Canonical Pathway Research Articles

ZBP1-mediated apoptosis and inflammation exacerbate steatotic liver ischemia-reperfusion injury.

There is increasing need to expand availability of donor liver grafts, including steatotic livers. However, the current use of steatotic grafts in liver transplantation is less acceptable due to their higher susceptibility to ischemia-reperfusion (I/R) injury. To investigate the mechanism underlying the susceptibility of steatotic liver to I/R injury, we detected cell death markers and inflammation in clinical donor livers and animal models. We found that caspase-8-mediated hepatic apoptosis is activated in steatotic liver I/R. However, ablation of caspase-8 only slightly mitigated steatotic liver I/R injury without affecting inflammation. We further demonstrated that RIPK1 kinase induces both caspase-8-mediated apoptosis and cell death-independent inflammation. Inhibition of RIPK1 kinase significantly protects against steatotic liver I/R injury by alleviating both hepatic apoptosis and inflammation. Additionally, we found that RIPK1 activation is induced by Z-DNA binding protein 1 (ZBP1) but not the canonical TNFα pathway during steatotic liver I/R. Deletion of ZBP1 substantially decreases the steatotic liver I/R injury. Mechanistically, ZBP1 is amplified by palmitic acid-activated JNK pathway in steatotic livers. Upon I/R, excessive reactive oxygen species trigger ZBP1 activation by inducing its aggregation independent of the Z-nucleic acids sensing action in steatotic livers, leading to the kinase activation of RIPK1 and the subsequent aggravation of liver injury. Thus, ZBP1-mediated RIPK1-driven apoptosis and inflammation exacerbate steatotic liver I/R injury, which could be targeted to protect steatotic donor livers during transplantation.

Aerobic exercise training engages the canonical wnt pathway to improve pulmonary function and inflammation in COPD

BackgroundWe studied whether the exercise improves cigarette smoke (CS) induced chronic obstructive pulmonary disease (COPD) in mice through inhibition of inflammation mediated by Wnt/β-catenin-peroxisome proliferator-activated receptor (PPAR) γ signaling.MethodsFirstly, we observed the effect of exercise on pulmonary inflammation, lung function, and Wnt/β-catenin-PPARγ. A total of 30 male C57BL/6J mice were divided into the control group (CG), smoke group (SG), low-intensity exercise group (LEG), moderate-intensity exercise group (MEG), and high-intensity exercise group (HEG). All the groups, except for CG, underwent whole-body progressive exposure to CS for 25 weeks. Then, we assessed the maximal exercise capacity of mice from the LEG, MEG, and HEG, and performed an 8-week treadmill exercise intervention. Then, we used LiCl (Wnt/β-catenin agonist) and XAV939 (Wnt/β-catenin antagonist) to investigate whether Wnt/β-catenin-PPARγ pathway played a role in the improvement of COPD via exercise. Male C57BL/6J mice were randomly divided into six groups (n = 6 per group): CG, SG, LiCl group, LiCl and exercise group, XAV939 group, and XAV939 and exercise group. Mice except those in the CG were exposed to CS, and those in the exercise groups were subjected to moderate-intensity exercise training. All the mice were subjected to lung function test, lung histological assessment, and analysis of inflammatory markers in the bronchoalveolar lavage fluid, as well as detection of Wnt1, β-catenin and PPARγ proteins in the lung tissue.ResultsExercise of various intensities alleviated lung structural changes, pulmonary function and inflammation in COPD, with moderate-intensity exercise exhibiting significant and comprehensive effects on the alleviation of pulmonary inflammation and improvement of lung function. Low-, moderate-, and high-intensity exercise decreased β-catenin levels and increased those of PPARγ significantly, and only moderate-intensity exercise reduced the level of Wnt1 protein. Moderate-intensity exercise relieved the inflammation aggravated by Wnt agonist. Wnt antagonist combined with moderate-intensity exercise increased the levels of PPARγ, which may explain the highest improvement of pulmonary function observed in this group.ConclusionsExercise effectively decreases COPD pulmonary inflammation and improves pulmonary function. The beneficial role of exercise may be exerted through Wnt/β-catenin-PPARγ pathway.

Spastin and alsin protein interactome analyses begin to reveal key canonical pathways and suggest novel druggable targets

Abstract Developing effective and long-term treatment strategies for rare and complex neurodegenerative diseases is challenging. One of the major roadblocks is the extensive heterogeneity among patients. This hinders understanding the underlying disease- causing mechanisms and building solutions that have implications for a broad spectrum of patients. One potential solution is to develop personalized medicine approaches based on strategies that target the most prevalent cellular events that are perturbed in patients. Especially in patients with a known genetic mutation, it may be possible to understand how these mutations contribute to problems that lead to neurodegeneration. Protein-protein interaction analyses offer great advantages for revealing how proteins interact, which cellular events are primarily involved in these interactions, and how they become affected when key genes are mutated in patients. This line of investigation also suggests novel druggable targets for patients with different mutations. Here, we focus on alsin and spastin, two proteins that are identified as “causative” for amyotrophic lateral sclerosis and hereditary spastic paraplegia, respectively, when mutated. Our review analyzes the protein interactome for alsin and spastin, the canonical pathways that are primarily important for each protein domain, as well as compounds that are either Food and Drug Administration- approved or are in active clinical trials concerning the affected cellular pathways. This line of research begins to pave the way for personalized medicine approaches that are desperately needed for rare neurodegenerative diseases that are complex and heterogeneous.

