Ras Signaling Research Articles

Differential expression of miR-21, miR-222, and Let-7b in serum and follicular fluid of polycystic ovary syndrome: Correlation with biochemical parameters

BackgroundPolycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age. Recently, it was discovered that microRNAs (miRNAs), which circulate freely in the circulation or biofluids, have diagnostic or prognostic potential. The current study looked at the levels of expression of three miRNAs in the serum and follicular fluid (FF) of PCOS patients and healthy women. MethodWe have shortlisted the most studied miRNAs—miR-21, miR-222, and Let-7b—from the previous literature, followed by individual quantification using real-time polymerase chain reaction (PCR). ResultsThe present study results showed differences in the expression of miR-21, miR-222, and Let-7b in PCOS patients compared to controls. The area under the receiver operating characteristic (ROC) area under the curve (AUC) for miR-21, miR-222, and Let-7b was 0.70, 0.65, and 0.75 in serum and 0.55, 0.66, and 0.59 in FFs, respectively. The combined ROC of the three miRNAs showed an AUC 0.86 for serum and 0.62 for FF samples. miR-21 showed a positive correlation with body mass index (BMI), and luteinizing hormone (LH) level; miR-222 showed a positive correlation with LH, fasting blood sugar (FBS), and 2-h plasma glucose (PG); while anti-mullerian hormone (AMH), age, erythrocyte sedimentation rate (ESR) and 2-h PG were found to be negatively correlated with Let-b expression. Regression analyses showed a negative association of miR-21 with menarche, progesterone, menses, and antral follicle count (AFC), while serum miR-222 was associated with BMI and age. Let 7-b had a negative association with serum, oocytes, and the LH/FSH ratio. Bioinformatics indicates their predicted targets are potentially involved in microRNA in cancer, diabetes, the P53 signaling pathway, prolactin signaling, the Hippo signaling pathway, Ras signaling, and pathways in cancer. ConclusionThe study found that serum Let-7b, miR-222, and miR-21 were differentially expressed in PCOS patients, suggesting potential involvement in the pathogenesis or progression of PCOS.

Exploring the therapeutic mechanism of curcumin in prostate cancer using network pharmacology and molecular docking

ObjectiveCurcumin, a phenolic compound extracted from turmeric rhizomes, exhibits antitumour effects in preclinical models of tumours. However, its mechanism of action in prostate cancer remains unclear. Exploring the molecular mechanisms of curcumin in prostate cancer based on network pharmacology and molecular docking provides a new theoretical basis for prostate cancer treatment. MethodUsing tools such as PharmMapper, SuperPred, TargetNet, and SwissTargetPrediction, we obtained information on curcumin-related targets. We comprehensively collected prostate cancer-related targets from several databases, including GeneCards, CTD, DisGeNET, OMIM, and PharmGKB. Cross-cutting drug-disease targets were then derived by screening using the Venny 2.1.0 tool. Subsequently, we used the DAVID platform to perform in-depth GO and KEGG enrichment analyses of the drug-disease-shared targets. To construct a PPI network map of the cross-targets and screen the 10 core targets, we combined the STRING database and Cytoscape 3.7.2. Molecular docking experiments were performed using AutoDockTools 1.5.7 software. Finally, we used several databases such as GEPIA, HPA, cBioPortal, and TIMER to further analyse the screened core targets in detail. ResultWe identified 307 key targets of curcumin in cancer treatment. After GO functional enrichment analysis, we obtained 1119 relevant entries, including 782 biological progression (BP) entries, 112 cellular component (CC) entries, and 225 molecular function (MF) entries. In addition, KEGG pathway enrichment analysis revealed 126 signalling pathways, which were mainly involved in the cancer pathway, such as lipid and atherosclerosis pathway, PI3K-Akt signal pathway, MAPK signal pathway, Ras signal pathways, and chemical carcinogenesis-reactive oxygen species. By applying Cytoscape 3.7.2 software, we identified SRC, PIK3R1, STAT3, AKT1, HSP90AA1, ESR1, EGFR, HSP90AB1, MAPK8, and MAPK1 as core targets. Molecular docking experiments showed that the binding energies of curcumin to these core targets were all below −1.85 kJ mol−1, which fully demonstrated that curcumin could spontaneously bind to these core targets. Finally, these results were validated at multiple levels, including mRNA expression, protein expression, and immune infiltration. ConclusionThrough in-depth network pharmacology and molecular docking studies, we have found that curcumin may have anticancer potential by upregulating the expression of PIK3R1 and STAT3, and downregulating the binding ability of molecules such as SRC, AKT1, HSP90AA1, ESR1, EGFR, HSP90AB1, MAPK8, and MAPK1. In addition, curcumin may interfere with the cyclic process of prostate cancer cells by inhibiting key signalling pathways such as the PI3K-Akt signalling pathway, MAPK signalling pathway, and Ras, thereby inhibiting their growth. This study not only reveals the potential molecular mechanism of curcumin in the treatment of prostate cancer but also provides an important theoretical basis for subsequent research.

