the PC cell lines, SW1990 and PaTu-8988t. A xenograft model was established by injecting nude mice with either DDX6-overexpressing or DDX6-silenced SW1990 cells. DDX6 overexpression promoted the proliferation and cell cycle transition, inhibited the cell apoptosis of PC cells, and accelerated tumor formation, whereas DDX6 knockdown elicited the opposite effects. DDX6 exerted positive effects on PC. RNA immunoprecipitation assay showed that DDX6 bound to kinesin family member C1 (KIFC1) mRNA, which was further confirmed by RNA pull-down assay. These results suggested that DDX6 positively regulated the expression of KIFC1. KIFC1 overexpression enhanced the proliferative capability of PC cells with DDX6 knockdown and inhibited their apoptosis. By contrast, DDX6 overexpression reversed the inhibitory effect of KIFC1 silencing on tumor proliferation. Subsequently, the transcription factor Yin Yang 1 (YY1) was shown to negatively regulate DDX6 at both the mRNA and protein levels. Dual-luciferase reporter assay verified that YY1 targeted the promoter of DDX6 and inhibited its transcription. High expression levels of YY1 decreased the proliferation of PC cells and promoted cell apoptosis, although these effects were reversed by DDX6 overexpression. Taken together, YY1 may target the DDX6/KIFC1 axis, thereby negatively regulating its expression, leading to an inhibitory effect on pancreatic tumor.

IFIT1 modulates the proliferation, migration and invasion of pancreatic cancer cells via Wnt/β-catenin signaling.

Previously, Interferon-induced Protein with Tetratricopeptide Repeats 1 (IFIT1) has been shown to promote cancer development. Here, we aimed to explore the role of IFIT1 in the development and progression of pancreatic cancer, including the underlying mechanisms. We explored IFIT1 expression in pancreatic cancer samples using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Cell Counting Kit-8 (CCK8), colony formation, scratch wound-healing and Transwell assays were performed to assess the proliferation, migration and invasion abilities of pancreatic cancer cells. Gene Set Enrichment Analysis (GSEA) and Western blotting were performed to assess the regulatory effect of IFIT1 on the Wnt/β-catenin pathway. We found that upregulation of IFIT1 expression is common in pancreatic cancer and is negatively associated with overall patient survival. Knockdown of IFIT1 expression led to decreased proliferation, migration and invasion of pancreatic cancer cells. We also found that IFIT1 could regulate Wnt/β-catenin signaling, and that a Wnt/β-catenin agonist could reverse this effect. In addition, we found that IFIT1 can promote epithelial-mesenchymal transition (EMT) of pancreatic cancer cells. Our data indicate that IFIT1 increases pancreatic cancer cell proliferation, migration and invasion by activating the Wnt/β-catenin pathway. In addition, we found that EMT could be regulated by IFIT1. IFIT1 may serve as a potential therapeutic target for pancreatic cancer.

Transcription factor KLF9 inhibits the proliferation, invasion, and migration of pancreatic cancer cells by repressing KIAA1522.

Pancreatic cancer (PC) has a poor prognosis and high mortality. Kruppel-like factor 9 (KLF9), a transcription factor, is aberrantly expressed in various neoplasms. The current study sought to analyze the functional role of KLF9 in the proliferation, invasion, and migration of PC cells. The expression patterns of KLF9 and KIAA1522 in normal pancreatic cells (HPDE-C7) and PC cells (Panc 03.27, BxPc3, SW1990) were determined by real-time quantitative polymerase chain reaction and Western blot assay. After treatment of KLF9 overexpression, proliferation, invasion, and migration were evaluated by cell counting kit-8, 5-ethynyl-2'-deoxyuridine staining, and Transwell assays. The binding of KLF9 to the KIAA1522 promoter was analyzed by dual-luciferase assay and chromatin immunoprecipitation. The rescue experiment was conducted to analyze the role of KIAA1522. KLF9 was downregulated, while KIAA1522 was upregulated in PC cells. KLF9 overexpression mitigated the proliferation, invasion, and migration of PC cells. Enrichment of KLF9 led to inhibition of the KIAA1522 promoter and repressed KIAA1522 expression. KIAA1522 overexpression neutralized the inhibitory role of KLF9 in PC cell functions. KLF9 is enriched in the KIAA1522 promoter and negatively regulates KIAA1522 expression, thereby mitigating the proliferation, invasion, and migration of PC cells.

Abstract 3336: Regulation of liver X receptor protein stability by novel ligands in pancreatic cancer cells

