PI3K Catalytic Subunits α and ß Modulate Cell Death and IL-6 Secretion Induced by Talc Particles in Human Lung Carcinoma Cells.

Hydrated magnesium silicate (or "talc" particles) is a sclerosis agent commonly used in the management of malignant pleural effusions, a common symptom of metastatic diseases, including lung cancers. However, the direct effects of talc particles to lung carcinoma cells, which can be found in the malignant pleural effusion fluids from patients with lung cancer, are not fully understood. Here, we report a study of the signaling pathways that can modulate the cell death and IL-6 secretion induced by talc particles in human lung carcinoma cells. We found that talc-sensitive cells have higher mRNA and protein expression of PI3K catalytic subunits α and ß. Further experiments confirmed that modulation (inhibition or activation) of the PI3K pathway reduces or enhances cellular sensitivity to talc particles, respectively, independent of the inflammasome. By knocking down specific PI3K isoforms, we also confirmed that both PI3Kα and -ß mediate the observed talc effects. Our results suggest a novel role of the PI3K pathway in talc-induced cell death and IL-6 secretion in lung carcinoma cells. These cellular events are known to drive fibrosis, and thus further studies of the PI3K pathway may provide a better understanding of the mechanisms of talc sclerosis in the malignant pleural space.

Building predictive in vitro pulmonary toxicity assays using high-throughput imaging and artificial intelligence.

Human lungs are susceptible to the toxicity induced by soluble xenobiotics. However, the direct cellular effects of many pulmonotoxic chemicals are not always clear, and thus, a general in vitro assay for testing pulmonotoxicity applicable to a wide variety of chemicals is not currently available. Here, we report a study that uses high-throughput imaging and artificial intelligence to build an in vitro pulmonotoxicity assay by automatically comparing and selecting human lung-cell lines and their associated quantitative phenotypic features most predictive of in vivo pulmonotoxicity. This approach is called "High-throughput In vitro Phenotypic Profiling for Toxicity Prediction" (HIPPTox). We found that the resulting assay based on two phenotypic features of a human bronchial epithelial cell line, BEAS-2B, can accurately classify 33 reference chemicals with human pulmonotoxicity information (88.8% balance accuracy, 84.6% sensitivity, and 93.0% specificity). In comparison, the predictivity of a standard cell-viability assay on the same set of chemicals is much lower (77.1% balanced accuracy, 84.6% sensitivity, and 69.5% specificity). We also used the assay to evaluate 17 additional test chemicals with unknown/unclear human pulmonotoxicity, and experimentally confirmed that many of the pulmonotoxic reference and predicted-positive test chemicals induce DNA strand breaks and/or activation of the DNA-damage response (DDR) pathway. Therefore, HIPPTox helps us to uncover these common modes-of-action of pulmonotoxic chemicals. HIPPTox may also be applied to other cell types or models, and accelerate the development of predictive in vitro assays for other cell-type- or organ-specific toxicities.

Fundus Photography in the 21st Century--A Review of Recent Technological Advances and Their Implications for Worldwide Healthcare.

BACKGROUND: The introduction of fundus photography has impacted retinal imaging and retinal screening programs significantly. LITERATURE REVIEW: Fundus cameras play a vital role in addressing the cause of preventive blindness. More attention is being turned to developing countries, where infrastructure and access to healthcare are limited. One of the major limitations for tele-ophthalmology is restricted access to the office-based fundus camera. RESULTS: Recent advances in access to telecommunications coupled with introduction of portable cameras and smartphone-based fundus imaging systems have resulted in an exponential surge in available technologies for portable fundus photography. Retinal cameras in the near future would have to cater to these needs by featuring a low-cost, portable design with automated controls and digitalized images with Web-based transfer. CONCLUSIONS: In this review, we aim to highlight the advances of fundus photography for retinal screening as well as discuss the advantages, disadvantages, and implications of the various technologies that are currently available.

Spatial omics techniques and data analysis for cancer immunotherapy applications.