Hsa-miR-342-3p and hsa-miR-360 may be the key molecules that promote periodontitis in type 2 diabetes mellitus

BackgroundPeriodontitis (PD) has been acknowledged as a complication associated with type 2 diabetes mellitus (T2DM). However, the precise mechanism through which T2DM fosters the development of PD remains elusive. Our objective is to elucidate the connection between these two conditions by conducting bioinformatics analysis. MethodsIn this study, we analyzed miRNA datasets pertaining to T2DM and PD sourced from GEO. Through differential expression analysis, we identified common differentially expressed miRNAs (DE-miRNAs) and subsequently analyzed the functional enrichment of these common DE-miRNAs. We further leveraged the PD transcriptome database to select DE-miRNA-targeted mRNAs and examined their association with immune infiltration. Finally, machine learning was used to further screen hub DE-miRNA-targeted mRNAs and validate our data in external datasets. ResultsTwo common DE-miRNAs, namely hsa-miR-342-3p and hsa-miR-360, were identified from the miRNA datasets of PD and T2DM. Functional enrichment analysis indicated that these two common DE-miRNAs predominantly participate in Ras, PI3K-Akt, p53, and MAPK signaling pathways. Integration of the PD transcriptome dataset revealed a total of 21 DE-miRNA-targeted mRNAs in PD, with strong correlations observed with plasma cells and dendritic cells. Finally, three hub DE-miRNA-targeted mRNAs (hsa-miR-342-3p-/hsa-miR-360-RASAL2, hsa-miR-360-ENTPD1/PLXDC2) were identified. ENTPD1 exhibited a robust positive correlation with plasma cells and a negative correlation with resting dendritic cells. ConclusionsTherefore, hsa-miR-342-3p-/hsa-miR-360-RASAL2, as well as hsa-miR-360-ENTPD1/PLXDC2, may serve as diagnostic and therapeutic targets for T2DM-associated PD.

Modulation of Ras signaling pathway by exosome miRNAs in T-2 toxin-induced chondrocyte injury

This study aims to investigate the impact of T-2 toxin on the regulation of downstream target genes and signaling pathways through exosome-released miRNA in the development of cartilage damage in Kashin-Beck disease (KBD). Serum samples from KBD patients and supernatant from C28/I2 cells treated with T-2 toxin were collected for the purpose of comparing the differential expression of exosomal miRNA using absolute quantitative miRNA-seq. Target genes of differential exosomal miRNAs were identified using Targetscan and Miranda databases, followed by GO and KEGG enrichment analyses. Validation of key indicators of chondrocyte injury in KBD was conducted using Real-time quantitative PCR (RT-qPCR) and Immunohistochemical staining (IHC). A total of 20 exosomal miRNAs related to KBD were identified in serum, and 13 in chondrocytes (C28/I2). The identified exosomal miRNAs targeted 48,459 and 60,612 genes, primarily enriched in cell organelles and membranes, cell differentiation, and cytoskeleton in the serum, and the cytoplasm and nucleus, metal ion binding in chondrocyte (C28/I2). The results of the KEGG enrichment analysis indicated that the Ras signaling pathway may play a crucial role in the pathogenesis of KBD. Specifically, the upregulation of hsa-miR-181a-5p and hsa-miR-21–3p, along with the downregulation of hsa-miR-152–3p and hsa-miR-186–5p, were observed. Additionally, T-2 toxin intervention led to a significant downregulation of RALA, REL, and MAPK10 expression. Furthermore, the protein levels of RALA, REL, and MAPK10 were notably decreased in the superficial and middle layers of cartilage tissues from KBD. The induction of differential expression of chondrocyte exosomal miRNAs by T-2 toxin results in the collective regulation of target genes RALA, REL, and MAPK10, ultimately mediating the Ras signaling pathway and causing a disruption in chondrocyte extracellular matrix metabolism, leading to chondrocyte injury.

