Inhibition Of Cell Proliferation Research Articles

Dietary Phytoactives in the Management of Gastric Cancer - A Mini Review

Abstract: Gastric cancer is the fourth leading cause of morbidity worldwide and is known to affect various gastric organs, including the esophagus, intestine, colon, and pancreas. There are variable factors responsible for the development of gastric tumors, such as environmental factors, EPV, hepatitis B and C, and other genetic factors. Phytoactives, or secondary metabolites of plants, have a wide range of biological impacts, including the capacity to prevent cancer in humans. They have this anticancerous impact because of their multi-target mechanism of action, which also includes antioxidant, anti-proliferative, cell apoptosis, and anti-mutagenic effects. To epitomize the role of vital dietary phytoactives as a new approach for treating gastric cancer and explains various mechanisms for their anticancerous activity. H. pylori-associated gastric tumors are the most common, and Phytoactives such as curcumin, piperine, walnut oil, tea, and others possess anticancer properties. They have shown anticancer activity against gastric tumor cell lines via different mechanisms like cell cycle arrest, apoptosis, inhibition of cell proliferation, adhesion, colonization of cancer cells, inhibition of inflammatory mediators, tumor cell bioenergetics, up and down-regulation of various gene and protein expression, and chelation and scavenging of ROS.

HHLA2 deficiency inhibits pancreatic cancer progression and THP-1 macrophage M2 polarization via EGFR/MAPK/ERK and mTOR/AKT pathway

BackgroundHuman endogenous retrovirus subfamily H long terminal repeat associating protein 2, (HHLA2), a member of B7 family, exhibits heightened expression in various malignant tumors. However, the exact functions of HHLA2 in pancreatic cancer (PC) remain incompletely elucidated.MethodsWe initially conducted an analysis of the B7 family members’ expression pattern in pancreatic tumor samples and adjacent normal tissues using The Cancer Genome Atlas (TCGA) database. Subsequently, immunohistochemistry, RT-qPCR and western blot methods were used to assess HHLA2 expression levels in PC tissues and cell lines. Furthermore, after silencing HHLA2 in PC cell lines, cell migration and proliferation of PC cells were detected by wound healing and CCK-8 assays, and cell invasion of PC cells was detected by transwell assays. We also investigated the regulation of epithelial-mesenchymal transition (EMT) markers and levels of EGFR, MEK, ERK1/2, mTOR and AKT via western blot analysis. Finally, the correlation between HHLA2 expression and immune infiltration was further explored.ResultsSilencing of HHLA2 resulted in the inhibition of PC cell proliferation, migration and invasion, potentially through the suppression of the EGFR/MAPK/ERK and mTOR/AKT signaling pathway. Additionally, silencing HHLA2 led to the inhibition of M2-type polarization of tumor associated macrophages (TAMs).ConclusionThe knockdown of HHLA2 was observed to inhibit the migration and invasion of PC cells through the regulation of the EMT process and EGFR/MAPK/ERK and mTOR/AKT pathway. Furthermore, silencing HHLA2 was found to modulate M2 polarization of TAMs. These finding suggest that HHLA2 could be a promising therapeutic target for Pancreatic cancer.

An Improved PDE6D Inhibitor Combines with Sildenafil To Inhibit KRAS Mutant Cancer Cell Growth.

The trafficking chaperone PDE6D (or PDEδ) was proposed as a surrogate target for K-Ras, leading to the development of a series of inhibitors that block its prenyl binding pocket. These inhibitors suffered from low solubility and suspected off-target effects, preventing their clinical development. Here, we developed a highly soluble, low nanomolar PDE6D inhibitor (PDE6Di), Deltaflexin3, which has the lowest off-target activity as compared to three prominent reference compounds. Deltaflexin3 reduces Ras signaling and selectively decreases the growth of KRAS mutant and PDE6D-dependent cancer cells. We further show that PKG2-mediated phosphorylation of Ser181 lowers K-Ras binding to PDE6D. Thus, Deltaflexin3 combines with the approved PKG2 activator Sildenafil to more potently inhibit PDE6D/K-Ras binding, cancer cell proliferation, and microtumor growth. As observed previously, inhibition of Ras trafficking, signaling, and cancer cell proliferation remained overall modest. Our results suggest reevaluating PDE6D as a K-Ras surrogate target in cancer.

METTL5 promotes cell proliferation, invasion, and migration by up-regulating Toll-like receptor 8 expression in colorectal cancer.