Abstract Liver X Receptors (LXRs) are nuclear receptors that act as ligand-modulated transcription factors. LXRs are overexpressed in various cancers, including pancreatic cancer, and targeting LXR with small molecule ligands has emerged as a promising therapeutic strategy in cancer. Current small molecule LXR ligands were originally developed for the treatment of atherosclerosis and other metabolic diseases. To identify ligands specifically targeting cancer cells, we screened a focused library of drug-like molecules predicted to bind to LXR using molecular docking. In the screen, novel LXR ligand GAC0001E5 (1E5) exhibited significant anti-proliferative effects in human pancreatic ductal adenocarcinoma (PDAC) cell lines. Functionally, 1E5 is an LXR inverse agonist which decreases LXR target gene expression and a “degrader” of LXR proteins following prolonged treatment. We posit that this novel ligand inhibits PDAC cells by downregulating the expression of LXR target genes. To test this hypothesis, we treated cancer cells with the previously described LXR inverse agonist SR9238 and examined its effect on cancer cell proliferation. Interestingly, treatments with SR9238 had no effect on pancreatic cancer cell proliferation as compared to the novel ligand 1E5 and the 1E5 derivative KD-95, suggesting that LXR inverse agonism was insufficient to inhibit cell proliferation. To determine whether SR9238 has similar effects on LXR protein levels as novel ligands, we performed western analysis of protein expression following ligand treatment. In contrast to the novel ligands 1E5 and KD-95, treatments with SR9238 increased LXR protein levels. Furthermore, time-course studies in cycloheximide treated cells indicated that novel ligands decreased protein stability whereas SR9238 had the opposite effect. Inhibition of proteasomal degradation and autophagy abolished the effects of 1E5 and KD-95 in a cell line-dependent manner. Related to these observations, previously published data revealed that knockdown of LXR expression greatly diminished PDAC cell proliferation. Taken together, these findings indicate that LXR expression is essential in pancreatic cancer cells and suggest that the novel ligands may disrupt its involvement in protein-protein interactions and non-genomic mechanisms. Citation Format: Abhinav Bagchi, Kasuni T. Gamage, Scott R. Gilbertson, Chin-Yo Lin. Regulation of liver X receptor protein stability by novel ligands in pancreatic cancer cells [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 3336.

Abstract 5692: Posttranscriptional molecular network facilitates KRAS-linked tumorigenesis via XRN1-miR-21 signaling in pancreatic cancers

Abstract Aberrant expression of mutant KRAS and miRNA-21 (miR-21) are commonly attribute to malignancy and tumorigenic progression of pancreatic cancers. However, few studies have explored the molecular mechanism linking these two prognostic factors. In this study, we explored the underlying molecular bridge between KRAS and miR-21 regulation in pancreatic cancer. Functional integrative analysis of KRAS, an essential oncogenic driver in pancreatic cancer progression, and together with miR-21 revealed XRN1, a 5′-3′ exoribonuclease, as RNA-linked top-scored molecule with a strong survival correlation and malignancy in pancreatic cancer patients. Depletion of XRN1 led to severe reduction in mature miR-21 expression, while unaltered precursor miR-21 levels; indicating that the XRN1 is critical in miR-21 maturation at post-Drosha step in conjunction with Dicer. Moreover, miR-21 sensor-based experiment also confirmed the critical role of XRN1 on miR-21 biogenesis. Depletion experiments showed that XRN1 positively regulates proliferation and self-renewal capacity of pancreatic cancer cells via its 5′-3′ exoribonuclease activity and concomitantly elevated the expression of PTEN and PDCD4 which are the key miR-21 cellular downstream targets. shRNA-mediated XRN1 depletion also yielded smaller tumor volume and longer survival in pancreatic cancer xenografts and syngeneic model demonstrating the therapeutic potential of targeting XRN1. Subsequent silencing of KRAS reduced XRN1 expression in pancreatic cancer cells, suggesting that KRAS plays a key role in this XRN1-miR-21 axis. Furthermore, drug-resistant pancreatic cells showed elevated expression of both KRAS and XRN1-miR-21 indicating that this axis is also associated with drug sensitivity. In sum, these results suggest that a novel KRAS-XRN1 posttranscriptional regulatory axis plays a pivotal role in tumor progression by mediating miR-21-directed signaling, and thus may suggest a novel targeting strategy for pancreatic cancer therapeutics. Significance: This study identifies molecular regulatory interplay between KRAS and XRN1-miR-21 signaling in pancreatic cancers, elucidates how KRAS-XRN1 modulates tumorigenesis via miR-21, and demonstrates that targeting this signaling can contribute to therapeutic strategies for pancreatic cancers. [Y. Seo and S. E. Oh contributed equally to this work.] Citation Format: Yoona Seo, Seung Eon Oh, Jong Heon Kim. Posttranscriptional molecular network facilitates KRAS-linked tumorigenesis via XRN1-miR-21 signaling in pancreatic cancers [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 5692.

