Reduced Tumor Cell Proliferation Research Articles

P0144 Unveiling the role of the MFSD2A protein in contributing to the resolution of colitis-associated cancer inflammation

Abstract Background The intrinsic connection between inflammation and tumor promotion is well-established in colorectal cancer (CRC), where chronic inflammation, like ulcerative colitis (UC), plays a significant role in intestinal carcinogenesis (1). UC patients are at higher risk of colitis-associated cancer, emphasizing the inflammation-cancer connection. Previous studies from our laboratory revealed that UC patients with active inflammation have impaired production of inflammation-resolving DHA-derived metabolites due to a defect in the endothelial Major Facilitator Superfamily Domain containing 2A (MFSD2A) (2). Given the MFSD2A’s role in reducing colonic inflammation, we hypothesize it also counteracts cancer-associated inflammation. Methods Human Intestinal Microvascular Endothelial Cells (HIMEC) isolated from CRC and healthy samples were transduced with lentiviruses carrying either the GFP-tagged MFSD2A encoding sequence (MFSD2A-OE), or the relative GFP control, or the MFSD2A-targeting shRNAs, or the scramble as control, then analyzed by transcriptomics and lipidomics. A colon-adenocarcinoma cell line (Caco-2) was cocultured with either MFSD2A-OE or GFP CRC HIMEC to evaluate their proliferation rate. An orthotopic model of CRC was performed by intrarectally injecting CD1 nude mice with a cell mixture of Caco-2 and CRC HIMEC overexpressing either MFSD2A or GFP as control. Collected tumors were analyzed by flow cytometry and lipidomics (3). Results CRC HIMEC displayed a pro-resolving signature at the lipidomic level, consistently with higher MFSD2A expression as compared to the healthy. However, gene expression analysis shows a pro-inflammatory profile in CRC HIMEC, suggesting that MFSD2A levels alone are insufficient to induce a fully pro-resolving phenotype. We performed loss of function experiments to assess whether the pro-resolving profile in CRC HIMEC depends on MFSD2A. MFSD2A silencing reduces pro-resolving functions, confirming its role in balancing pro-inflammatory and pro-resolving signals in CRC regarding lipid production. Caco-2 co-cultured with MFSD2A-OE CRC HIMEC displayed a decrease in their proliferation rate after 48 and 72 hours of co-culture, indicating that endothelial MFSD2A overexpression reduces tumor cell proliferation. By comparison with animals injected with Caco-2 and GFP HIMEC, mice injected with a Caco-2/MFSD2A-OE CRC HIMEC mixture developed tumors with reduced weight and size, and increased pro-resolving lipid release. Additionally, flow cytometry showed increased M2 macrophage polarization, indicating a shift toward a pro-resolving immune response. Conclusion These data suggested that MFSD2A contrasts cancer-associated inflammation, ensuring the correct balance between pro-inflammatory and pro-resolving milieu.

Efficacy of metformin and verteporfin treatment alone or in combination in a murine head and neck cancer xenograft model.

Despite treatment advances, head and neck squamous cell carcinoma (HNSCC) still poses a significant global health challenge. Combination therapies have emerged as more effective strategies than traditional chemotherapy in clinical practice by improving tumor response rates and patient survival while minimizing treatment-related toxicity. This study investigates the anticancer effects of metformin and verteporfin (Yes-associated protein 1 [YAP1] inhibitor) alone or in combination in HNSCC using vitro and in vivo approaches. Quantitative RT-PCR analysis of HNSCC cell lines reveals upregulation of YAP1 and related genes in the Hippo signaling pathway. Cell viability assays demonstrate a beneficial synergistic effect between metformin and verteporfin in inhibiting HNSCC tumor growth. In male BALB/cAJcl-nu/nu mice harboring HNSCC tumor xenografts, intraperitoneal administration of metformin and verteporfin enhances the inhibitory effect on tumor growth and suppresses YAP1 nuclear translocation when compared to vehicle or monotherapies. Furthermore, combination of these drugs reduces tumor cell proliferation (marked by Ki-67) and inhibits phosphoS6 ribosomal protein, as observed through immunofluorescent and immunohistochemical (IHC) analyses. The in vivo study underscores the therapeutic potential of the dual targeting approach with metformin and verteporfin in treating HNSCC, with minimal toxicity.

Hsa-miR-214-3p inhibits breast cancer cell growth and improves the tumor immune microenvironment by downregulating B7H3.

