Cell Signaling Research Articles

Abstract PO4-16-01: EZH2 is Overexpressed in Invasive Carcinoma and DCIS in both BRCA1 and BRCA2 Mutation Carriers

Abstract Background The transcriptional repressor EZH2 (Polycomb Group Protein Enhancer of Zeste Homologue 2) is overexpressed in multiple neoplasms including breast cancer where it associates with high proliferation, aggressive features and poor prognosis and is of interest as a potential therapeutic target. EZH2 nuclear expression in breast cancer has been shown to correlate specifically with high tumor grade and basal-like histologic features but has not been well studied in cancers from BRCA carriers. Here we evaluate the expression of EZH2 protein in BRCA-associated invasive breast carcinomas and precursor lesions. Methods Eighteen cases of invasive breast cancer in consented BRCA carriers (8 BRCA1 and 10 BRCA2) treated at Dana Farber Cancer Institute from 2001-2019 were reviewed by two pathologists (TAD and RBPP). Histopathologic features (histologic subtype, nuclear grade, mitotic score) and presence of precursor lesions were recorded. Immunohistochemistry for EZH2 was performed on freshly cut formalin-fixed paraffin-embedded tissue sections (1-3 sections/case) using a monoclonal antibody against EZH2 (D2C9, Cell Signaling). Nuclear EZH2 expression was scored as negative (score=1, no staining); weak (score 2, < 25%); moderate (score=3, 25–75%); and strong (score=4, >75%), of any intensity following published criteria. EZH2 overexpression was defined as scores 3 and 4. Results The majority of the invasive carcinomas showed grade III histology (13/18, 72%), as expected. Of 8 BRCA1 cases, 6 were classified as grade III (75%), 1 as grade II, and 1 as grade I (7 cases of invasive ductal carcinoma and 1 case of invasive tubular carcinoma). Of 10 BRCA2 cases, 7 were classified as grade III (70%) and 3 as grade II (30%) (5 cases of invasive ductal carcinoma and 5 cases of invasive carcinoma with ductal and lobular features). The majority of cases (13 of 18, 72%) showed high expression of EZH2 in the invasive carcinoma (6 BRCA1 and 7 BRCA2 cases; 2 grade II and 11 grade III). Of 13 cases with EZH2 overexpression, 6 were ER+/PR+/HER2- and 7 were triple negative, with 9 invasive ductal carcinomas and 4 invasive carcinomas with ductal and lobular features. Of 5 cases with no/low EZH2 overexpression, 1 was ER+/PR+/HER2-, 3 were ER+/PR-/HER2- and 1 was triple negative, with 3 invasive ductal carcinomas, 1 invasive carcinoma with ductal and lobular features and 1 invasive tubular carcinoma. In all cases with adjacent ductal carcinoma in situ (DCIS) (n=14), the pattern of EZH2 expression in the DCIS was identical to that seen in the invasive carcinoma (11 high and 3 low expression). Overexpression of EZH2 was not observed in any other precursor lesions (LCIS, ADH, ALH) or in normal tissue. Conclusion EZH2 protein overexpression is seen in the majority of invasive carcinomas in BRCA carriers, both in BRCA1 and in BRCA2. In all cases, the associated DCIS shows the same pattern of expression as the invasive carcinoma, suggesting that overexpression occurs early in tumorigenesis, while tumor cells are still confined within the ducts. The high rate of overexpression of EZH2 in BRCA1- and BRCA2-associated tumors suggests a potential role for EZH2 as a target and/or a biomarker in these cancers. Citation Format: Tabata Alves Domingos, Roberto Bonfim Pimenta Peixoto, Rodrigo Fonseca Abreu, Ashka Patel, Krishan Taneja, McKenna Moore, Celina Kleer, Deborah Dillon. EZH2 is Overexpressed in Invasive Carcinoma and DCIS in both BRCA1 and BRCA2 Mutation Carriers [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 PO4-16-01.