Abstract 5742: Therapeutic efficacy of CA102N in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a challenging cancer to treat due to its aggressive nature and late-stage diagnosis. Considering the lack of effective therapies, there is an urgent need to develop new and effective therapeutics to improve the prognosis of patients. CA102N is a carrier-mediated novel anticancer product where hyaluronic acid is conjugated to Nimesulide-NH2. CA102N is currently under development for the treatment of solid tumors, including colorectal cancer. CA102N was safe and well tolerated in the first-in-human trial in patients with locally advanced or metastatic solid tumors. In the present study, the in vitro anticancer activity of CA102N was investigated in K-ras mutant PANC-1 pancreatic cancer cells. The antitumor efficacy was also tested in vivo in the PANC-1 mice model. Results demonstrated that CA102N inhibits cell growth, cell migration, angiogenesis, and cell proliferation in both K-ras mutant PANC-1 pancreatic cancer cells or tumors. Administration of CA102N in PANC-1 xenografts resulted in a dose-dependent and statistically significant decrease in tumor growth at 9.8 mg/kg and 14.7 mg/kg Nim equivalents, respectively, compared to the vehicle control group. A significant decrease (80%) in liver metastasis was also noted in the PANC-1 orthotopic mice model at 19.6 mg/kg Nim equivalents dose level. Mechanistically, CA102N was found to suppress the PI3k/Akt/mTOR and Raf/MEK/ERK signaling pathways, in addition to reducing the expressions of the N6-methyladenosine (m6A) reader and writer proteins, OCT3/4 transcription factor and RAS levels in vivo. CA102N also modified the tumor stroma and improved drug permeability in vivo. Based on this compelling data, CA102N shows promise as a potential treatment for pancreatic cancer. Combination therapies and regimens are currently being explored in in vivo models as a strategy for clinical development. Citation Format: Louis Lin, Shuling Tu, Eskouhie Tchaparian. Therapeutic efficacy of CA102N in pancreatic cancer [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 5742.

Abstract 6001: Repurposing dihydroergotamine mesylate as a potential MCL-1 inhibitor and its antitumor effect in pancreatic cancer cells

Abstract Background: Myeloid cell leukemia 1 (MCL-1) is an anti-apoptotic member of the BCL-2 protein family which is involved in intrinsic (mitochondrial) pathway of apoptosis and is deregulated in several cancers. Current MCL-1 specific inhibitors have limitations in terms of efficiency, tolerability, and off-target effects and challenges associated with shallow binding groove of MCL-1. In this study, dihydroergotamine mesylate (DHE), an approved anti-migraine drug, was found as a potential ligand of the MCL-1 BH3-binding pocket and was further evaluated for its potential MCL-1 inhibition and effect on pancreatic cancer cells. Methods: PubChem Database was used for library preparation and virtual screening (OpenBabel and AutoDock Vina). The 55 crystal structures available for MCL-1 in Protein Data Bank (PDB) were clustered using principal component analysis with Bio3D R-package to select representative PDBs for further screening. Consensus voting and k-means clustering was used to shortlist compounds for Molecular dynamics (MD) simulations. MD simulations (100 nanoseconds) by GROMACS were employed to measure stability of ligands in MCL-1 binding pocket. DHE efficacy on cell proliferation was evaluated with MTS and clonogenic assays and on apoptosis using the FITC/Annexin V flow cytometry assay in MiaPaCa-2 and PANC-1 pancreatic cancer cell lines. Effect of DHE treatment on MCL-1 gene and protein expression was analyzed using qRT-PCR and western blotting, respectively. Results: Computational screening identified DHE (PubChem ID: 12308974) as a top hit exhibiting consistent interactions within the MCL-1 BH3-binding pocket. Notably, DHE displayed stable RMSD and formed four hydrogen bonds throughout MD simulations. In vitro results showed that DHE significantly reduced viability of MiaPaCa-2 and PANC-1 pancreatic cancer cells with IC50 values of 31 μM and 47 μM, respectively. DHE significantly (P<0.001) suppressed colony formation by pancreatic cancer cells in a dose-dependent (10μM, 20μM, and 40μM) manner. The percentage of Annexin V-positive and Propidium iodide-negative fraction in MiaPaCa-2 cells treated with DHE also increased in a dose-dependent manner. DHE did not have significant effect on MCL-1 mRNA expression. However, western blotting demonstrated MCL-1 protein levels were significantly reduced in both pancreatic cancer cell lines upon DHE treatment. Conclusion: Our findings showed DHE suppresses the pancreatic cancer cells viability and proliferation, and significantly increased early apoptotic cells. DHE treatment had no effect on MCL-1 at the transcription level but significantly reduced the protein, suggesting posttranslational modifications, and thus future mechanism of action studies is required. The present study reports that the anti-migraine drug DHE might be a novel therapeutic agent for pancreatic cancer and needs to be explored further. Citation Format: Pooja Mittal, Jae Ho Lo, Shivani Soni, Francesca Battaglin, Shradheya RR Gupta, Lesly Torres-Gonzalez, Yan Yang, Sandra Algaze, Priya Jayachandran, Karam Ashouri, Alexandra Wong, Wu Zhang, Jian Yu, Lin Zhang, Joshua Millstein, Indrakant Kumar Singh, Heinz Josef Lenz. Repurposing dihydroergotamine mesylate as a potential MCL-1 inhibitor and its antitumor effect in pancreatic cancer cells [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 6001.