Molecular determinants of Ras-mTORC2 signaling

Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested the involvement of the Switch I (SWI) effector domain of Ras in binding mTORC2 components, the regulation of the Ras-mTORC2 pathway is not entirely understood. In Dictyostelium, mTORC2 is selectively activated by the Ras protein RasC, and the RasC-mTORC2 pathway then mediates chemotaxis to cAMP and cellular aggregation by regulating the actin cytoskeleton and promoting cAMP signal relay. Here, we investigated the role of specific residues in RasC's SWI, C-terminal allosteric domain, and hypervariable region (HVR) related to mTORC2 activation. Interestingly, our results suggest that RasC SWI residue A31, which was previously implicated in RasC-mediated aggregation, regulates RasC’s specific activation by the Aimless RasGEF. On the other hand, our investigation identified a crucial role for RasC SWI residue T36, with secondary contributions from E38 and allosteric domain residues. Finally, we found that conserved basic residues and the adjacent prenylation site in the HVR, which are crucial for RasC’s membrane localization, are essential for RasC-mTORC2 pathway activation by allowing for both RasC’s own cAMP-induced activation and its subsequent activation of mTORC2. Therefore, our findings revealed new determinants of RasC-mTORC2 pathway specificity in Dictyostelium, contributing to a deeper understanding of Ras signaling regulation in eukaryotic cells.

The Therapeutic Target of IBD and the Mechanism of Dipyridamole in Treating IBD Explored by Geo Gene Chips, Network Pharmacology, and Molecular Docking.

Inflammatory Bowel Disease (IBD) is a refractory disease with repeated attacks, and there is no accurate treatment target at present. Dipyridamole, a phosphodiesterase (PDE) inhibitor, has been proven to be an effective treatment for IBD in a pilot study. This study explored the therapeutic target of IBD and the pharmacological mechanism of dipyridamole for the treatment of IBD. The candidate targets of dipyridamole were obtained by searching the pharmMapper online server and Swiss Target Prediction Database. The IBD-related targets were selected from four GEO chips and three databases, including Genecards, DisGeNET, and TTD database. A protein-protein interaction (PPI) network was constructed, and the core targets were identified according to the topological structure. KEGG and GO enrichment analysis and BioGPS location were performed. Finally, molecular docking was used to verify dipyridamole and the hub targets. We obtained 112 up-regulated genes and 157 down-regulated genes, as well as 105 composite targets of Dipyridamole-IBD. Through the PPI network analysis, we obtained the 7 hub targets, including SRC, EGFR, MAPK1, MAPK14, MAPK8, PTPN11, and LCK. The BioGPS showed that these genes were highly expressed in the immune system, digestive system, and endocrine system. In addition, the 7 hub targets had good intermolecular interactions with dipyridamole. The therapeutic effect of dipyridamole on IBD may involve immune system activation and regulation of inflammatory reactions involved in the regulation of extracellular matrix, perinuclear region of cytoplasm, protein kinase binding, and positive regulation of programmed cell death through cancer pathway (proteoglycans in cancer), lipid metabolism, Ras signaling pathway, MAPK signaling pathway, PI3K-AKT signaling pathway, Th17 cell differentiation, and other cellular and innate immune signaling pathways. This study predicted the therapeutic target of IBD and the molecular mechanism of dipyridamole in treating IBD, providing a new direction for the treatment of IBD and a theoretical basis for further research.

The Significance of Key Proteins in the RAS Signaling Pathway: Implications for Cancer and Therapeutic Targets

The RAS signaling pathway is a crucial cell transduction pathway central to transmitting signals from outside the cell to the cell nucleus and influencing fundamental biological mechanisms like cell growth, division, and specialization. This signaling pathway has recently received much attention in scientific research because of its involvement in various diseases, especially carcinogenesis. Our study identified the significance of crucial proteins in the RAS signaling cascade in cancer development and progression. We found that proteins such as PDGFRB, PDGFB, IGF1, HRAS, HGF, FGF10, and ABL1 are involved in various types of cancer and could serve as potential therapeutic targets. Misregulation of these proteins may result in unregulated cell proliferation and contribute to cancer development and maintenance. The study also emphasizes the importance of oncogenes in cancer development, with RAS being identified as a pivotal oncogene. In addition, the findings indicate several proteins, including PDGFRA, NRAS, HRAS, CSF1R, KIT, MET, ABL1, FGFR2, FGFR3, and KRAS, function as oncogenes and are related to different forms of cancer and diseases. Targeted therapies for these proteins are being investigated in various cancer types, including gastrointestinal stromal tumors, chronic myelogenous leukemia, and bladder cancer. Moreover, we identified NF1 as a critical tumor suppressor gene essential in regulating cellular proliferation. Mutations in the NF1 gene lead to neurofibromatosis category 1. This paper emphasizes the significance of crucial proteins implicated in the RAS signaling pathway in cancer growth and advancement. Understanding the complexity of these proteins and their dysregulation could offer essential insights into the progression of practical treatment approaches that enhance and refine cancer therapies. These findings provide promising avenues for further research and advances in cancer treatment and give us hope for better outcomes in the fight against this challenging disease.