N6-methyladenosine (m6A) modification represents the predominant alteration found in eukaryotic messenger RNA and plays a crucial role in the progression of various tumors. However, despite its significance, the comprehensive investigation of METTL5, a key m6A methyltransferase, in colorectal cancer (CRC) remains limited. To investigate the role of METTL5 in CRC. We assessed METTL5 expression levels in clinical samples obtained from CRC patients as well as in CRC cell lines. To elucidate the downstream targets of METTL5, we performed RNA-sequencing analysis coupled with correlation analysis, leading us to identify Toll-like receptor 8 (TLR8) as a potential downstream target. In vitro functional assessments of METTL5 and TLR8 were conducted using CCK-8 assays, scratch assays, as well as assays measuring cell migration and invasion. Our findings reveal a pronounced upregulation of METTL5 expression in both CRC cells and tissues, which correlated significantly with an unfavorable prognosis. In vitro experiments unequivocally demonstrated the oncogenic role of METTL5, as evidenced by its promotion of CRC cell proliferation, invasion, and migration. Notably, we identified TLR8 as a downstream target of METTL5, and subsequent down-regulation of TLR8 led to a significant inhibition of CRC cell proliferation, invasion, and tumor growth. The heightened expression of METTL5 in CRC is strongly associated with clinicopathological features and a poor prognosis, thereby underscoring its potential utility as a critical marker for facilitating early diagnosis and prognostication in CRC.

Exploring Melezitose as a Potential Therapeutic Agent in Lung Cancer: Inhibitory Effects on Cell Proliferation and EMT-Mediated Signaling in A549 Cells

Background Lung cancer is one of the most common cancers worldwide and a leading cause of cancer-related deaths. The study delves into melezitose, a naturally occurring compound known for its biocompatibility. Purpose This study aims to uncover its therapeutic potential and molecular mechanisms within lung cancer, particularly in A549 cells. Melezitose’s impact on inhibiting cell proliferation and influencing epithelial-mesenchymal transitions (EMT) was the primary focus. Material and Methods In a time-dependent manner, A549 cells, representative of lung adenocarcinoma (LUAD), underwent melezitose treatment. Analysis of cytotoxicity by MTT assay, cell migration assay, and its responsible genes were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) kit method. Results The MTT assay indicated a significant reduction in A549 cell growth after 48 hours of melezitose treatment. Additionally, melezitose induced G1 phase cell arrest and stimulated apoptosis in A549 cells. Subsequent determination of IC50 values represents the concentration at which melezitose inhibits 50% of cell growth. The study also investigated EMT-related gene expression like claudin 1 (CLD1), E-cadherin (ECADH), SNAIL1, SLUG, and vimentin (VIM) through RT-PCR. The findings revealed strong binding associations between melezitose and these EMT targets, suggesting a potential regulatory role of melezitose in impeding EMT processes. Conclusion Overall, this study illuminates the significant role of melezitose in lung cancer. Its observed inhibition of lung cancer cell proliferation and its influential impact on EMT-related gene expression highlights. Melezitose has potential as a therapeutic agent, particularly in the context of NSCC. The multifaceted effects of melezitose on A549 cells open promising avenues for advancing our understanding of this disease and developing innovative therapeutic strategies.

Therapeutic antibody engineering for efficient targeted degradation of membrane proteins in lysosomes

Targeted degradation of pathological proteins is a promising approach to enhance the effectiveness of therapeutic monoclonal antibodies (mAbs) in cancer therapy. In this study, we demonstrate that this objective can be efficiently achieved by the grafting of mannose 6-phosphate analogues called AMFAs22AMFA: Analogues of the Mannose 6-phosphate Functionalized at Anomeric Position onto the therapeutic antibodies trastuzumab and cetuximab, both directed against membrane antigens. The grafting of AMFAs confers to these antibodies the novel property of being internalized via the mannose 6-phosphate receptor (M6PR) pathway. AMFA conjugation to these mAbs significantly increases their cellular uptake and leads to enhanced degradation of the target antigens in cancer cells. This results in a drastic inhibition of cancer cell proliferation compared to unconjugated mAbs, as demonstrated in various cancer cell lines, and an increased therapeutic efficacy in mouse and zebrafish xenografted models. These findings highlight the potential of this technology to improve therapeutic outcomes in cancer treatment.

A β-Carboline Derivate PAD4 Inhibitor Reshapes Neutrophil Phenotype and Improves the Tumor Immune Microenvironment against Triple-Negative Breast Cancer.

