Pathway Activity Research Articles

Transcriptional Profiling Underscores the Role of Preprocurement Allograft Metabolism and Innate Immune Status on Outcomes in Human Liver Transplantation

Objective: The adverse effects of ischemia-reperfusion injury (IRI) remain a principal barrier to a successful outcome after lifesaving orthotopic liver transplantation (OLT). Gene expression during different phases of IRI is dynamic and modified by individual exposures, making it attractive for identifying potential therapeutic targets for improving the number of suitable organs for transplantation and patient outcomes. However, data remain limited on the functional landscape of gene expression during liver graft IRI, spanning procurement to reperfusion and recovery. Therefore, we sought to characterize transcriptomic profiles of IRI during multiple phases in human OLT. Methods: We conducted clinical data analyses, histologic evaluation, and RNA sequencing of 17 consecutive human primary OLT. We performed liver allograft biopsies at 4 time points: baseline (B, before donor cross-clamp), at the end of cold ischemia (CI), during early reperfusion (ER, after revascularization), and during late reperfusion (LR). Data were generated and then recipients grouped by post-OLT outcomes categories: immediate allograft function (IAF; n = 11) versus early allograft dysfunction (EAD; n = 6) groups. Results: We observed that CI (vs B) modified a transcriptomic landscape enriched for a metabolic and immune process. Expression levels of hallmark inflammatory response genes were higher transitioning from CI to ER and decreased from ER to LR. IAF group predominantly showed higher bile and fatty acid metabolism activity during LR compared with EAD group, while EAD group maintained more immunomodulatory activities. Throughout all time points, EAD specimens exhibited decreased metabolic activity in both bile and fatty acid pathways. Conclusions: We report transcriptomic profiles of human liver allograft IRI from prepreservation in the donor to posttransplantation in the recipient. Immunomodulatory and metabolic landscapes across ER and LR phases were different between IAF and EAD allografts. Our study also highlights marker genes for these biological processes that we plan to explore as novel therapeutic targets or surrogate markers for severe allograft injury in clinical OLT.

CARMIL1 regulates liver cancer cell proliferation by activating the ERK/mTOR pathway through the TRIM27/p53 axis

Capping protein regulatory factor and myosin 1 linker 1 is termed CARMIL1. CARMIL1 is involved in several physiological processes; it forms an actin filament network and plasma membrane-bound cellular projection tissues and positively regulates the cellular components and tissues. CARMIL1 exhibits important biological functions in cancer; nonetheless, these functions have not been completely explored. We aimed to investigate the novel functions of CARMIL1 in liver cancer, particularly in cell proliferation. The cell counting kit-8, 5-ethynyl-2′-deoxyuridine, Component A experiments, and subcutaneous tumor formation model suggest that CARMIL1 is central to the proliferation of liver cancer cells both in vivo and in vitro. We extracted CARMIL1 samples from The Cancer Genome Atlas Program and analyzed its enrichment. CARMIL1 regulated the pathway activity by affecting the expression of star molecular proteins of the extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR). Moreover, it influenced the proliferation ability of liver cancer cells. Western blotting suggested that CARMIL1 downregulation could affect ERK and mTOR phosphorylation. Results of the co-immunoprecipitation demonstrated that CARMIL1 binds to tripartite motif (TRIM)27, which in turn binds to p53. Subsequently, CARMIL1 can regulate p53 stability and promote its degradation through TRIM27. Additionally, CARMIL1 inhibition enhanced the sensitivity of liver cancer cells to sorafenib. Tumor growth was significantly inhibited in the group treated with sorafenib and CARMIL1, compared with the group treated with CARMIL1 alone. Sorafenib is a first-line targeted chemotherapeutic drug for hepatocellular carcinoma treatment. It increases the long-term survival of hepatocellular carcinoma by 44%. In this study, downregulated CARMIL1 combined with sorafenib significantly reduced the tumor volume and weight of the mouse subcutaneous tumor model, indicating the potential possibility of combining CARMIL1 with sorafenib in hepatocellular carcinoma treatment. In summary, CARMIL1 promotes liver cancer cell proliferation by regulating the TRIM27/p53 axis and activating the ERK/mTOR pathway.

Mass spectrometry and Nuclear magnetic resonance spectroscopy profiles of red and pink Rhododendron flower petals establish them as rich sources of bioactive secondary metabolites