C. elegans Hedgehog-related proteins are tissue- and substructure-specific components of the cuticle and pre-cuticle.

In C. elegans, expanded families of divergent Hedgehog-related and Patched-related proteins promote numerous processes ranging from epithelial and sense organ development to pathogen responses to cuticle shedding during the molt cycle. The molecular functions of these proteins have been mysterious since nematodes lack a canonical Hedgehog signaling pathway. Here we show that Hedgehog-related proteins are components of the cuticle and pre-cuticle apical extracellular matrices that coat, shape, and protect external epithelia. Of four Hedgehog-related proteins imaged, two (GRL-2 and GRL-18) stably associated with the cuticles of specific tubes and two (GRL-7 and WRT-10) labelled pre-cuticle substructures such as furrows or alae. We found that wrt-10 mutations disrupt cuticle alae ridges, consistent with a structural role in matrix organization. We hypothesize that most nematode Hedgehog-related proteins are apical extracellular matrix components, a model that could explain many of the reported functions for this family. These results highlight ancient connections between Hedgehog proteins and the extracellular matrix and suggest that any signaling roles of C. elegans Hedgehog-related proteins will be intimately related to their matrix association.

Characterizing dysregulations via cell-cell communications in Alzheimer’s brains using single-cell transcriptomes

BackgroundAlzheimer’s disease (AD) is a devastating neurodegenerative disorder affecting 44 million people worldwide, leading to cognitive decline, memory loss, and significant impairment in daily functioning. The recent single-cell sequencing technology has revolutionized genetic and genomic resolution by enabling scientists to explore the diversity of gene expression patterns at the finest resolution. Most existing studies have solely focused on molecular perturbations within each cell, but cells live in microenvironments rather than in isolated entities. Here, we leveraged the large-scale and publicly available single-nucleus RNA sequencing in the human prefrontal cortex to investigate cell-to-cell communication in healthy brains and their perturbations in AD. We uniformly processed the snRNA-seq with strict QCs and labeled canonical cell types consistent with the definitions from the BRAIN Initiative Cell Census Network. From ligand and receptor gene expression, we built a high-confidence cell-to-cell communication network to investigate signaling differences between AD and healthy brains.ResultsSpecifically, we first performed broad communication pattern analyses to highlight that biologically related cell types in normal brains rely on largely overlapping signaling networks and that the AD brain exhibits the irregular inter-mixing of cell types and signaling pathways. Secondly, we performed a more focused cell-type-centric analysis and found that excitatory neurons in AD have significantly increased their communications to inhibitory neurons, while inhibitory neurons and other non-neuronal cells globally decreased theirs to all cells. Then, we delved deeper with a signaling-centric view, showing that canonical signaling pathways CSF, TGFβ, and CX3C are significantly dysregulated in their signaling to the cell type microglia/PVM and from endothelial to neuronal cells for the WNT pathway. Finally, after extracting 23 known AD risk genes, our intracellular communication analysis revealed a strong connection of extracellular ligand genes APP, APOE, and PSEN1 to intracellular AD risk genes TREM2, ABCA1, and APP in the communication from astrocytes and microglia to neurons.ConclusionsIn summary, with the novel advances in single-cell sequencing technologies, we show that cellular signaling is regulated in a cell-type-specific manner and that improper regulation of extracellular signaling genes is linked to intracellular risk genes, giving the mechanistic intra- and inter-cellular picture of AD.

A dose-response study on functional and transcriptomic effects of FSH on ex vivo mouse folliculogenesis.

Follicle-stimulating hormone (FSH) binds to its membrane receptor (FSHR) in granulosa cells to activate various signal transduction pathways and drive the gonadotropin-dependent phase of folliculogenesis. Poor female reproductive outcomes can result from both FSH insufficiency owing to genetic or non-genetic factors and FSH excess as encountered with ovarian stimulation in assisted reproductive technology (ART), but the underlying molecular mechanisms remain elusive. Herein, we conducted single-follicle and single-oocyte RNA sequencing analysis along with other approaches in an ex vivo mouse folliculogenesis and oogenesis system to investigate the effects of different concentrations of FSH on key follicular events. Our study revealed that a minimum FSH threshold is required for follicle maturation into the high estradiol-secreting preovulatory stage, and such threshold is moderately variable among individual follicles between 5-10 mIU/mL. FSH at 5, 10, 20, and 30 mIU/mL induced distinct expression patterns of follicle maturation-related genes, follicular transcriptomics, and follicular cAMP levels. RNA-seq analysis identified FSH-stimulated activation of G proteins and downstream canonical and novel signaling pathways that may critically regulate follicle maturation, including the cAMP/PKA/CREB, PI3K-AKT/FOXO1, and glycolysis pathways. High FSH at 20 and 30 mIU/mL resulted in non-canonical FSH responses including premature luteinization, high production of androgen and proinflammatory factors, and reduced expression of energy metabolism-related genes in oocytes. Together, this study improves our understanding of gonadotropin-dependent folliculogenesis and provides crucial insights into how high doses of FSH used in ART may impact follicular health, oocyte quality, pregnancy outcome, and systemic health.