Abstract PO5-17-07: MUCINOUS BREAST CARCINOMA: REAL WORLD DATA IN A REFERENCE CENTER IN SÃO PAULO

Abstract BACKGROUND: Mucinous breast carcinoma (MC) is a subtype of invasive carcinoma that corresponds to 1 to 6% of all types of breast cancer. It occurs when the mucinous component accounts for more than 50% of the tumor producing extracellular mucin in large scale. Mucin is a complex carbohydrate engaged in processes such as epithelial differentiation, adhesion modulation and cell signaling. Along with other special types of breast neoplasia, MC appears to have an unique behavior and, since it’s low amount of cases, a detailed clinical evaluation has been difficult. OBJECTIVES: To describe the profile of patients with breast MC that were treated in a reference center in São Paulo, Brazil as well as evaluate its management and outcomes. METHODS: A descriptive longitudinal observation study conducted at São Paulo’s Women’s Health Reference Center - Hospital da Mulher from July 2022 to January 2023, based on 192 medical records of patients diagnosed with MC from 2003 to 2018. RESULTS: 192 patient’s records were included. The age range was 31-93 years, out of which 74,3% of the patients were older than 50 when diagnosed. 48,7% identified as white and 43,7% showed earlier diagnoses of systemic hypertension. The time range between diagnosis and first treatment was 4,5 months. Out of the histopathological features, the nuclear grade was 3 in 69 patients (34,6% of the cases), and histological grade 1 in 52,7%. About pathological staging, the size of the tumors went form 0,5 to 9,5 cm, with an average of ± 4,7cm, the majority of them showed positive ER (89,4%), negative PR (85%), and were negative for Her-2 (76%). Greater number of patients (92,4%) went through both clinical and surgical treatment. Only 15 (7,5%) had isolated clinical treatment. 31% of the patients received systemic chemotherapy treatment, 25% of those were neoadjuvant. 81,9% of patients received aromatase inhibitor and only 17% radiotherapy. Distant metastasis were diagnosed in 27 patients (13,5%) and the local recurrence was observed in 15,3%. Out of distant metastasis, pulmonary type was more prevalent, registered in 45,7% of patients, followed by osseous (22,88%) and hepatic (14,2%). 33 patients (16,5%) progressed to death, however, the specific cause was not determined. CONCLUSION: MC is a rare subtype of breast cancer, more prevalent in post-menopausal and white women. Frequently shows low histological grade and positive hormonal receptors, which reinforces the idea of a indolent behavior. The treatment is mostly surgical, with adjuvance if necessary. Metastasis are infrequent and knowing the profile of patients with MC, as well as it’s behavior, allows the better management when facing this diagnosis. Citation Format: Marina Fleury De Figueiredo, Andre Mattar, Felipe Cavagna, Ana Lígia Minatel da Silva, Rafaella Nadai, Juliana Medeiros de Oliveira, Luma Fleury de Figueiredo. MUCINOUS BREAST CARCINOMA: REAL WORLD DATA IN A REFERENCE CENTER IN SÃO PAULO [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 PO5-17-07.

Abstract PO3-13-08: D17104: Aryl Hydrocarbon Receptor Signaling and Other Biomarkers of Fat Metabolism in Human Mammary Adipose Tissue from Women with Malignant Breast Disease

Abstract Background: Obesity is a recognized risk factor for the development of metabolic disease and breast cancer possibly due to chronic inflammation mediated by cytokines secreted by adipose tissue. Given this, our group examined differential expression of several markers of chronic inflammation in adipose tissue of both benign and malignant breast tissue from patients. The aryl hydrocarbon receptor (AhR) is an important cytosolic, ligand-dependent transcription factor found within hepatocytes and adipocytes that is upregulated in many types of cancer. AhR promotes the initiation, progression, and metastasis of cancer cells allowing certain cancers to evade immune recognition (Xue et al. Front. Immunology 2018). Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme that generates kynurenine (KYN) from tryptophan (TRP), which acts as an AhR agonist. We sought to determine expression of IDO1 via immunohistochemistry (IHC) in mammary adipose tissue associated with benign or malignant breast. We hypothesized that adipose tissue adjacent to malignant breast tissue may have heightened expression of IDO1 in addition to Cyp1b1, and SPP1; both of which are also markers of increased aryl-hydrocarbon transcriptional activity, compared to adipose tissue noncontiguous to malignant lesions. KYN affects gene expression for insulin resistance and levels are elevated in plasma of older obese women compared to lean women; however, the relationship to breast malignancy is not known. Methods: IDO1 (Cell Signaling, 1:200), Cyp1b1 (Thermo Fisher, 1:300), and SPP1 (Thermo Fisher, 1:200) IHC was performed according to the manufacturers' protocols. Adipose tissue immediately adjacent to, and >2 mm away from, tumors (three DCIS, 11 invasive carcinomas) was evaluated in 14 patients. Adipose tissue in separate tissue blocks from benign breast tissue (three ipsilateral, 11 contralateral) in the same patients was also evaluated. Human plasma KYN levels were assayed by HPLC in the Dartmouth Clinical Pharmacology Shared Resource as described previously (Rojas et al. Obesity 2021). Results: In this preliminary pilot study, there was no difference in staining pattern for IDO1 in adipose tissue present in malignant and benign samples. Two patients showed increased Cyp1b1 and SPP1 staining immediately adjacent to the invasive carcinoma (Figure 1). Patients with benign breast disease did not show increased Cyp1b1 or SPP1 staining. Results will be confirmed using qPCR for mRNA. Plasma samples from patients with benign disease showed a lower level of KYN expression on average (0.370 ug/mL, N = 8) compared to plasma from patients with malignant disease (0.424, N = 10). Additionally, the ratio of KYN:TRP was lower in the benign group (0.065) compared to the cohort with malignancy (0.080). Univariate linear regression will be used to further assess relationships between continuous variables in the study population (e.g., patient age, BMI, KYN plasma levels, benign vs malignant breast disease). Regression lines and the associated slope estimates (b-value) and p-values for regression models will be provided on scatter plots. Conclusions: While there was no difference in IDO1 staining patterns in adipose tissue of malignant and benign samples in this group, tissue samples from patients with invasive carcinoma demonstrated increased Cyp1b1 and SPP1 staining adjacent to the malignant tumor; while benign tissue samples did not show a positive stain. This suggests increased aryl-hydrocarbon transcriptional activity in invasive breast carcinoma—further linking AhR signaling with adipose tissue immune dysregulation and invasive malignancy. Additionally, KYN plasma levels were higher in patients with malignant disease indicating a possible relationship between insulin resistance and breast malignancy. Additional research may offer new insights relating to this important pathway and ultimately treatments aimed at blunting its nefarious effects. Citation Format: Adam Ephraim, Jonathan Pirruccello, Scott Palisoul, Jonathan Marotti, Mary Chamberlin. D17104: Aryl Hydrocarbon Receptor Signaling and Other Biomarkers of Fat Metabolism in Human Mammary Adipose Tissue from Women with Malignant Breast Disease [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 PO3-13-08.