Triple-negative breast cancer is a highly aggressive and heterogeneous breast cancer subtype characterized by early metastasis, poor prognosis, and high recurrence. Targeting histone citrullination-mediated chromatin dysregulation to induce epigenetic alterations shows great promise in TNBC therapy. We report the synthesis, optimization, and evaluation of a novel series of β-carboline-derived peptidyl arginine deiminase 4 inhibitors that exhibited potent inhibition of TNBC cell proliferation. The most outstanding PAD4 inhibitor, compound 28, hindered the PAD4-H3cit-NET signaling pathway and inhibited the growth of solid tumors and pulmonary metastatic nodules in the 4T1 in situ mouse model. Furthermore, 28 improved the tumor immune microenvironment by reshaping neutrophil phenotype, upregulating the proportions of dendritic cells and M1 macrophages, and reducing the amount of myeloid-derived suppressor cells. In conclusion, our work offered 28 as an efficacious PAD4 inhibitor that exerts a combination of conventional chemotherapy and immune-boosting effects, which represents a potential therapy strategy for TNBC.

Phytochemical profiling, cytotoxic, anti-migration, and anti-angiogenic potential of phenolic-rich fraction from Peganum harmala: in vitro and in ovo studies.

Peganum harmala has been extensively employed in Algerian traditional medicine practices. This study aimed to explore the impact of n-butanol (n-BuOH) extract sourced from Peganum harmala seeds on cell proliferation, cell migration, and angiogenesis inhibition. Cytotoxic potential of n-BuOH extract was evaluated using MTT (3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide) assay against human breast adenocarcinoma MCF-7 cells, cell migration was determined using scratch assay, and anti-angiogenic effect was evaluated through macroscopic and histological examinations conducted on chick embryo chorioallantoic membrane. Additionally, this research estimated the phytochemical profile of n-BuOH extract. Fifteen phenolic compounds were identified using Ultra-performance liquid chromatography UPLC-ESI-MS-MS analysis. In addition, the n-BuOH extract of P. harmala exhibited potent antioxidant and free radical scavenging properties. The n-BuOH extract showed potent cytotoxicity against MCF-7 cell with an IC50 value of 8.68 ± 1.58 μg/mL. Furthermore, n-BuOH extract significantly reduced migration. A strong anti-angiogenic activity was observed in the groups treated with n-BuOH extract in comparison to the negative control. Histological analysis confirmed the anti-angiogenic effect of the n-BuOH extract. This activity is probably a result of the synergistic effects produced by different polyphenolic classes.

Antibody and siRNA Nanocarriers to Suppress Wnt Signaling, Tumor Growth, and Lung Metastasis in Triple‐Negative Breast Cancer

AbstractThe paucity of targeted therapies for triple‐negative breast cancer (TNBC) causes patients with this aggressive disease to suffer a poor clinical prognosis. A promising target for therapeutic intervention is the Wnt signaling pathway, which is activated in TNBC cells when extracellular Wnt ligands bind overexpressed Frizzled7 (FZD7) transmembrane receptors. This stabilizes intracellular β‐catenin proteins that in turn promote transcription of oncogenes that drive tumor growth and metastasis. To suppress Wnt signaling in TNBC cells, this work develops therapeutic nanoparticles (NPs) functionalized with FZD7 antibodies and β‐catenin small interfering RNAs (siRNAs). The antibodies enable TNBC cell specific binding and inhibit Wnt signaling by locking FZD7 receptors in a ligand unresponsive state, while the siRNAs suppress β‐catenin through RNA interference. Compared to NPs coated with antibodies or siRNAs individually, NPs coated with both agents more potently reduce the expression of several Wnt related genes in TNBC cells, leading to greater inhibition of cell proliferation, migration, and spheroid formation. In two murine models of metastatic TNBC, the dual antibody/siRNA nanocarriers outperformed controls in terms of inhibiting tumor growth, metastasis, and recurrence. These findings demonstrate suppressing Wnt signaling at both the receptor and mRNA levels via antibody/siRNA nanocarriers is a promising approach to combat TNBC.

MicroRNA-150-5p-mediated Inhibition of Cell Proliferation, G1/S Transition, and Migration in Bladder Cancer through Targeting NEDD4-binding Protein 2-like 1 Gene

MicroRNA-150-5p-mediated Inhibition of Cell Proliferation, G1/S Transition, and Migration in Bladder Cancer through Targeting NEDD4-binding Protein 2-like 1 Gene

WDR61 ablation triggers R-loop accumulation and suppresses breast cancer progression.