Cutaneous inflammasome driving ASC / gasdermin-D activation and IL-1β-secreting macrophages in severe atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin disease with intense pruritus, and chronic skin colonization by Staphylococcus aureus. To understand the inflammatory status in AD, we investigated the inflammasome complex, that activates ASC (Apoptosis-associated speck-like protein containing a CARD), caspase-1 and GSDMD (gasdermin-D), and production of IL-1β and IL-18. We aimed to evaluate the expression of the inflammasome pathway in the skin of adults with AD. Thirty patients with moderate to severe AD and 20 healthy controls were enrolled in the study. We performed the analysis of the inflammasome components NLRP1, NLRP3, AIM-2, IL-1β, IL-18, Caspase-1, ASC, GSDMD, and CD68 expression (macrophage marker) by immunohistochemistry and immunofluorescence. The main findings included increased expression of NLRP3, NLRP1 and AIM-2 at dermal level of severe AD; augmented IL-18 and IL-1β expression at epidermis of moderate and severe patients, and in the dermis of severe AD; augmented expression of ASC, caspase-1 and GSDMD in both epidermis and dermis of moderate and severe AD. We detected positive correlation between caspase-1, GSDMD and IL-1β (epidermis) and caspase-1 (dermis) and AD severity; NLRP3, AIM-2 and IL-1β, and NLRP3 with IL-18 in the epidermis; ASC, GSDMD and IL-1β, and NLRP3, AIM-2, caspase-1, and IL-18 in the dermis. We also evidenced the presence of CD68+ macrophages secreting GSDMD, ASC and IL-1β in moderate and severe AD. Cutaneous macrophages, early detected in moderate AD, have its role in the disease inflammatory mechanisms. Our study indicates a canonical activation pathway of inflammasomes, reinforced by the chronic status of inflammation in AD. The analysis of the inflammasome complex evidenced an imbalance in its regulation, with increased expression of the evaluated components, which is remarkably in severe AD, emphasizing its relevance as potential disease biomarkers and targets for immunomodulatory interventions.

WNT3 promotes chemoresistance to 5-Fluorouracil in oral squamous cell carcinoma via activating the canonical β-catenin pathway

Background5-Fluorouracil (5FU) is a primary chemotherapeutic agent used to treat oral squamous cell carcinoma (OSCC). However, the development of drug resistance has significantly limited its clinical application. Therefore, there is an urgent need to determine the mechanisms underlying drug resistance and identify effective targets. In recent years, the Wingless and Int-1 (WNT) signaling pathway has been increasingly studied in cancer drug resistance; however, the role of WNT3, a ligand of the canonical WNT signaling pathway, in OSCC 5FU-resistance is not clear. This study delved into this potential connection.Methods5FU-resistant cell lines were established by gradually elevating the drug concentration in the culture medium. Differential gene expressions between parental and resistant cells underwent RNA sequencing analysis, which was then substantiated via Real-time quantitative PCR (RT-qPCR) and western blot tests. The influence of the WNT signaling on OSCC chemoresistance was ascertained through WNT3 knockdown or overexpression. The WNT inhibitor methyl 3-benzoate (MSAB) was probed for its capacity to boost 5FU efficacy.ResultsIn this study, the WNT/β-catenin signaling pathway was notably activated in 5FU-resistant OSCC cell lines, which was confirmed through transcriptome sequencing analysis, RT-qPCR, and western blot verification. Additionally, the key ligand responsible for pathway activation, WNT3, was identified. By knocking down WNT3 in resistant cells or overexpressing WNT3 in parental cells, we found that WNT3 promoted 5FU-resistance in OSCC. In addition, the WNT inhibitor MSAB reversed 5FU-resistance in OSCC cells.ConclusionsThese data underscored the activation of the WNT/β-catenin signaling pathway in resistant cells and identified the promoting effect of WNT3 upregulation on 5FU-resistance in oral squamous carcinoma. This may provide a new therapeutic strategy for reversing 5FU-resistance in OSCC cells.

Wnt signaling in cardiac development and heart diseases.

The Wnt signaling pathway is a fundamental cellular communication system with extensive implications in various organs including the heart. In cardiac homeostasis, it governs essential processes like cellular proliferation, differentiation, and apoptosis, ensuring the heart's structural and functional integrity from embryonic stages and throughout life. Both canonical and non-canonical Wnt signaling pathways play a critical role during embryonic heart development in a region- and stage-specific manner. Canonical Wnt signaling also plays a significant role in heart diseases such as myocardial infarction and heart failure. However, the role of non-canonical Wnt signaling in heart diseases has not been fully elucidated. Wnt5a is a major ligand that activates non-canonical Wnt pathway, and recent studies start to clarify the role of the Wnt5a signaling axis in cardiac health and disease. In this review, we will briefly summarize the previous findings on the role of Wnt signaling pathways in heart development and diseases, and then focus on the role of Wnt5a signaling in heart failure progression. The multifaceted roles of the Wnt signaling pathway highlight its therapeutic potential for various types of heart diseases.

Lung adenocarcinomas without driver genes converge to common adaptive strategies through diverse genetic, epigenetic, and niche construction evolutionary pathways

Somatic evolution selects cancer cell phenotypes that maximize survival and proliferation in dynamic environments. Although cancer cells are molecularly heterogeneous, we hypothesized convergent adaptive strategies to common host selection forces can be inferred from patterns of epigenetic and genetic evolutionary selection in similar tumors. We systematically investigated gene mutations and expression changes in lung adenocarcinomas with no common driver genes (n = 313). Although 13,461 genes were mutated in at least one sample, only 376 non-synonymous mutations evidenced positive evolutionary selection with conservation of 224 genes, while 1736 and 2430 genes exhibited ≥ two-fold increased and ≥ 50% decreased expression, respectively. Mutations under positive selection are more frequent in genes with significantly altered expression suggesting they often “hardwire” pre-existing epigenetically driven adaptations. Conserved genes averaged 16-fold higher expression in normal lung tissue compared to those with selected mutations demonstrating pathways necessary for both normal cell function and optimal cancer cell fitness. The convergent LUAD phenotype exhibits loss of differentiated functions and cell–cell interactions governing tissue organization. Conservation with increased expression is found in genes associated with cell cycle, DNA repair, p53 pathway, epigenetic modifiers, and glucose metabolism. No canonical driver gene pathways exhibit strong positive selection, but extensive down-regulation of membrane ion channels suggests decreased transmembrane potential may generate persistent proliferative signals. NCD LUADs perform niche construction generating a stiff, immunosuppressive microenvironment through selection of specific collagens and proteases. NCD LUADs evolve to a convergent phenotype through a network of interconnected genetic, epigenetic, and ecological pathways.