The mechanism of high mobility group box-1 protein and its bidirectional regulation in tumors.

High-mobility group box-1 protein (HMGB1) is a nonhistone chromatin-related protein widely found in eukaryotic cells. It is involved in the transcription, replication, and repair of DNA to maintain nuclear homeostasis. It participates in cell growth, differentiation, and signal transduction. Recent studies showed that HMGB1 has a bidirectional regulatory effect on tumors by regulating TLR4/MYD88/NF-κB and RAGE/AMPK/mTOR signaling pathways. On the one hand, it is highly expressed in a variety of tumors, promoting tumor proliferation and invasion, while on the other hand, it induces autophagy and apoptosis of tumor cells and stimulates tumor-infiltrating lymphocytes to produce an anti-tumor immune response. At present, HMGB1 could be used as a target to regulate the drug resistance and prognostication in cancer. Clinical applications of HMGB1 in cancer need further in-depth studies.

A versatile delivery vehicle for cellular oxygen and fuels, or metabolic sensor? - A review and perspective on the functions of myoglobin.

A canonical view of the primary physiological function of myoglobin (Mb) is that it is an oxygen (O2) storage protein supporting mitochondrial oxidative phosphorylation, especially as the tissue O2 partial pressure (pO2) drops and Mb offloads O2. Besides O2 storage/transport, recent findings support functions for Mb in lipid trafficking and sequestration, interacting with cellular glycolytic metabolites such as lactate (LAC) and pyruvate (PYR) , and "ectopic" expression in some types of cancer cells and in brown adipose tissue (BAT). Data from Mb knockout (Mb-/-) mice and biochemical models suggest additional metabolic roles for Mb, especially regulation of nitric oxide (NO) pools, modulation of BAT bioenergetics, thermogenesis, and lipid storage phenotypes. From these and other findings in the literature over many decades, Mb's function is not confined to delivering O2 in support of oxidative phosphorylation, but also to serve as an O2-sensor that modulates intracellular pO2- and NO-responsive molecular signaling pathways. This paradigm reflects a fundamental change in how oxidative metabolism and cell regulation are viewed in Mb-expressing cells such as skeletal muscle, heart, brown adipocytes, and select cancer cells. Herein, we review historic and emerging views related to the physiological roles for Mb, and present working models illustrating the possible importance of interactions between Mb, gases, and small molecule metabolites in regulation of cell signaling and bioenergetics.

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 PO2-25-07: PEGylated Functional Upstream Domain (PEG-FUD): an anti-cancer therapy for breast Cancer

Abstract Background: Breast cancer (BC) is the most common cancer diagnosed in women. In breast cancers, the extracellular matrix (ECM) is known to play a key role in disease progression by mediating cell signaling processes that drive cancer cell proliferation, migration and invasion. Work by our group identified that aligned collagen fibers perpendicular to the tumor boundary are prognostic of poor patient prognosis. Further, through matrix targeted proteomics we determined that the ECM protein, Fibronectin (FN), organizes with aligned collagen fibers in BC tissues. FN is known as the master regulator of ECM assembly because FN deposition precedes and regulates the deposition of several other ECM proteins. Clinical evidence shows that high expression of FN levels in BC patients correlates with an increased mortality risk. Due to the abundant expression of FN in the tumor microenvironment, FN has been a useful biomarker for cancer imaging and therapy. Despite multiple efforts in developing therapies that target the ECM, currently there are no effective ECM treatments available due to toxicity and lack of specificity. Methods: To target abnormal FN deposition, we used a FN binding peptide, Functional Upstream Domain (FUD), derived from the F1 adhesin from Streptococcus pyogenes. PEGylated-FUD (PEG-FUD) is a potent inhibitor of FN assembly which binds the 70 kDa N-terminal region of FN with high affinity. We hypothesize that PEG-FUD will localize to the tumor site and that targeted disruption of FN assembly with PEG-FUD will reduce ECM deposition, thus block tumor progression in-vivo. In this study, we used an in vivo imaging system to assess the biodistribution of 20-kDa PEG-FUD following subcutaneous injection of Cy5 labeled peptide in 4T1 mammary tumor bearing mice. Additionally, in a second therapeutic experiment, we treated 4T1 mammary tumor bearing mice with 20-kDa PEG-FUD every 48 hr for a total of 10 treatments and measured tumor volume as an indicator of primary tumor burden. Results: As anticipated, we observed PEG-FUD’s accumulation and localization in 4T1 tumors. Additionally, PEG-FUD treatment significantly reduced tumor growth compared to saline treatment. There were no observed changes in standard toxicity assessments such as body weight and spleen weight among treatment groups. After PEG-FUD’s therapeutic treatment, we observed a significant reduction in the deposition of FN matrix and an increase in cleaved caspase-3, a known marker for apoptosis by western blotting providing a potential mechanism of action of PEG-FUD inducing changes in tumor growth. Conclusions: PEG-FUD’s localization in tumors suggests its utility as a solo cancer imaging agent while providing indications that PEG-FUD can be used to deliver other therapeutics to the tumor site as a conjugate in the future. In addition, therapeutic treatment of PEG-FUD inhibited tumor growth providing preliminary evidence for PEG-FUD’s use as a tumor targeting anti-cancer agent. Citation Format: Metti Gari. PEGylated Functional Upstream Domain (PEG-FUD): an anti-cancer therapy for 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 PO2-25-07.