AbstractRhododendron petals are considered high‐value owing to their commercial utility, national/state flower status in certain countries, and bioactive potential from recent studies. Profiling and quantitative analysis of the bioactive metabolites would evaluate if they can be natural sources. This study is focused on the comprehensive profiling of secondary metabolites in the hot aqueous petal extracts of Red and Pink Rhododendron flowers (Rhododendron arboreum and Rhododendron campanulatum) using separation and fragmentation patterns from mass spectrometry (MS) (gas chromatography‐MS and liquid chromatography‐tandem MS) and chemical shifts from proton‐nuclear magnetic resonance spectroscopy. The complementary analytical platforms highlighted the petals to be rich in promising bioactive molecules such as quinic acid, chlorogenic acid (caffeoyl quinic acid), protocatechuic acid, coumaroyl quinic acids, catechin, epigallocatechin, and shikimic acid. The analysis also reflected the activity of shikimic acid, phenolic acid, and flavonoid biosynthetic pathways in Rhododendron flowers. These metabolites are well reported for their bioactive potential as anti‐oxidative, anti‐viral, anti‐cancerous, anti‐diabetic, anti‐inflammatory, etc. While the quantitative and multivariate analysis showed variations in the levels of phenolic acids and flavonoids, it is established that red and pink Rhododendron flower petals are a rich source of bioactive phytochemicals of interest to the phytochemical Industry.

Glutamine Metabolism Heterogeneity in Glioblastoma Unveils an Innovative Combination Therapy Strategy.

Treatment of glioblastoma multiforme (GBM) remains challenging. Unraveling the orchestration of glutamine metabolism may provide a novel viewpoint on GBM therapy. The study presented a full and comprehensive comprehending of the glutamine metabolism atlas and heterogeneity in GBM for facilitating the development of a more effective therapeutic choice. Transcriptome data from large GBM cohorts were integrated in this study. A glutamine metabolism-based classification was established through consensus clustering approach, and a classifier by LASSO analysis was defined for differentiating the classification. Prognosis, signaling pathway activity, tumor microenvironment, and responses to immune checkpoint blockade (ICB) and small molecular drugs were characterized in each cluster. A combinational therapy of glutaminase inhibitor CB839 with dihydroartemisinin (DHA) was proposed, and the influence on glutamine metabolism, apoptosis, reactive oxygen species (ROS), and migration was measured in U251 and U373 cells. We discovered that GBM presented heterogeneous glutamine metabolism-based clusters, with unique survival outcomes, activity of signaling pathways, tumor microenvironment, and responses to ICB and small molecular compounds. In addition, the classifier could accurately differentiate the two clusters. Strikingly, the combinational therapy of CB839 with DHA synergistically attenuated glutamine metabolism, triggered apoptosis and ROS accumulation, and impaired migrative capacity in GBM cells, demonstrating the excellent preclinical efficacy. Altogether, our findings unveil the glutamine metabolism heterogeneity in GBM and propose an innovative combination therapy of CB839 with DHA for this malignant disease.

Cancer-associated fibroblast-derived gene signature discriminates distinct prognoses by integrated single-cell and bulk RNA-seq analyses in breast cancer.

Cancer-associated fibroblasts (CAFs) are one of the most predominant cellular subpopulations in the tumor stroma and play an integral role in cancer occurrence and progression. However, the prognostic role of CAFs in breast cancer remains poorly understood. We identified a number of CAF-related biomarkers in breast cancer by combining single-cell and bulk RNA-seq analyses. Based on univariate Cox regression as well as Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis, a novel CAF-associated prognostic model was developed. Breast cancer patients were grouped according to the median risk score and further analyzed for outcome, clinical characteristic, pathway activity, genomic feature, immune landscape, and drug sensitivity. A total of 341 CAF-related biomarkers were identified from single-cell and bulk RNA-seq analyses. We eventually screened eight candidate prognostic genes, including CERCAM, EMP1, SDC1, PRKG1, XG, TNN, WLS, and PDLIM4, and constructed the novel CAF-related prognostic model. Grouped by the median risk score, high-risk patients showed a significantly worse prognosis and exhibited distinct pathway activities such as uncontrolled cell cycle progression, angiogenesis, and activation of glycolysis. In addition, the combined risk score and tumor mutation burden significantly improved the ability to predict patient prognosis. Importantly, patients in the high-risk group had a higher infiltration of M2 macrophages and a lower infiltration of CD8+ T cells and activated NK cells. Finally, we calculated the IC50 for a range of anticancer drugs and personalized the treatment regimen for each patient. Integrating single-cell and bulk RNA-seq analyses, we identified a list of compositive CAF-associated biomarkers and developed a novel CAF-related prognostic model for breast cancer. This robust CAF-derived gene signature acts as an excellent predictor of patient outcomes and treatment responses in breast cancer.

Chalcomoracin promotes apoptosis and endoplasmic reticulum stress in hepatocellular carcinoma cells.