Comprehensive transcriptomic and metabolomic analysis of porcine intestinal epithelial cells after PDCoV infection

IntroductionPorcine deltacoronavirus (PDCoV), an emerging swine enteropathogenic coronavirus with worldwide distribution, mainly infects newborn piglets with severe diarrhea, vomiting, dehydration, and even death, causing huge economic losses to the pig industry. However, the underlying pathogenic mechanisms of PDCoV infection and the effects of PDCoV infection on host transcripts and metabolites remain incompletely understood.MethodsThis study investigated a combined transcriptomic and metabolomic analysis of porcine intestinal epithelial cells (IPEC-J2) following PDCoV infection by LC/MS and RNA-seq techniques. A total of 1,401 differentially expressed genes and 254 differentially accumulated metabolites were detected in the comparison group of PDCoV-infected vs. mock-infected.Results and discussionWe found that PDCoV infection regulates gene sets associated with multiple signaling pathways, including the neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, MAPK signaling pathway, chemokine signaling pathway, ras signaling pathway and so on. Besides, the metabolomic results showed that biosynthesis of cofactors, nucleotide metabolism, protein digestion and absorption, and biosynthesis of amino acid were involved in PDCoV infection. Moreover, integrated transcriptomics and metabolomics analyses revealed the involvement of ferroptosis in PDCoV infection, and exogenous addition of the ferroptosis activator erastin significantly inhibited PDCoV replication. Overall, these unique transcriptional and metabolic reprogramming features may provide a better understanding of PDCoV-infected IPEC-J2 cells and potential targets for antiviral treatment.

Ephrin type-A receptor 2-antisense RNA1/2 promote proliferation and migration of MDA-MB-231 cells through EPHA2-dependent Ras signaling pathway mediated by MAPK8/JNK1, MAPK9/JNK2-NFATC2/NFAT1 and JUND

Ephrin type-A receptor 2 (EPHA2) is a receptor tyrosine kinase that is overexpressed in a variety of cancers, including breast cancer. EPHA2 expression may be causally related to tumorigenesis; therefore, it is important to understand how EPHA2 expression is regulated. We previously reported that EPHA2 antisense RNA (EPHA2-AS), a natural antisense transcript, is an important modulator of EPHA2 mRNA levels and hence production of EPHA2 protein. EPHA2-AS encodes two splice variants, EPHA2-AS1 and EPHA2-AS2. The two variants are constitutively expressed in a concordant manner with EPHA2 mRNA in human breast adenocarcinoma cell lines and in patient samples, with the highest levels detected in the basal-like/triple-negative molecular subtype of breast cancer cells. In this study, we investigated the mechanism of EPHA2-AS1/2 in triple-negative breast cancer using MDA-MB-231 cells. We performed RNA-seq transcriptome analyses of MDA-MB-231 cells treated with AHCC®, which suppressed expression of EPHA2-AS1/2 and EPHA2 mRNA, and EPHA2-AS1/2-silenced MDA-MB-231 cells. Bioinformatics analyses identified 545 overlapping differentially expressed genes that were significantly up- or down-regulated by these treatments. Subsequent functional enrichment analyses of the overlapping genes in combination with in vitro assays indicated that EPHA2-AS1/2 may promote the proliferation and migration of MDA-MB-231 cells through the EPHA2-dependent Ras signaling pathways mediated by MAPK8/JNK1, MAPK9/JNK2-NFATC2/NFAT1 (proliferation and migration) and JUND (migration). These results thus suggest that EPHA2-AS1/2 may represent a potential molecular target for triple-negative breast cancer treatment.

Endogenous oncogenic KRAS expression increases cell proliferation and motility in near-diploid hTERT RPE-1 cells

About 18% of all human cancers carry a mutation in the KRAS gene making it among the most sought-after anti-cancer targets. However, mutant KRas protein has proved remarkably undruggable. The recent approval of the first generation of RAS inhibitors therefore marks a seminal milestone in the history of cancer research. Inevitably though, it also raises the predictable challenges of limited drug efficacies and acquired resistance. Hence, new approaches that improve our understanding of the tumorigenic mechanisms of oncogenic RAS within more physiological settings continue to be essential. Here, we have employed the near-diploid human hTERT RPE-1 cells to generate isogenic cell lines in which one of the endogenous KRAS alleles carries an oncogenic KRAS mutation at glycine 12. Cells with a KRASG12V/+, KRASG12C/+, or KRASG12D/+ genotype, together with wild-type KRASG12G(WT)/+ cells, reveal that oncogenic KRAS.G12X mutations increase cell proliferation rate and cell motility and reduced focal adhesions in KRASG12V/+ cells. EGF-induced phosphorylation of ERK and AKT was comparable between KRASG12V/+, KRASG12C/+, KRASG12D/+, and KRASG12G(WT)/+ cells. Interestingly, KRASG12X/+ cells showed varying responses to distinct inhibitors with the KRASG12V/+ and KRASG12D/+ cells more sensitive to hydroxyurea and MEK inhibitors, U0126 and trametinib, but more resistant to PI3K inhibitor, PIK-90, than the KRASG12G(WT)/+ cells. A combination of low doses of hydroxyurea and U0126 showed an additive inhibition on growth rate that was greater in KRASG12V/+ than wild-type cells. Collectively, these cell lines will be a valuable resource for studying oncogenic RAS signalling and developing effective anti-KRAS reagents with minimum cytotoxicity on wild-type cells.