Abstract RF02-02: BBO-10203, a first-in-class, orally bioavailable, selective covalent small molecule that inhibits RAS-driven PI3Kalpha activity without affecting glucose metabolism

Abstract PI3Kα is the most mutated kinase and the second most mutated oncogene in human cancer. Activation of PI3Ka can be achieved by receptor tyrosine kinases such as insulin receptor and insulin-like growth factor receptor 1 and/or by directly interacting with RAS family members. Previous elegant preclinical studies have established that RAS-driven PI3Ka activation is important in tumor cells but may not be involved in cell types controlling glucose metabolism. Alpelisib, a small molecule inhibitor of the kinase activity of PI3Ka, has been approved for the treatment of ER+ PIK3CA mutant breast cancer in combination with fulvestrant after endocrine therapy in advanced or metastatic breast cancer based on an improvement in PFS versus fulvestrant alone. Inhibition of PI3Ka activity by alpelisib in normal tissues resulted in a severe (G3/4) hyperglycemia rate of 37% with frequent dose interruptions and discontinuations. Additionally, preclinical studies have demonstrated that the dysregulation of glucose homeostasis resulting from PI3Ka kinase inhibition leads to hyperinsulinemia that increases pathway flux, rendering kinase inhibitors less effective. Here, we report on a novel covalent small molecule designed to inhibit RAS-mediated activation of the AKT pathway via PI3Ka without the resultant hyperglycemia associated with direct inhibition of PI3Ka kinase activity. BBO-10203 disrupts the physical interaction between RAS and PI3Ka in tumor cells resulting in potent signaling pathway inhibition. This agent selectively binds to PI3Ka and disrupts its interaction with K-,H-, and N-RAS with low single digit nanomolar potency (~5 nM). Breaking the interaction between these two oncogenes inhibits basal pAKT cellular levels (BT-474/KYSE-410 IC50: ~5 nM) in HER2 amplified (HER2amp) and wild-type or mutant PI3Ka cell lines. Even though BBO-10203 does not inhibit the kinase activity of PI3Ka, its effects on cancer cell signaling inhibition and transcriptional regulation highly resemble those of alpelisib. BBO-10203 displays excellent drug-like properties and oral bioavailability. Single dose treatment of KYSE-410 (HER2amp/KRASG12C) tumor bearing mice with increasing doses (1-100 mg/kg, PO) of BBO-10203 results in dose and time dependent inhibition of pAKT in vivo. Maximal inhibition (~80%) is achieved at 30 mg/kg and lasts for 24 hours. Repeated dose treatment of tumor bearing mice with BBO-10203 is well tolerated and results in significant efficacy in PIK3CA mutant as well as HER2amp human xenograft models. In the KYSE-410 xenograft model, BBO-10203 daily oral dosing of 30 mg/kg results in significant tumor regressions. Importantly, treatment with BBO-10203 does not affect insulin signaling in differentiated adipocytes in vitro, nor does it impact glucose metabolism in vivo at 3-times the maximal efficacious dose level in xenograft studies. In conclusion, we have identified a novel approach to inhibit the PI3Ka signaling pathway by blocking its interaction with, and activation by RAS. This approach can achieve strong pAKT inhibition in tumor cells without changes in glucose metabolism. Clinical investigation of BBO-10203 for the treatment of both ER+/PIK3CA mutant and HER2amp breast cancer is warranted. Citation Format: Pedro Beltran, Simanshu Dhirendra, Rui Xu, Ming Chen, Daniel Czyzyk, Sofia Donovan, Siyu Feng, Cindy Feng, Lijuan Fu, Felice Lightstone, Ken Lin, Anna Maciag, Dwight Nissley, Erin Riegler, Kerstin Sinkevicius, Andrew Stephen, James Stice, David Turner, Bin Wang, Keshi Wang, Yue Yang, Cathy Zhang, Frank McCormick, Eli Wallace. BBO-10203, a first-in-class, orally bioavailable, selective covalent small molecule that inhibits RAS-driven PI3Kalpha activity without affecting glucose metabolism [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 RF02-02.

Phage-Displayed SH2 Domain Libraries: From Ultrasensitive Tyrosine Phosphoproteome Probes to Translational Research.

Tyrosine phosphorylation is a critical regulator of cell signaling. A large fraction of the tyrosine phosphoproteome, however, remains uncharacterized, largely due to a lack of robust and scalable methods. The Src homology 2 (SH2) domain, a structurally conserved protein domain present in many intracellular signal-transducing proteins, naturally binds phosphorylated tyrosine (pTyr) residues, providing an ideal scaffold for the development of sensitive pTyr probes. Its modest affinity, however, has greatly limited its application. Phage display is an in vitro technique used for identifying ligands for proteins and other macromolecules. Using this technique, researchers have been able to engineer SH2 domains to increase their affinity and customize their specificity. Indeed, highly diverse phage display libraries have enabled the engineering of SH2 domains as affinity-purification (AP) tools for proteomic analysis as well as probes for aberrant tyrosine signaling detection and rewiring, and represent a promising class of novel diagnostics and therapeutics. This review describes the unique structure-function characteristics of SH2 domains, highlights the fundamental contribution of phage display in the development of technologies for the dissection of the tyrosine phosphoproteome, and highlights prospective uses of SH2 domains in basic and translational research.