Abstract 4495: Structure-based screening for potential ROR1 inhibitors and their potential as antitumor agents in pancreatic cancer cells

Abstract Background: The identification of novel compounds that specifically target cancer cells while sparing normal tissues is crucial. The orphan tyrosine kinase-like receptor 1 (ROR1) is one such target expressed exclusively in various human malignancies, including pancreatic cancer, but not in normal adult tissues. Inhibiting ROR1 signaling shows promise in halting pancreatic cancer cell proliferation and invasion. In this study, lead compounds CSF1R-IN-1 and MCULE-8715589337 were found as potential inhibitors of the ROR1 and were further evaluated for its potential effect on pancreatic cancer cell viability. Method: We curated a compound library of 6 million from PubChem, 2 million lead-like compounds from ZINC15, and 2 million compounds from diverse vendors. Employing Glide’s HTVS followed by XP, we refined the selection based on docking scores lower than the reference molecule, Ponatinib. Compounds were shortlisted by pre-simulation MMGBSA calculations with Schrödinger Prime MM-GBSA module, focusing on compounds with energies below -100 kcal/mol. Then we performed MD simulation for 100ns. Post-simulation MMGBSA on the last 1 ns of simulation assessed change in ligands’ binding energies. The selected leads underwent further validation utilizing two pancreatic cancer cell lines, MiaPaCa-2 and PANC-1. Half-maximal inhibitory concentration (IC50) was assessed using MTS cell viability assay. Result: Comparing pre- and post-simulation MMGBSA energies, the reference compound showed increased, while CSF1R-IN-1 and MCULE-8715589337 exhibited decreased energies. The reference ligand's RMSD showed stability with fluctuations of 3 to 3.5 Å, whereas CSF1R-IN-1 and MCULE-8715589337 had stable and lower RMSD values (approximately 3 Å and 2.4 Å). Test compounds, CSF1R-IN-1 and MCULE-8715589337 showed better molecular interactions with ROR1 compared to reference. The reference compound formed hydrogen bonds with ILE555 and ASP633, along with pi-pi stacking with PHE552. CSF1R-IN-1 forms a hydrogen bond with ILE555 and ASP633, pi-pi stacking with PHE552, and interaction with SER632 through a water bridge. Additionally, compound MCULE-8715589337 forms hydrogen bonds with ASP633, SER632, LYS506, and GLU523. CSF1R-IN-1 notably inhibited proliferation in MiaPaCa-2 cells (IC50: 1.29 μM) while no efficacy in PANC-1 cells (IC50: 78.41 μM). MCULE-8715589337 showed relatively similar IC50 values of 39.06 μM and 37.73 μM in MiaPaCa-2 and PANC-1 cells, respectively. Conclusion: The reduction in MMGBSA energies, low RMSD values, and a higher number of interactions highlight the better characteristics of both compounds compared to the reference. Our findings show CSF1R-IN-1 and MCULE-8715589337 have efficacy in pancreatic cancer cells, but further studies are required to validate and to understand the mechanism of action. Citation Format: Indrakant Kumar Singh, Shradheya RR Gupta, Pooja Mittal, Shivani Soni, Jae Ho Lo, Francesca Battaglin, Yan Yang, Lesly Torres-Gonzalez, Sandra Algaze, Priya Jayachandran, Karam Ashouri, Alexandra Wong, Wu Zhang, Joshua Millstein, Heinz Josef Lenz. Structure-based screening for potential ROR1 inhibitors and their potential as antitumor agents in pancreatic cancer cells [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 4495.

Abstract 2903: CTHRC1 induces PSCs (pancreatic stellate cells) into myCAFs (myofibroblast-like cancer-associated fibroblasts)

Abstract Collagen triple helix repeat containing-1 (CTHRC1) is a secreted protein that contributes to the progression of various cancers, including pancreatic cancer. The higher expression of CTHRC1 in tumor tissues is associated with poorer survival outcomes. However, its specific roles in tumor extracellular matrix (ECM) remodeling remain unclear. Our study aims to investigate the influences of CTHRC1 on pancreatic stellate cells (PSCs), a main source of ECM production in pancreatic cancer. The analyses of the publicly available pancreatic cancer patient data revealed that CTHRC1 is mainly expressed in cancer stroma and highly correlated with ECM-related genes. An in vitro study showed that more than 40% of these genes can be upregulated by CTHRC1. CTHRC1 specifically activated PSC into myofibroblast-like cancer-associated fibroblasts (myCAFs), which are characterized by a significantly upregulated POSTN gene expression. Periostin (coded by the POSTN gene) has a central role in the CTHRC1-PSCs-cancer metastasis axis. Furthermore, CTHRC1 promoted pancreatic cancer cell proliferation through PSC activation to a greater extent than via direct stimulation. Proof-of-concept experiments showed that the long-term (4-week) inhibition of CTHRC1 led to significant tumor suppression and ECM reduction, and also resulted in an unexpected shift in the CAF subtype from myCAFs to inflammatory CAFs (iCAFs). PSC activation was demonstrated to be the key molecular mechanism responsible for the tumor-promoting effects of CTHRC1, and CTHRC1 has a critical role in CAF subtype differentiation and tumor microenvironment (TME) remodeling. The inhibition of CTHRC1 as a therapeutic strategy for the treatment of pancreatic cancer warrants further investigation. Citation Format: Jin Gyeong Lee, Min Kyung Kang, Fen Jiang, Kyoungjin Ryu, Juwon Kim, Yun-Yong Park, Sang Seok Koh. CTHRC1 induces PSCs (pancreatic stellate cells) into myCAFs (myofibroblast-like cancer-associated fibroblasts) [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 2903.