Although, superkiller complex protein 8 (SKI8), previously known as WDR61 has been identified and mapped in breast tumor, little is currently known about its function. This study aims to elucidate the role of WDR61 in breast tumor development and its potential as a therapeutic target. Here, we show that tamoxifen-induced knockout of Wdr61 reduces the risk of breast tumors, resulting in smaller tumor size and weight, and improved overall survival. Furthermore, we show that knockdown of WDR61 compromises the proliferation of breast tumor cells with reduced colony-forming capacity. Further investigations demonstrate that the protective effect of WDR61 loss on breast tumor development is due to genomic instability. Mechanistic studies reveal that WDR61 interacts with the R-loop, and loss of WDR61 leads to R-loops accumulation in breast tumor cells, causing DNA damage and subsequent inhibition of cell proliferation. In summary, this study highlights the critical dependence of breast tumors on WDR61, which suppresses R-loop and counteracts endogenous DNA damage in tumor cells.

Muscone restores anoikis sensitivity in TMZ-resistant glioblastoma cells by suppressing TOP2A via the EGFR/Integrin β1/FAK signaling pathway

BackgroundTemozolomide (TMZ) resistance is the main obstacle faced by glioblastoma multiforme (GBM) treatment. Muscone, one of the primary active pharmacological ingredients of Shexiang (Moschus), can cross the blood-brain barrier (BBB) and is being investigated as an antineoplastic medication. However, muscone treatment for GBM has received little research, and its possible mechanisms are still unclear. PurposeThis study aims to evaluate the effect and the potential molecular mechanism of muscone on TMZ-resistant GBM cells. MethodsThe differentially expressed genes (DEGs) between TMZ-resistant GBM cells and TMZ-sensitive GBM cells were screened using GEO2R. By progressively raising the TMZ concentration, a relatively stable TMZ-resistant human GBM cell line was established. The drug-resistance traits of U251-TR cells were assessed via the CCK-8 assay and Western Blot analysis of MGMT and TOP2A expression. Cell viability, cell proliferation, cell migration ability, and drug synergism were detected by the CCK-8 assay, colony formation assay, wound healing assay, and drug interaction relationship test, respectively. Anoikis was quantified by Calcein-AM/EthD-1 staining, MTT assay, and flow cytometry. Measurements of cell cycle arrest, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were performed using cell cycle staining, Annexin V-FITC/PI labeling, JC-1 assay, and ROS assay, respectively. DNA damage was measured by TUNEL assay, alkaline comet assay, and γ-H2AX foci assay. GEPIA was used to investigate the link between the anoikis marker (FAK)/drug resistance gene and critical proteins in the EGFR/Integrin β1 signaling pathway. Molecular docking was used to anticipate the probable targets of muscone. The intracellular co-localization and expression of EGFR and FAK were shown using immunofluorescence. The U251-TR cell line stably overexpressing EGFR was constructed using lentiviral transduction to assess the involvement of EGFR-related signaling in anoikis resistance. Western Blot was employed to detect the expression of migration-related proteins, cyclins, anoikis-related proteins, DNA damage/repair-related proteins, and associated pathway proteins. ResultsDEGs analysis identified 97 deregulated chemotherapy-resistant genes and 3779 upregulated genes in TMZ-resistant GBM cells. Subsequent experiments verified TMZ resistance and the hyper-expression of DNA repair-related genes (TOP2A and MGMT) in continuously low-dose TMZ-induced U251-TR cells. Muscone exhibited dose-dependent inhibition of U251-TR cell migration and proliferation, and its co-administration with TMZ showed the potential for enhanced therapeutic efficacy. By downregulating FAK, muscone reduced anoikis resistance in anchorage-independent U251-TR cells. It also caused cell cycle arrest in the G2/M phase by upregulating p21 and downregulating CDK1, CDK2, and Cyclin E1. Muscone-induced anoikis was accompanied by mitochondrial membrane potential collapse, ROS production, an increase in the BAX/Bcl-2 ratio, as well as elevated levels of Cytochrome c (Cyt c), cleaved caspase-9, and cleaved caspase-3. These findings indicated that muscone might trigger mitochondrial-dependent anoikis via ROS generation. Moreover, significant DNA damage, DNA double-strand breaks (DSBs), the formation of γ-H2AX foci, and a reduction in TOP2A expression are also associated with muscone-induced anoikis. Overexpression of EGFR in U251-TR cells boosted the expression of Integrin β1, FAK, β-Catenin, and TOP2A, whereas muscone suppressed the expression levels of EGFR, Integrin β1, β-Catenin, FAK, and TOP2A. Muscone may influence the expression of the key DNA repair enzyme, TOP2A, by suppressing the EGFR/Integrin β1/FAK pathway. ConclusionWe first demonstrated that muscone suppressed TOP2A expression through the EGFR/Integrin β1/FAK pathway, hence restoring anoikis sensitivity in TMZ-resistant GBM cells. These data suggest that muscone may be a promising co-therapeutic agent for enhancing GBM treatment, particularly in cases of TMZ-resistant GBM with elevated TOP2A expression.