In-depth profiling of cancer cells/tissues is expanding our understanding of the genomic, epigenomic, transcriptomic, and proteomic landscape of cancer. However, the complexity of the cancer microenvironment, particularly its immune regulation, has made it difficult to exploit the potential of cancer immunotherapy. High-throughput spatial omics technologies and analysis pipelines have emerged as powerful tools for tackling this challenge. As a result, a potential revolution in cancer diagnosis, prognosis, and treatment is on the horizon. In this review, we discuss the technological advances in spatial profiling of cancer around and beyond the central dogma to harness the full benefits of immunotherapy. We also discuss the promise and challenges of spatial data analysis and interpretation and provide an outlook for the future.

Chemical probe mediated visualization of protein S-palmitoylation in patient tissue samples.

While protein palmitoylation has been studied for decades, our understanding of its clinical importance is minimal compared to other post translational modifications. As a result of the inherent challenges preventing the production of antibodies to palmitoylated epitopes we are unable to correlate levels of protein palmitoylation in biopsied tissues at a meaningful resolution. The most common method for detecting palmitoylated proteins without metabolic labelling is through chemical labeling of palmitoylated cysteines with the acyl-biotinyl exchange (ABE) assay. We have adapted the ABE assay to detect protein palmitoylation in formalin fixed paraffin embedded (FFPE) tissue sections. The assay is sufficient to detect subcellular regions of cells with increased labeling which indicates areas enriched in palmitoylated proteins. To visualize specific palmitoylated proteins in both cultured cells and in FFPE preserved tissue arrays we have integrated the ABE assay with a proximity ligation assay (ABE-PLA). Our findings demonstrate for the first time that FFPE preserved tissues can be labelled with unique chemical probes to detect either areas enriched in palmitoylated proteins or the localization of specific palmitoylated proteins using our ABE-PLA methodology.

MALT1 Is a Targetable Driver of Epithelial-to-Mesenchymal Transition in Claudin-Low, Triple-Negative Breast Cancer.

MALT1 is the effector protein of the CARMA/Bcl10/MALT1 (CBM) signalosome, a multiprotein complex that drives pro-inflammatory signaling pathways downstream of a diverse set of receptors. Although CBM activity is best known for its role in immune cells, emerging evidence suggests that it plays a key role in the pathogenesis of solid tumors, where it can be activated by selected G protein-coupled receptors (GPCR). Here, we demonstrated that overexpression of GPCRs implicated in breast cancer pathogenesis, specifically the receptors for Angiotensin II and thrombin (AT1R and PAR1), drove a strong epithelial-to-mesenchymal transition (EMT) program in breast cancer cells that is characteristic of claudin-low, triple-negative breast cancer (TNBC). In concert, MALT1 was activated in these cells and contributed to the dramatic EMT phenotypic changes through regulation of master EMT transcription factors including Snail and ZEB1. Importantly, blocking MALT1 signaling, through either siRNA-mediated depletion of MALT1 protein or pharmacologic inhibition of its activity, was effective at partially reversing the molecular and phenotypic indicators of EMT. Treatment of mice with mepazine, a pharmacologic MALT1 inhibitor, reduced growth of PAR1+, MDA-MB-231 xenografts and had an even more dramatic effect in reducing the burden of metastatic disease. These findings highlight MALT1 as an attractive therapeutic target for claudin-low TNBCs harboring overexpression of one or more selected GPCRs. IMPLICATIONS: This study nominates a GPCR/MALT1 signaling axis as a pathway that can be pharmaceutically targeted to abrogate EMT and metastatic progression in TNBC, an aggressive form of breast cancer that currently lacks targeted therapies.

Regulation of EGFR signalling by palmitoylation and its role in tumorigenesis.