Dermal Dive: An Overview of Cutaneous Wounding Techniques in Zebrafish

Cutaneous wounds are common injuries that affect millions of people around the world. In vulnerable populations such as the elderly and those with diabetes, defects in wound healing can lead to the development of chronic open wounds. Although mammalian models are commonly used to study cutaneous wound healing, the challenges of invivo imaging in mammals have hampered detailed observation of cell coordination and cell signaling during wound healing. The zebrafish is becoming increasingly popular for studying cutaneous wound healing owing to its genetic accessibility, suitability for experimental manipulation, and the ability to perform live, invivo imaging with cellular or even subcellular resolution. In this paper, we review some of the techniques that have been developed for eliciting cutaneous wounds in the zebrafish, including an economical method we recently developed using a rotary tool that generates consistent and reproducible full-thickness wounds. Combined with the thousands of transgenic lines and experimental assays available in zebrafish, the ability to generate reproducible cutaneous wounds makes it possible to study key cellular and molecular events during wound healing using this powerful experimental model organism.

Interview with Surendra Ghaskadbi – President of the Indian Society of Cell Biology

ABSTRACT Surendra Ghaskadbi is a cell and developmental biologist who held the positions of Senior and Emeritus Scientist at the MACS–Agharkar Research Institute in Pune, India. His lab has been particularly interested in cell signalling and pattern formation during early development, focusing on using Hydra as a research organism. More recently, Surendra has played a role in teaching and outreach in India, and he is also the current President of the Indian Society of Cell Biology. We chatted to him to find out more about his career, his advice for junior researchers and his role as society President.

Shear Stress Induces Morphology Changes in Human Lung Microvascular Endothelial Cells via Calcium Influx

Introduction: The pulmonary vascular endothelium is in contact with laminar blood flow, resulting in a physiologic degree of shear stress exerted on endothelial cells (ECs). In vitro cell culture of ECs is typically performed under static conditions, leading to underappreciation of the impact of shear stress. Calcium signaling is among the most ubiquitous cell signaling mechanisms, and changes in intracellular calcium concentration ([Ca2+]i) have been linked to shear stress. The impact of shear stress on human lung microvascular endothelial cell (hLMVEC) morphology and cell signaling is not well described. We hypothesized that hLMVECs exposed to shear stress would undergo Ca2+-dependent morphologic changes. Methods: The effect of shear stress on [Ca2+]i was first tested using ratiometric measurement. Early passage (p≤8) primary hLMVECs from three unique donor lots were plated in gelatin-coated Ibidi 0.2 uM depth plates for 16 h, then incubated with the calcium indicator Fura-2 AM for 1 h. Measurement of [Ca2+]i was obtained over 15 minutes under static and physiologic shear stress (0 and 12 dyn/cm2, respectively), using validated flow rates from the Ibidi system. Measured [Ca2+]i was compared between cells treated vehicle or an inhibitor of the mechanosensitive Ca2+-permeable channel TRPV4 (HC-067047). HC treatment was for 1 h prior to and throughout the duration of shear exposure. Morphology changes were then assessed in hLMVECs from the same three cell lots grown in standard 6-well plates; one plate kept in static culture, while another was exposed to 24 h of 12 dyn/cm2 shear stress using an orbital shaker. Treatments in each plate were control, vehicle, and HC. Representative photos of each well were taken at 0 h and at 24 h. Average ratios of shortest to longest dimensions from 30 cells/field were measured using ImageJ and compared between treatments and shear conditions. A 2-way ANOVA with post-test was used to test for statistically significant changes in morphology. Results: Shear stress increased [Ca2+]i within 15 minutes, and both baseline [Ca2+]i and observed change in response to shear stress were decreased by TRPV4 inhibition with HC. Morphologic changes were observed after exposure to shear stress on microscopic examination, with cells grown in static culture having typical cobblestone appearance and shear-adapted cells having an elongated, spindle like appearance aligned in the direction of flow. The calculated ratio (shortest to longest dimension) of all wells at 0 h was ~0.70 with no significant differences between groups. 24 h of shear stress caused a nearly 80% decrease in ratio compared to static culture in untreated cells (p<0.05), with a decrease of only 30% in HC-treated cells after 24 h of shear (p<0.05). Conclusions: Exposure to shear stress at physiological levels results in a rapid increase [Ca2+]i through mechanosensitive, Ca2+-permeable TRPV4 channels as well as notable morphologic changes to hLMVECs with prolonged exposure to shear stress that appear to be mediated in large part by TRPV4. Further investigation into the impact of shear stress on hLMVECs is warranted to elucidate biochemical pathways and other downstream functional consequences of increased [Ca2+]i. NIH-T32HL007534. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Paneth Cell TCF4 Deficiency Alters Gut Microbiota and Host Tissue Gene Expression

