CBP/P300 BRD Inhibition Reduces Neutrophil Accumulation and Activates Antitumor Immunity in TNBC.

Tumor-associated neutrophils (TANs) have been shown to promote immunosuppression and tumor progression, and a high TAN frequency predicts poor prognosis in triple-negative breast cancer (TNBC). Dysregulation of CREB binding protein (CBP)/P300 function has been observed with multiple cancer types. The bromodomain (BRD) of CBP/P300 has been shown to regulate its activity. In this study, we found that IACS-70654, a novel and selective CBP/P300 BRD inhibitor, reduced TANs and inhibited the growth of neutrophil-enriched TNBC models. In the bone marrow, CBP/P300 BRD inhibition reduced the tumor-driven abnormal differentiation and proliferation of neutrophil progenitors. Inhibition of CBP/P300 BRD also stimulated the immune response by inducing an IFN response and MHCI expression in tumor cells and increasing tumor-infiltrated CTLs. Moreover, IACS-70654 improved the response of a neutrophil-enriched TNBC model to docetaxel and immune checkpoint blockade. This provides a rationale for combining a CBP/P300 BRD inhibitor with standard-of-care therapies in future clinical trials for neutrophil-enriched TNBC.

Hydrogel-Embedded Precision-Cut Lung Slices Model Lung Cancer Premalignancy Ex Vivo.

Lung cancer is the leading global cause of cancer-related deaths. Although smoking cessation is the best prevention, 50% of lung cancer diagnoses occur in people who have quit smoking. Research into treatment options for high-risk patients is constrained to rodent models, which are time-consuming, expensive, and require large cohorts. Embedding precision-cut lung slices (PCLS) within an engineered hydrogel and exposing this tissue to vinyl carbamate, a carcinogen from cigarette smoke, creates an in vitro model of lung cancer premalignancy. Hydrogel formulations are selected to promote early lung cancer cellular phenotypes and extend PCLS viability to six weeks. Hydrogel-embedded PCLS are exposed to vinyl carbamate, which induces adenocarcinoma in mice. Analysis of proliferation, gene expression, histology, tissue stiffness, and cellular content after six weeks reveals that vinyl carbamate induces premalignant lesions with a mixed adenoma/squamous phenotype. Putative chemoprevention agents diffuse through the hydrogel and induce tissue-level changes. The design parameters selected using murine tissue are validated with hydrogel-embedded human PCLS and results show increased proliferation and premalignant lesion gene expression patterns. This tissue-engineered model of human lung cancer premalignancy is the foundation for more sophisticated ex vivo models that enable the study of carcinogenesis and chemoprevention strategies.

Targeting eIF4A triggers an interferon response to synergize with chemotherapy and suppress triple-negative breast cancer.

Protein synthesis is frequently dysregulated in cancer and selective inhibition of mRNA translation represents an attractive cancer therapy. Here, we show that therapeutically targeting the RNA helicase eIF4A with zotatifin, the first-in-class eIF4A inhibitor, exerts pleiotropic effects on both tumor cells and the tumor immune microenvironment in a diverse cohort of syngeneic triple-negative breast cancer (TNBC) mouse models. Zotatifin not only suppresses tumor cell proliferation but also directly repolarizes macrophages toward an M1-like phenotype and inhibits neutrophil infiltration, which sensitizes tumors to immune checkpoint blockade. Mechanistic studies revealed that zotatifin reprograms the tumor translational landscape, inhibits the translation of Sox4 and Fgfr1, and induces an interferon (IFN) response uniformly across models. The induction of an IFN response is partially due to the inhibition of Sox4 translation by zotatifin. A similar induction of IFN-stimulated genes was observed in breast cancer patient biopsies following zotatifin treatment. Surprisingly, zotatifin significantly synergizes with carboplatin to trigger DNA damage and an even heightened IFN response, resulting in T cell-dependent tumor suppression. These studies identified a vulnerability of eIF4A in TNBC, potential pharmacodynamic biomarkers for zotatifin, and provide a rationale for new combination regimens consisting of zotatifin and chemotherapy or immunotherapy as treatments for TNBC.

Comparison of three radiographic assessment methods for detecting slipped capital femoral epiphyses in cats: Klein's line, modified Klein's line and the S-sign.