The epidermal growth factor receptor (EGFR) is an essential driver of oncogenic signalling, and EGFR inhibitors are some of the earliest examples of successful targeted therapies in multiple types of cancer. The tractability of EGFR as a therapeutic target is overshadowed by the inevitable drug resistance that develops. Overcoming resistance mechanisms requires a deeper understanding of EGFR regulation in cancer cells. In this review, we discuss our recent discovery that the palmitoyltransferase DHHC20 palmitoylates EGFR on the C-terminal domain and plays a critical role in signal regulation during oncogenesis. Inhibiting DHHC20 expression or mutating the palmitoylation site on EGFR alters the EGF-induced signalling kinetics from a transient signal to a sustained signal. The change in signalling is accompanied by a decrease in cell proliferation in multiple human cancer cell lines. Our in vivo studies demonstrate that ablating the gene Zdhhc20 by CRISPR/Cas9-mediated inhibition in a mouse model of oncogenic Kras-driven lung adenocarcinoma potently inhibits tumorigenesis. The negative effect on tumorigenesis is mediated by EGFR since the expression of a palmitoylation-resistant mutant form of EGFR also inhibits Kras-driven lung adenocarcinoma. Finally, reducing EGFR palmitoylation increases the sensitivity of multiple cancer cell lines to existing inhibitors of EGFR and downstream signalling effector pathways. We will discuss the implications of these effects and strategies for targeting these new vulnerabilities.

Leveraging advances in immunopathology and artificial intelligence to analyze in vitro tumor models in composition and space.

Cancer is the leading cause of death worldwide. Unfortunately, efforts to understand this disease are confounded by the complex, heterogenous tumor microenvironment (TME). Better understanding of the TME could lead to novel diagnostic, prognostic, and therapeutic discoveries. One way to achieve this involves in vitro tumor models that recapitulate the in vivo TME composition and spatial arrangement. Here, we review the potential of harnessing in vitro tumor models and artificial intelligence to delineate the TME. This includes (i) identification of novel features, (ii) investigation of higher-order relationships, and (iii) analysis and interpretation of multiomics data in a (iv) holistic, objective, reproducible, and efficient manner, which surpasses previous methods of TME analysis. We also discuss limitations of this approach, namely inadequate datasets, indeterminate biological correlations, ethical concerns, and logistical constraints; finally, we speculate on future avenues of research that could overcome these limitations, ultimately translating to improved clinical outcomes.

Group VIII Metal Carbonyl Cluster-Boronic Acid Conjugates: Cytotoxicity and Mode of Action Studies.

A set of metal carbonyl cluster-boronic acid conjugates of the group VIII metals (Fe, Ru, and Os) were synthesized and their antiproliferative effects measured against two breast cancer cell lines (MCF-7 and MDA-MB-231) and a noncancerous breast epithelial (MCF-10A) cell line. The cytotoxicity followed the order Ru > Os > Fe for the MDA-MB-231 cells, although the latter two exhibited similar cytotoxicity against MCF-7 and MCF-10A cells. The osmium species {Os3(CO)10(µ-H)[µ-SC6H4-p-B(OH)2]} (2) could be chemically oxidized to its hydroxy analogue [Os3(CO)10(µ-H)(µ-SC6H4 -p-OH)] (2-OH), which showed comparable cytotoxicity. Mode of action studies pointed to an apoptotic pathway for cell death.

Utility of In Vitro Bioactivity as a Lower Bound Estimate of In Vivo Adverse Effect Levels and in Risk-Based Prioritization.

Use of high-throughput, in vitro bioactivity data in setting a point-of-departure (POD) has the potential to accelerate the pace of human health safety evaluation by informing screening-level assessments. The primary objective of this work was to compare PODs based on high-throughput predictions of bioactivity, exposure predictions, and traditional hazard information for 448 chemicals. PODs derived from new approach methodologies (NAMs) were obtained for this comparison using the 50th (PODNAM, 50) and the 95th (PODNAM, 95) percentile credible interval estimates for the steady-state plasma concentration used in in vitro to in vivo extrapolation of administered equivalent doses. Of the 448 substances, 89% had a PODNAM, 95 that was less than the traditional POD (PODtraditional) value. For the 48 substances for which PODtraditional < PODNAM, 95, the PODNAM and PODtraditional were typically within a factor of 10 of each other, and there was an enrichment of chemical structural features associated with organophosphate and carbamate insecticides. When PODtraditional < PODNAM, 95, it did not appear to result from an enrichment of PODtraditional based on a particular study type (eg, developmental, reproductive, and chronic studies). Bioactivity:exposure ratios, useful for identification of substances with potential priority, demonstrated that high-throughput exposure predictions were greater than the PODNAM, 95 for 11 substances. When compared with threshold of toxicological concern (TTC) values, the PODNAM, 95 was greater than the corresponding TTC value 90% of the time. This work demonstrates the feasibility, and continuing challenges, of using in vitro bioactivity as a protective estimate of POD in screening-level assessments via a case study.