Immune checkpoint inhibitors play an important role in the treatment of solid tumors, but the currently used immune checkpoint inhibitors targeting programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), and cytotoxic T-lymphocyte antigen-4 (CTLA-4) show limited clinical efficacy in many breast cancers. B7H3 has been widely reported as an immunosuppressive molecule, but its immunological function in breast cancer patients remains unclear. We analyzed the expression of B7H3 in breast cancer samples using data from the Cancer Genome Atlas Program (TCGA) and the Gene Expression Omnibus (GEO) databases. MicroRNAs were selected using the TarBase, miRTarBase, and miRBase databases. The regulatory role of the microRNA hsa-miR-214-3p on B7H3 was investigated through dual-luciferase reporter assays, which identified the specific action sites of interaction. The expression levels of B7H3 and hsa-miR-214-3p in human breast cancer tissues and adjacent normal tissues were quantified using Western blotting and quantitative PCR (qPCR). In vitro experiments were performed to observe the effects of modulating the expression of B7H3 or hsa-miR-214-3p on breast cancer cell proliferation and apoptosis. Additionally, the regulatory impact of hsa-miR-214-3p on B7H3 was examined. Enzyme-linked immunosorbent assays (ELISA) and flow cytometry were employed to assess the effects of co-cultured breast cancer cells and normal human peripheral blood mononuclear cells (PBMCs) on immune cells and associated cytokines. In breast cancer tissues, the expression level of B7H3 is inversely correlated with that of hsa-miR-214-3p, as well as with the regulatory effects on breast cancercell behavior. Hsa-miR-214-3p was found to inhibit breast cancer cell growth by downregulating B7H3. Importantly, our research identified, for the first time, two binding sites for hsa-miR-214-3p on the 3' UTR of B7H3, both of which exert similar effects independently. Co-culture experiments revealed that hsa-miR-214-3p obstructs the suppressive function of B7H3 on CD8+ T cells and natural killer cells. This study confirms the existence of two hsa-miR-214-3p binding sites on the 3' UTR of B7H3, reinforcing the role of hsa-miR-214-3p as a regulatory factor for B7H3. In breast cancer, hsa-miR-214-3p reduces tumor cell proliferation and enhances the tumor immune microenvironment by downregulating B7H3. These findings suggest new potential targets for the clinical treatment of breast cancer.

Single-cell RNA-sequencing and spatial transcriptomic analysis reveal a distinct population of APOE- cells yielding pathological lymph node metastasis in papillary thyroid cancer.

Thyroid cancer is one of the most common endocrine tumors worldwide, especially among women and the metastatic mechanism of papillary thyroid carcinoma remains poorly understood. Thyroid cancer tissue samples were obtained for single-cell RNA-sequencing and spatial transcriptomics, aiming to intratumoral and antimetastatic heterogeneity of advanced PTC. The functions of APOE in PTC cell proliferation and invasion were confirmed through in vivo and in vitro assays. Pseudotime analysis and CellChat were performed to explore the the molecular mechanisms of the APOE in PTC progression. We identified a subpopulation of tumor cells with lower expression levels of APOE, associated with advanced stages of PTC and cervical metastasis. APOE overexpression significantly reduced tumor cell proliferation and invasion, both in vitro and in vivo, by activating the ABCA1-LXR axis. APOE- tumor cells may promote tumor growth by interacting with dendritic cells and CD4+ T cells via CD99- rather than CD6-regulated signaling. We established a machine learning-based scRNA-seq data, 13-gene signature predictive of lymph node metastasis. We identified a distinct APOE- tumor cell population associated with cervical metastasis and poor prognosis. Our results and models have potential clinical, prognostic, and therapeutic implications for advanced PTC. A subpopulation of tumor cells with lower expression levels of APOE was strongly associated with more advanced stages and metastasis of PTC. APOE-negative (APOE-) cellsoverall exhibited weaker interactions with immune cells. A machine-learning bioinformatics model based on scRNA-seq data of in-situ thyroid cancer tissue was established to predict lymph node metastasis.

Targeting ACAT1 for Precision Chemo‐Immunoprevention in Lung Cancer

AbstractLung cancer (LC) is a leading cause of cancer‐related deathworldwide, and altered cholesterol metabolism is a hallmark of cancer cells. Acyl‐CoA:cholesterol acyltransferase 1(ACAT1), or Sterol O‐acyltransferase 1 (SOAT1), isa key cholesterol esterification enzyme. Its overexpression promotes tumorprogression by accumulating cholesterol esters. Inhibition of ACAT1 also potentiatesCD8+ T cells medicated anti‐tumor immunity by increasing plasma membranecholesterol level. This study, as the first of its kind, shows the ACAT1/SOAT1 overexpressioncorrelates with poor prognosis in early‐stage lung adenocarcinoma (LUAD) patients.Long‐term treatment with ACAT1 inhibitor avasimibe suppresses tumorigenesis inboth Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growthfactor receptor (EGFR) mutation‐induced LC mouse models without overttoxicity. ACAT1 inhibition reduces tumor cell proliferation, migration, andinvasion and causes G0/G1 cell cycle arrest, while boosting CD8+ T cells'effector function and memory phenotype. Single‐cell RNA sequencing reveals thatACAT1 inhibition downregulates cholesterol biosynthesis and central carbon andnitrogen metabolism pathways in tumor cells, while upregulating genes relatedto oxidative phosphorylation and fatty acid oxidation in CD8+ T cells. Finally, avasimibe improves the efficacy of a human EGFR vaccine in preventing LCprogression. These novel findings suggest potential strategies for cancer preventionand therapy.