Abstract 5934: Ferroptosis induction therapy for castration resistant prostate cancer

Abstract Prostate cancer (PCa) is the most common non-skin cancer in men, and second leading cause of death. The fundamental treatment for treating PCa is androgen deprivation therapy which is an effective form of treatment initially. Nevertheless, the disease reoccurs in most men; this is referred to as castration-resistant PCa (CRPC), for which the standard treatment is enzalutamide. However, some develop resistance to this enzalutamide anti-androgen agent. Ferroptosis is an iron-dependent lipid peroxidation form of regulated cell death being investigated as a novel potential therapy for PCa using ferroptosis inducers such as RSL3; they have shown promise in selectively triggering cell death in cancer cells while sparing healthy tissues. We hypothesize that RSL3- induced ferroptosis will overcome enzalutamide resistance in PCa cells. To test this, we utilized C4-2B MDVR cells which represents a cell model for aggressive PCa that metastasized to the bone and is resistant to both castration therapy and the enzalutamide drug treatment. C42B-MDVR cells were treated with increasing concentrations of RSL3 followed by analysis of cell viability with MTS assay. A dose dependent reduction in cell viability was observed which was reversed by ferroptosis inhibitor, ferrostatin-1. Western blot analysis revealed a decreased expression of the antioxidant enzyme glutathione peroxidase 4 (GPX4) with ferroptosis induction indicating a mechanism of the RSL3-induced cell death. The limitations to our research are that the research was performed in vitro and have not been tested on mouse models to visualize the effects of the treatments in animals. In conclusion, ferroptosis inducer RSL3 decreases GPX4 expression and decreases proliferation of PCa cells while ferrostatin-1 reverses the effects of the RSL3 treatment. Thus, ferroptosis inducers present a potential novel therapeutic approach for CRPC patients who have developed resistance to conventional androgen therapies. NIH NIGMS RISE 5R25GM058904, NIH/NIMHD U54MD007590; U54MD013376 Citation Format: Mojisoluwa Awolowo, Precious Elechi Dike, Valerie Odero-Marah. Ferroptosis induction therapy for castration resistant prostate cancer [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 5934.

Abstract 7270: Combination targeting of SKP2 and KDM5B induces senescence in castration-resistant prostate cancer cells via AKT signaling

Abstract Prostate cancer (PCa) is the second-leading cause of cancer mortalities and morbidities in the United States and is the most commonly diagnosed malignancy in men. Despite the fact that androgen deprivation therapy is the first-line option to initial responses, most PCa patients invariably develop castration-resistant prostate cancer (CRPC). CRPC is a lethal type of malignancy, therefore, novel and effective treatment strategies are needed. The goal of this study was to evaluate the combination treatment of small molecule inhibitors, SZL-P1-41, a SKP2 target and PBIT, a KDM5B target on PCa growth and progression; as well as to delineate the underlying mechanisms of suppressing CRPC. Literature reports that S-Phase Kinase Associated Protein 2 (SKP2) is upregulated in PCa and Lysine-specific demethylase 5B (KDM5B) serves as a histone demethylase with a crucial role in cancers. Studies showed that KDM5B is increased in human PCa and KDM5B knockout decreases carcinogenic properties of PCa cells. We previously reported that SKP2 loss partially decreases the growth of prostate tumors and that KDM5B levels are reversely regulated by SKP2 in PCa cells. However, the mechanisms of KDM5B and SKP2 interplay on PCa malignancy is unknown. We hypothesize that combination of PBIT and SZL-P1-41 treatment will enhance anti-cancer effects on PCa progression by inducing cellular senescence. Here, we showed that inhibition of KDM5B and SKP2 decreased the proliferation of PCa cells, and KDM5B KO cells were more vulnerable to SKP2 inhibition. More importantly, a combined inhibition of KDM5B and SKP2 significantly blocked malignant progression and migration of PCa cells. Mechanistically, combined inhibition of KDM5B and SKP2 in PCa cells abrogated AKT activation, resulting in an induction of cellular senescence, which was measured via Western blot analysis and senescence-associated β-galactosidase (SA-β-Gal) staining. Taken together, our results show that combined inhibition of KDM5B and SKP2 is more efficacious in inhibiting proliferation and migration in CRPC cells, and this regimen would be an ideal therapeutic approach for treating CRPC. Citation Format: LaKendria K. Brown, Thanigaivelan Kanagasabai, Guoliang Li, Sherly I. Celada, Zhenbang Chen. Combination targeting of SKP2 and KDM5B induces senescence in castration-resistant prostate cancer cells via AKT signaling [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 7270.

Abstract 7192: Teriflunomide overcomes gemcitabine resistance by inhibiting the GSK3β/mTOR pathway in pancreatic cancer