Introduction: The mucosal immune system is crucial in regulating gut microbiota composition. Paneth cells, the specialized secretory cells in the small intestine, produce α-defensins that regulate gut microbiota. The Wnt signaling pathway with T-cell factor-4 (TCF4) as the terminal signaling molecule regulates Paneth cell differentiation and function. Wnt signaling plays a role in Paneth cell α-defensin expression. TCF4 binds to the α-defensin promoter and increases the transcriptional activity. In this study, we investigated the role of Paneth cell TCF4 in regulating gut microbiota and the host intestinal gene expression. Materials and methods: We generated the Paneth cell-specific TCF4 knockout mice by crossing Tcf4flox/flox mice with the Defa6Cre mice. Tcf4flox/flox-Defa6Cre (KO) were compared with Tcf4flox/flox and Defa6Cre mice (WT). Colonic flushing from these mice was subjected to metatranscriptomics, metagenomics, and proteomics. Colonic tissue was analyzed for transcriptomics. Plasma lipopolysaccharide (LPS) was measured to evaluate endotoxemia. Intestinal mucosal inflammatory response and Paneth cell function were assessed by RT-PCR for cytokines, defensins, and lysozyme. Bioinformatics was applied to assess the taxonomic diversity and functional activity of the gut microbiome, colonic luminal peptides, and colonic mucosal gene expression. Results: The metatranscriptome profile of colonic contents showed an altered microbial diversity in KO mice compared to WT mice. PCoA analyses indicated that the microbial composition in the three strains was distinctly different from each other. At the phylum level, Proteobacteria and viruses such as Pisuviricota, Cressdnaviricota, and Artiverviricota were higher in KO mice than in WT mice. LEfSe analyses showed that the abundance of numerous bacterial and viral populations, such as Firmicutes, Clostridia, and Geminiviridae, was high in KO mice. Functional profiling further confirmed the alteration of microbial diversity in the KO mice. The increased abundance of bacteria in KO mice was related to human diseases, infectious diseases, and bacterial toxin production. Proteomic analyses indicated that nearly 36% of proteins identified in the KO mice were significantly altered compared to WT mice. The Reactome analyses indicated that the altered proteins in the KO mice were related to the immune system, cell cycle, cell signaling, and protein metabolism. RT-PCR of intestinal tissue showed that the expression of Defa6, lysozyme, and Reg3γ was low in KO mice compared to WT mice. On the other hand, the expression of IL-1β, IL-6, and MCP1 in the intestine was higher in KO mice compared to WT mice. These changes in microbiota and mucosa in the KO mice were associated with increased plasma LPS. Finally, there was a significant sex-dependent differences in the Paneth TCF4 activity on gut microbiota, intestinal gene expression and endotoxemia. Conclusion: This study indicates that the Paneth cell TCF4 is essential in regulating the gut microbial composition and host intestinal gene expression. NIH/NIAID, UO1-AI170019 and U01-AI172991. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Oxylipin biosynthesis via an unprecedented 16-hydroperoxide lyase pathway in green tissues of cucumber (Cucumis sativus L.) plants

The plant lipoxygenase cascade is a source of various regulatory oxylipins that play a role in cell signalling, stress adaptation, and immune response. Recently, we detected an unprecedented 16(S)-lipoxygenase, CsLOX3, in the leaves and fruit pericarp of cucumber (Cucumis sativus L.). In the present work, an array of products biosynthesized through the conversions of α-linolenic acid 16-hydroperoxide (16-HPOT) was detected. Firstly, a prominent 15-hydroxy-9,12-pentadecadienoic acid (Me/TMS) was detected, the product of hydroperoxide lyase (HPL) chain cleavage of 16-HPOT and further reduction of aldehyde 15-oxo-9,12-pentadecadienoic acid to alcohol. Besides, the presence of dicarboxylic acid, 3,6-pentadecadiene-1,15-dioic acid, was deduced from the detection of its catalytic hydrogenation product, pentadecane-1,15-dioic acid. Finally, 12,15-dihydroxypentadecanoic acid (Me/TMS) was detected amongst the hydrogenated products, thus indicating the presence of the parent 12,15-dihydroxy-9,13-pentadecadienoic acid. To confirm the proposed HPL chain cleavage, the 16(S)-HPOT was prepared and incubated with the recombinant cucumber HPL CYP74B6 enzyme. The CYP74B6 possessed high activity towards 16-HPOT. Chain cleavage yields the (9Z,12Z)-15-oxo-9,12-pentadecadienoic acid, undergoing a spontaneous isomerization into (9Z,13E)-15-oxo-9,13-pentadecadienoic acid. Thus, the cucumber plants as well as the recombinant cucumber HPL CYP74B6 possessed unprecedented 16-HPL activity, cleaving 16-HPOT into a C15 fragment, 15-oxo-9,12-pentadecadienoic acid, and a complementary volatile C3 fragment, propionic aldehyde. The 16-LOX/16-HPL route of oxylipin biosynthesis presents a novel facet of the plant LOX pathway.

Na+/H+ exchanger isoform 1 (NHE1) confers resistance to apoptosis in pulmonary arterial smooth muscle cells (PASMCs)