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.

Biomimetic bone-periosteum scaffold for spatiotemporal regulated innervated bone regeneration and therapy of osteosarcoma

The complexity of repairing large segment defects and eradicating residual tumor cell puts the osteosarcoma clinical management challenging. Current biomaterial design often overlooks the crucial role of precisely regulating innervation in bone regeneration. Here, we develop a Germanium Selenium (GeSe) co-doped polylactic acid (PLA) nanofiber membrane-coated tricalcium phosphate bioceramic scaffold (TCP-PLA/GeSe) that mimics the bone-periosteum structure. This biomimetic scaffold offers a dual functionality, combining piezoelectric and photothermal conversion capabilities while remaining biodegradable. When subjected to ultrasound irradiation, the US-electric stimulation of TCP-PLA/GeSe enables spatiotemporal control of neurogenic differentiation. This feature supports early innervation during bone formation, promoting early neurogenic differentiation of Schwann cells (SCs) by increasing intracellular Ca2+ and subsequently activating the PI3K-Akt and Ras signaling pathways. The biomimetic scaffold also demonstrates exceptional osteogenic differentiation potential under ultrasound irradiation. In rabbit model of large segment bone defects, the TCP-PLA/GeSe demonstrates promoted osteogenesis and nerve fibre ingrowth. The combined attributes of high photothermal conversion capacity and the sustained release of anti-tumor selenium from the TCP-PLA/GeSe enable the synergistic eradication of osteosarcoma both in vitro and in vivo. This strategy provides new insights on designing advanced biomaterials of repairing large segment bone defect and osteosarcoma.

Ras-Targeting Stabilized Peptide Increases Radiation Sensitivity of Cancer Cells.

Radiation therapy is one of the most common treatments for cancer. However, enhancing tumors' radiation sensitivity and overcoming tolerance remain a challenge. Previous studies have shown that the Ras signaling pathway directly influences tumor radiation sensitivity. Herein, we designed a series of Ras-targeting stabilized peptides, with satisfactory binding affinity (KD = 0.13 μM with HRas) and good cellular uptake. Peptide H5 inhibited downstream phosphorylation of ERK and increased radio-sensitivity in HeLa cells, resulting in significantly reduced clonogenic survival. The stabilized peptides, designed with an N-terminal nucleation strategy, acted as potential radio-sensitizers and broadened the applications of this kind of molecule. This is the first report of using stabilized peptides as radio-sensitizers, broadening the applications of this kind of molecule.

Evaluation of KRAS inhibitor-directed therapies for pancreatic cancer treatment.

Despite significant advancements in the treatment of other cancers, pancreatic ductal adenocarcinoma (PDAC) remains one of the world's deadliest cancers. More than 90% of PDAC patients harbor a Kirsten rat sarcoma (KRAS) gene mutation. Although the clinical potential of anti-KRAS therapies has long been realized, all initial efforts to target KRAS were unsuccessful. However, with the recent development of a new generation of KRAS-targeting drugs, multiple KRAS-targeted treatment options for patients with PDAC have entered clinical trials. In this review, we provide an overview of current standard of care treatment, describe RAS signaling and the relevance of KRAS mutations, and discuss RAS isoform- and mutation-specific differences. We also evaluate the clinical efficacy and safety of mutation-selective and multi-selective inhibitors, in the context of PDAC. We then provide a comparison of clinically relevant KRAS inhibitors to second-line PDAC treatment options. Finally, we discuss putative resistance mechanisms that may limit the clinical effectiveness of KRAS-targeted therapies and provide a brief overview of promising therapeutic approaches in development that are focused on mitigating these resistance mechanisms.