Abstract Background: Pancreatic cancer (PC) is a lethal malignancy that is partly attributable to the acquisition of chemoresistance to conventional chemotherapies. Hence, developing strategies to overcome such chemoresistance is essential. Teriflunomide, an active metabolite of leflunomide, has been widely utilized in managing autoimmune disorders, and emerging evidence suggests that it can augment the efficacy of chemotherapy in cancer. We investigated the therapeutic effect of teriflunomide in overcoming resistance to Gemcitabine-based therapy in PC. Methods: We performed docking and genomewide transcriptomic profiling analyses to identify therapeutic targets of Teriflunomide associated with Gemcitabine resistance (Gem-R) in PC. Subsequently, we analyzed the TCGA dataset to determine the prognostic significance of candidate genes in PC patients. A series of experiments were performed in PC cells, and the key findings were validated in patient-derived 3D organoids and a Gem-R Mia-PaCa2 xenograft model. Results: Docking and transcriptomic profiling studies identified GSK3β, MAPK8, and MAPK9 as potential targets of Teriflunomide, which were significantly upregulated in Gem-R cell lines (P < 0.05). GSK3β was independently associated with poor overall and disease-free survival in PC patients. Combined Teriflunomide and Gemcitabine treatment exhibited superior synergistic anti-tumor potential, as evidenced by reducing PC cell proliferation, clonogenicity, invasion, and migration. The combined treatment with Teriflunomide and Gemcitabine primarily inhibited GSK3β phosphorylation, resulting in the silencing of the downstream AKT/mTOR pathway. Lentiviral-based silencing of GSK3β inhibited the AKT/mTOR pathway and reduced the IC50s of Gemcitabine in PC cells. Furthermore, co-administrating both drugs to Gem-R Mia-PaCa-2 xenograft mice significantly reduced tumor growth rates and volumes. These findings were recapitulated in patient-derived 3D-organoids, where the combined treatment resulted in fewer and smaller organoids vs. each drug individually (P < 0.05). Conclusions: We provide novel evidence that Teriflunomide helps overcome resistance to Gemcitabine by inhibiting the GSK3β/AKT/mTOR pathway, which could improve treatment outcomes in PC. Citation Format: Caiming Xu, Yuan Li, Silei Sui, Meiling Chen, Laleh Melstorm, Haiyong Han, Steven T. Rosen, Ajay Goel. Teriflunomide overcomes gemcitabine resistance by inhibiting the GSK3β/mTOR pathway in pancreatic cancer [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 7192.

Abstract 5685: MiR-345-5p attenuates pancreatic cancer metastasis through KLK7/E-Cad/β-Catenin/Rap1 axis

Abstract Background: Pancreatic cancer (PC) is a highly lethal malignancy with a 5-year survival rate of 10.8%, primarily attributed to early invasion and metastasis. Epithelial-to-mesenchymal transition (EMT) plays a crucial role in the metastatic progression of PC. Existing therapies, such as Gemcitabine, Abraxane, and FOLFIRINOX, have limited efficacy, emphasizing the need for identifying molecular targets to enhance PC patient survival. Recent advancements in miRNA therapies show promise, but a detailed understanding of miRNAs regulating PC growth and metastasis remains elusive. This study aims to uncover the role of miR-345-5p in regulating PC metastasis. Methods: A miRNA microarray analysis of KC mouse data identified miR-345-5P as one of the significantly down-regulated miRNA, targeting multiple metastasis-related genes. Expression of miR-345-5p in PC was assessed using published databases, validated through qPCR and ISH in PC cell lines, serum samples, and patient tissue. In vitro overexpression and in vivo assays elucidated the functional roles of miR-345-5p in PC metastasis. A computational approach and dual-luciferase assays were used to identify the target of miR-345-5p. Western blots, immunohistochemistry, and immunofluorescence were utilized to decipher miR-345-5p mediated molecular mechanisms. Results: MiR-345-5p expression was downregulated in PC tissue, serum, and cell lines compared to non-tumor tissues. Restoring miR-345-5p reduced PC cell proliferation, migration, and invasion in vitro and in vivo. KLK 7 was confirmed as a direct target, and functional experiments revealed miR-345-5p's acting as a tumor-suppressor by inhibiting the E-Cad/B-catenin/Rap1 axis signaling pathway. MiR-345-5p overexpression stabilized the E-Cadherin-β-catenin complex, leading to reduced β-catenin accumulation in the nucleus and thus leading to declined transcription of crucial genes (C-myc, cyclin D1 & MMP7) responsible for PC progression. Given the pivotal role of the c-myc gene in metabolism, we performed a metabolomic analysis. The findings unveiled significant metabolic rewiring, marked by reductions in glucose and amino acid metabolism, attributable to decreased C-myc expression due to restoration of miR-345 in PC cells. Conclusion: MiR-345-5p overexpression negatively regulates PC proliferation and EMT by directly targeting KLK7/E-Cad/β-catenin/Rap1 axis. Restoring miR-345-5p with standard care therapies could be a promising strategy for improving survival in metastatic PC patients. Citation Format: Nagabhishek Sirpu Natesh, Susheel Kumar Nethi, Brianna M White, Jussuf T. Kaifi, Surinder K. Batra, Surya Mallapragada, Satyanarayana Rachagani. MiR-345-5p attenuates pancreatic cancer metastasis through KLK7/E-Cad/β-Catenin/Rap1 axis [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 5685.