Multifunctional System with Camptothecin and [12]aneN3 Units for Effective In Vivo Anti Pancreatic Cancer through Synergistic Chemotherapy, Gene Therapy, and Photodynamic Therapy.

Maximizing drug cargo carrying capacity in blood circulation, controlling the in vivo fate of nanoparticles, and precisely drug release to tumor targets are the main aims of multifunctional nanomedicine-based antitumor therapy. Here we combined macrocyclic polyamine di(triazole-[12]aneN3) (M) and chemical drug camptothecin (CPT, C) through photosensitizer 1,1-dicyano-2-phenyl-2-(4-diphenylamino) phenyl-ethylene (DT) containing the cleavable disulfide (S) linkage as an all-in-one theranostic nanoprodrug, MDTSC. The corresponding compound with carbon chain (C) linkage, MDTCC, was also prepared for a comparison study. MDTSC showed the ability to carry plasmids, including the p53 tumor suppressor gene, to form lipoplexes with a size of ∼150 nm. Further addition of DOPE-PEG2k resulted in the hybrid lipoplexes MDTSC/DOPE-PEG2k@DNA, which showed good stability in blood circulation and good biocompatibility to normal cell lines. Experiments demonstrated that the hybrid lipoplexes were able to realize the successful cellular uptake and endosomal escape, to generate ROS under visible light irradiation as well as to trigger the localized release of CPT and the plasmid encoding p53 in tumor cells. In vitro, the hybrid lipoplexes showed better EGFP expression than the commercial Lipo2000, and markedly reduced tumor cell proliferation and migration rate irrespective of whether the BxPC-3 cell lines were grown on plates or 3D tumor spheroids. In vivo, the hybrid lipoplexes showed effective anticancer activity by reducing the BxPC-3 pancreatic tumor growth by 99% through the synergetic combination of chemotherapy, photodynamic therapy, and gene therapy. This research represented the first example of using a cocktail of three therapeutic approaches to achieve cooperative and effective anti pancreatic cancer treatment in vivo and in vitro.

GNAO1 overexpression promotes neural differentiation of glioma stem-like cells and reduces tumorigenicity through TRIM21/CREB/HES1 axis.

Inducing tumor cell differentiation is a promising strategy for treating malignant cancers, including glioma, yet the critical regulator(s) underlying glioma cell differentiation is poorly understood. Here, we identify G Protein Subunit Alpha O1 (GNAO1) as a critical regulator of neural differentiation of glioma stem-like cells (GSCs). GNAO1 expression was lower in gliomas than in normal neuronal tissues and high expression of GNAO1 correlated with a better prognosis. GNAO1 overexpression markedly promoted neural differentiation of GSCs, leading to decreased cell proliferation and colony formation. Mechanistically, GNAO1 recruited TRIM21 and facilitated TRIM21-mediated ubiquitination. This ubiquitination resulted in the degradation of CREB and further reduced p300-mediated H3K27ac levels of the HES1 promoter. As a result, GNAO1 overexpression downregulated HES1 expression, which reinforced neuronal differentiation. In addition, knockdown of METTL3, a key writer of the N6-methyladenosine (m6A), enhanced GNAO1 mRNA stability. Treatment with GNAO1 adenovirus increased neuronal differentiation of tumor cells and reduced tumor cell proliferation in orthotopic GSC xenografts and temozolomide further enhanced GNAO1 adenovirus effects, resulting in extended animal survival. Our study presents that engineering GNAO1 overexpression-inducing neural differentiation of GSCs is a potential therapy strategy via synergistic inhibition of malignant proliferation and chemotherapy resistance.

