Interrogating the Impact of Protease Activity on Tumor Progression Using 3D Spheroid Models.

Cancers have a complex relationship with the surrounding environment that regulates everything from progression to response to treatment. Cell-cell and cell-matrix interactions are heavily influenced by protease biology. Studies on the tumor microenvironment have revealed a new complexity for proteases, describing novel substrates for classic proteases, and protease-independent roles for these enzymes. The rapid expansion of 3D in vitro model systems provides excellent tools to study the intricate influence of proteases on the tumor microenvironment. Here we describe a spheroid invasion assay, providing a platform to interrogate key protease-matrix interactions in the context of early-stage breast cancer. Incorporation of pharmacological inhibition and RNAi techniques enables the elucidation of key protease-dependent pathways and can be complemented with immunofluorescence analysis to visualize matrix cleavage events and visualize cell behavior during collective cell invasion.

Opposing roles for ADAMTS2 and ADAMTS14 in myofibroblast differentiation and function.

Crosstalk between cancer and stellate cells is pivotal in pancreatic cancer, resulting in differentiation of stellate cells into myofibroblasts that drives tumour progression. To assess cooperative mechanisms in a 3D context, we generated chimeric spheroids using human and mouse cancer and stellate cells. Species-specific deconvolution of bulk-RNA sequencing data revealed cell type-specific transcriptomes underpinning invasion. This dataset highlighted stellate-specific expression of transcripts encoding the collagen-processing enzymes ADAMTS2 and ADAMTS14. Strikingly, loss of ADAMTS2 reduced, while loss of ADAMTS14 promoted, myofibroblast differentiation and invasion independently of their primary role in collagen-processing. Functional and proteomic analysis demonstrated that these two enzymes regulate myofibroblast differentiation through opposing roles in the regulation of transforming growth factor ß availability, acting on the protease-specific substrates, Serpin E2 and fibulin 2, for ADAMTS2 and ADAMTS14, respectively. Showcasing a broader complexity for these enzymes, we uncovered a novel regulatory axis governing malignant behaviour of the pancreatic cancer stroma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A covalent fragment-based strategy targeting a novel cysteine to inhibit activity of mutant EGFR kinase.

Several generations of ATP-competitive anti-cancer drugs that inhibit the activity of the intracellular kinase domain of the epidermal growth factor receptor (EGFR) have been developed over the past twenty years. The first-generation of drugs such as gefitinib bind reversibly and were followed by a second-generation such as dacomitinib that harbor an acrylamide moiety that forms a covalent bond with C797 in the ATP binding pocket. Resistance emerges through mutation of the T790 gatekeeper residue to methionine, which introduces steric hindrance to drug binding and increases the Km for ATP. A third generation of drugs, such as osimertinib were developed which were effective against T790M EGFR in which an acrylamide moiety forms a covalent bond with C797, although resistance has emerged by mutation to S797. A fragment-based screen to identify new starting points for an EGFR inhibitor serendipitously identified a fragment that reacted with C775, a previously unexploited residue in the ATP binding pocket for a covalent inhibitor to target. A number of acrylamide containing fragments were identified that selectively reacted with C775. One of these acrylamides was optimized to a highly selective inhibitor with sub-1 µM activity, that is active against T790M, C797S mutant EGFR independent of ATP concentration, providing a potential new strategy for pan-EGFR mutant inhibition.

A Pilot Study Investigating Plasma Protein Electrophoresis in One Anuran and Six Urodelan Species.