Abstract PO3-03-01: Impact of Environmental Temperature on Clinical Outcomes and Tumor Microenvironment of Early-Stage Breast Cancer

Abstract Background Preclinical evidence using mouse model suggests that thermal/cold stress increases tumor growth by modulating the tumor microenvironment (TME); however, the clinical relevance of temperature on breast cancer (BC) outcomes is unknown. Studies show that residing in cold regions is associated with higher incidence of BC. We aim to study the impact of environmental temperature on the pathological complete response (pCR) and survival of early-stage BC patients (pts). Methodology A multi-institutional study was conducted within the Oncology Research Information Exchange Network (ORIEN). We analyzed the clinical and genomic data for early-stage BC pts from 12 centers in different environment zones (5 warm and 7 cold) (based on average annual regional temperature obtained from National Centers for Environmental Information). Cox regression was used to measure the association of climate and overall/relapse-free survival (OS/RFS) after adjusting for age, race, body mass index, grade, stage, treatments, comorbidities, and subtype. Raw feature counts were normalized, and differential expression analysis was carried out using DESeq2, while adjusting for all of the covariates. Differential expression rank order was used for subsequent gene set enrichment analysis (GSEA), performed using the cluster profile package in R. Gene sets queried included the Hallmark, Canonical pathways, and GO Biological Processes Ontology collections available through the Molecular Signatures Database (MSigDB). Results Out of the 1,304 early-stage BC pts, 271 pts received neoadjuvant chemotherapy (NAC) (186 warm, 85 cold). Higher clinical T- and N-stages were observed in pts from warm compared to cold regions (p< 0.001). Pts residing in cold regions had more comorbidities (57.6% vs 4.8%, p< 0.001). Pts in warm regions had higher pCR, though not statistically significant (8% vs 2.5%, p= 0.1). In the overall population, the OS (univariate (UV) HR= 0.48, 95% CI 0.27-0.64, p < 0.001; adjusted HR (aHR)= 0.56, 95% CI 0.32 - 0.96, p= 0.03) and RFS (UV HR= 0.51, 95% CI 0.38 - 0.68, p< 0.001; aHR= 0.52, 95% CI 0.36 - 0.75, p= 0.0005) were higher in pts from warm compared to cold regions (Table 1). Among the patient subgroup treated with NAC, the OS (aHR= 0.35, p= 0.02) and RFS (aHR= 0.49, p= 0.02) of pts from warmer regions was also higher than cold regions. RNA sequencing was performed on 826 tumors (443 warm, 383 cold) using pre-treatment samples. Naïve B-cells (mean: 0.09 vs 0.08, p=0.004), CD4 naïve T cells (0.03 vs 0.02, p= 0.0008), CD4+ memory T cells (0.03 vs 0.02, p= 0.04), gamma-delta T-cells (0.007 vs 0.004, p= 0.006) were higher in pts residing in cold compared to warm regions while CD8 T cells, T regulatory cells, macrophages, dendritic cells, natural killer cells were similar in both regions. Macrophage signaling, cholesterol metabolism and the pathway for negative regulation of cell migration in angiogenesis were upregulated in pts living in warm compared to cold regions. Conclusion Early-stage BC pts living in cold have worse OS and RFS compared to warm regions. Our study is the first to report significant differences in the TME among these pts. However, it is important to acknowledge that the limited availability of comprehensive data regarding patients' relocation to different environmental temperature zones during treatments, and individual temperature preferences may be considered a potential limitation of our study. To ensure the validity and robustness of these intriguing findings, larger multi-center studies should be conducted, encompassing detailed information on the duration of exposure to both warm and cold weather conditions. Table 1: OS and RFS of patients living in warm vs cold environments Citation Format: Arya Mariam Roy, Spencer Rosario, Anthony George, Qiang Hu, Brian Czerniecki, John Carpten, Virginia Borges, Bryan Schneider, Jill Kolesar, Christopher Moskaluk, Craig Shriver, Cindy Matsen, Shridar Ganesan, Sneha Phadke, Wajeeha Razaq, Kristopher Attwood, Shipra Gandhi. Impact of Environmental Temperature on Clinical Outcomes and Tumor Microenvironment of Early-Stage 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 PO3-03-01.

Abstract PO1-14-10: Comprehensive clinicogenomic characterization of inflammatory breast cancer