A dual gene combination therapy approach by KRAS-G12V gene editing and SLC25A22 gene silencing for KRAS-mutant colorectal cancer

KRAS gene is mutated in 40% of colorectal cancers (CRC), which induces malignant proliferation by regulating cellular nutrient metabolism and biosynthesis. It has been found that malignant proliferation of KRAS-mutant colorectal cancer relies on the upregulation of SLC25A22 protein expression, suggesting that inhibition the expression of both KRAS and SLC25A22 is a potential CRC therapeutics. Stably knocking down the oncogenic KRAS-G12V gene can achieve long-term gene therapy effects, while transient downregulation of SLC25A22, a normal functional gene most of the time, is preferred to kill tumor cells and minimize the side impact on normal cells. Here, two lipid nanoparticles (LNP) were designed to encapsulate KRAS-G12V CRISPR/Cas9 gene editing plasmids (pKRAS-LNPs) and SLC25A22 siRNA (siSLC-LNPs), respectively. Therapeutic effects of both nanoparticles alone and in combination on KRAS-G12V mutant colorectal cancer cells in vitro were first examined. The result showed that delivery of pKRAS-LNPs or siSLC-LNPs alone could effectively achieve KRAS-G12V gene editing or SLC25A22 gene silencing and inhibit tumor cell proliferation, while co-delivery of both LNPs could achieve stronger inhibition of tumor cell proliferation by inducing stronger apoptosis. Furthermore, we found that co-delivery of pKRAS-LNPs and siSLC-LNPs induced stronger apoptosis and cell proliferation inhibition compared to pKRAS&siSLC-LNPs that were constructed by pre-mixing pKRAS and siSLC and then encapsulating them. Finally, we validated that co-delivery of pKRAS-LNPs and siSLC-LNPs can achieve KRAS-G12V colorectal cancer treatment in vivo with a tumor inhibition rate of 61.15%. In summary, the delivery vectors constructed for nucleic acids targeting KRAS and SLC25A22 achieved therapeutic targeting of KRAS-G12V colorectal cancer in vitro and in vivo.

Verification of In Vitro Anticancer Activity and Bioactive Compounds in Cordyceps Militaris-Infused Sweet Potato Shochu Spirits.

Many liqueurs, including spirits infused with botanicals, are crafted not only for their taste and flavor but also for potential medicinal benefits. However, the scientific evidence supporting their medicinal effects remains limited. This study aims to verify in vitro anticancer activity and bioactive compounds in shochu spirits infused with Cordyceps militaris, a Chinese medicine. The results revealed that a bioactive fraction was eluted from the spirit extract with 40% ethanol. The infusion time impacted the inhibitory effect of the spirit extract on the proliferation of colon cancer-derived cell line HCT-116 cells, and a 21-day infusion showed the strongest inhibitory effect. Furthermore, the spirit extract was separated into four fractions, A-D, by high-performance liquid chromatography (HPLC), and Fractions B, C, and D, but not A, exerted the effects of proliferation inhibition and apoptotic induction of HCT-116 cells and HL-60 cells. Furthermore, Fractions B, C, and D were, respectively, identified as adenosine, cordycepin, and N6-(2-hydroxyethyl)-adenosine (HEA) by comprehensive chemical analyses, including proton nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FT-IR), and electrospray ionization mass spectrometry (ESI-MS). To better understand the bioactivity mechanisms of cordycepin and HEA, the agonist and antagonist tests of the A3 adenosine receptor (A3AR) were performed. Cell viability was suppressed by cordycepin, and HEA was restored by the A3AR antagonist MR1523, suggesting that cordycepin and HEA possibly acted as agonists to activate A3ARs to inhibit cell proliferation. Molecular docking simulations revealed that both adenosine and cordycepin bound to the same pocket site of A3ARs, while HEA exhibited a different binding pattern, supporting a possible explanation for the difference in their bioactivity. Taken together, the present study demonstrated that cordycepin and HEA were major bioactive ingredients in Cordyceps militaries-infused sweet potato shochu spirits, which contributed to the in vitro anticancer activity.