Abstract 2077: Exploring the multifaceted efficacy of de novo lipogenesis inhibitor in cancer therapy

Abstract Cancer cells frequently exhibit altered lipid metabolism to sustain their rapid growth and proliferation.Sterol Regulatory Element-Binding Proteins (SREBPs) are key transcription factors that orchestratelipid biosynthesis and uptake in cells. Dysregulation of SREBPs is a common feature of many cancertypes, making them attractive targets for therapeutic intervention. In this study, we present acomprehensive exploration of the potential and rationale for targeting lipid metabolism in cancerthrough the use of a small molecule inhibitor that targets activation of SREBP. We illustrate that oursmall molecule inhibitor (SMI) exhibits potent anticancer activity and effectively targets SREBPsignaling in a variety of prostate cancer models as well as in non-prostate cancer models that arelipogenesis driven. Our novel SMI induces apoptosis and reduces PCa cell proliferation, suppressescell invasion and migration. Furthermore, we illustrate that in addition to suppressing the transcriptionfactor activity of SREBPs, these novel SMIs are highly effective in suppressing both the full length andvariant androgen receptor and their signaling in prostate cancer cells. Notably, in cell and organoidmodels, these novel inhibitors exhibit synergistic anticancer activity in combination with the ARsignaling inhibitor, Enzalutamide. The anticancer activity of these inhibitors extend beyond ARdependent prostate cancer and are also effective in organoid models of neuroendocrine PCa, aparticularly drug-resistant and aggressive PC subtype. Lastly, we illustrate excellent pharmacokineticsand potent anti-tumor effects of these novel SREBP activation inhibitors using in vivo castration-resistant prostate cancer CDX tumors. Given the urgent need for novel targets in PCa, our first-in-class inhibitor represents a promising therapeutic strategy with the potential to effectively targetdysregulated lipogenesis in cancer patients. Citation Format: Dallin Lowder, Jin Na Shin, Maria E. Ruiz-Echartea, Jenny Deng, Aleksandra Rusin, Darlene Skapura, Michael Ittmann, Salma Kaochar. Exploring the multifaceted efficacy of de novo lipogenesis inhibitor in cancer therapy [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 2077.

Abstract 1501: Engineering tissue models to elucidate prostate cancer bone metastasis dynamics

Abstract Prostate cancer (PCa) ranks as the second most frequently diagnosed cancer in men and the fifth leading cause of male cancer-related deaths globally Advanced PCa exhibits a propensity for bone metastasis which significantly contributes to morbidity. It is challenging to understand its mechanisms and develop effective treatments due to complicated tumor microenvironments. The current study addresses this gap by focusing on the construction of in vitro and in vivo models to mimic tissue-specific microenvironments resembling primary prostate tumor sites and metastatic bone tissue, shedding light on the complex processes of cancer cell dissemination, osteomimicry, and bone metastasis. To recapitulate the complex interplay between primary tumor sites and bone microenvironments, the study employs a combination of tunable extracellular matrix mechanics, bone-derived growth factors, and bone marrow-derived stromal cells. These engineered niches emulate the contexts of both primary tumor and bone tissue. LNCAP-C4-2B prostate cancer cells are introduced into these niches to analyze various aspects, including cell proliferation, osteoblastic differentiation, dissemination, and in vivo tumor formation. The study introduces a 3D organoid culture that mimics the pre-metastatic niche, providing a platform for studying PCa cell proliferation and dissemination. A negative relationship is observed between 3D matrix stiffness and cell proliferation, while a positive link is identified with cell dissemination. Bone matrix-derived soluble factors (BMSF) are identified as key modulators of LNCAP-C4-2B cell behavior, suppressing LNCAP-C4-2B proliferation but enhancing osteomimicry in vitro, Notably, bone marrow-derived stromal cells, primed with bone matrix-derived growth factors, amplify tumor cell proliferation and chemotaxis in vitro, contributing to increased tumor formation in vivo. In vivo experiments demonstrate BMSFs eliciting local inflammatory responses and inhibiting in vivo tumor formation. Moreover, osteogenic bone marrow stromal cells are shown to play a promotive role in LNCAP-C4-2B proliferation and migration, emphasizing the influence of the bone microenvironment on the prostate-to-bone progression of PCa cells. In conclusion, this study highlights the potential of the engineered tissue models for dissecting tumor progression and screening therapeutic interventions. By leveraging tissue-engineered soft and hard tissues, our approach offers a versatile toolbox for comprehensively investigating PCa progression and bone metastasis. These insights extend to potential therapeutic screening applications, providing a valuable resource for advancing our understanding of cancer pathogenesis and treatment strategies. Citation Format: Bo Han, Zhi Yang, Shuqing Zhao, Gangning Liang, Ba Xuan Hoang, Josephine Fang. Engineering tissue models to elucidate prostate cancer bone metastasis dynamics [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 1501.

Abstract 5897: Understanding and targeting fatty acid CoA ligase ACSL4 in human prostate cancer