Chalcomoracin (CMR), a Diels-Alder adduct obtained from mulberry leaves, demonstrated wide-spectrum anti-cancer activity. Herein, we aimed to explore the function of CMR and how it works in hepatocellular carcinoma (HCC). Human HCC cell lines Hep3B and SNU-387 were cultured and treated with various concentrations of CMR (1.5, 3, and 6 µM). Subsequently, the effects of CMR on cell viability, colony formation, apoptosis, migration, and invasion abilities were studied in vitro. Furthermore, the levels of endoplasmic reticulum (ER) stress-related proteins and mitogen-activated protein kinase (MAPK) pathway-related proteins in cells under CMR exposure were detected using western blot. Experiments in vivo were conducted to examine the effects of CMR on tumor growth in HCC. CMR administration inhibited the viability and clonogenic, migration, and invasion abilities, as well as promoted cell apoptosis and ER stress in Hep3B and SNU-387 cells. In addition, CMR treatment reduced the phosphorylation levels of ERK, P38, and JNK in the MAPK pathway. Moreover, an in vivo study showed that CMR administration could inhibit tumorigenesis and MAPK pathway activity in HCC. Our data indicate that CMR has the potential to inhibit the development of HCC, potentially through the inhibition of the MAPK pathway. These findings suggest that CMR may have promising applications as an anticancer agent in future therapeutics for HCC.

Network-based elucidation of colon cancer drug resistance mechanisms by phosphoproteomic time-series analysis

Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. Leveraging progress in proteomic technologies and network-based methodologies, we introduce Virtual Enrichment-based Signaling Protein-activity Analysis (VESPA)—an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations—and use it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogating tumor-specific enzyme/substrate interactions accurately infers kinase and phosphatase activity, based on their substrate phosphorylation state, effectively accounting for signal crosstalk and sparse phosphoproteome coverage. The analysis elucidates time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring, experimentally confirmed by CRISPR knock-out assays, suggesting broad applicability to cancer and other diseases.

Troxerutin suppress inflammation response and oxidative stress in jellyfish dermatitis by activating Nrf2/HO-1 signaling pathway

BackgroundStomolophus meleagris envenomation causes severe cutaneous symptoms known as jellyfish dermatitis. The potential molecule mechanisms and treatment efficiency of dermatitis remain elusive because of the complicated venom components. The biological activity and molecular regulation mechanism of Troxerutin (TRX) was firstly examined as a potential treatment for jellyfish dermatitis. MethodsWe examined the inhibit effects of the TRX on tentacle extract (TE) obtained from S. meleagris in vivo and in vitro using the mice paw swelling models and corresponding assays for Enzyme-Linked Immunosorbent Assay (ELISA) Analysis, cell counting kit-8 assay, flow cytometry, respectively. The mechanism of TRX on HaCaT cells probed the altered activity of relevant signaling pathways by RNA sequencing and verified by RT-qPCR, Western blot to further confirm protective effects of TRX against the inflammation and oxidative damage caused by TE. ResultsTE significantly induced the mice paw skin toxicity and accumulation of inflammatory cytokines and reactive oxygen species in vivo and vitro. Moreover, a robust increase in the phosphorylation of mitogen-activated protein kinase (MAPKs) and nuclear factor-kappa B (NF-κB) signaling pathways was observed. While, the acute cutaneous inflammation and oxidative stress induced by TE were significantly ameliorated by TRX treatment. Notablly, TRX suppressed the phosphorylation of MAPK and NF-κB by initiating the nuclear factor erythroid 2-related factor 2 signaling pathway, which result in decreasing inflammatory cytokine release. ConclusionTRX inhibits the major signaling pathway responsible for inducing inflammatory and oxidative damage of jellyfish dermatitis, offering a novel therapy in clinical applications.

Effects of polyethylene and biodegradable microplastics on the physiology and metabolic profiles of dandelion

Biodegradable plastics, such as poly(butylene adipate terephthalate) (PBAT) and polylactic acid (PLA), are potential alternatives to conventional polyethylene (PE), both of which are associated with the production of microplastics (MPs). However, the toxicity of these compounds on medicinal plants and their differential effects on plant morphophysiology remain unclear. This study supplemented soils with MPs sized at 200 μm at a rate of 1% w/w and incubated them for 50 days to investigate the impact of MPs on the growth and metabolites of dandelion (Taraxacum mongolicum Hand.-Mazz.). The results demonstrated that the investigated MPs decreased the growth of dandelion seedlings, induced oxidative stress, and altered the activity of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase). Based on the comprehensive toxicity assessment results, the ecological toxicity was in the following order: PE MPs > PBAT MPs > PLA MPs. Metabolomics analyses revealed metabolic reprogramming in dandelion plants, leading to the enrichment of numerous differentially accumulated metabolites (DAMs) in the leaves. These pathways include carbohydrate metabolism, energy metabolism, and biosynthesis of secondary metabolites, suggesting that dandelions respond to MP stress by enhancing the activity of sugar, organic acid, and amino acid metabolic pathways. In addition, phenolic acids and flavonoids are critical for maintaining the balance in the antioxidant defense system. Our results provide substantial insights into the toxicity of biodegradable MPs to plants and shed light on plant defense and adaptation strategies. Further assessment of the safety of biodegradable MPs in terrestrial ecosystems is essential to provide guidance for environmentally friendly management.