EXTH-06. DEVELOPMENT OF ANTISENSE OLIGONUCLEOTIDE THERAPY AGAINST ZFTA-RELA EPENDYMOMA

Abstract Treatment for ependymoma (EPN), the third most common pediatric brain tumor, has not changed in the last 30 years, consisting of surgery and radiotherapy. When EPNs relapse, there are very limited therapeutic options, thus highlighting a dire need for more effective treatment for these tumors. Over 95% of supratentorial EPN are driven by ZFTA fusion oncoproteins, the most common variant being ZFTA-RELA. ZFTA fusion oncoproteins (FO) alone are sufficient to drive tumors in murine models. We hypothesize that: these tumors continue to depend on ZFTA-RELA expression, and that the FO represents a lead target for therapeutic development. RESULTS To assess this dependency, we utilized CRISPR-Cas9 mediated knockout (KO) of ZFTA-RELA oncoproteins from a series of patient derived cell lines as compared to control cells. We found that ZFTA FO driven patient models were dependent upon ZFTA FO expression, and KO led to impaired cellular proliferation. To determine whether a reduction in FO protein levels would also be sufficient to reduce tumor growth, we screened a series of antisense oligonucleotides (ASOs) for ZFTA FO knockdown efficiency in patient-derived cell lines. We quantified knockdown efficiency by western blot and selected the top-performing ASOs targeting a functionally important zinc finger domain of ZFTA. Top-performing ASOs reduced tumor cell proliferation in single dose experiments by 50%, while a non-targeting ASO treated population was unaffected. Repeated ASO doses over 96 hours saw a 75% reduction compared to a 10% reduction in the non-targeting group. CONCLUSION Our findings suggest that ZFTA-RELA drives tumor initiation and continued maintenance and proliferation of EPN. Tumor proliferation can be slowed by reducing FO levels below a required threshold. ASO therapy, potentially in combination with translation inhibitors, is one promising route for targeted treatments in this aggressive EPN subtype and could be expanded against other EPN driver proteins such as EZHIP.

Photo-Thermally Controllable Tumor Metabolic Modulation to Assist T Cell Activation for Boosting Immunotherapy.

Glycolysis is crucial for tumor cell proliferation, supporting their energy needs and influencing the tumor microenvironment (TME). On one hand, increased lactate levels produced by glycolysis acidifies the TME, inhibiting T cell activity. On the other hand, glycolysis promotes the expression of PD-L1 through various mechanisms, facilitating immune evasion. Therefore, controlled modulation of glycolysis in tumor cells to subsequently improve the immune tumor microenvironment holds significant implications for clinical cancer treatment and immune regulation. To reverse the immunosuppressive microenvironment caused by tumor glycolysis and reduce tumor immune escape, we developed a photo-thermal-controlled precision drug delivery platform to regulate tumor metabolism and aid in the activation of T cells, thereby enhancing immunotherapy. First, hollow mesoporous Prussian blue (HPB) was prepared, and the glycolysis inhibitor 3-bromopyruvate (3-BrPA) was encapsulated within HPB using the phase-change material 1-tetradecanol, resulting in B/T-H. This product was then modified with tumor cell membranes to obtain a photo-thermal controllable regulator (B/T-H@Membrane, B/T-HM). Due to the excellent drug loading and photo-thermal properties of HPB, upon reaching the tumor, B/T-HM can rapidly heat under 808 nm irradiation, causing the 1-tetradecanol to transition to a liquid phase and release 3-BrPA, which effectively inhibits tumor glycolysis through the HK2 pathway, thereby reducing tumor cell proliferation, decreasing lactate production, and downregulating tumor PD-L1 expression. In synergy with photo-thermal and αPD-1, this photo-thermally controllable metabolic-immune therapy effectively activates T cells to eliminate tumor. In response to the changes in immune microenvironment caused by tumor metabolism, a photo-thermal precision-controlled drug delivery platform was successfully developed. This platform reshapes the tumor immunosuppressive microenvironment, providing a new approach for T cell-based tumor immunotherapy. It also opens new avenues for photo-thermal controllable metabolic-immune therapy.

Construction of a tumor mutational burden-derived LncRNA prognostic computational framework associated with therapy sensitivity in skin cutaneous melanoma

BackgroundSkin cutaneous melanoma (SKCM) poses a significant public health challenge due to its aggressive nature and limited treatment options. To address this, the study introduces the Tumor Mutational Burden-Derived Immune lncRNA Prognostic Index (TILPI) as a potential prognostic tool for SKCM.MethodsTILPI was developed using a combination of gene set variation analysis, differential expression analysis, and COX regression analysis. Additionally, functional experiments were conducted to validate the findings, focusing on the effects of STARD4-AS1 knockdown on SKCM tumor cell behavior. These experiments encompassed assessments of tumor cell proliferation, gene and protein expression, migration, invasion, and in vivo tumor growth.ResultsThe results demonstrated that knockdown of STARD4-AS1 led to a significant reduction in tumor cell proliferation and impaired migration and invasion abilities. Moreover, it resulted in the downregulation of ADCY4, PRKACA, and SOX10 gene expression, as well as decreased protein expression of ADCY4, PRKACA, and SOX10. In vivo experiments further confirmed the efficacy of STARD4-AS1 knockdown in reducing tumor growth.ConclusionsThis study elucidates the mechanistic role of STARD4-AS1 and its downstream targets in SKCM progression, highlighting the importance of the ADCY4/PRKACA/SOX10 pathway. The integration of computational analysis with experimental validation enhances the understanding of TILPI and its clinical implications. Overall, the findings underscore the potential of novel computational frameworks like TILPI in predicting and managing SKCM, particularly through targeting the ADCY4/PRKACA/SOX10 pathway.