MALT1 is a critical mediator of PAR1-driven NF-κB activation and metastasis in multiple tumor types.

Protease-activated receptor 1 (PAR1), a thrombin-responsive G protein-coupled receptor (GPCR), is implicated in promoting metastasis in multiple tumor types, including both sarcomas and carcinomas, but the molecular mechanisms responsible remain largely unknown. We previously discovered that PAR1 stimulation in endothelial cells leads to activation of NF-κB, mediated by a protein complex comprised of CARMA3, Bcl10, and the MALT1 effector protein (CBM complex). Given the strong association between NF-κB and metastasis, we hypothesized that this CBM complex could play a critical role in the PAR1-driven metastatic progression of specific solid tumors. In support of our hypothesis, we demonstrate that PAR1 stimulation results in NF-κB activation in both osteosarcoma and breast cancer, which is suppressed by siRNA-mediated MALT1 knockdown, suggesting that an intact CBM complex is required for the response in both tumor cell types. We identify several metastasis-associated genes that are upregulated in a MALT1-dependent manner after PAR1 stimulation in cancer cells, including those encoding the matrix remodeling protein, MMP9, and the cytokines, IL-1ß and IL-8. Further, exogenous expression of PAR1 in MCF7 breast cancer cells confers highly invasive and metastatic behavior which can be blocked by CRISPR/Cas9-mediated MALT1 knockout. Importantly, we find that PAR1 stimulation induces MALT1 protease activity in both osteosarcoma and breast cancer cells, an activity that is mechanistically linked to NF-κB activation and potentially other responses associated with aggressive phenotype. Several small molecule MALT1 protease inhibitors have recently been described that could therefore represent promising new therapeutics for the prevention and/or treatment of PAR1-driven tumor metastasis.

The CARMA3-Bcl10-MALT1 Signalosome Drives NFκB Activation and Promotes Aggressiveness in Angiotensin II Receptor-Positive Breast Cancer.

The angiotensin II receptor AGTR1, which mediates vasoconstrictive and inflammatory signaling in vascular disease, is overexpressed aberrantly in some breast cancers. In this study, we established the significance of an AGTR1-responsive NFκB signaling pathway in this breast cancer subset. We documented that AGTR1 overexpression occurred in the luminal A and B subtypes of breast cancer, was mutually exclusive of HER2 expression, and correlated with aggressive features that include increased lymph node metastasis, reduced responsiveness to neoadjuvant therapy, and reduced overall survival. Mechanistically, AGTR1 overexpression directed both ligand-independent and ligand-dependent activation of NFκB, mediated by a signaling pathway that requires the triad of CARMA3, Bcl10, and MALT1 (CBM signalosome). Activation of this pathway drove cancer cell-intrinsic responses that include proliferation, migration, and invasion. In addition, CBM-dependent activation of NFκB elicited cancer cell-extrinsic effects, impacting endothelial cells of the tumor microenvironment to promote tumor angiogenesis. CBM/NFκB signaling in AGTR1+ breast cancer therefore conspires to promote aggressive behavior through pleiotropic effects. Overall, our results point to the prognostic and therapeutic value of identifying AGTR1 overexpression in a subset of HER2-negative breast cancers, and they provide a mechanistic rationale to explore the repurposing of drugs that target angiotensin II-dependent NFκB signaling pathways to improve the treatment of this breast cancer subset.Significance: These findings offer a mechanistic rationale to explore the repurposing of drugs that target angiotensin action to improve the treatment of AGTR1-expressing breast cancers. Cancer Res; 78(5); 1225-40. ©2017 AACR.