LncRNA FOXD2-AS1 promotes the growth, invasion and migration of OSCC cells by regulating the MiR-185–5p/PLOD1/Akt/mTOR pathway

Although lncRNAs are recognized to contribute to the development of oral squamous-cell carcinoma (OSCC), their exact function in invasion and cell migration is not clear. In this research, we explored the molecular and cellular mechanisms of FOXD2-AS1 in OSCC. Prognostic and bioinformatics analyses were used to test for the differential expression of FOXD2-AS1-PLOD1. Following FOXD2-AS1 suppression or overexpression, changes in cell viability were measured using the CCK-8 test; changes in cell migration and invasion abilities were measured using the migration and the Transwell assay. The expression of associated genes and proteins was found using Western blot and RT-qPCR. Analysis of luciferase reporter genes was done to look for regulatory connections between various molecules. The FOXD2-AS1-PLOD1 pair, which was highly expressed in OSCC, was analyzed and experimentally verified to be closely related to the prognosis of OSCC, and a nomogram model and correction curve were constructed. The inhibition of FOXD2-AS1 resulted in the reduction of cell activity, migration, invasion ability and changes in genes related to invasion and migration. In vivo validation showed that inhibition of FOXD2-AS1 expression slowed tumor growth, and related proteins changed accordingly. The experiments verified that FOXD2-AS1 negatively regulated miR-185–5 p and that miR-185–5 p negatively regulated PLOD1. In addition, it was found that the expression of PLOD1, p-Akt and p-mTOR proteins in OSCC cells was reduced by the inhibition of FOXD2-AS1, and FOXD2-AS1 and PLOD1 were closely related to the Akt/mTOR pathway. Increased expression of FOXD2-AS1 promotes OSCC growth, invasion and migration, which is important in part by targeting miR-185–5 p/PLOD1/Akt/mTOR pathway activity.

Exercise-induced changes in myocardial glucose utilization during periods of active cardiac growth

Exercise training can promote physiological cardiac growth, which has been suggested to involve changes in glucose metabolism to facilitate hypertrophy of cardiomyocytes. In this study, we used a dietary, in vivo isotope labeling approach to examine how exercise training influences the metabolic fate of carbon derived from dietary glucose in the heart during acute, active, and established phases of exercise-induced cardiac growth. Male and female FVB/NJ mice were subjected to treadmill running for up to 4 weeks and cardiac growth was assessed by gravimetry. Cardiac metabolic responses to exercise were assessed via in vivo tracing of [13C6]-glucose via mass spectrometry and nuclear magnetic resonance. We found that the half-maximal cardiac growth response was achieved by approximately 1 week of daily exercise training, with near maximal growth observed in male mice with 2 weeks of training; however, female mice were recalcitrant to exercise-induced cardiac growth and required a higher daily intensity of exercise training to achieve significant, albeit modest, increases in cardiac mass. We also found that increases in the energy charge of adenylate and guanylate nucleotide pools precede exercise-induced changes in cardiac size and were associated with higher glucose tracer enrichment in the TCA pool and in amino acids (aspartate, glutamate) sourced by TCA intermediates. Our data also indicate that the activity of collateral biosynthetic pathways of glucose metabolism may not be markedly altered by exercise. Overall, this study provides evidence that metabolic remodeling in the form of heightened energy charge and increased TCA cycle activity and cataplerosis precedes cardiac growth caused by exercise training in male mice.

Cachd1 interacts with Wnt receptors and regulates neuronal asymmetry in the zebrafish brain.

Neurons on the left and right sides of the nervous system often show asymmetric properties, but how such differences arise is poorly understood. Genetic screening in zebrafish revealed that loss of function of the transmembrane protein Cachd1 resulted in right-sided habenula neurons adopting left-sided identity. Cachd1 is expressed in neuronal progenitors, functions downstream of asymmetric environmental signals, and influences timing of the normally asymmetric patterns of neurogenesis. Biochemical and structural analyses demonstrated that Cachd1 can bind simultaneously to Lrp6 and Frizzled family Wnt co-receptors. Consistent with this, lrp6 mutant zebrafish lose asymmetry in the habenulae, and epistasis experiments support a role for Cachd1 in modulating Wnt pathway activity in the brain. These studies identify Cachd1 as a conserved Wnt receptor-interacting protein that regulates lateralized neuronal identity in the zebrafish brain.

Vaspin facilitates the proliferation and osteogenic differentiation of periodontal ligament stem cells.