OBJECTIVES: The aim of the present study was to investigate whether diagnostic assessment methods used on radiographs in humans with slipped capital femoral epiphysis (SCFE) can be used in cats. METHODS: The ventrodorsal (VD) extended-leg and VD frog-leg pelvic radiographs of 20 cats with SCFE without fully displaced femoral capital epiphyses (FCE), eight cats with fully displaced FCE and five control cats with normal pelvic anatomy were assessed by five observers on two separate occasions 3 months apart. The Klein's line and modified Klein's line were assessed on each VD extended-leg radiograph, and the S-sign was assessed on each VD extended-leg and VD frog-leg radiograph. RESULTS: Excluding cases of fully displaced FCE, the S-sign on the VD frog-leg radiographs more accurately diagnosed SCFE than the S-sign on the VD extended-leg radiographs and the Klein's line (92.4% vs 88.8% vs 60.6%, respectively), and had the greatest sensitivity (93.9% vs 79.2% vs 30.6%, respectively). The S-sign on the VD extended-leg radiographs had greater specificity than the Klein's line and S-sign on the VD frog-leg radiographs (99.2% vs 97.9% vs 90.9%, respectively). The modified Klein's line detected SCFE in 40.2% of cases that were negative for the Klein's line. CONCLUSIONS AND RELEVANCE: The S-sign in both VD extended-leg and VD frog-leg views successfully detected SCFE in cats and can be used to increase early diagnosis and treatment in cats with SCFE that have only subtle radiographic changes.

Targeting EIF4A triggers an interferon response to synergize with chemotherapy and suppress triple-negative breast cancer.

Protein synthesis is frequently dysregulated in cancer and selective inhibition of mRNA translation represents an attractive cancer therapy. Here, we show that therapeutically targeting the RNA helicase eIF4A by Zotatifin, the first-in-class eIF4A inhibitor, exerts pleiotropic effects on both tumor cells and the tumor immune microenvironment in a diverse cohort of syngeneic triple-negative breast cancer (TNBC) mouse models. Zotatifin not only suppresses tumor cell proliferation but also directly repolarizes macrophages towards an M1-like phenotype and inhibits neutrophil infiltration, which sensitizes tumors to immune checkpoint blockade. Mechanistic studies revealed that Zotatifin reprograms the tumor translational landscape, inhibits the translation of Sox4 and Fgfr1, and induces an interferon response uniformly across models. The induction of an interferon response is partially due to the inhibition of Sox4 translation by Zotatifin. A similar induction of interferon-stimulated genes was observed in breast cancer patient biopsies following Zotatifin treatment. Surprisingly, Zotatifin significantly synergizes with carboplatin to trigger DNA damage and an even heightened interferon response resulting in T cell-dependent tumor suppression. These studies identified a vulnerability of eIF4A in TNBC, potential pharmacodynamic biomarkers for Zotatifin, and provide a rationale for new combination regimens comprising Zotatifin and chemotherapy or immunotherapy as treatments for TNBC.

Precision Cut Lung Slices as a Preclinical Model for Non-Small Cell Lung Cancer Chemoprevention.

Lung cancer chemoprevention is critical to addressing cancer burden in high-risk populations. Chemoprevention clinical trials rely on data from preclinical models; however, in vivo studies have high financial, technical, and staffing requirements. Precision cut lung slices (PCLS) provide an ex vivo model that maintains the structure and function of native tissues. This model can be used for mechanistic investigations and drug screenings and reduces the number of animals and time required to test hypotheses compared with in vivo studies. We tested the use of PCLS for chemoprevention studies, demonstrating recapitulation of in vivo models. Treatment of PCLS with the PPARγ agonizing chemoprevention agent iloprost produced similar effects on gene expression and downstream signaling as in vivo models. This occurred in both wild-type tissue and Frizzled 9 knockout tissue, a transmembrane receptor required for iloprost's preventive activity. We explored new areas of iloprost mechanisms by measuring immune and inflammation markers in PCLS tissue and media, and immune cell presence with immunofluorescence. To demonstrate the potential for drug screening, we treated PCLS with additional lung cancer chemoprevention agents and confirmed activity markers in culture. PCLS offers an intermediate step for chemoprevention research between in vitro and in vivo models that can facilitate drug screening prior to in vivo studies and support mechanistic studies with more relevant tissue environments and functions than in vitro models. PREVENTION RELEVANCE: PCLS could be a new model for premalignancy and chemoprevention research, and this work evaluates the model with tissue from prevention-relevant genetic and carcinogen exposed in vivo mouse models, in addition to evaluating chemoprevention agents.