LncRNA IL21-AS1 facilitates tumour progression by enhancing CD24-induced phagocytosis inhibition and tumorigenesis in ovarian cancer

CD24 is overexpressed in various tumours and considered a regulator of cell migration, invasion, and proliferation. Recent studies have found that CD24 on ovarian cancer (OC) and triple-negative breast cancer cells interacts with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10) on tumour-associated macrophages (TAMs) to inhibit phagocytosis by macrophages. Because of its multiple roles in regulating the immune response and tumorigenesis, CD24 is a very promising therapeutic target. However, the regulatory mechanism of CD24 in OC remains unclear. Here, we found that the long noncoding RNA (lncRNA) IL21-AS1, which was upregulated in OC, inhibited macrophage-mediated phagocytosis and promoted OC cell proliferation and apoptosis inhibition. More importantly, after IL21-AS1 knockdown, a significant survival advantage was observed in mice engrafted with tumours. Mechanistically, we identified IL21-AS1 as a hypoxia-induced lncRNA. Moreover, IL21-AS1 increased HIF1α-induced CD24 expression under hypoxic conditions. In parallel, we found that IL21-AS1 acted as a competing endogenous RNA (ceRNA) for miR-561-5p to regulate CD24 expression. Finally, IL21-AS1 increased CD24 expression in OC and facilitated OC progression. Our findings provide a molecular basis for the regulation of CD24, thus highlighting a potential strategy for targeted treatment of OC.

Quality by design empowered preparation of itraconazole albumin nanoparticles for prostate cancer.

The current advent explores the potential of itraconazole (ITR) in prostate cancer (PCa), by its incorporation into albumin nanoparticles (NP). ITR as a repurposed moiety has displayed tremendous potential in various cancers. However, poor aqueous solubility poses hurdles towards its clinical translation. Amorphisation of ITR was observed post-incorporation within NP matrix which could prevent its precipitation in aqueous media. ITR NP was developed using quality by design and multivariate analysis and evaluated for cellular uptake, cell proliferation inhibition and the mechanism of PCa cell inhibition. Time and concentration-dependent serum stability and hemolytic potential revealed safety of ITR NP. Morphological changes and nuclear staining studies revealed the efficacy of ITR and ITR NP in promoting growth inhibition of PC-3 cells. Superior qualitative and quantitative uptake, reactive oxygen species (ROS) and mitochondrial impairment for ITR NP in comparison with ITR and control group was observed. Cell cycle study revealed remarkable G2/M phase inhibition in PC-3 cells. ITR NP demonstrated superior anticancer potential in 3D tumoroids mimicking the micro-metastatic lesions compared to control and ITR. Hence, ITR NP can be a favorable alternative therapeutic alternative in PCa.