To investigate whether visceral adipose tissue-derived serine protease inhibitor (vaspin) can alleviate the inhibitory effect of high-glucose (HG) culture on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) and to preliminarily explore the underlying mechanisms. High glucose produces damage to the regeneration of periodontal tissue of PDLSCs. The expression level of vaspin in periodontal tissue is high in periodontitis patients and effectively reduced after initial therapy of periodontal diseases. However, the effect of vaspin on PDLSCs remains unknown. PDLSCs were cultured in media augmented with 5.5 or 25.0 mM concentrations of glucose to elucidate the impact and mechanism of vaspin on PDLSCs under high glucose invitro. Proliferation was measured by Cell Counting Kit-8 (CCK8) assay. Osteogenesis of PDLSCs was assessed by alkaline phosphatase (ALP) staining, ALP activity, and Alizarin Red staining. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) were used to investigate the osteo-specific markers. Then, the molecular impact of vaspin in the presence/absence of HG on PDLSCs physiology was determined with TGF-β1/Smad signaling pathway as the main focus. It was revealed that the proliferation and osteogenic differentiation (OD) of PDLSCs under HG was reduced, and by adding vaspin the anti-osteogenic impact of HG was relieved. Moreover, vaspin enhanced TGF-β1/Smad signaling pathway activity. Pretreatment with TGF-β1 inhibitor blocked vaspin-triggered TGF-β1/Smad signal activation and minimized the vaspin-induced protective effect against HG-inhibited growth and OD. In summary, vaspin observably reduces HG-mediated inhibition of PDLSCs OD by modulating the TGF-β1/Smad signaling pathway. Vaspin may be a potential therapeutic for periodontal tissue regeneration in diabetic patients.

40S Ribosomal Protein S6 Kinase Integrates Daylength Perception and Growth Regulation in Arabidopsis thaliana.

Plant growth occurs via the interconnection of cell growth and proliferation in each organ following specific developmental and environmental cues. Therefore, different photoperiods result in distinct growth patterns due to the integration of light and circadian perception with specific Carbon (C) partitioning strategies. In addition, the TARGET OF RAPAMYCIN (TOR) kinase pathway is an ancestral signaling pathway that integrates nutrient information with translational control and growth regulation. Recent findings in Arabidopsis (Arabidopsis thaliana) have shown a mutual connection between the TOR pathway and the circadian clock. However, the mechanistical network underlying this interaction is mostly unknown. Here, we show that the conserved TOR target, the 40S ribosomal protein S6 kinase (S6 K) is under circadian and photoperiod regulation both at the transcriptional and post-translational level. Total S6 K (S6K1 and S6K2) and TOR-dependent phosphorylated-S6 K protein levels were higher during the light period and decreased at dusk especially under short day conditions. Using chemical and genetic approaches we found that the diel pattern of S6 K accumulation results from 26S proteasome-dependent degradation and is altered in mutants lacking the circadian F-box protein ZEITLUPE (ZTL), further strengthening our hypothesis that S6 K could incorporate metabolic signals via TOR, which are also under circadian regulation. Moreover, under short days when C/energy levels are limiting, changes in S6K1 protein levels affected starch, sucrose and glucose accumulation and consequently impacted root and rosette growth responses. In summary, we propose that S6K1 constitutes a missing molecular link where day-length perception, nutrient availability and TOR pathway activity converge to coordinate growth responses with environmental conditions.

Abstract PO5-21-06: Pathway profiling for prediction of response to neoadjuvant Letrozole therapy in ER positive postmenopausal breast cancer: gaining new insights for targeted treatment

Abstract Introduction: The NEOLBC (NCT03283384) study included postmenopausal patients with hormone receptor positive (ER≥50%, PR any), HER2 negative, stage II/III breast cancer. A baseline biopsy was taken prior to start letrozole treatment and after two weeks of treatment. Based on the two week Ki67-IHC, patients were randomized (Ki67 ≥1%) to receive either Letrozole + Ribociclib or standard chemotherapy until surgery, or continued (Ki67 < 1%) Letrozole mono therapy. Here we describe the signal transduction pathway profiles to better understand the molecular mechanisms of response to neoadjuvant letrozole. Methods: Signal transduction pathway activities for the ER, AR, MAPK, PI3K, HH, TGFβ and Notch pathways were measured on baseline and two weeks samples using the OncoSIGNal qPCR test (InnoSIGN). High pathway activity in a tumor sample was defined as pathway activity value above the 95th percentile of reference normal epithelial breast tissue. The pathway activity patterns of the Ki67 < 1% (n=30) and Ki67 > 1% (n=63) groups were compared between baseline and two weeks of treatment. Results: Although all measured baseline samples (n=93) were ER IHC staining positive (≥50%), there is a large range in baseline ER signal transduction pathway activity (32 to 78 on a scale from 0 – 100) and 20% of the samples show an ER-activity range (32 - 45) that normally falls in the range of ER IHC stained negative breast tissue. The group with Ki67 > 1% after 2 weeks Letrozole treatment showed non elevated ER pathway activity at baseline more often (25%) compared to the Ki67 < 1% group (10%). 92% of the patients showed a decrease in ER pathway (11.6 ± 8.6) activity after 2 week letrozole treatment compared to baseline (p=8.5e-13). Interestingly, baseline AR pathway activity has a good differentiating predictive value for the two week response to letrozole treatment and the overall AR pathway activity score was higher in the low Ki67 group (51.4 ± 7.4) than in the Ki67 ≥ 1% group (46.7 ± 6.4; p=0.0053) and more often classified as high in 30% of the low Ki67 group vs. 13% of the Ki67 ≥ 1% group. The Ki67 > 1% group showed activation of non-hormonal pathways (PI3K, MAPK and/or HH) more frequent (38%) at baseline compared to the < 1% Ki67 group (19%). Conclusion: Positive ER staining does not always relate to a high ER pathway activity, possibly explaining that not all patients respond equally well to ER inhibition therapy. Activation of hormonal pathways (ER, but also AR) appear to be predictive for early (2 weeks) response towards Letrozole as assessed by Ki67 staining. Involvement of non-hormonal pathways is associated with less effective response towards Letrozole alone. Pathway activity patterns could improve insight to underlying hormonal treatment escape mechanisms which could lead to alternative treatment options in patients with ER-positive breast cancer. Citation Format: Nadia de Gruil, Anne Florine de Groot, Yvonne Wesseling-Rozendaal, Diederick Keizer, Sigi Neerken, Judith Kroep. Pathway profiling for prediction of response to neoadjuvant Letrozole therapy in ER positive postmenopausal breast cancer: gaining new insights 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 PO5-21-06.