Abstract BACKGROUND: Inflammatory breast cancer (IBC) is a rare type of breast cancer associated with a unique clinical presentation and overall poor outcomes, recognized as a distinct category by the AJCC staging system. The biological mechanisms driving the IBC phenotype are relatively undefined—partially due to a lack of comprehensive, large-scale genomic studies and limited clinical cohorts. Here, we report one of the largest, subtype-informed clinicogenomic characterizations of IBC to date. METHODS: A retrospective analysis of 2457 patients with metastatic breast cancer who underwent targeted tumor-only DNA-sequencing (OncoPanel, up to 447 cancer-associated genes) was performed at Dana-Farber Cancer Institute. Clinicopathologic, single nucleotide variant (SNV), copy number variant (CNV) and tumor mutational burden (TMB) comparisons were made between IBC and non-IBC cases. Median follow-up was 28.1 months. RESULTS: Our profiled cohort included 140 patients with IBC specimens (n = 68 primary tumors, 72 metastatic tumors) and 2317 patients with non-IBC specimens (n = 702 primaries, 65 local recurrences, 1550 metastases). Of these, 87.4% of patients were White, 4.7% Black, 3.6% Asian or Pacific Islander, and 4.3% other/unknown. Clinicopathologic differences between IBC and non-IBC cases were consistent with previous reports—including younger age at diagnosis of metastatic disease (51 vs 54 years, p = 0.04), and a higher proportion of grade 3, estrogen receptor-negative and HER2-positive tumors (p < 0.001). Among the hormone-receptor (HR)-positive subtype, IBC tumors showed a significant enrichment in Luminal B (LumB)-inferred disease (62.5% vs 39.8%, p < 0.005), defined as tumors with grade 3 or progesterone receptor staining < 10%. The most recurrent somatic alterations spanning all subtypes in IBC were TP53 (72%), ERBB2 (32%), PIK3CA (24%), CCND1 (12%), MYC (9%), FGFR1 (8%) and GATA3 (8%). A multivariate logistic regression analysis accounting for HR and HER2 status revealed a significant enrichment in TP53 SNVs in IBC vs non-IBC (OR 2.08 [95% CI 1.34-3.24], adjusted p-value 0.04). Frequency of TP53 alterations in IBC vs non-IBC cases was 85.1% vs 64.3% in HER2-positive and 50.0% vs 27.7% in HR-positive disease—with an enrichment of TP53 mutations in IBC LumB-inferred tumors vs non-LumB (64% vs 26%, p < 0.05). When comparing HR+ IBC LumB vs HR+ non-IBC LumB cases, TP53 mutations were again enriched (p < 0.05)—suggesting LumB-like histopathology is not the only driving feature of TP53 enrichment in HR+ IBC. TMB did not differ substantially between IBC and non-IBC and no other statistically significant enrichments were observed, including when grouping mutations into six canonical cancer pathways (cell cycle, Notch signaling, PI3K pathway, RTK/RAS signaling, TP53 pathway and WNT signaling). CONCLUSIONS: Taken together, this study provides a comprehensive landscape of somatic alterations in a large cohort of patients with metastatic IBC and non-IBC. Our data support a lack of major genomic differences other than enrichments in TP53 mutations and an associated LumB-like histopathology. These results both reinforce the importance of TP53 mutations in IBC biology and suggest additional analyses beyond somatic DNA-level changes are warranted. Future efforts with the DFCI IBC cohort will assess germline-somatic interactions, non-genomic or transcriptomic characterizations, and potential environmental influences to better understand the mechanisms driving this unique disease. Citation Format: Nolan Priedigkeit, Beth Harrison, Melissa Hughes, Robert Shue, Yvonne Li, Gregory Kirkner, Claire Remolano, Sarah Strauss, Janet Files, Anne-Marie Feeney, Ayesha Mohammed-Abreu, Ana Garrido-Castro, Romualdo Barroso-Sousa, Brittany Bychkovsky, Faina Nakhlis, Jennifer Bellon, Tari King, Bruce Johnson, Lynette Sholl, Deborah Dillon, Beth Overmoyer, Sara Tolaney, Andrew Cherniack, Nancy Lin, Filipa Lynce. Comprehensive clinicogenomic characterization of inflammatory 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 PO1-14-10.

Abstract PS14-07: Exploring Circulating Leukocyte RNA Expression: Implications for Treatment Outcomes and Immune-Related Adverse Events in Patients with Triple Negative Breast Cancer Enrolled in the GeparNuevo Trial

Abstract Abstract Exploring Circulating Leukocyte RNA Expression: Implications for Treatment Outcomes and Immune-Related Adverse Events in Patients with Triple Negative Breast Cancer Enrolled in the GeparNuevo Trial Background: Significant research has been conducted on the influence of immune checkpoint inhibitor therapy on tumor microenvironment, particularly with regard to tumor-infiltrating immune cells. Nevertheless, our understanding of the circulating immune repertoire and its association with treatment outcomes remains limited. Consequently, our subproject of the GeparNuevo trial aimed to explore the RNA phenotype of circulating leukocytes and its impact on overall survival (OS), and adverse events of patients enrolled in the GeparNuevo trial (Loibl S et al. Annals Oncol 2022). Methods: The GeparNuevo phase II trial focused on the effects of neoadjuvant nab-paclitaxel followed by epirubicin/cyclophosphamide (nabP-EC) chemotherapy combined with the anti-PD-L1 immune checkpoint inhibitor durvalumab versus placebo in patients with non-metastatic triple-negative breast cancer. RNA-stabilizing PAXgene tubes were used to collect blood samples prior to treatment initiation. RNA was extracted from circulating leukocytes of 117 patients and analyzed using a custom NanoString nCounter CodeSet, including 290 immune-related target genes. The associations between 16 immune cell scores, 26 immune signaling scores, 31 individual gene expression patterns, OS, and immune-related adverse events (irAEs) were analyzed. Results: Univariate Cox regression analysis using continuous scores revealed a significant correlation between PIP3 activates AKT signaling, T cells, CDK2, and TIMP1 expression with OS in the placebo arm. Higher expression of PIP3 activates AKT signaling, T cells, and CDK2, as well as lower expression of TIMP1, were associated with prolonged survival. Notably, T cell scores and CDK2 expression exhibited a significant interaction with the treatment arm (p=0.0489 and 0.0210, respectively). Multivariate Cox regression analysis demonstrated a significant association of DPP4, ICOS and MYC expression with OS. Additionally, CDK2, CDKN2A, F5 and HLA-DRA expression were linked to the presence of irAEs. In the durvalumab arm, TNFR2 non canonical NFkB pathway signaling, CDK2 and CDKN2A expression showed an inverse association with the occurrence of irAEs. Conclusions: Our study provides preliminary evidence that RNA derived from circulating leukocytes may serve as a potential biomarker for predicting treatment outcomes and identifying patients prone to develop side effects during standard-of-care chemotherapy or immune checkpoint therapy. These findings highlight the potential utility of peripheral immune cell RNA profiling in improving treatment strategies and patient management. Further research and validation are necessary to fully comprehend the clinical significance and broader implications of these findings. Citation Format: Hanna Hübner, Matthias Ruebner, Andreas Schneeweiss, Carsten Denkert, Hans-Peter Sinn, Michael Braun, Thomas Karn, Bruno V. Sinn, Dirk-Michael Zahm, Jörg Thomalla, Jens Huober, Claus Hanusch, Michael Untch, Christine Solbach, Theresa Link, Natalie Filmann, Julia Rey, Johannes Holtschmidt, Sibylle Loibl, Peter A. Fasching. Exploring Circulating Leukocyte RNA Expression: Implications for Treatment Outcomes and Immune-Related Adverse Events in Patients with Triple Negative Breast Cancer Enrolled in the GeparNuevo Trial [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 PS14-07.