Assessment of retinal vascular calibres as a biomarker of disease activity in birdshot chorioretinopathy.

PURPOSE: Birdshot chorioretinopathy (BCR) is a potentially blinding ocular disorder involving the retinal vasculature and choroid without any systemic manifestations. The objective of the study was to describe vascular calibre changes in BCR and analyse the possibility of this optical biomarker for staging and monitoring disease activity in BCR. METHODS: This retrospective case-control study at a tertiary referral eye centre in the UK included 33 eyes from 21 patients with BCR and equal number of eyes from control subjects. Diagnosis of BCR was confirmed on fundus fluorescein and indocyanine green angiography. Vascular calibres were measured using validated semiautomated software. RESULTS: Patients with BCR had smaller retinal venular calibres central retinal venular equivalent (CRVE) than controls (211.3 versus 227.9 µm, p = 0.008). After adjusting for variables, the difference between the two groups for CRVE at baseline was statistically significant based on two different analysis methods. Central retinal venular equivalent (CRVE) was lower at the 6-month follow-up visit (206.2 versus 213.8 µm, p-value = 0.03), and arteriole-to-venule ratio was larger (0.74 versus 0.71, p = 0.04) in subjects with BCR. Arteriolar calibre (CRAE) remained the same. CONCLUSION: This study provides novel insight into the pattern of vascular involvement in BCR. There was significant difference in the CRVE in patients with BCR. More studies are needed to correlate this data with visual function and treatment outcome and to validate the findings.

Controversies in the pathophysiology and management of hyphema.

Traumatic hyphemas present dilemmas to physicians. There are numerous controversies pertaining to the optimal approach to traumatic hyphema and no standardized guidelines for its management. We address some of these controversies and present a pragmatic approach. We discuss various medical agents and surgical techniques available for treatment, along with the indications for their use. We address the complications associated with hyphema and how to diagnose and manage them and consider the management of hyphema in special situations such as in children and sickle-cell anemia and in rare clinical syndromes such as recurrent hyphema after placement of anterior chamber intraocular lenses.

Pan-erbB inhibition potentiates BRAF inhibitors for melanoma treatment.

The BRAF inhibitor vemurafenib is currently used for treating patients with BRAF V600E mutant melanoma. However, the responses to vemurafenib are generally partial and of relatively short duration. Recent evidence suggests that activation of the epidermal growth factor receptor (EGFR)/erbB signaling pathway may be responsible for the development of BRAF inhibitor resistance in melanoma patients. In this study, we characterized the erbB family of receptors and ligands in melanoma cell lines and examined whether targeting both BRAF and erbB provided enhanced antitumor activity in BRAF mutant melanoma. Variable levels of erbB2, erbB3, and truncated erbB4 were expressed in both BRAF wildtype and mutant melanoma cells with no significant differences between wildtype and mutant lines. EGFR was rarely expressed. Neuregulin 3 and neuregulin 4 were the major erbB ligands released by melanoma cells. Multi-erbB targeting with the irreversible tyrosine kinase inhibitor canertinib exerted a more effective growth inhibitory effect in both BRAF wildtype and mutant melanoma cells compared with the single-erbB or dual-erbB targeting inhibitors, gefitinib, erlotinib, and lapatinib. Canertinib inhibited both EGF-induced and neuregulin 1-induced erbB downstream signaling in both mutant and wildtype cell lines. However, canertinib induced apoptosis and sub-G1 arrest only in mutant cells. Canertinib statistically increased the antiproliferative effects of vemurafenib in the BRAF mutant melanoma cell lines while little or no enhanced effect was observed with the combination treatment in the wildtype cell lines. A combined inhibition strategy targeting BRAF together with multiple erbB family kinases is potentially beneficial for treating BRAF V600E mutant melanoma. Wildtype BRAF melanoma may also benefit from a multi-erbB kinase inhibitor.