Impact of Optimized Ku-DNA Binding Inhibitors on the Cellular and In Vivo DNA Damage Response.

Background: DNA-dependent protein kinase (DNA-PK) is a validated cancer therapeutic target involved in DNA damage response (DDR) and non-homologous end-joining (NHEJ) repair of DNA double-strand breaks (DSBs). Ku serves as a sensor of DSBs by binding to DNA ends and activating DNA-PK. Inhibition of DNA-PK is a common strategy to block DSB repair and improve efficacy of ionizing radiation (IR) therapy and radiomimetic drug therapies. We have previously developed Ku-DNA binding inhibitors (Ku-DBis) that block in vitro and cellular NHEJ activity, abrogate DNA-PK autophosphorylation, and potentiate cellular sensitivity to IR. Results and Conclusions: Here we report the discovery of oxindole Ku-DBis with improved cellular uptake and retained potent Ku-inhibitory activity. Variable monotherapy activity was observed in a panel of non-small cell lung cancer (NSCLC) cell lines, with ATM-null cells being the most sensitive and showing synergy with IR. BRCA1-deficient cells were resistant to single-agent treatment and antagonistic when combined with DSB-generating therapies. In vivo studies in an NSCLC xenograft model demonstrated that the Ku-DBi treatment blocked IR-dependent DNA-PKcs autophosphorylation, modulated DDR, and reduced tumor cell proliferation. This represents the first in vivo demonstration of a Ku-targeted DNA-binding inhibitor impacting IR response and highlights the potential therapeutic utility of Ku-DBis for cancer treatment.

Inhibiting circ_0000673 blocks the progression of colorectal cancer through downregulating CPSF6 via targeting miR-548b-3p.

Colorectal cancer (CRC) is one of the most common cancers, and its progression is regulated by several factors, including circular RNA (circRNA). The objective of this study was to determine the role, or roles, of circ_0000673 in CRC. We used quantitative real-time polymerase chain reaction (qPCR) to detect the expression of circ_0000673, miR-548b-3p and cleavage and polyadenylation specific factor 6 (CPSF6) in DLD-1 and RKO cells. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to determine circ_0000673 roles in proliferation. Wound healing and transwell assays were used to detect cell migration and invasion abilities. Expression of CPSF6 protein and stem cell-associated proteins were examined using western blot. The putative relationship between miR-548b-3p and circ_0000673 or CPSF6 was verified with dual-luciferase reporter assay. The role of circ_0000673 in CRC was also investigated in a tumor xenograft assay in nude mice. Circ_0000673 expression was increased in CRC tissues and cancer cells. Silencing circ_0000673 reduced tumor cell proliferation, migration and invasion, while also decreasing cell stemness. MiR-548b-3p was found to be a target of circ_0000673, while CPSF6 was a downstream target of miR-548b-3p. The tumor-regulatory effects of si-circ_0000673, anti-miR-548b-3p and oe-CPSF6 were partially reversed by anti-miR-548b-3p, si-CPSF6 and si-circ_0000673, respectively, in rescue assays. Downregulation of circ_0000673 reduced solid tumor growth in vivo. Circ_0000673 inhibition reduced CPSF6 expression by targeting miR-548b-3p, thereby blocking proliferation, migration and invasion of CRC tumor cells.

Biodegradable magnesium based metal materials inhibit the growth of cervical cancer cells

Traditional chemotherapy drugs for cervical cancer often cause significant toxic side effects and drug resistance problems, highlighting the urgent need for more innovative and effective treatment strategies. Magnesium alloy is known to be degradable and biocompatible. The release of degradation products Mg2+, OH−, and H2 from magnesium alloy can alter the tumor microenvironment, providing potential anti-tumor properties. We explored the innovative use of magnesium alloy biomaterials in the treatment of cervical cancer, investigating how various concentrations of Mg2+ on the proliferation and cell death of cervical cancer cells. The results revealed that varying concentrations of Mg2+ significantly inhibited cervical cancer by arresting the cell cycle in the G0/G1 phase and inducing apoptosis in SiHa cells, effectively reducing tumor cell proliferation. In vivo experiments demonstrated that 20 mM Mg2+ group had the smallest tumor volume, exhibiting a potent inhibitory effect on the biological characteristics of cervical cancer. This enhances the therapeutic potential of this biomaterial as a local anti-tumor therapy and lays a theoretical foundation for the potential application of magnesium in the treatment of cervical cancer.