Abstract PO5-13-10: Determining tumor-driving cell signaling pathways in breast cancer: Support for targeted therapy selection

Abstract Introduction: Success of therapeutic interventions largely rely on the pathological and biological characteristics of the tumor and varies due to the heterogeneous nature of breast cancers. Signal transduction pathways play an important role and are often used as target for specific therapies like hormone treatment, PI3K inhibitors etc. Immunohistochemistry (IHC) is currently the first assessment to divide tumors for subtyping and treatment and, especially in more advanced disease, mutation analyses are often used. Both IHC and mutation analyses only analyze a specific part of a signal transduction pathway and do not correlate well with outcome. Therefore, there is a need for new approaches to look at signaling pathways in cancer. We have developed a methodology that measures and quantifies signal transduction pathways (STPs) activities to reveal potential tumor-driving STPs to identify new options for targeted therapy in breast cancer. Method: Using the mRNA-based OncoSIGNal pathway activity profiling qPCR test (InnoSIGN), STP activities of 7 pathways (AR, ER, PI3K, MAPK, HH, TGFβ and Notch) were measured and quantified on a scale from 0-100. By using a set of non-tumor epithelial breast tissue samples, a reference range for normal physiological pathway activity was defined. A threshold was set on the 95th percentile of this range. A pathway activity score above this threshold was considered to be high (activated). Using this methodology, 201 ER positive (based on IHC) and 64 triple negative FFPE breast tumor samples obtained from different study sites and commercial biobanks were analyzed, including samples from primary and metastatic lesions, to determine for each individual tumor its pathway activity profile and identify activated tumor-driving pathways. Results: In the 195 primary ER positive tumors, the ER pathway was found to be activated in 78% of the samples, even though all samples were strongly ER immunohistochemistry stain positive. In addition to ER pathway activation, occasionally other pathways were activated: PI3K in 37%, HH in 25%, MAPK in 19%, AR in 20%, TGFβ in 6%, and Notch in 1%, of these samples. Analysis of 27 metastatic samples of ER positive tumors revealed in only 63% an active ER pathway. Additionally, more frequently PI3K pathway was observed in 70% of the samples. Other frequencies of pathway activation were: AR in 7% and MAPK in 4%, No Notch and TGFβ activity was found in this cohort. In 76 of the 78 triple negative tumors (TNBC) the ER pathway was inactive as expected. Remarkably, 2 samples appeared to have an activated ER pathway. An activated MAPK pathway was found in 50%, PI3K in 49%, HH in 18%, AR in 14%, TGFβ in 5% and Notch in 3% of these TNBC tumors. Conclusion: Compared to ER positive primary tumors we observed in metastatic ER positive tumors less frequent activation of the ER and more frequent activation of the PI3K pathway, suggesting that the latter pathway is associated with a more aggressive tumor phenotype. The triple negative subtype is characterized predominantly by activation of the PI3K, MAPK, AR and/or HH pathways, which may create new options for personalized targeted therapy of this subtype. OncoSIGNal can be used to determine the tumor driving signaling pathways in breast cancer patients, guiding selection of personalized targeted therapies. Citation Format: Paul van de Wiel, Yvonne Wesseling-Rozendaal, Anke Pierik, Dianne van Strijp, Saskia Vermeer, Fatema Tashrifwala, Josette Hartnett, Robert Babkowski, Suzanne Rose. Determining tumor-driving cell signaling pathways in breast cancer: Support for targeted therapy selection [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 PO5-13-10.