Abstract PO5-05-02: Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer

Abstract CDK4/6 inhibitors (CDK4/6i) such as palbociclib, abemaciclib and ribociclib are used to treat HR+/HER2- breast cancer, but patients can develop resistance via many mechanisms, several of which converge on the upregulation of cyclin D-CDK4/6 signaling node. This has been shown to limit the effectiveness of CDK4/6i in ER+ breast cancer with up to 20% patients exhibiting innate resistance and up to 70% patients developing acquired resistance after 3 years on therapy (Scheidemann, 2021). To address acquired resistance, we sought a degrader approach. We utilized our PRODEGY platform of Cereblon (CRBN) binders to synthesize CRBN mediated CDK4/6 bifunctional degraders and identified BTX-9341 as a development candidate due to its potency in vitro, in CDK4/6i resistant models and its effectiveness at inhibiting tumor growth in vivo. Degradation of CDK4/6 by immunoblot analysis of the triple negative breast cancer (TNBC) cell line, MDA-MB-231, treated with BTX-9341 for 6 hours showed up to 85% degradation of CDK4 and CDK6 with DC50s of < 1nM. Similar degradation depth and DC50 values were seen in HR+ breast cancer cells including T47D, MCF7 and ZR-751. CDK4/6 phosphorylates the protein RB which releases the transcription factor E2F, inducing the expression of genes which promote cell cycle progression. To determine the effect of CDK4/6 degradation on downstream signaling, we examined RB phosphorylation by in-cell western. Twenty-four hours treatment with BTX-9341 blocked RB phosphorylation robustly, with phospho-RB IC50s at < 30nM in HR+ breast cancer cells as well as MDA-MB-231 cells. Cell cycle analysis by staining with propidium iodide after 24 hours of treatment with BTX-9341 caused G0/G1 cell cycle arrest at concentrations as low as 10nM. We used a 2D colony formation assay (CFA) as a readout for inhibition of proliferation by cell cycle arrest. BTX-9341 showed potent inhibition of cell proliferation with CFA IC50s of < 50nM in TNBC cell lines and < 20nM in HR+ cell lines. We demonstrated that BTX-9341 was significantly more potent in vitro than the CDK4/6i, which had CFA values between 50 and 700nM in HR+ cell lines and between 100 and 1000nM in TNBC. This increased activity was due to CRBN mediated target degradation, as demonstrated by a shift in CFA IC50 values in a CRBN knockout cell line towards the values seen with the inhibitors. In palbociclib-resistant HR+/HER2- cell lines models which exhibit high CDK6 expression, BTX-9341 maintained a low CFA IC50 (< 150nM) while other CDK4/6 inhibitors displayed micromolar CFA IC50s. BTX-9341 displays excellent pharmacokinetic properties and oral bioavailability, which allowed for oral dosing in xenograft studies. MCF7 xenograft results with BTX-9341 showed dose-dependent tumor growth inhibition, tumor regression at higher doses and overall greater potency compared to the clinical CDK4/6i. BTX-9341 shows good exposure in the brain and exhibits tumor growth inhibition in an intracranial MCF-7 xenograft model, indicating that BTX-9341 could inhibit the growth of brain metastases. These results show that BTX-9341 displays excellent single agent activity in vitro and in vivo particularly in comparison to clinically approved CDK4/6i and that this activity is maintained in CDK4/6i resistant models. This indicates that a degrader approach to targeting this pathway may be more effective than current therapies, and that using this modality in a post CDK4/6i setting may be more effective than switching CDK4/6 inhibitors. Reference: Scheidemann, Erin R, and Ayesha N Shajahan-Haq. “Resistance to CDK4/6 Inhibitors in Estrogen Receptor-Positive Breast Cancer.” International journal of molecular sciences vol. 22,22 12292. 14 Nov. 2021, doi:10.3390/ijms222212292 Citation Format: Hannah Majeski, Akinori Okano, Kirti Chahal, Angela Pasis, Casey Carlson, Arvind Shakya, Qiao Liu, Shenlin Huang, Aparajita Hoskote Chourasia, Leah Fung. Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- 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 PO5-05-02.

Abstract PO5-24-01: Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models

Abstract Despite improvement of outcomes for HR+, HER2- metastatic breast cancer by cyclin dependent kinase 4 and 6 (CDK4/6) inhibitors, safety concerns and disease progression raise a critical need to identify novel approaches. Narazaciclib (ON123300), a novel CDK4/6i, designed to enhance efficacy and safety by its multi-targeted kinase inhibitor activity. Narazaciclib is in Ph I trials; NCT04739293 and CXHL1900340; studying different administration regimens and in endometrial cancer in combination with letrozole (NCT05705505). Our study explored the activity of narazaciclib and its metabolite ON1232580 in comparisons to the FDA approved CDK4/6i and identify additional targets engaged by narazaciclib. To identify direct and secondary targets engaged by narazaciclib and palbociclib, proteome wide Cellular Thermal Shift Assay (CETSA) and Integrative Inferred Kinase Activity (INKA) analysis were performed. Both CETSA and INKA analysis identified additional targets engaged by narazaciclib compared to palbociclib in both MDA-MB-231 lysates and intact cells such as BUB1, CHEK1, AURKA, GSK3α and GSK3β. In TNBC patients with BUB1 overexpression, bioinformatics analysis indicates a low survival correlation. Molecular docking data showed a higher affinity of narazaciclib with BUB1 compared to palbociclib and abemaciclib. The differential activity of narazaciclib was explored by using cohorts of human and mouse breast cancer cell lines and cell based assays. Comparison of narazaciclib’s in vitro IC50 profile to other CDK4/6i was studied against a panel of 370 kinases (HotSpot) and Kd values were determined by KINOMEscan. Intracellular IC50 kinase values were determined by NanoBret technology. Narazaciclib and abemaciclib were found to be the most promiscuous in vitro kinase inhibitors and ribociclib the most specific. Abemaciclib and narazaciclib had similar profiles against the CDK family members. Kd values of CDK4/cyclinD1 binding show similar trends; abemaciclib (0.08 nM), narazaciclib (0.18 nM), palbociclib (0.75 nM) and ribociclib (1.3 nM). Although narazaciclib displayed nM IC50 values in the in vitro assays against many CDKs, narazaciclib was very specific in cellular kinase assays with highest activity against CDK4/6, CSF1R and NUAK1. A stronger inhibition of cell proliferation, as well as induction of apoptosis and senescence were detected in narazaciclib and its metabolite ON1232580 treated PYMT cells, a murine mammary carcinoma model which recapitulates luminal BC subtype, compared to the other CDK4/6i. Both narazaciclib and ON1232580 enhanced CCL5 and CXCL10 mRNA levels, while a higher reduction of PD-L1 and pRb protein levels and a promoting effect on the H2D1 and B2M mRNA levels was shown in narazaciclib and its metabolite treated PYMT cells. Narazaciclib showed significant synergy with anti-PD1 in the EMT-6 syngeneic breast cancer model. A differential effect of narazaciclib on the expression levels of 62 cytokines was detected compared to abemaciclib and palbociclib. Lastly, inhibition of autophagy sensitizes cancer cells to both ON123300 and ON1232580, and induces irreversible cell proliferation inhibition, providing a novel therapeutic approach. Our findings identify important differences generated from assay models studying kinase inhibition, binding and pathway engagement. Understanding the role of the differential targets engaged by narazaciclib, the potential enhanced antitumor immunity and the sensitization by autophagy inhibitors to cell death, will guide future clinical studies. Citation Format: Petros Kechagioglou, Debomita Chakraborty, Camille Dupont, Hajime Yurugi, Ute Distler, Stefan Tenzer, Alexey Chernobrovkin, Kristina Riegel, Julianne Mooz, Mahil Lamber, Volker Dötsch, Stephen Cosenza, Steven Fruchtman, Krishnaraj Rajalingam. Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models [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 PO5-24-01.