Abstract PO4-15-05: Genomic characterization and molecular predictive biomarkers for chemotherapy in a real-world population with metastatic triple-negative breast cancer

Abstract Background: The genomic landscape of metastatic triple-negative breast cancer (mTNBC) in a real-world population is poorly defined and despite recent advances in precision oncology, molecular biomarkers for chemotherapy are largely lacking. The presence of specific genomic alterations and alterations in oncogenic pathways have the potential to provide prognostic as well as predictive information for chemotherapy, which remains the backbone treatment strategy for mTNBC. Methods: We conducted next-generation sequencing (NGS) with the FoundationOne CDx panel on tissue from the primary tumor and/or metastasis of 112 consecutive patients with mTNBC that were treated between 1998 and 2019 at 4 different Swedish hospitals. Every genomic alteration was, if applicable, subdivided into 1 of 10 canonical oncogenic pathways and noted for its involvement in the homologous recombination pathway (HRP). Frequently altered genes and pathways were then correlated with overall survival (OS) and evaluated regarding their association with progression-free survival (PFS) and response rate (RR) in patients treated with different chemotherapy agents. Tumor samples of patients with rapid progression or exceptional response to chemotherapy underwent exploratory comparison with regards to frequently altered genes and pathways and known clinical prognostic factors were compared between the two patient groups. Results: After excluding 15 patients due to insufficient quantity/quality of tumor tissue or absence of clinical data, 97 patients were analyzed. The median age at diagnosis of metastatic disease was 61 (range: 28-90), 80 % had visceral disease and 26 % received platinum-based chemotherapy in the first line setting. The most frequently altered genes were: TP53 (82 %), RAD21 (25 %), PIK3CA (23 %), MYC (22 %) and BRCA 1 or 2 (16 %). The most frequently altered pathways were TP53 (86 %), PI3K (60 %), RTK/RAS (47 %) and cell cycle (36 %). In total, 26 % of patients had an alteration in the HRP. None of the most frequently occurring genomic alterations were associated with OS. Variants of clinical significance in the HRP and BRCA1/BRCA2 genes were associated with a longer PFS in patients treated with platinum-based chemotherapy in the first line setting (HR 0.12-0.84 and 0.12-0.92). Exceptional responders to chemotherapy exhibited a more favorable clinical profile than rapid progressors (median age 51.5 vs 66.5, median Charlson comorbidity index 0.5 vs 3) whereas some numerical difference in the genomic profiling in terms of genomic alterations in MYC and RAS/RTK pathways (more often in exceptional responders) could be observed. Conclusions: We found no genomic alteration with prognostic significance in a cohort of mTNBC patients treated with chemotherapy in a real-world setting. Somatic variants of clinical significance in BRCA 1 and 2 and other HRP-related genes seem to define subgroups of patients with mTNBC that respond favorably to platinum-based chemotherapy. Further research into the genomic landscape of tumors from patients with rapid progression or exceptional response to specific treatment strategies can provide insights into mechanisms of resistance and identify new predictive biomarkers. Citation Format: Erik Olsson, Henrik Lindman, Evangelos Digkas, Viktoria Thurfjell, Haidar Mir Ali, Ute Krüger, Anna-Karin Wennstig, Marie Sundqvist, Antonis Valachis. Genomic characterization and molecular predictive biomarkers for chemotherapy in a real-world population with metastatic 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 PO4-15-05.

Verteporfin inhibits TGF-β signaling by disrupting the Smad2/3-Smad4 interaction.

Transforming growth factor-β (TGF-β) signaling plays a crucial role in pathogenesis, such as accelerating tissue fibrosis and promoting tumor development at the later stages of tumorigenesis by promoting epithelial-mesenchymal transition, cancer cell migration, and invasion. Targeting TGF-β signaling is a promising therapeutic approach, but non-specific inhibition may result in adverse effects. In this study, we focus on the Smad2/3-Smad4 complex, a key component in TGF-β signaling transduction, as a potential target for cancer therapy. Through a phase-separated condensate-aided biomolecular interaction system, we identified verteporfin (VP) as a small-molecule inhibitor that specifically targets the Smad2/3-Smad4 interaction. VP effectively disrupted the interaction between Smad2/3 and Smad4 and thereby inhibited canonical TGF-β signaling, but not the interaction between Smad1 and Smad4 in BMP signaling. Furthermore, VP exhibited inhibitory effects on TGF-β-induced epithelial-mesenchymal transition and cell migration. Our findings indicate a novel approach to develop protein-protein interaction inhibitors of the canonical TGF-β signaling pathway for treatments of related diseases.