Pulmonary hypertension (PH) is a condition caused by remodeling of the lung vasculature due to abnormal cell turnover involving increased cell proliferation and resistance to apoptosis. The mechanisms controlling abnormal cell growth and survival are incompletely understood. We previously found that Na+/H+ exchanger (NHE) activity is increased in PASMCs from PH patients and the Sugen Hypoxia (SuHx) animal model of PH. NHE isoform 1 (NHE1) is a membrane protein that regulates intracellular pH (pHi) and cell volume via export of H+ and import of Na+. This protein also has a cytosolic tail region shown to be involved in non-canonical functions such as cytoskeletal interactions and cell signaling via protein-protein interactions. NHE1 has been associated with increased PASMC proliferation; however, it is unclear if increased NHE activity, dysregulation of pHi or cytoskeletal interactions are involved in resistance to apoptosis. In this study, we tested the hypothesis that increased expression of NHE1 leads to apoptosis resistance in PASMCs via dysregulation of pHi. We first used the rat SuHx model of PH in which male Wistar rats receive a single subcutaneous injection of the vascular endothelial growth factor receptor inhibitor, SU5416, followed by 3 wks of exposure to hypoxia (10% O2) and 2 wks of normoxia. PASMCs were isolated from distal pulmonary arteries of control and SuHx Wistar rats and treated with the NHE inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 1 uM), or vehicle for 24 h before analyzing cell apoptosis via Hoechst staining under baseline and stimulated conditions (250 uM H2O2). Endoplasmic reticulum stress was measured using the fluorescent stain, Thioflavin T (ThT). In some experiments, cells from control rats were infected with adenovirus containing green fluorescent protein (AdGFP; control for infection), wild-type NHE1 (AdNHE1-WT) or NHE1 mutants: AdNHE1-E262I lacking ion translocation activity and AdNHE1-EM lacking the ezrin binding site which prevents cytoskeletal interactions. NHE1 protein expression and localization were assessed via immunoblotting (total and surface proteins). Effcacy of Na+/H+ translocation was assessed from Na+-dependent recovery following an ammonium pulse. At baseline, despite increased ThT levels in SuHx cells, apoptosis was not different compared to control cells, indicating SuHx cells have suppressed apoptotic response to increased ER stress. Following treatment with EIPA, apoptosis was increased in SuHx cells, possibly indicating they became sensitized to the high levels of ER stress and were able to proceed into the apoptotic pathway. Similarly, following treatment with H2O2, SuHx PASMCs showed resistance to apoptosis compared to control cells, which was alleviated with pre-treatment with EIPA. In control cells, wild-type NHE1 and NHE1 mutants were expressed in similar amounts which led to an expected increase in NHE activity for WT and NHE1-EM, but not for NHE1-E262I when compared to GFP. We found that enhanced expression of NHE1-WT was suffcient to inhibit apoptosis following treatment with H2O2. Interestingly, both NHE1 mutants also significantly decreased PASMC apoptosis following H2O2 stimulation. Our results indicate NHE1 is necessary for apoptosis resistance in PASMCs from animals with PH and that increased expression is suffcient to confer resistance to apoptosis in control PASMCs. Additionally, these results suggest neither pHi regulation nor interaction between NHE1 and ezrin are essential for NHE1-induced apoptosis resistance, indicating another mechanism through which NHE1 confers resistance to apoptosis in PH. Based on these findings, we speculate that development of NHE1-related therapeutic targets could potentially restore cell apoptosis and reverse disease. AHA 23PRE1022720, HL159906, HL073859. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lighting ATR/Chk1 by mesoscale TopBP1 condensates

Biomolecular condensation has gained considerable attention as a fundamental mechanism in cell signaling and various biological processes. A recent study by Egger et al. provides valuable insights into the constituents of topoisomerase IIβ binding protein 1 (TopBP1) condensates and sheds light on the mechanism of Chk1 activation by ataxia telangiectasia-mutated and Rad3-related (ATR) at the interface of these condensates.

An essential role for EROS in redox-dependent endothelial signal transduction

The chaperone protein EROS (“Essential for Reactive Oxygen Species”) was recently discovered in phagocytes. EROS was shown to regulate the abundance of the ROS-producing enzyme NADPH oxidase isoform 2 (NOX2) and to control ROS-mediated cell killing. Reactive oxygen species are important not only in immune surveillance, but also modulate physiological signaling responses in multiple tissues. The roles of EROS have not been previously explored in the context of oxidant-modulated cell signaling. Here we show that EROS plays a key role in ROS-dependent signal transduction in vascular endothelial cells. We used siRNA-mediated knockdown and developed CRISPR/Cas9 knockout of EROS in human umbilical vein endothelial cells (HUVEC), both of which cause a significant decrease in the abundance of NOX2 protein, associated with a marked decrease in RAC1, a small G protein that activates NOX2. Loss of EROS also attenuates receptor-mediated hydrogen peroxide (H2O2) and Ca2+ signaling, disrupts cytoskeleton organization, decreases cell migration, and promotes cellular senescence. EROS knockdown blocks agonist-modulated eNOS phosphorylation and nitric oxide (NO●) generation. These effects of EROS knockdown are strikingly similar to the alterations in endothelial cell responses that we previously observed following RAC1 knockdown. Proteomic analyses following EROS or RAC1 knockdown in endothelial cells showed that reduced abundance of these two distinct proteins led to largely overlapping effects on endothelial biological processes, including oxidoreductase, protein phosphorylation, and endothelial nitric oxide synthase (eNOS) pathways. These studies demonstrate that EROS plays a central role in oxidant-modulated endothelial cell signaling by modulating NOX2 and RAC1.

Gestational Long Term High-Altitude Hypoxia and Metabolomic Reprogramming in Uterine Arteries of Adult Sheep