Abstract PO4-26-06: Identifying mutation-independent signaling pathway activation in molecular subtypes of triple negative breast cancers

Abstract Introduction Triple negative breast cancer (TNBC) is an aggressive cancer defined by the absence of estrogen receptors (ER), progesterone receptors (PR), and lack of human epidermal growth factor receptor 2 (HER-2). We previously identified molecular subgroups with distinct mutational, epigenetic, and clinicopathologic signatures. Here, we sought to identify signaling pathways using targeted RNA expression-based pathway analysis. Methods We analyzed 44 TNBC cases diagnosed at NYU Langone Health between March 2011 and April 2018. Samples were compared based on histology, immunohistochemistry, and epigenetics. We assessed mRNA expression levels of pathway-specific target genes with RT-qPCR using the OncoSIGNal pathway profiling assay. Signal transduction pathway analyses were used to measure activity of the estrogen receptor (ER), androgen receptor (AR), phosphoinositide-3-kinase (PI3K), mitogen-activated protein kinase (MAPK), Hedgehog (HH), Notch, and transforming growth factor beta (TGF- β) pathways. Results DNA methylation Cluster 1, which is enriched for apocrine histology and PI3K/Akt mutations, showed no increased activity in PI3K pathway by RNA expression, but showed increase of AR and MAPK signaling, with a single case of increased ER signaling in the absence of ESR1 activating mutations. In contrast, both DNA methylation Cluster 2 (no recurrent mutations) and Cluster 3 (DNA instability cluster) showed upregulation in PI3K pathway in 4/7 (57%) and 11/25 (44%) of cases, respectively. PI3K pathway activation was invariably associated with high Ki-67 >15%, however not all high Ki-67 cases showed high PI3K activity. Invasive ductal carcinoma (IDC) morphology was characterized by a nearly even split between MAPK (13/28, 46%) and PI3K (12/28, 44%), with single HH and TGF- β driven cases. In 5/28 (18%) of IDC we did not identify an upregulated pathway. Discussion We show that RNA expression-based pathway activity and mutational and epigenetic analyses provide complimentary information about the molecular landscape of TNBC. PI3K mutations are early drivers and lead to distinct epigenetic signatures, however they are not associated with increased PI3K pathway activity by RNA expression. In contrast, TNBC without PI3K mutations may show activation of the PI3K pathway in the absence of PI3K/Akt mutations. Our observation suggests that distinct biomarkers may be differently suited to predict response to PI3K inhibitors in TNBC. This may include expanding the number of patients for which PI3K/Akt pathway inhibition might be a therapeutic option, as well as explaining the lack of response in PI3K mutated tumors. Citation Format: Chanel Schroff, Lawrence Lin, Dianne van Strijp, Daniel Roses, Sigi Neerken, Freya Schnabel, Farbod Darvishian, Matija Snuderl. Identifying mutation-independent signaling pathway activation in molecular subtypes of triple negative breast cancers [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 PO4-26-06.

Abstract PO2-28-08: A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy

Abstract TNBC is one of the most aggressive subtypes of breast cancer. Combating chemotherapy resistance is critical to improving quality of care and reducing fatality among TNBC patients. In order to understand the mechanisms of chemoresistance in TNBC tumors, it has been proposed that the spatial interactions between cell types in the tumor microenvironment (TME) could offer insights into the differential response to therapy among tumors, and metastatic potential of certain tumors over others. Thus, this study utilizes spatially resolved transcriptomic (SRT) technology to profile the spatial interactions in the TNBC TME. With the goal of better understanding chemoresistance in TNBC and providing a proof-of-concept for new technology, we have launched a longitudinal SRT study of a TNBC PDX of residual disease before, during, and after adriamycin and cyclophosphamide (AC) treatment (Tx). SRT by 10X Genomics Visium was performed on vehicle, AC-treated residual tumor (21 days post-Tx), and AC-treated regrown tumor (50 days post-Tx when tumors regrew to starting tumor volume). This study is divided into two parts: 1) development of a computational pipeline; 2) spatial colocalization analysis of residual and regrown tumors to understand chemoresistance. PART 1: Due to the lack of specialized tools for processing and sorting xenograft reads from SRT data, we developed the Xenomake pipeline, which combines a xenograft sorting algorithm (Xengsort) and spatial barcode demultiplexing pipeline to assign reads into the host and graft organisms for each spatial spot. RESULTS: Xenomake permits clustering the spatial spots into stroma-rich (enriched for mouse mRNAs), and epithelial-rich (enriched for human mRNAs) regions. We show that Xenomake can find differential cytokine production in the stroma and epithelium. Since PDX data separate the tumor into stroma and epithelium by organism, the pipeline enables fine-tuned downstream analysis such as stromal-stromal and stromal-epithelial interactions. Xenomake is thus generally applicable for SRT involving PDX samples. PART 2: Previously we detailed patterns of tumor regression into a residual tumor state, followed by uncontrolled regrowth in the absence of treatment in multiple PDX models of TNBC. Although using PDX models necessitates immune-compromised mice, several types of stromal populations are present and analyzable in these models. Thus, using organism-assigned reads processed by Xenomake, we computationally inferred the localization pattern of stromal cell types in Visium spots including cancer associated fibroblasts (CAF), macrophages (MP), endothelial cells (ENDO), monocytes, and perivascular-like cells. We compared the spatial localization profiles (SLP) of stromal cell types across samples. With human reads, we computed the spatial pathway activity map (SPAM) for the ~30 HALLMARK pathways across samples. We correlated the patterns of SPAM with stroma cell-type SLP to survey the stroma-epithelial interactions across samples. RESULTS: Stroma cells are generally distributed in the tumor periphery in vehicle and AC50, while in AC21 there is a notable increase in stroma abundance and stroma infiltration within tumor mass. SLP of MP (Cd68+ and Csf1r+) is correlated with ENDO (Pecam1+), and with CAF (Acta2+ and Pdgfrb+). Additionally, all 3 cell types are correlated with Vim and Cd44 expression. Although all samples show enrichment of MP, ENDO, and CAF, they interact differently with pathway activities of adjacent epithelial cells. In vehicle and AC50, the 3 cell types are colocalized with OXPHOS, MYC targets, E2F pathway, while in AC21, a switch to colocalization with EMT is observed. Furthermore, hypoxia response and glycolysis display anti-correlation with stroma cell types, meaning that these pathway activities are further away from stroma and occupy distinct territories. The results suggest stroma-tumor metabolic crosstalk and ways of targeting residual disease. Citation Format: Benjamin Strope, Katherine Pendleton, William Bowie, Gloria Echeverria, Qian Zhu. A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy [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 PO2-28-08.