Loss of Frizzled 9 in Lung Cells Alters Epithelial Phenotype and Promotes Premalignant Lesion Development.

The transmembrane receptor Frizzled 9 (FZD9) is important for fetal neurologic and bone development through both canonical and non-canonical WNT/FZD signaling. In the adult lung, however, Fzd9 helps to maintain a normal epithelium by signaling through peroxisome proliferator activated receptor γ (PPARγ). The effect of FZD9 loss on normal lung epithelial cells and regulators of its expression in the lung are unknown. We knocked down FZD9 in human bronchial epithelial cell (HBEC) lines and found that downstream EMT targets and PPARγ activity are altered. We used a FZD9-/- mouse in the urethane lung adenocarcinoma model and found FZD9-/- adenomas had more proliferation, increased EMT signaling, decreased activation of PPARγ, increased expression of lung cancer associated genes, increased transformed growth, and increased potential for invasive behavior. We identified PPARγ as a transcriptional regulator of FZD9. We also demonstrated that extended cigarette smoke exposure in HBEC leads to decreased FZD9 expression, decreased activation of PPARγ, and increased transformed growth, and found that higher exposure to cigarette smoke in human lungs leads to decreased FZD9 expression. These results provide evidence for the role of FZD9 in lung epithelial maintenance and in smoking related malignant transformation. We identified the first transcriptional regulator of FZD9 in the lung and found FZD9 negative lesions are more dangerous. Loss of FZD9 creates a permissive environment for development of premalignant lung lesions, making it a potential target for intervention.

PPARgamma agonism inhibits progression of premalignant lesions in a murine lung squamous cell carcinoma model.

The N-nitroso-trischloroethylurea (NTCU)-induced mouse model of squamous lung carcinoma recapitulates human disease from premalignant dysplasia through invasive tumors, making it suitable for preclinical chemoprevention drug testing. Pioglitazone is a peroxisome proliferator-activated receptor γ (PPARγ) agonist shown to prevent lung tumors in preclinical models. We investigated pioglitazone's effect on lesion development and markers of potential preventive mechanisms in the NTCU model. Female FVB/N mice were exposed to vehicle, NTCU or NTCU + oral pioglitazone for 32 weeks. NTCU induces the appearance of basal cells in murine airways while decreasing/changing their epithelial cell makeup, resulting in development of bronchial dysplasia. H&E and keratin 5 (KRT5) staining were used to detect and grade squamous lesions in formalin fixed lungs. mRNA expression of epithelial to mesenchymal transition (EMT) markers and basal cell markers were measured by qPCR. Dysplasia persistence markers desmoglein 3 and polo like kinase 1 were measured by immunohistochemistry. Basal cell markers KRT14 and p63, club cell specific protein and ciliated cell marker acetylated tubulin were measured by immunofluorescence. Pioglitazone treatment significantly reduced squamous lesions and the presence of airway basal cells, along with increasing normal epithelial cells in the airways of NTCU-exposed mice. Pioglitazone also significantly influenced EMT gene expression to promote a more epithelial, and less mesenchymal, phenotype. Pioglitazone reduced the presence of squamous dysplasia and maintained normal airway cell composition. This work increases the knowledge of mechanistic pathways in PPARγ agonism for lung cancer interception and provides a basis for further investigation to advance this chemoprevention strategy.

Iloprost requires the Frizzled-9 receptor to prevent lung cancer.