Chemical constituents from uncaria laevigata and their vasodilatory activities in vitro and action mechanism

A new triterpenoid compound 1* (scandine A1) was obtained from 95% ethanol extract of Uncaria laevigata. Meanwhile, eleven described compounds were also isolated for the first time from Uncaria laevigata. Herein, compound 2 exhibited strong diastolic cardio-cerebrovascular activity (EC50BA = 9.22 μM and EC50CA = 14.65 μM), which was not been previously described. Compound 1* also showed certain diastolic cardio-cerebrovasculary activity. Network pharmacology indicated that the diastolic cardio-cerebrovascular activity of compound 2 was most correlated with the Ras signalling pathway. Molecular docking confirmed that it exhibited strong binding activity with target protein (matrix metalloproteinase inhibitor-1). Moreover, compound 2 demonstrated significant potential on cardio-cerebrovascular activity in vitro. Overall, compounds 1* and 2 with good diastolic cardio-cerebrovascular activity were discovered in this work.

Abstract PO3-23-11: Multiomics analysis of ER+ breast cancer with matched primaries, lymph node metastases and recurrent cancers on adjuvant endocrine therapy

Abstract Background: 80% of all breast cancers (BCs) are ER-positive (ER+). A significant number develop endocrine therapy resistance (ETR) . Clones develop in lymph nodes (LNs) which differ from the primary and these are implicated in ETR. A multiomics analysis of primary ER+ breast cancers, matched LN metastasis (LN+), if present, and matched recurrences (primary, nodal or distant) has been performed. Patients and Methods: The patients included are: Cohort A: 95 patients with matched primary and recurrent ER+ BC on different endocrine therapies. 35% had involved LNs at diagnosis. Recurrences: local in 59/95, concurrent local and nodal in 33/95, LN-only in 3/95. Median time to recurrence 4.5 years. Cohort B: 18 patients with ER+ LN+ BC who developed multiple recurrences (local and/or nodal, >3 consecutive recurrences/patient) on different lines of sequential endocrine therapy. Cohort C: 7 patients with ER+ breast cancer with multiple involved LNs treated with 3-6 months of neoadjuvant ET before surgery. Biopsies of primary and matched LNs performed at diagnosis and throughout treatment (3-4 timepoints/patient). Clinical response by serial ultrasound. Primary and LN samples (mean 12 samples/patient) were analysed together with normal DNA. Targeted RNAseq and DNA exome sequencing and whole-genome expression analysis was performed. Somatic mutations and copy number alterations (CNA) were determined, and differential gene expression analysis was performed. Validation of pathways implicated in ETR was by spatial NanoString GeoMx. All patients have long term follow-up. Results: Cohort A: A mean of 6 potential driver mutations identified by DNA sequencing in each primary BC (range 1-16). Most commonly mutated genes were PIK3CA, TP53, ERBB4, MAP3K1 and ERBB3. Multiple aberrations and a highly diverse mutational landscape were observed in all recurrences. In the majority of recurrences significant changes in variant allele frequency (VAF) were identified across several ETR-implicated genes and in over 50% new gains/losses were identified. Most frequently, losses were seen in ERBB3, ARID1A, HRAS and FOXA1 with gains in CDH1, NF1, PIK3CA, ESR1, TBX3 and GATA3 in recurrences vs primaries. Transcriptomic analysis revealed differentially expressed pathways including ER, HER2, GATA3, AKT, RAS and p63 signalling. Integrated analysis revealed several driver mutations with corresponding changes in upstream pathway activity. 33 patients had concurrent local and nodal recurrences and 52% had similar somatic mutation profiles in both with only minor variance in VAF. In 48%, 1 or more new potential driver mutations was identified in axillary nodes compared to recurrent primary cancers with losses in AKT1, CDH1 and TP53 being most common. Multiomics profiling of Cohorts B and C is complete and analysis underway. Discussion: This multiomics study is the largest cohort to-date of matched early and recurrent ER+/HER2- cancers. It sheds new light on the complex somatic and transcriptomic changes in local and nodal recurrent disease. The mechanisms of ETR are diverse and underlying mechanisms and clinically meaningful biomarkers including potentially actionable mutations and targets have been identified. Cohort C will uniquely capture and compare clonal evolution of breast cancer in primary and multiple matched lymph during neoadjuvant ET. Citation Format: Carlos Martinez-Perez, Charlene Kay, Rebecca Swan, Lorna Renshaw, J Michael Dixon, Arran K Turnbull. Multiomics analysis of ER+ breast cancer with matched primaries, lymph node metastases and recurrent cancers on adjuvant endocrine therapy [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 PO3-23-11.