As threats to amphibian health increase, there is a growing need for diagnostic tools to assess and monitor their health status. Plasma protein electrophoresis has proven to be useful in other nonmammalian species. It enables quantification of protein fractions in plasma that may be altered in various disease processes, and is therefore useful in narrowing down differential diagnoses and detecting inflammation, in combination with other modalities such as biochemical and hematologic testing. The amphibian electrophoretogram must be defined before baseline reference intervals are obtained across species. Agarose gel electrophoresis was performed on plasma samples collected from presumed clinically normal individuals of one anuran and six urodelans: Osteopilus septentrionalis (n=2), Gyrinophilus porphyriticus (n=1), Notophthalmus viridescens (n=1), Eurycea guttolineata (n=2), Amphiuma tridactylum (n=2), Cryptobranchus alleganiensis (n=5), and Siren lacertina (n=6). The electrophoretograms varied in number of fractions between each species; however, the number of fractions was consistent within a species. An albumin migrating fraction was consistently observed in all species. A prealbumin migrating fraction was identified in species that primarily use organs other than skin for respiration. This study provides preliminary examples of a normal plasma protein electrophoretogram for seven amphibian species. Further studies quantifying reference intervals and identification of protein fractions will help establish protein electrophoresis as a useful tool in amphibian health investigations.

ADAMTS3 restricts cancer invasion in models of early breast cancer progression through enhanced fibronectin degradation.

Proteases have long been associated with cancer progression, due to their ability to facilitate invasion upon matrix remodelling. However, proteases are not simply degraders of the matrix, but also play fundamental roles in modulating cellular behaviour through the proteolytic processing of specific substrates. Indeed, proteases can elicit both pro- and anti- tumorigenic effects depending on context. Using a heterocellular spheroid model of breast cancer progression, we demonstrate the repressive function of myoepithelial ADAMTS3, with its loss directing myoepithelial-led invasion of luminal cells through a physiologically relevant matrix. Degradomic analysis, using terminal amine isotopic labelling of substrates (TAILS), combined with functional assays, implicate ADAMTS3 as a mediator of fibronectin degradation. We show further that loss of ADAMTS3 enhances levels of fibronectin in the microenvironment, promoting invasion through canonical integrin α5ß1 activation. Our data highlight a tumour suppressive role for ADAMTS3 in early stage breast cancer, and contribute to the growing evidence that proteases can restrain cancer progression.

Broad host susceptibility of North American amphibian species to Batrachochytrium salamandrivorans suggests high invasion potential and biodiversity risk.

Batrachochytrium salamandrivorans (Bsal) is a fungal pathogen of amphibians that is emerging in Europe and could be introduced to North America through international trade or other pathways. To evaluate the risk of Bsal invasion to amphibian biodiversity, we performed dose-response experiments on 35 North American species from 10 families, including larvae from five species. We discovered that Bsal caused infection in 74% and mortality in 35% of species tested. Both salamanders and frogs became infected and developed Bsal chytridiomycosis. Based on our host susceptibility results, environmental suitability conditions for Bsal, and geographic ranges of salamanders in the United States, predicted biodiversity loss is expected to be greatest in the Appalachian Region and along the West Coast. Indices of infection and disease susceptibility suggest that North American amphibian species span a spectrum of vulnerability to Bsal chytridiomycosis and most amphibian communities will include an assemblage of resistant, carrier, and amplification species. Predicted salamander losses could exceed 80 species in the United States and 140 species in North America.

HDAC Inhibition Restores Response to HER2-Targeted Therapy in Breast Cancer via PHLDA1 Induction.

The downregulation of Pleckstrin Homology-Like Domain family A member 1 (PHLDA1) expression mediates resistance to targeted therapies in receptor tyrosine kinase-driven cancers. The restoration and maintenance of PHLDA1 levels in cancer cells thus constitutes a potential strategy to circumvent resistance to inhibitors of receptor tyrosine kinases. Through a pharmacological approach, we identify the inhibition of MAPK signalling as a crucial step in PHLDA1 downregulation. Further ChIP-qPCR analysis revealed that MEK1/2 inhibition produces significant epigenetic changes at the PHLDA1 locus, specifically a decrease in the activatory marks H3Kme3 and H3K27ac. In line with this, we show that treatment with the clinically relevant class I histone deacetylase (HDAC) inhibitor 4SC-202 restores PHLDA1 expression in lapatinib-resistant human epidermal growth factor receptor-2 (HER2)+ breast cancer cells. Critically, we show that when given in combination, 4SC-202 and lapatinib exert synergistic effects on 2D cell proliferation and colony formation capacity. We therefore propose that co-treatment with 4SC-202 may prolong the clinical efficacy of lapatinib in HER2+ breast cancer patients.