Disrupting glioblastoma networks with tumor treating fields (TTFields) in in vitro models

PurposeThis study investigates the biological effect of Tumor Treating Fields (TTFields) on key drivers of glioblastoma’s malignancy—tumor microtube (TM) formation—and on the function and overall integrity of the tumor cell network.MethodUsing a two-dimensional monoculture GB cell network model (2DTM) of primary glioblastoma cell (GBC) cultures (S24, BG5 or T269), we evaluated the effects of TTFields on cell density, interconnectivity and structural integrity of the tumor network. We also analyzed calcium (Ca2+) transient dynamics and network morphology, validating findings in patient-derived tumoroids and brain tumor organoids.ResultsIn the 2DTM assay, TTFields reduced cell density by 85–88% and disrupted network interconnectivity, particularly in cells with multiple TMs. A “crooked TM” phenotype emerged in 5–6% of treated cells, rarely seen in controls. Ca2+ transients were significantly compromised, with global Ca2+ activity reduced by 51–83%, active and periodic cells by over 50%, and intercellular co-activity by 52% in S24, and almost completely in BG5 GBCs. The effects were more pronounced at 200 kHz compared to a 50 kHz TTFields. Similar reductions in Ca2+ activity were observed in patient-derived tumoroids. In brain tumor organoids, TTFields significantly reduced tumor cell proliferation and infiltration.ConclusionOur comprehensive study provides new insights into the multiple effects of Inovitro-modeled TTFields on glioma progression, morphology and network dynamics in vitro. Future in vivo studies to verify our in vitro findings may provide the basis for a deeper understanding and optimization of TTFields as a therapeutic modality in the treatment of GB.

Molecular Mechanisms of Anticarcinogenic Potential of Hydrocotyle umbellata and Its Major Components

Despite the development of several anticancer treatments, there remains a need for new drugs that can overcome resistance and reduce side effects. While the medicinal herb Hydrocotyle umbellata (H. umbellata) has been used to relieve pain and inflammation, its antitumor properties have not yet been explored. In this study, we investigated the anticarcinogenic potential of H. umbellata extract (HUE) and its major components, as well as the underlying molecular mechanisms. Our results showed that HUE inhibited the growth of various tumor cell lines, including B16F10, without affecting non-cancer cells. Furthermore, HUE was effective in treating and preventing tumor growth in mice. Our mechanistic studies revealed that HUE inhibited cellular respiration, thereby reducing tumor cell proliferation. When combined with 2-deoxy-D-glucose, HUE demonstrated an enhanced anticancer effect by increasing the rate apoptosis. Analysis of the ethyl acetate and n-butanol fractions of HUE identified 1,3,4-trihydroxy-2-butanyl-α-d-glucopyranoside and caffeoylquinic acid derivatives as the major components responsible for the observed anticancer effects. In conclusion, our findings suggest that HUE and its two major components have the potential to be developed as effective therapeutic agents for a wide range of tumors by targeting cancer cell metabolism.

DIPG-35. OVERCOMING THE BLOOD-BRAIN BARRIER CHALLENGE IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMGs) are aggressive paediatric brainstem tumours with no known cure. The development of effective treatments is greatly impeded by the failure of most anti-tumour agents to penetrate the blood-brain barrier (BBB). Our research, along with that of others, has revealed that the BBB remains intact in DMG. We employed DMG orthotopic models to explore potential region-specific variations in response to the therapeutic agent temsirolimus as well as confocal microscopy and single-cell RNA sequencing (scRNAseq) to elucidate the impact of DMGs on brain endothelial morphology and pathways governing BBB integrity. We first assessed the effectiveness of the mTOR inhibitor temsirolimus when administered in cortex-injected DMG models compared to brainstem-injected DMG. We found significantly increased survival rates, elevated temsirolimus levels, and reduced tumour cell proliferation in the cortex versus the brainstem. Confocal imaging analysis indicated no structural differences between DMG and Matrigel-injected animals. However, scRNAseq unveiled distinct transcriptomic changes: brainstem-injected DMG exhibited downregulation of genes related to inflammatory and apoptotic pathways, whereas cortex-injected DMG showed downregulation of interferon pathways. To overcome the tightened barrier in the brainstem, we explored three MCL1 inhibitors and SNGR-TNFa (targeting CD13) as potential blood-brain barrier modulators. These inhibitors significantly reduced transendothelial electrical resistance, increased tracer dye leakage in an in vitro DMG BBB model, and decreased the protein expression of claudin-5. Moreover, in vivo, we found that administration of a single dose of either SNGR-TNFa or the MCL1 inhibitor S63845 improved penetration of Texas-Red (3KDa) in DMG-engrafted animals, indicating the effective opening of the BBB. In summary, our study revealed that DMG directly affects BBB pathways in endothelial cells, tightening the barrier and reducing treatment efficacy. MCL1 inhibitors and SNGR-TNFa show promise in modulating the BBB and have the potential to enhance therapeutic activity.

Dicerandrol C Suppresses Proliferation and Induces Apoptosis of HepG2 and Hela Cancer Cells by Inhibiting Wnt/β-Catenin Signaling Pathway.