Abstract Prostate cancer (PCa) is the most common non-cutaneous malignancy among men. Although localized prostate cancer is curable, recurrence of metastatic castration-resistant prostate cancer (mCRPC) is almost inevitable and is the major cause of death in human PCa. Recent findings from our lab and others demonstrate that RB1 loss drives lineage plasticity, metastasis, and lethality of prostate tumors initiated by PTEN loss. A downstream target upregulated by RB1 loss is acyl CoA synthetase long chain family member 4 (ACSL4), a fatty acid ligase with an essential role in utilization of long chain fatty acids. ACSL4 has been reported to be upregulated in cancers of various histological origins, including prostate cancer, and its high expression is correlated with aggressiveness of cancer. We hypothesize that ACSL4 is a critical downstream effector of RB1 loss-driven prostate tumorigenesis and pharmacological inhibition of ACSL4 could represent a novel therapeutic approach to the treatment of lethal RB1 deficient or ACSL4 overexpressing prostate cancer. Here we aim to develop novel ACSL4 inhibitors for preclinical studies. Through a structure based virtual screening that targeted ATP binding domains of ACSL4, we successfully identified 15 potential ACSL4 inhibitors. To validate the result, we performed an in vitro screening using various parental and isogenic human PCa cell lines, and found that 4 out of 15 compounds effectively inhibited PCa cell proliferation in a dose-response manner and their inhibitory effects positively correlated with cellular levels of ACSL4. Next, we will perform isotope-labeling enzyme activity assays to determine the specificity of the 4 candidate drugs, and PDPK analyses followed by characterizing their in vivo efficacies in preclinical studies, with a final goal to develop a well-validated ACSL4 inhibitor. Successful completion of this work will provide great insights into contributions of ACSL4 to prostate tumorigenesis as well as preclinical data of ACSL4 inhibitors to treat lethal prostate cancer. Citation Format: Jinjin Wu, Mu-En Wang, Ming Chen, Pyeong Hwa Jeong, Jiyong Hong, Lixin Wan. Understanding and targeting fatty acid CoA ligase ACSL4 in human prostate cancer [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 5897.

Mitochondrial energy metabolism-related gene signature as a prognostic indicator for pancreatic adenocarcinoma.

Background: Pancreatic adenocarcinoma (PAAD) is a highly malignant gastrointestinal tumor and is associated with an unfavorable prognosis worldwide. Considering the effect of mitochondrial metabolism on the prognosis of pancreatic cancer has rarely been investigated, we aimed to establish prognostic gene markers associated with mitochondrial energy metabolism for the prediction of survival probability in patients with PAAD. Methods: Gene expression data were obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases, and the mitochondrial energy metabolism-related genes were obtained from the GeneCards database. Based on mitochondrial energy metabolism score (MMs), differentially expressed MMRGs were established for MMs-high and MMs-low groups using ssGSEA. After the univariate Cox and least absolute and selection operator (LASSO) analyses, a prognostic MMRG signature was used in the multivariate Cox proportional regression model. Survival and immune cell infiltration analyses were performed. In addition, a nomogram based on the risk model was used to predict the survival probability of patients with PAAD. Finally, the expression of key genes was verified using quantitative polymerase chain reaction and immunohistochemical staining. Intro cell experiments were performed to evaluated the proliferation and invasion of pancreatic cancer cells. Results: A prognostic signature was constructed consisting of two mitochondrial energy metabolism-related genes (MMP11, COL10A1). Calibration and receiver operating characteristic (ROC) curves verified the good predictability performance of the risk model for the survival rate of patients with PAAD. Finally, immune-related analysis explained the differences in immune status between the two subgroups based on the risk model. The high-risk score group showed higher estimate, immune, and stromal scores, expression of eight checkpoint genes, and infiltration of M0 macrophages, which might indicate a beneficial response to immunotherapy. The qPCR results confirmed high expression of MMP11 in pancreatic cancer cell lines, and IHC also verified high expression of MMP11 in clinical pancreatic ductal adenocarcinoma tissues. In vitro cell experiments also demonstrated the role of MMP11 in cell proliferation and invasion. Conclusion: Our study provides a novel two-prognostic gene signature-based on MMRGs-that accurately predicted the survival of patients with PAAD and could be used for mitochondrial energy metabolism-related therapies in the future.

Investigating the structural basis of piperine targeting AKT1 against prostate cancer through in vitro and molecular dynamics simulations

AkT1, significantly impacts many tumours cell functions, like transcription, apoptosis, glucose metabolism, cell proliferation, and cell migration. For tumours to develop and spread, aberrant activation of AKT1 is essential. Therefore, a major focus of molecularly targeted PCa treatment is AKT1. The present study investigates the effect of piperine compared to SDF using in-vitro studies, viz colony formation assay, comet assay and AKT1 gene expression studies using human PCa cell line PC3. A cluster of approximately at least 50 cells constitutes a colony. The clonogenic assay showed that the number and size of colonies significantly decreased when treated with compounds (SDF and piperine) in comparison to the untreated cells which effectively proliferated to form more colonies. Piperine treatment showed significant inhibition of colony formation than SDF. Effective genotoxicity was observed in piperine-treated PC3 cells with an increased Tail length of 120 µm and it was moderately observed in SDF with a Tail length of 30 µm treated on PC3 cells. The control group did not show any considerable genotoxicity with a Tail length of 6 µm. Our data, both in vitro and in silico, suggested that piperine would be a good starting point for developing novel drugs for the treatment of PCa. The downstream functions of Akt1 may be inhibited by these effects, which could impede the proliferation of PCa cells. High stability of the piperine-AKT1 complex was found by the MD simulation. Higher hydrophilic residues like Lys268 and Ser205 at the active pocket may be the cause of the binding stability. Overall, the observed results confirmed the anti-PCa effect of piperine by causing effective DNA damage and proved to be genotoxic in nature against the human PCa. These effects may impede the downstream activities of Akt1 and result in PCa cell growth regression. Communicated by Ramaswamy H. Sarma