TGFß-mediated MMP13 secretion drives myoepithelial cell dependent breast cancer progression.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer. Virtually all women with DCIS are treated, despite evidence suggesting up to half would remain with stable, non-threatening, disease. Overtreatment thus presents a pressing issue in DCIS management. To understand the role of the normally tumour suppressive myoepithelial cell in disease progression we present a 3D in vitro model incorporating both luminal and myoepithelial cells in physiomimetic conditions. We demonstrate that DCIS-associated myoepithelial cells promote striking myoepithelial-led invasion of luminal cells, mediated by the collagenase MMP13 through a non-canonical TGFß - EP300 pathway. In vivo, MMP13 expression is associated with stromal invasion in a murine model of DCIS progression and is elevated in myoepithelial cells of clinical high-grade DCIS cases. Our data identify a key role for myoepithelial-derived MMP13 in facilitating DCIS progression and point the way towards a robust marker for risk stratification in DCIS patients.

Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion.

Pancreatic ductal adenocarcinoma (PDAC) continues to show no improvement in survival rates. One aspect of PDAC is elevated ATP levels, pointing to the purinergic axis as a potential attractive therapeutic target. Mediated in part by highly druggable extracellular proteins, this axis plays essential roles in fibrosis, inflammation response, and immune function. Analyzing the main members of the PDAC extracellular purinome using publicly available databases discerned which members may impact patient survival. P2RY2 presents as the purinergic gene with the strongest association with hypoxia, the highest cancer cell-specific expression, and the strongest impact on overall survival. Invasion assays using a 3D spheroid model revealed P2Y2 to be critical in facilitating invasion driven by extracellular ATP. Using genetic modification and pharmacological strategies, we demonstrate mechanistically that this ATP-driven invasion requires direct protein-protein interactions between P2Y2 and αV integrins. DNA-PAINT super-resolution fluorescence microscopy reveals that P2Y2 regulates the amount and distribution of integrin αV in the plasma membrane. Moreover, receptor-integrin interactions were required for effective downstream signaling, leading to cancer cell invasion. This work elucidates a novel GPCR-integrin interaction in cancer invasion, highlighting its potential for therapeutic targeting.

Everybody needs good neighbours: the progressive DCIS microenvironment.

Ductal carcinoma in situ (DCIS) is a pre-invasive form of breast cancer where neoplastic luminal cells are confined to the ductal tree. While as many as 70% of DCIS cases will remain indolent, most women are treated with surgery, often combined with endocrine and radiotherapies. Overtreatment is therefore a major issue, demanding new methods to stratify patients. Somewhat paradoxically, the neoplastic cells in DCIS are genetically comparable to those in invasive disease, suggesting the tumour microenvironment is the driving force for progression. Clinical and mechanistic studies highlight the complex DCIS microenvironment, with multiple cell types competing to regulate progression. Here, we examine recent studies detailing distinct aspects of the DCIS microenvironment and discuss how these may inform more effective care.

Low HER2 expression in normal breast epithelium enables dedifferentiation and malignant transformation via chromatin opening.