Abstract PO3-26-12: Targeting Lipocalin-2 in HER2+ Inflammatory Breast Cancer Using Herceptin-Conjugated Liposomes

Abstract Inflammatory breast cancer (IBC) is a rare and aggressive subtype of BC that accounts for 1 to 5% of all types of BC. Due to its inflammatory characteristics and the absence of a palpable mass, IBC is usually confounded with a mastitis. Unfortunately, once properly diagnosed, IBC has already metastasized. A high portion of IBC tumors overexpress human epidermal growth factor receptor 2 (HER2). These IBC patients (HER2+) are treated with Trastuzumab, a monoclonal antibody. However, many IBCs become resistant to this therapy. Thus, there is not an optimal treatment against this horrendous disease. One potential target against IBC is Lipocalin-2 (LCN2), a secreted protein involved in iron homeostasis, immune responses, transport of siderophores, and epithelial cell differentiation. In many aggressive tumors, high levels of LCN2 have been associated with increased cancer cell motility, proliferation, angiogenesis, invasion, and metastasis. LCN2 is aberrantly abundant in IBCs compared with non-IBC patients and cell lines. This information indicates that LCN2 could be a target for IBC therapy. Our research team showed that small interference RNAs (siRNAs) targeting LCN2 effectively reduced the cell proliferation and invasion ability of IBC cells. To systemically deliver siRNA specifically to HER2+ IBC cells, we prepared Herceptin-conjugated liposome loaded with siRNA against LCN2. Trastuzumab conjugated liposomes werecharacterized by using dynamic light scattering to measure the size distribution and the zeta potential. Our formulation displayed sizes of 18.3 and 73.7 nm which could correspond to free DSPE-PEG-(2000) trastuzumab and the trastuzumab-liposome, respectively. To determine the optimal timepoint of liposome internalization into IBC cell lines, we incubated HER2+ IBC3 cells with liposomes for different time-points. Optimal internalization was observed at 24 and 48-hr. To further understand the downstream effectors of LCN2 in IBC cells, we performed RNAseq in siRNA-mediated LCN2 knockdown cells. Further bioinformatics with the RNAseq data, revealed 138 dysregulated genes following LCN2 knockdown as compared with a negative control (NC) siRNA. Particularly, increased STAT1 levels has been associated with to distant metastasis and poor clinical prognosis in cancer. In our study, STAT1 levels decreased after LCN2 silencing. Ingenuity Pathway Analysis (IPA) showed 25 canonical pathways altered in the LCN2 knockdown cells as compared with the NC-siRNA. These results indicate that LCN2 activate molecular pathways involved in cell proliferation, invasion and metastasis in IBC cells. Citation Format: Marienid Flores-Colón, Pablo Vivas-Rivera, Pablo E. Vivas-Mejía. Targeting Lipocalin-2 in HER2+ Inflammatory Breast Cancer Using Herceptin-Conjugated Liposomes [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 PO3-26-12.

Angiotensin (1-7) Inhibits Transforming Growth Factor-Β1-Induced Epithelial-Mesenchymal Transition of Human Keratinocyte Hacat Cells in vitro.

Angiotensin (1-7) (Ang-(1-7)) is an emerging component of the renin-angiotensin system (RAS) with effective anti-fibrosis properties and has been shown to interfere with epithelial-mesenchymal transition (EMT) by numerous studies. In recent years, EMT has been proposed as a new therapeutic target for skin fibrotic diseases such as keloids. However, the effect of Ang-(1-7) on EMT in skin is still unclear. Hence, the purpose of this study was to explore the effect of Ang-(1-7) on Transforming growth factor-β1(TGF-β1)-induced EMT of human immortalized keratinocytes HaCaT in vitro. The study involved the use of the human immortalized keratinocyte cell line (HaCaT). The cells were cultured in high-glucose DMEM medium with 10% fetal bovine serum and 1% penicillin-streptomycin. Four groups were created for experimentation: control group (Group C), TGF-β1-treated group (Group T), Ang-(1-7)-treated group (Group A), and a group treated with both TGF-β1 and Ang-(1-7) (Group A + T). Various assays were conducted, including a cell proliferation assay using CCK-8 solution, a scratch wound healing assay to evaluate cell migration, and Western blotting to detect protein expressions related to cell characteristics. Additionally, quantitative real-time polymerase chain reaction (PCR) was performed to analyze epithelial-mesenchymal transition (EMT) related gene expression levels. The study aimed to investigate the effects of TGF-β1 and Ang-(1-7) on HaCaT cells. We found that Ang-(1-7) not only reduced the migration of HaCaT cells induced by TGF-β1 in vitro but also reduced the expression of α-SMA and vimentin, and restored the protein expression of E-cadherin and claudin-1. Mechanistically, Ang-(1-7) inhibits the phosphorylation levels of Smad2 and Smad3 in the TGF-β1 canonical pathway, and suppresses the expression of EMT-related transcription factors (EMT-TFs) such as SNAI2, TWIST1, and ZEB1. Taken together, our findings suggest that Ang-(1-7) inhibits TGF-β1-induced EMT in HaCaT cells in vitro by disrupting the TGF-β1-Smad canonical signaling pathway. These results may be helpful in the treatment of EMT in skin fibrotic diseases such as keloids.