Comprehensive analysis of ATP6V1s family member, ATP6V1C2, with prognostic and drug development values in colorectal cancer

Member of the V-type ATPase family have attracted vast attention in tumor progression. Nevertheless, the specific member of V-ATPase, ATP6V1C2, its regulatory function in colorectal cancer (CRC) progression was poorly understood. In this study, comprehensive analyses demonstrated the role of ATP6V1C2 in CRC progression and drug screening based on ATP6V1C2 was carried out. As a result, among the ATPV1s family, ATP6V1C2 was significantly highly expressed in CRC. Immuno-infiltration analysis suggests that, the interaction between CRC cells and immune cells resulting in reduced immune and estimate scores. GSEA analysis found that, ATP6V1C2 negatively correlates with immune cells，especially CD8T cells. Next, Ecotyper database queries indicated that ATP6V1C2 was negatively correlates with characteristic gene expression in CD8T cells. Then, COX regression analysis and survival curves made it clear that ATP6V1C2 is positively correlates with clinicopathological progression leading to poor CRC prognosis. CellMiner explore told us LOR-253 and Sonidegib may be effective in CRC cancer treatment. Molecular Docking between ATP6V1C2 and 9 first-line and 9 natural drugs showed that ATP6V1C2 was recognized by the best geometrical and energetic matching pattern of 2 First-line and 4 natural drugs. RT-PCR and immunoblotting confirmed that ATP6V1C2 was significantly overexpressed in CRC. Four natural drugs screened by molecular docking were effective in cell proliferation inhibition by CCK8 test. In summary, ATP6V1C2 may be a new therapeutic target for CRC. The illustration is shown in Figure 9.

Overexpression of Fatty Acid Synthase Upregulates Glutamine-Fructose-6-Phosphate Transaminase 1 and O-Linked N-Acetylglucosamine Transferase to Increase O-GlcNAc Protein Glycosylation and Promote Colorectal Cancer Growth.

Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly upregulated in CRC, and therapeutic approaches of targeting this enzyme are currently being tested in multiple clinical trials. However, the mechanisms behind the pro-oncogenic action of FASN are still not completely understood. Here, for the first time, we show that overexpression of FASN increases the expression of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) and O-linked N-acetylglucosamine transferase (OGT), enzymes involved in hexosamine metabolism, and the level of O-GlcNAcylation in vitro and in vivo. Consistently, expression of FASN significantly correlates with expression of GFPT1 and OGT in human CRC tissues. shRNA-mediated downregulation of GFPT1 and OGT inhibits cellular proliferation and the level of protein O-GlcNAcylation in vitro, and knockdown of GFPT1 leads to a significant decrease in tumor growth and metastasis in vivo. Pharmacological inhibition of GFPT1 and OGT leads to significant inhibition of cellular proliferation and colony formation in CRC cells. In summary, our results show that overexpression of FASN increases the expression of GFPT1 and OGT as well as the level of protein O-GlcNAcylation to promote progression of CRC; targeting the hexosamine biosynthesis pathway could be a therapeutic approach for this disease.