Overwhelming evidence points to an aberrant Wnt/β-catenin signaling as a critical factor in hepatocellular carcinoma (HCC) and cervical cancer (CC) pathogenesis. Dicerandrol C (DD-9), a dimeric tetrahydroxanthenone isolated from the endophytic fungus Phomopsis asparagi DHS-48 obtained from mangrove plant Rhizophora mangle via chemical epigenetic manipulation of the culture, has demonstrated effective anti-tumor properties, with an obscure action mechanism. The objective of the current study was to explore the efficacy of DD-9 on HepG2 and HeLa cancer cells and its functional mechanism amid the Wnt/β catenin signaling cascade. Isolation of DD-9 was carried out using various column chromatographic methods, and its structure was elucidated with 1D NMR. The cytotoxicity of DD-9 on HepG2 and HeLa cells was observed with respect to the proliferation, clonality, migration, invasion, apoptosis, cell cycle, and Wnt/β-catenin signaling cascade. We found that DD-9 treatment significantly reduced tumor cell proliferation in dose- and time-dependent manners in HepG2 and HeLa cells. The subsequent experiments in vitro implied that DD-63 could significantly suppress the tumor clonality, metastases, and induced apoptosis, and that it arrested the cell cycle at the G0/G1 phase of HepG2 and HeLa cells. Dual luciferase assay, Western blot, and immunofluorescence assay showed that DD-9 could dose-dependently attenuate the Wnt/β-catenin signaling by inhibiting β-catenin transcriptional activity and abrogating β-catenin translocated to the nucleus; down-regulating the transcription level of β-catenin-stimulated Wnt target gene and the expression of related proteins including p-GSK3-β, β-catenin, LEF1, Axin1, c-Myc, and CyclinD1; and up-regulating GSK3-β expression, which indicates that DD-9 stabilized the β-catenin degradation complex, thereby inducing β-catenin degradation and inactivation of the Wnt/β-catenin pathway. The possible interaction between DD-9 and β-catenin and GSK3-β protein was further confirmed by molecular docking studies. Collectively, DD-9 may suppress proliferation and induce apoptosis of liver and cervical cancer cells, possibly at least in part via GSK3-β-mediated crosstalk with the Wnt/β-catenin signaling axis, providing insights into the mechanism for the potency of DD-9 on hepatocellular and cervical cancer.

PD-L1 regulates tumor proliferation and T-cell function in NF2-associated meningiomas.

Programmed death-ligand 1 (PD-L1) expression is an immune evasion mechanism that has been demonstrated in many tumors and is commonly associated with a poor prognosis. Over the years, anti-PD-L1 agents have gained attention as novel anticancer therapeutics that induce durable tumor regression in numerous malignancies. They may be a new treatment choice for neurofibromatosis type 2 (NF2) patients. The aims of this study were to detect the expression of PD-L1 in NF2-associated meningiomas, explore the effect of PD-L1 downregulation on tumor cell characteristics and T-cell functions, and investigate the possible pathways that regulate PD-L1 expression to further dissect the possible mechanism of immune suppression in NF2 tumors and to provide new treatment options for NF2 patients. PD-L1 is heterogeneously expressed in NF2-associated meningiomas. After PD-L1 knockdown in NF2-associated meningioma cells, tumor cell proliferation was significantly inhibited, and the apoptosis rate was elevated. When T cells were cocultured with siPD-L1-transfected NF2-associated meningioma cells, the expression of CD69 on both CD4+ and CD8+ T cells was partly reversed, and the capacity of CD8+ T cells to kill siPD-L1-transfected tumor cells was partly restored. Results also showed that the PI3K-AKT-mTOR pathway regulates PD-L1 expression, and the mTOR inhibitor rapamycin rapidly and persistently suppresses PD-L1 expression. Invivo experimental results suggested that anti-PD-L1 antibody may have a synergetic effect with the mTOR inhibitor in reducing tumor cell proliferation and that reduced PD-L1 expression could contribute to antitumor efficacy. Targeting PD-L1 could be helpful for restoring the function of tumor-infiltrating lymphocytes and inducing apoptosis to inhibit tumor proliferation in NF2-associated meningiomas. Dissecting the mechanisms of the PD-L1-driven tumorigenesis of NF2-associated meningioma will help to improve our understanding of the mechanisms underlying tumor progression and could facilitate further refinement of current therapies to improve the treatment of NF2 patients.

Exploring the multi-targeting phytoestrogen potential of Calycosin for cancer treatment: A review.