Overexpression of the HER2 protein in breast cancer patients is a predictor of poor prognosis and resistance to therapies. We used an inducible breast cancer transformation system that allows investigation of early molecular changes. HER2 overexpression to similar levels as those observed in a subtype of HER2-positive breast cancer patients induced transformation of MCF10A cells and resulted in gross morphological changes, increased anchorage-independent growth of cells, and altered the transcriptional programme of genes associated with oncogenic transformation. Global phosphoproteomic analysis during HER2 induction predominantly detected an increase in protein phosphorylation. Intriguingly, this correlated with chromatin opening, as measured by ATAC-seq on acini isolated from 3D cell culture. HER2 overexpression resulted in opening of many distal regulatory regions and promoted reprogramming-associated heterogeneity. We found that a subset of cells acquired a dedifferentiated breast stem-like phenotype, making them likely candidates for malignant transformation. Our data show that this population of cells, which counterintuitively enriches for relatively low HER2 protein abundance and increased chromatin accessibility, possesses transformational drive, resulting in increased anchorage-independent growth in vitro compared to cells not displaying a stem-like phenotype.

Nuclear FGFR1 promotes pancreatic stellate cell-driven invasion through up-regulation of Neuregulin 1.

Pancreatic stellate cells (PSCs) are key to the treatment-refractory desmoplastic phenotype of pancreatic ductal adenocarcinoma (PDAC) and have received considerable attention as a stromal target for cancer therapy. This approach demands detailed understanding of their pro- and anti-tumourigenic effects. Interrogating PSC-cancer cell interactions in 3D models, we identified nuclear FGFR1 as critical for PSC-led invasion of cancer cells. ChIP-seq analysis of FGFR1 in PSCs revealed a number of FGFR1 interaction sites within the genome, notably NRG1, which encodes the ERBB ligand Neuregulin. We show that nuclear FGFR1 regulates transcription of NRG1, which in turn acts in autocrine fashion through an ERBB2/4 heterodimer to promote invasion. In support of this, recombinant NRG1 in 3D model systems rescued the loss of invasion incurred by FGFR inhibition. In vivo we demonstrate that, while FGFR inhibition does not affect the growth of pancreatic tumours in mice, local invasion into the pancreas is reduced. Thus, FGFR and NRG1 may present new stromal targets for PDAC therapy.

Incidence of iron deficiency Anaemia during pregnancy in Far North Queensland.

INTRODUCTION: The WHO estimates the incidence of iron deficiency anaemia in Australia is 25%. However there is considerable variation during pregnancy and in regional areas. OBJECTIVE: The aim of this investigation is to quantify the incidence of iron deficiency anaemia during pregnancy within Far North Queensland. DESIGN: This is a single-centre retrospective cohort study. Cairns Hospital is the main referral centre for complex maternity care in Far North Queensland with an estimated population of 280-000, which includes many people from rural and remote communities and a high proportion who identify as Aboriginal or Torres Strait Islander. This study included all births at the Cairns Hospital in 2018, a total of 2190 deliveries. FINDINGS: The study randomly sampled 551 mothers from the cohort, and the incidence of iron deficiency anaemia was 34.9%. 48.7% of women who identified as Aboriginal or Torres Strait Islander within the region were anaemic. This was significantly higher than an incidence of 28.9% for the rest of the population. Other risk factors include booking appointment after 28-weeks, Asian ethnicity and age less than 25-years. A BMI greater than 35 was protective DISCUSSION: This study will inform antenatal care providers within the region and improve obstetric outcomes by increasing awareness. Identifying risk factors will also facilitate prompt treatment and improve maternity care for vulnerable patient groups. On a broader level, the study provides new data to inform population health estimates both nationally and internationally. CONCLUSION: The incidence of iron deficiency anaemia during pregnancy in Far North Queensland is significantly higher than previous estimates.

Efficacy of Plant-Derived Fungicides at Inhibiting Batrachochytrium salamandrivorans Growth.