Prevention of premalignant lesion progression is a promising approach to reducing lung cancer burden in high-risk populations. Substantial preclinical and clinical evidence has demonstrated efficacy of the prostacyclin analogue iloprost for lung cancer chemoprevention. Iloprost activates peroxisome proliferator-activated receptor gamma (PPARG) to initiate chemopreventive signaling and in vitro, which requires the transmembrane receptor Frizzled9 (FZD9). We hypothesized a Fzd 9 -/- mouse would not be protected by iloprost in a lung cancer model. Fzd 9 -/- mice were treated with inhaled iloprost in a urethane model of lung adenoma. We found that Fzd 9 -/- mice treated with iloprost were not protected from adenoma development compared to wild-type mice nor did they demonstrate increased activation of iloprost signaling pathways. Our results established that iloprost requires FZD9 in vivo for lung cancer chemoprevention. This work represents a critical advancement in defining iloprost's chemopreventive mechanisms and identifies a potential response marker for future clinical trials.

Intranasal Iloprost Prevents Tumors in a Murine Lung Carcinogenesis Model.

Lung cancer chemoprevention with the prostacyclin analogue iloprost is the most promising approach to date for intercepting progression of premalignant lung lesions in former smokers. Previous preclinical studies of iloprost used oral delivery, but a study modeling delivery directly to the target organ was needed. In vivo and in vitro studies have identified gene expression changes following iloprost treatment, including increased e-cadherin and Ppargγ and decreased COX2 and vimentin. We used tumor counts and gene expression to demonstrate the effectiveness of intranasal delivery of iloprost in a murine model of premalignant adenomas. Intranasal delivery of iloprost reduced adenoma multiplicity 14 weeks after urethane exposure in FVB/N mice compared with untreated urethane controls. Intranasal iloprost reversed urethane-induced gene expression changes in tumors and whole lung. These results correspond to previous studies of oral iloprost and in vitro treatment of human bronchial epithelial cells. This study demonstrates that intranasal delivery of iloprost in a mouse model of lung premalignant lesions is effective chemoprevention. This will be an essential tool for exploring mechanisms and outcomes of iloprost chemoprevention, along with supporting ongoing clinical trials of inhaled iloprost chemoprevention. PREVENTION RELEVANCE: Iloprost is a promising chemoprevention agent for lung cancer and this work describes a new delivery approach in vivo.

Non-Coding RNA and Frizzled Receptors in Cancer.

Frizzled receptors have been long recognized for their role in Wnt/ß-catenin signaling, a pathway known for its tumorigenic effects. More recent studies of frizzled receptors include efforts to understand non-coding RNA (ncRNA) regulation of these receptors in cancer. It has become increasingly clear that ncRNA molecules are important for regulating the expression of both oncogenic and tumor-suppressive proteins. The three most commonly described ncRNA molecules are microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). Here, we review ncRNA molecules that directly or indirectly affect frizzled protein expression and downstream signaling. Exploring these interactions highlights the potential of incorporating ncRNA molecules into cancer prevention and therapy strategies that target frizzled receptors. Previous investigations of frizzled receptors and ncRNA have established strong promise for a role in cancer progression, but additional studies are needed to provide the substantial pre-clinical evidence required to translate findings to clinical applications.

Ocular Surface Potential Difference Measured in Human Subjects to Study Ocular Surface Ion Transport.

Purpose: The epithelium lining the ocular surface, which includes corneal and conjunctival epithelia, expresses the prosecretory chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) and the proabsorptive epithelial sodium channel (ENaC). Here, methodology was established to measure the millivolt (mV) potential differences at the ocular surface, called ocular surface potential difference (OSPD), in human subjects produced by ion transport. Methods: OSPD was measured in human subjects in which a fluid-filled measuring electrode contacted a fluid pool created by eversion of the lateral lower eyelid, with a reference electrode placed subcutaneously in the forearm. Through the use of a high-impedance voltmeter, OSPD was measured continuously over 10 to 15 minutes in response to a series of perfusate fluid exchanges. Results: Baseline OSPD (± SEM) in six normal human subjects was -21.3 ± 3.6 mV. OSPD depolarized by 1.7 ± 0.6 mV following the addition of the ENaC inhibitor amiloride, hyperpolarized by 6.8 ± 1.5 mV with a zero chloride solution, and further hyperpolarized by 15.9 ± 1.6 mV following CFTR activation by isoproterenol. The isoproterenol-induced hyperpolarization was absent in two cystic fibrosis subjects lacking functional CFTR. OSPD measurement produced minimal epithelial injury. Conclusions: Our results establish the feasibility and safety of OSPD measurement in humans and demonstrate robust CFTR activity, albeit minimal ENaC activity, at the ocular surface. OSPD measurement may be broadly applicable to investigate fluid transport mechanisms and test drug candidates to treat ocular surface disorders. Translational Relevance: To the best of our knowledge, this is the first measurement of the electrical potential generated by the ocular surface epithelium in human subjects, offering a new approach to study ocular surface function and health.