Sleep Fragmentation after Traumatic Brain Injury Impairs Behavior and Conveys Long-Lasting Impacts on Neuroinflammation

Traumatic brain injury (TBI) causes a prolonged inflammatory response in the central nervous system (CNS) driven by microglia. Microglial reactivity is exacerbated by stress, which often provokes sleep disturbances. We have previously shown that sleep fragmentation (SF) stress after experimental TBI increases microglial reactivity and impairs hippocampal function 30 days post-injury (DPI). The neuroimmune response is highly dynamic the first few weeks after TBI, which is also when injury induced sleep-wake deficits are detected. Therefore, we hypothesized that even a few weeks of TBI SF stress would synergize with injury induced sleep-wake deficits to promote neuroinflammation and impair outcome. Here, we investigated the effects of environmental SF in a lateral fluid percussion model of mouse TBI. Half of the mice were undisturbed, and half were exposed to 5 hours of SF around the onset of the light cycle, daily, for 14 days. All mice were then undisturbed 15-30 DPI, providing a period for SF stress recovery (SF-R). Mice exposed to SF stress slept more than those in control housing 7-14 DPI and engaged in more total daily sleep bouts during the dark period. However, SF stress did not exacerbate post-TBI sleep deficits. Testing in the Morris water maze revealed sex dependent differences in spatial reference memory 9-14 DPI with males performing worse than females. Post-TBI SF stress suppressed neurogenesis-related gene expression and increased inflammatory signaling in the cortex at 14 DPI. No differences in sleep behavior were detected between groups during the SF stress recovery period 15-30 DPI. Microscopy revealed cortical and hippocampal IBA1 and CD68 percent-area increased in TBI SF-R mice 30 DPI. Additionally, neuroinflammatory gene expression was increased, and synaptogenesis-related gene expression was suppressed in TBI-SF mice 30 DPI. Finally, IPA canonical pathway analysis showed post-TBI SF impaired and delayed activation of synapse-related pathways between 14 and 30 DPI. These data show that transient SF stress after TBI impairs recovery and conveys long-lasting impacts on neuroimmune function independent of continuous sleep deficits. Together, these finding support that even limited exposure to post-TBI SF stress can have lasting impacts on cognitive recovery and regulation of the immune response to trauma.

Mechanistic study of Huangqi Guizhi Wuwu decoction amelioration of doxorubicin-induced cardiotoxicity by reducing oxidative stress and inhibiting cellular pyroptosis

Huangqi Guizhi Wuwu Decoction (HQGZWWD) has shown promising potential in treating various cardiovascular diseases. This study aimed to elucidate the molecular basis and therapeutic role of HQGZWWD in the treatment of doxorubicin (DOX)-induced myocardial injury. The HPLC fingerprint of HQGZWWD was used to analyze the active components. A DOX-induced myocardial damage rat model was developed, and the therapeutic effects of HQGZWWD were evaluated using echocardiography, myocardial enzyme levels, and hematoxylin and eosin staining. Network pharmacology was used to screen treatment targets, and western blotting and immunohistochemistry were performed to assess cellular pyroptosis levels. Oxidative stress levels were measured using assay kits, and mitochondrial damage was examined using transmission electron microscopy. An in vitro model of DOX-induced cell damage was established, and treatment was administered using serum containing HQGZWWD and N-acetylcysteine (NAC). Oxidative stress levels were detected using assay kits and DCFH-DA, whereas cellular pyroptosis levels were assessed through WB, immunofluorescence, and ELISA assays. HQGZWWD ameliorated DOX-induced myocardial injury. Network pharmacology identified IL-1β and IL-18 as crucial targets. HQGZWWD downregulated the protein levels of the inflammatory factors IL-1β and IL-18, inhibited the expression of GSDMD-NT, and simultaneously suppressed the synthesis of Caspase-1, ASC, NLRP3, and Caspase-11. Additionally, HQGZWWD inhibited oxidative stress, and the use of NAC as an oxidative stress inhibitor resulted in significant inhibition of the GSDMD-NT protein in H9C2 cells. These findings highlight the myocardial protective effects of HQGZWWD by inhibiting oxidative stress and suppressing both canonical and non-canonical pyroptotic pathways.

Not just for lymphoid cells: The role of the noncanonical NF-κB signaling pathway in early and late myelopoiesis with a focus on hypereosinophilic disorders.

The noncanonical NF-κB pathway is involved in lymphoid organ development, B cell maturation, and cytokine production. However, new research has demonstrated that this pathway is also key for the orderly and sequential maturation of myeloid cells, including neutrophils and eosinophils. When this pathway is disrupted or constitutively activated, aberrations in hematopoietic stem and progenitor cell (HSPC) survival and proliferation, as well as subsequent granulopoiesis and eosinophilopoiesis are affected. Disturbance of such a coordinated and delicate process can manifest in devastating clinical disease including acute and chronic myeloid leukemias (AML and CML, respectively), pre-leukemic processes such as myelodysplastic syndrome (MDS) or hyperinflammatory conditions like Hypereosinophilic Syndrome (HES). In this review, we will discuss the molecular machinery within the noncanonical NF-κB pathway, crosstalk with the canonical NF-κB pathway, murine models of noncanonical signaling, as well as how aberrations in this pathway manifest in leukemic or hyperinflammatory disease with a focus on HES. Potential and promising drug therapies will also be discussed, emphasizing the noncanonical NF-κB pathway as a potential target for improved treatment for patients suffering from leukemia or idiopathic HES. The hope is that review of such mechanisms and treatments may eventually result in findings that aid physicians in rapidly diagnosing and more accurately classifying patients suffering from such complex and overlapping hematopoietic diseases.