The Role of Different TET Proteins in Cytosine Demethylation Revealed by Mathematical Modeling

In living cells, some reactions can be conducted by more than one enzyme and sometimes it is difficult to establish which enzyme is responsible. Such is the case with proteins from the TET family, capable of converting 5-methyl-2’-deoxycytidine (5-mdC) in DNA to 5-(hydroxymethyl)-2’-deoxycytidine (5-hmdC) and further to 5-formyl-2’-deoxycytidine (5-fdC) and 5-carboxy-2’-deoxycytidine (5-cadC). The estimation of the efficiency of particular TETs in particular oxidative reactions and different cell types is important but experimentally difficult. Here, we propose an approach with mathematical modeling in which methylation and known deoxycytidine modification pathways are presented by 343 possible model versions with assumed different combinations of TET1, 2, and 3 activities in different pathways. Model parameters were calculated on the basis of 5-mdC, 5-hmdC, 5-fdC, 5-cadC, and 5-hmdU levels experimentally assessed in five human cultured cell lines and previously published. Selection of the model versions that give in simulations the best average fit to experimental data suggested that not all TET proteins participate in all modification reactions and that TET3 activity may be especially important in the reaction of 5-fdC removal.

Abstract PO3-24-12: CRIPTO-regulated extracellular vesicles mediate heterotypic cellular crosstalk in triple negative breast cancers

Abstract Tumor progression involves the dynamic interaction between various cell types within the tumor microenvironment. Fibroblasts, which normally participate in wound healing, exhibit reciprocal communication with breast cancer cells that contributes to tumor cell plasticity and heterogeneity. Gaining insights into the molecular mechanisms underlying cell-to-cell communication, which drive cellular plasticity and play a pivotal role in breast cancer progression and metastasis, has the potential to unlock novel avenues for targeted therapeutic interventions. The small GPI-adaptor protein CRIPTO is a key player in this context. CRIPTO orchestrates the activity of TGF-β and growth factor-like signaling pathways that activate c-Src/MEK/AKT. CRIPTO is expressed during mammary development, wound healing, and breast cancer but is largely undetectable in homeostatic tissue, making it a promising therapeutic target. Our previous studies have demonstrated that the activity of CRIPTO is contingent upon cellular stress, with pronounced influence within pro-fibrotic, hypoxic domains of tumors. Importantly, we have shown that inhibition of CRIPTO using the soluble recombinant protein antagonist A4Fc effectively impedes the progression of triple negative breast cancer (TNBC) in murine xenograft models. Building upon these findings, we have been dissecting CRIPTO‘s role in coordinating fibroblast activation and cell-state reprogramming in TNBC. Notably, we have found that inhibition of CRIPTO with A4Fc suppresses cancer associated fibroblast (CAF) activation and collagen remodeling in vivo. And, by modeling tumor cell:fibroblast crosstalk using in vitro organoid co-cultures, we have demonstrated CRIPTO expression in tumor cells is necessary for extracellular vesicle (EV) uptake by fibroblasts, their SMAD pathway activation and their priming for reciprocal signaling back to tumor cells to for transcriptional reprogramming and cellular invasion. The significance of these findings lies in their potential to pave the way for the development of new therapies specifically targeting crucial tumor cell:fibroblast crosstalk mechanisms at primary and metastatic sites. Plasticity targeting therapies are urgently needed in the treatment of TNBC and other malignancies, and hold great promise for improving patient outcomes. Citation Format: David Freeman, Benjamin Spike, Brooke Gates. CRIPTO-regulated extracellular vesicles mediate heterotypic cellular crosstalk in triple negative breast cancers [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 PO3-24-12.