Cancer remains a significant challenge in the field of oncology, with the search for novel and effective treatments ongoing. Calycosin (CA), a phytoestrogen derived from traditional Chinese medicine, has garnered attention as a promising candidate. With its high targeting and low toxicity profile, CA has demonstrated medicinal potential across various diseases, including cancers, inflammation, and cardiovascular disease. Studies have revealed that CA possesses inhibitory effects against a diverse array of cancers. The underlying mechanism of action involves a reduction in tumor cell proliferation, induction of tumor cell apoptosis, and suppression of tumor cell migration and invasion. Furthermore, CA has been shown to enhance the efficacy of certain chemotherapeutic drugs, making it a potential component in treating malignant tumors. Given its high efficacy, low toxicity, and multi-targeting characteristics, CA holds considerable promise as a therapeutic agent for cancer treatment. The objective of this review is to present a synthesis of the current understanding of the antitumor mechanism of CA and its research progress.

Abstract PO5-18-11: Use of DiviTum®TKa as a biomarker assay for CDK4/6 inhibitor medication compliance and drug-drug interaction assessment in ER/PR positive metastatic breast cancer

Abstract Background: CDK4/6 inhibitor (CDK4/6i) with endocrine therapy (ET) is standard first line (1L) treatment for metastatic hormone positive (ER/PR+) HER2- negative breast cancer (mBC). Some patients have progression free survival (PFS) of 2 years or more whereas some patients have very short PFS on these medications. Thymidine kinase is a biomarker that reflects cell proliferation. DiviTum®TKa is an assay that was cleared by the FDA in July 2022 based on data that showed that thymidine kinase activity (TKa) value of < 250 DiviTum units (DuA) is associated with the decreased likelihood of disease progression within 30 days or 60 days post testing. The purpose of this study is to assess the use of TKa as a biomarker for identifying potential medical compliance issues and drug-drug interactions (DDI) in patients on CDK4/6i and to evaluate whether counseling and optimization of concurrent medications will lead to lower TKa levels in subsequent treatment cycles and possibly longer PFS. Trial design: This is a pilot study to assess whether TKa can serve as a biomarker to identify patients with a suboptimal reduction in tumor cell proliferation caused by a potential CDK4/6i compliance or DDI issue. We will also assess whether correction of the issue can improve suppression of TKa levels in ER/PR+ mBC patients on 1L CDK4/6i and ET. For screening, we are utilizing a novel automated real-time data driven cohort identification system designed by Dr. Guannan Gong at Yale. Patients will be treated and monitored as per standard of care. An aliquot of serum obtained during routine blood draws will be sent to a CLIA-certified Biovica lab for TKa testing. TKa results will be returned in real time. Patients with a detectable TKa baseline level (above 145 DuA), followed by a sustained TKa level (>145 DuA) at cycle 1 D15 and D28 will be assessed for medication compliance and DDI. TKa levels will be repeated at subsequent standard of care blood draws to assess if the intervention affected TKa levels. Patients will be followed for 2 years. Eligibility criteria: Participants must have ER+ mBC and starting 1L CDK4/6i + ET and be previously CDK4/6i -naïve. Specific aims: Primary objective: to estimate the rate of improvement in CDK4/6i response (change in TKa levels) after counseling for medication compliance and adjustment of potential deleterious DDIs. Secondary objectives: 1) estimate the rate of sub-optimal CDK 4/6i response in cycle 1 of treatment. 2) Compare clinical benefit rate (CBR) in patients with sub-optimal and optimal CDK4/6i response after both cycles 1 and cycle 3. 3) Compare PFS in patients with sub-optimal and optimal CDK 4/6i response after both cycles 1 and cycle 3. 4) Assess CDK4/6i response via TKa levels upon CDK4/6i dose reductions or changes in CDK4/6i regimens. 5) Compare CDK4/6i response profiles across the three CDK 4/6i among different patients and within the same patients if CDK4/6i is changed throughout treatment course. 6) Correlate TKa levels with tumor marker levels. 7) Assess plasma concentrations of CDK4/6i in patients with suboptimal TKa levels. Statistical methods: For binary endpoints measured at a given time point, we will estimate the two-sided 95% exact CI using Clopper-Pearson method. With a sample size of 120 patients, assuming 20% drop out rate, we predict that about 15% of patients will have suboptimal CDK4/6i response, estimating a 95% CI of 0.086-0.235 in the first cycle. Comparisons between paired outcomes will be completed using the paired t-test or Wilcoxon signed-rank test for continuous parameters of interest, or McNemar’s Chi-square test for categorical parameters of interest. Time to event endpoints will be estimated using Kaplan-Meier method. Present accrual and target accrual: This trial has just been activated. Target accrual is 120 patients. Contact information for people with a specific interest in the trial: Mariya.Rozenblit@yale.edu Citation Format: Mariya Rozenblit, Adriana Kahn, Guannan Gong, Amy Williams, Lajos Pusztai. Use of DiviTum®TKa as a biomarker assay for CDK4/6 inhibitor medication compliance and drug-drug interaction assessment in ER/PR positive metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-18-11.