The emerging fungal amphibian pathogen, Batrachochytrium salamandrivorans (Bsal), is currently spreading across Europe and given its estimated invasion potential, has the capacity to decimate salamander populations worldwide. Fungicides are a promising in situ management strategy for Bsal due to their ability to treat the environment and infected individuals. However, antifungal drugs or pesticides could adversely affect the environment and non-target hosts, thus identifying safe, effective candidate fungicides for in situ treatment is needed. Here, we estimated the inhibitory fungicidal efficacy of five plant-derived fungicides (thymol, curcumin, allicin, 6-gingerol, and Pond Pimafix®) and one chemical fungicide (Virkon® Aquatic) against Bsal zoospores in vitro. We used a broth microdilution method in 48-well plates to test the efficacy of six concentrations per fungicide on Bsal zoospore viability. Following plate incubation, we performed cell viability assays and agar plate growth trials to estimate the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of each fungicide. All six fungicides exhibited inhibitory and fungicidal effects against Bsal growth, with estimated MIC concentrations ranging from 60 to 0.156 µg/mL for the different compounds. Allicin showed the greatest efficacy (i.e., lowest MIC and MFC) against Bsal zoospores followed by curcumin, Pond Pimafix®, thymol, 6-gingerol, and Virkon® Aquatic, respectively. Our results provide evidence that plant-derived fungicides are effective at inhibiting and killing Bsal zoospores in vitro and may be useful for in situ treatment. Additional studies are needed to estimate the efficacy of these fungicides at inactivating Bsal in the environment and treating Bsal-infected amphibians.

Omental patch for lower uterine segment repair during caesarean section.

Increasing rates of caesarean section have contributed to a higher number of complications such as vesicouterine fistula. A G6P5 woman in her early forties presented for her third elective repeat caesarean section. At the time of delivery, there was uterine dehiscence and the fetus was visible through a large 7×5 cm transparent window in the lower segment. After delivery of the baby, the uterus was unable to be repaired with sutures so an omental patch was fixed on the lower segment to facilitate healing. It is hypothesised that the angiogenic properties of the omentum may have promoted healing of the uterine defect and reduced the risk of vesicouterine fistula by providing a protective barrier. The patient recovered well and by 4 months post partum, her menstrual cycle had returned. Thereby, the use of an omental patch during uterine repair may reduce long-term complications associated with repeat caesarean section.

Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumor invasion.

In pancreatic ductal adenocarcinoma (PDAC), differentiation of pancreatic stellate cells (PSCs) into myofibroblast-like cancer-associated fibroblasts (CAFs) can both promote and suppress tumor progression. Here, we show that the Rho effector protein kinase N2 (PKN2) is critical for PSC myofibroblast differentiation. Loss of PKN2 is associated with reduced PSC proliferation, contractility, and alpha-smooth muscle actin (α-SMA) stress fibers. In spheroid co-cultures with PDAC cells, loss of PKN2 prevents PSC invasion but, counter-intuitively, promotes invasive cancer cell outgrowth. PKN2 deletion induces a myofibroblast to inflammatory CAF switch in the PSC matrisome signature both in vitro and in vivo. Further, deletion of PKN2 in the pancreatic stroma induces more locally invasive, orthotopic pancreatic tumors. Finally, we demonstrate that a PKN2KO matrisome signature predicts poor outcome in pancreatic and other solid human cancers. Our data indicate that suppressing PSC myofibroblast function can limit important stromal tumor-suppressive mechanisms, while promoting a switch to a cancer-supporting CAF phenotype.

Effect of surgeon volume on long-term quality of life outcomes following tension-free vaginal tape surgery.