Superresolution Imaging of Aquaporin-4 Cluster Size in Antibody-Stained Paraffin Brain Sections.

The water channel aquaporin-4 (AQP4) forms supramolecular clusters whose size is determined by the ratio of M1- and M23-AQP4 isoforms. In cultured astrocytes, differences in the subcellular localization and macromolecular interactions of small and large AQP4 clusters results in distinct physiological roles for M1- and M23-AQP4. Here, we developed quantitative superresolution optical imaging methodology to measure AQP4 cluster size in antibody-stained paraffin sections of mouse cerebral cortex and spinal cord, human postmortem brain, and glioma biopsy specimens. This methodology was used to demonstrate that large AQP4 clusters are formed in AQP4(-/-) astrocytes transfected with only M23-AQP4, but not in those expressing only M1-AQP4, both in vitro and in vivo. Native AQP4 in mouse cortex, where both isoforms are expressed, was enriched in astrocyte foot-processes adjacent to microcapillaries; clusters in perivascular regions of the cortex were larger than in parenchymal regions, demonstrating size-dependent subcellular segregation of AQP4 clusters. Two-color superresolution imaging demonstrated colocalization of Kir4.1 with AQP4 clusters in perivascular areas but not in parenchyma. Surprisingly, the subcellular distribution of AQP4 clusters was different between gray and white matter astrocytes in spinal cord, demonstrating regional specificity in cluster polarization. Changes in AQP4 subcellular distribution are associated with several neurological diseases and we demonstrate that AQP4 clustering was preserved in a postmortem human cortical brain tissue specimen, but that AQP4 was not substantially clustered in a human glioblastoma specimen despite high-level expression. Our results demonstrate the utility of superresolution optical imaging for measuring the size of AQP4 supramolecular clusters in paraffin sections of brain tissue and support AQP4 cluster size as a primary determinant of its subcellular distribution.

Aggregation state determines the localization and function of M1- and M23-aquaporin-4 in astrocytes.

The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23-AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays. Here, we demonstrate that the AQP4 aggregation state determines its subcellular localization and cellular functions. Individually expressed M1-AQP4 was freely mobile in the plasma membrane and could diffuse into rapidly extending lamellipodial regions to support cell migration. In contrast, M23-AQP4 formed large arrays that did not diffuse rapidly enough to enter lamellipodia and instead stably bound adhesion complexes and polarized to astrocyte end-feet in vivo. Co-expressed M1- and M23-AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1-AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23-AQP4-enriched arrays with the extracellular matrix. Our results therefore demonstrate an aggregation state-dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1- and M23-AQP4.

Cholesterol-dependent kinase activity regulates transmitter release from cerebellar synapses.

Changes in membrane cholesterol content can alter protein kinase activity, however, it is not known whether kinases regulating transmitter release are sensitive to membrane cholesterol content. Here we have used the cholesterol extracting agent methyl-beta-cyclodextrin to measure the effects of acute cholesterol reduction on transmitter release from cultured cerebellar neurons. Cholesterol depletion increased the frequency of spontaneous transmitter release without altering the amplitude and time course of mEPSCs. Evoked transmitter release was decreased by cholesterol extraction and the paired pulse ratio was also decreased. Alterations in synaptic transmission were not associated with failure of action potential generation or changes in presynaptic Ca(2+) signaling. Both the increase in mEPSC frequency and the change in paired pulse ratio were blocked by the broad spectrum protein kinase inhibitor staurosporine. The increase in mEPSC frequency was also sensitive to selective inhibitors of PKC and PKA. Our results therefore demonstrate that the activity of presynaptic protein kinases that regulate spontaneous and evoked neurotransmitter release is sensitive to changes of membrane cholesterol content.