Abstract PO1-08-09: Drynaria fortunie, a nutritional herb for prevention in triple negative breast cancer

Abstract Study Rationale: The triple negative breast cancer (TNBC) lacks the expression of hormone and growth factor receptors and responds only to anthracycline/taxol-based conventional chemotherapy. Major therapeutic limitations include systemic toxicity and acquired resistance to chemo-therapeutics. Relatively non-toxic nutritional herbs from traditional Chinese medicine (TCM) may represent testable alternatives against TNBC. These herbs effectively target multiple signaling pathways (Yang et al: J. Ethno-pharmacology 264: 113249, 2021). Growth inhibitory efficacy of nutritional herbs in a cellular model for TNBC is associated with downregulation of RB, RAS, PI3K and AKT signaling pathways (Telang et al: Pharmaceuticals 14: 1318, 2021). Drynaria fortunie (DF) is a nutritional herb that is used in many TCM herbal formulations. The present study is designed to examine the effects of DF in a cellular model for TNBC and to identify mechanistic leads for its efficacy. Experimental Model, herbal extract and biomarkers: Estrogen receptor negative, progesterone receptor negative and human epidermal growth factor receptor-2 negative MDA-MB-231 human breast cancer cell line represented a cellular model for TNBC. Non-fractionated aqueous extract from the bark of DF represented the test agent. Cell cycle progression, RB signaling and caspase 3/7 activity represented quantitative end point biomarkers. Study Outcome: Treatment with DF at cytostatic concentrations induced S phase arrest and inhibited RB signaling via downregulated expression of cyclin E, CDK2, pRB and E2F1. DF treatment also induced caspase 3/7 activity, a marker for cellular apoptosis. Collectively, these data provide potential mechanistic leads for anti-proliferative and pro-apoptotic effects of DF in the present model. Conclusion: Present data validate a mechanism-driven experimental approach to prioritize efficacious nutritional herbs as testable alternatives for prevention/therapy of TNBC. Citation Format: Nitin TELANG, Hareesh Nair, George Wong. Drynaria fortunie, a nutritional herb for prevention in triple negative 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 PO1-08-09.

Abstract PO2-14-11: Proteogenomic approaches for the identification of NF1/neurofibromin-depleted estrogen receptor positive breast cancers for targeted treatment

Abstract NF1 is a key tumor suppressor that represses both RAS and estrogen receptor-α (ER) signaling in breast cancer. Blocking both pathways by fulvestrant (F), a selective ER degrader (SERD), together with binimetinib (B), a MEK inhibitor, promotes tumor regression in NF1-depleted ER+ models. We aimed to establish approaches to determine how NF1 protein levels impact B+F treatment response to improve our ability to identify B+F sensitive tumors. We examined a panel of ER+ PDX models by DNA and mRNA sequencing and found that more than half of these models carried an NF1 shallow deletion and generally have low mRNA levels. Consistent with RAS and ER activation, RET and MEK levels in NF1-depleted tumors were elevated when profiled by mass spectrometry (MS) after kinase inhibitor bead pull-down. MS showed that NF1 can also directly and selectively bind to palbociclib-conjugated beads, aiding quantification. An immunohistochemistry (IHC) assay was also established to measure NF1, but the MS-based approach was more quantitative. Combined IHC and MS analysis defined a threshold of NF1 protein loss in ER+ breast PDX, below which tumors regressed upon treatment with B+F. These results suggest that we now have a MS-verified NF1 IHC assay that can be used for patient selection as a complement to somatic genomic analysis. Citation Format: Beom-Jun Kim, Ze-Yi Zheng, Jonathan Lei, Matthew Holt, Anran Chen, Jianheng Peng, Diana Fandino, Purba Singh, Hilda Kennedy, Yongchao Dou, M Rosario Chica-Parrado, Emmanuel Bikorimana, Dan Ye, Yunguan Wang, Ariella Hanker, Nada Mohamed, Susan Hilsenbeck, Bora Lim, Jaya Ruth Asirvatham, Arun Sreekumar, Bing Zhang, George Miles, Meenakshi Anurag, Matthew Ellis, Eric Chang. Proteogenomic approaches for the identification of NF1/neurofibromin-depleted estrogen receptor positive breast cancers for targeted treatment [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 PO2-14-11.