INTRODUCTION AND HYPOTHESIS: The role of the general obstetrician/gynaecologist completing routine urogynaecology procedures is controversial, and some research has suggested that these patients should be referred to high-volume subspecialists. In response to recent public and regulatory scrutiny of vaginal mesh procedures, credentialling guidelines have been released in Australia requiring surgeons to demonstrate a minimum caseload prior to performing tension-free vaginal tape (TVT) surgery for incontinence. Hence, a retrospective cohort study was conducted to evaluate the long-term quality of life outcomes of TVT procedures for high- and low-volume surgeons. METHODS: One hundred seventy patients who had undergone TVT surgery between 1 May 2011 and 1 May 2016 in the Sunshine Coast health district were invited to complete the UDI-6 (Urinary Distress Inventory) and IIQ-7 (Incontinence Impact Questionnaire) surveys. Perioperative information was accessed from available health records. Mean UDI-6 and IIQ-7 scores were compared for high- and low-volume groups, and the groups were assessed for confounding factors. RESULTS: Of the 170 patients eligible, 83 completed the surveys (47.2%). No differences in UDI-6 or IIQ-7 scores were found between high- and low-volume surgeons (p > 0.05). High-volume surgeons completed more concomitant procedures amongst survey respondents (p < 0.05), though this was not reproduced when considering all 170 patients eligible for the study. There were no significant differences in age, ASA (American Society of Anaesthesiologists) score or complication rate amongst survey respondents. CONCLUSIONS: Amongst the patients surveyed, high- and low-volume surgeons had similar long-term quality of life outcomes for TVT surgery, without any significant difference in complication rate.

Frequency-dependent transmission of Batrachochytrium salamandrivorans in eastern newts.

Transmission is the fundamental process whereby pathogens infect their hosts and spread through populations, and can be characterized using mathematical functions. The functional form of transmission for emerging pathogens can determine pathogen impacts on host populations and can inform the efficacy of disease management strategies. By directly measuring transmission between infected and susceptible adult eastern newts (Notophthalmus viridescens) in aquatic mesocosms, we identified the most plausible transmission function for the emerging amphibian fungal pathogen Batrachochytrium salamandrivorans (Bsal). Although we considered a range of possible transmission functions, we found that Bsal transmission was best explained by pure frequency dependence. We observed that >90% of susceptible newts became infected within 17 days post-exposure to an infected newt across a range of host densities and initial infection prevalence treatments. Under these conditions, we estimated R0  = 4.9 for Bsal in an eastern newt population. Our results suggest that Bsal has the capability of driving eastern newt populations to extinction and that managing host density may not be an effective management strategy. Intervention strategies that prevent Bsal introduction or increase host resistance or tolerance to infection may be more effective. Our results add to the growing empirical evidence that transmission of wildlife pathogens can saturate and be functionally frequency-dependent.

Elucidating the role of the kinase activity of endothelial cell focal adhesion kinase in angiocrine signalling and tumour growth.

A common limitation of cancer treatments is chemotherapy resistance. We have previously identified that endothelial cell (EC)-specific deletion of focal adhesion kinase (FAK) sensitises tumour cells to DNA-damaging therapies, reducing tumour growth in mice. The present study addressed the kinase activity dependency of EC FAK sensitisation to the DNA-damaging chemotherapeutic drug, doxorubicin. FAK is recognised as a therapeutic target in tumour cells, leading to the development of a range of inhibitors, the majority being ATP competitive kinase inhibitors. We demonstrate that inactivation of EC FAK kinase domain (kinase dead; EC FAK-KD) in established subcutaneous B16F0 tumours improves melanoma cell sensitisation to doxorubicin. Doxorubicin treatment in EC FAK-KD mice reduced the percentage change in exponential B16F0 tumour growth further than in wild-type mice. There was no difference in tumour blood vessel numbers, vessel perfusion or doxorubicin delivery between genotypes, suggesting a possible angiocrine effect on the regulation of tumour growth. Doxorubicin reduced perivascular malignant cell proliferation, while enhancing perivascular tumour cell apoptosis and DNA damage in tumours grown in EC FAK-KD mice 48 h after doxorubicin injection. Human pulmonary microvascular ECs treated with the pharmacological FAK kinase inhibitors defactinib, PF-562,271 or PF-573,228 in combination with doxorubicin also reduced cytokine expression levels. Together, these data suggest that targeting EC FAK kinase activity may alter angiocrine signals that correlate with improved acute tumour cell chemosensitisation. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.