Long term hypoxic stress during pregnancy causes a reduction in uterine blood flow, compromising oxygen and nutrient delivery to the fetus. There are multiple epigenetic factors involved in the structural and functional impairments that underlie the impairments in uterine arterial function. Evidence indicates that oxidative stress plays an important role to the decrements in blood flow following high altitude pregnancy. The purpose of the current study was to test the hypothesis that changes in oxylipin and endocannabinoid signaling are important to the increases in oxidative stress along with changes in glucose and fat metabolism. To further understand the mechanisms associated with disruption of uterine artery blood flow due to high altitude pregnancy, we examined a high altitude pregnant sheep model. Pregnant and non-pregnant sheep were housed either at low (700m) or high altitude (3801 meters) for 110+ days of pregnancy. Uterine arteries were excised from pregnant and non-pregnant sheep and targeted as well as untargeted metabolomic analyses were performed to test the hypothesis that hypoxia increases oxidative stress and reduces oxidative metabolism. Enrichment analysis of the metabolites show that hypoxia impacted several key biochemical pathways. At high altitude there were decreases in Alpha Linolenic Acid and Linoleic Acid Metabolism along with reductions in Pyruvate Metabolism and antioxidants. Further to this, there was an increase in the metabolism of Arachidonic Acid, Glycerolipids, and Methionine, which are linked to inflammation, cell signaling, and cell growth. Fully understanding how increased oxidative stress and inflammatory pathways along with decreased antioxidant capacity and changes in cellular metabolism is tied to malformations in uterine arterial structure and function following high altitude gestation is an active area of investigation. This work was supported by the National Institutes of Health Grants NIH R01HL155295, R01HL149608, R03HD098477, P01HD083132, and U24DK097154. Additional support was provided by the Loma Linda University School of Medicine and West Coast Metabolomics Center. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lipidomics of Phospholipase A2 Reveals Exquisite Specificity in Macrophages

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that hydrolyze phospholipids at their sn-2 fatty acyl position. Our laboratory has demonstrated that PLA2 enzymes regulate membrane remodeling and cell signaling by their specificity toward their phospholipid substrates at the molecular level. Recent in vitro studies show that each type of PLA2, including GIVA cytosolic PLA2 (cPLA2), GV secreted PLA2 (sPLA2), GVIA calcium independent PLA2 (iPLA2) and GVIIA lipoprotein-associated PLA2 (LpPLA2), also known as platelet-activating factor acetyl hydrolase (PAFAH), can discriminate exquisitely between fatty acids at the sn-2 position. Thus, these enzymes regulate the production of diverse polyunsaturated fatty acid (PUFA) precursors of inflammatory metabolites. We now determined PLA2 specificity in macrophage cells grown in cell culture, where the amounts and localization of the phospholipid substrates play a role in which specific phospholipids are hydrolyzed by each enzyme type. We employ PLA2 stereospecific inhibitors in tandem with a novel UPLC-MS/MS based lipidomics platform to quantify more than a thousand unique phospholipid molecular species demonstrating cPLA2, sPLA2, and iPLA2 activity and specificity toward the phospholipids in living cells. The observed specificity follows the in vitro capability of the enzymes and can reflect the enrichment of certain phospholipid species in specific membrane locations where particular PLA2's associate. For assaying, we target 20:4-PI for cPLA2, 22:6-PG for sPLA2 and 18:2-PC for iPLA2. These new results provide great insight into the physiological role of PLA2 enzymes in cell membrane remodeling and could shed light on how PLA2 enzymes underpin inflammation and other lipid-related diseases.

Molecular mechanisms of the effects of mulberry (Morus Rubra) extracts on UV-radiation and oxidative stress-induced skin cell damage

Solar ultraviolet (UV) radiation, while beneficial for the homeostasis of human skin at proper doses, causes skin cell damage under extreme conditions, with a result of photoaging and skin cancer. With the depletion of the ozone layer, the prevalence of UV-induced skin cancer including melanoma has increased dramatically worldwide. Unfortunately, that poses a public health challenge for almost all countries. Over the past two decades, we have been working extensively on understanding the cellular and molecular mechanisms through which UV radiation induces skin aging and skin cancer. We have observed that UV radiation activates cell surface receptors, more pronouncedly EGFR, leading to a cascade of activation of MAP kinase pathways, with a result of MMP activation and in turn matrix degradation, and apoptosis. We have also found that UV radiation induces the downregulation of water channel aquaporins and skin cell dehydration. This novel discovery together with other reports has resulted in the emergence of numerous skin care products. Moreover, UV radiation induces the generation of reactive oxygen species, which exacerbates skin cell damage. With this important line of discovery, more skin care products with added antioxidants have been developed. Mulberries ( Morus Rubra) have been known to be rich in antioxidants and applied to cosmetic products in Asian countries with reported effcacy although the cellular and molecular mechanisms of its actions remain elusive. The objective of this study was to investigate these mechanisms from various perspectives. We hypothesize that the mulberry extracts affect cell signaling pathways that lead to protection against UV radiation or oxidative stressed-induced cell damage. First, we extracted the antioxidants with methanol from two types of mulberries, red and white. Human skin keratinocytes (HaCaT cells) were treated with UV radiation and hydrogen peroxide (H2O2), with and without mulberry extracts. Confocal microscopy and Western blot analysis were employed to measure various cell signaling components. The results showed that mulberry extracts protect against UV radiation and H2O2 induced cell death, with red extracts showing better protection than white extracts. Mulberry extracts attenuate H2O2-induced mitochondrial activity as tested by MitoTracker™ Red CMXRos. Our previous studies have shown that UV radiation induces mTOR pathway activation evidenced by elevation of phosphorylated S6. Interestingly, in this study, Western blot analysis data indicated that mulberry extracts potentiate UV-induced S6 phosphorylation and H2O2-induced MEK activation. Furthermore, confocal data demonstrated that mulberry extract pretreatment inhibits IL1β induced NFκB p65 translocation from the cytoplasm to the nucleus. Taken all together, our data suggest that mulberry extracts containing abundant antioxidants affect MAPK kinase and mTOR pathways and modulate cell signaling pathways associated with skin cell survival and inflammation under UV radiation and oxidative stress. Funding from the Committee on Aid to Faculty Research (CAFR) from Providence College. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.