Abstract GS01-08: CDK4/6 inhibition is a potential vulnerability in NF1-depleted ER+ breast cancer

Abstract CDK4/6 control a key checkpoint by phosphorylating Rb, leading to Rb degradation and subsequent S-phase entry. CDK4/6 inhibition, together with endocrine therapy, is standard of care for advanced ER+ breast cancer; however, CDK4/6 inhibition typically is cytostatic. This study aims to identify breast cancer patients who may better respond to treatments targeting CDK4/6. NF1/neurofibromin is a key tumor suppressor that we have shown can represses not only RAS as a GAP but also ER as a transcriptional co-repressor. Dual activation of RAS and ER promotes endocrine therapy resistance. We computed kinase activity scores from phosphor-proteomic data in the CPTAC breast cancer cohort and found that both CDK4/6 activity scores and Rb-pS780 levels negatively correlated with NF1 protein levels. Furthermore, NF1 mutations co-occur with mutations in CDK4/6 and CCND1-3 in breast tumors. These results suggest a functional dependency between NF1 loss and activation of the CDK4/6-Rb pathway. To delineate how NF1 and CDK4 interact molecularly, we depleted NF1 in ER+ breast cancer cells and found an increase in Rb-pS780 and in phosphorylation in CDK4’s activation loop (CDK4-pT172). Cyclin-D binding is required for CDK4 activation. CCND1 expression is under the direct control by ER, and upon NF1-depletion, Cyclin D1 expression was increased, partly due to enhanced ER recruitment to an ERE in CCND1. Cyclin-D-bound CDK4 must be phosphorylated at T172 to be fully active; however, what kinase is responsible for this is largely unknown. We have evidence that CDK4-T172 phosphorylation is RAF, but not ERK, dependent. When the NF1-depleted ER+ cells were seeded in the presence of fulvestrant together with either palbociclib or abemaciclib, apoptosis readily occurred. Remarkably, fulvestrant plus palbociclib efficiently caused durable tumor regression in two ER+ PDX models, in which NF1 is undetectable by IHC. In contrast, no tumor regression was seen in a PDX model with detectable NF1. Finally, greater growth inhibition and apoptosis were detected in NF1-low tumors in the neoadjuvant NeoPalAna trial when palbociclib was added after anastrozole. These data support a model whereby ER and RAS signaling converge upon CDK4/6, and CDK4/6 activation is a key survival mechanism when ER signaling is attenuated by treatment in NF1-depleted ER+ breast cancer cells. This apparent addiction for CDK4/6 activity makes NF1-depleted ER+ breast tumors vulnerable to CDK4/6 inhibition, thus creating a potential therapeutic opportunity to match CDK4/6 inhibition with patients who can benefit the most. Citation Format: Ze-Yi Zheng, Anran Chen, Eric Jaehnig, Meenakshi Anurag, Jonathan Lei, Chenwei Wang, Long Feng, Purba Singh, Hilda Kennedy, Jin Cao, Ghazal Yadav, Jill Tsai, Xi Chen, Charles Foulds, Bora Lim, Matthew Ellis, Bing Zhang, Eric Chang. CDK4/6 inhibition is a potential vulnerability in NF1-depleted ER+ 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 GS01-08.

A novel microRNA miR-4433a-3p as a potential diagnostic biomarker for lung adenocarcinoma

BackgroundLung adenocarcinoma is one of the leading causes of cancer-related deaths because of the lack of early specific clinical indicators. MicroRNAs (miRNAs) have become the focus in lung cancer diagnosis. Further studies are required to explore miRNA expression in the serum of lung adenocarcinoma patients and their correlation with therapy and analyse specific messenger RNA targets to improve the specificity and sensitivity of early diagnosis. MethodsThe Toray 3D-Gene miRNA array was used to compare the expression levels of various miRNAs in the sera of patients with lung adenocarcinoma and healthy volunteers. Highly expressed miRNAs were selected for further analysis. To verify the screening results, serum and pleural fluid samples were analysed using qRT-PCR. Serum levels of the miRNAs and their correlation with the clinical information of patients with lung adenocarcinoma were analysed. The functions of miRNAs were further analysed using the Kyoto Encyclopedia of Gene and Genomes and Gene Ontology databases. ResultsMicroarray analysis identified 60 and 50 miRNAs with upregulated and downregulated expressions, respectively, in the serum of patients with lung adenocarcinoma compared to those in healthy individuals. Using qRT-qPCR to detection of miRNAs expression in the serum or pleural effusion of patients with early and advanced lung adenocarcinoma, we found that miR-4433a-3p could be used as a diagnostic marker and therapeutic evaluation indicator for lung adenocarcinoma. Serum of miR-4433a-3p levels significantly correlated with the clinical stage. miR-4433a-3p may be more suitable than other tumour markers for the early diagnosis and evaluation of therapeutic effects in lung adenocarcinoma. miR-4433a-3p may affect tumour growth and metastasis by acting on target genes (PIK3CD, UBE2J2, ICMT, PRDM16 and others) and regulating tumour-related signalling pathways (MAPK signal pathway, Ras signalling pathway and others). ConclusionmiR-4433a-3p may serve as a biomarker for the early diagnosis of lung adenocarcinoma and monitoring of therapeutic effects.