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.

The Future of Precision Oncology.

Our understanding of the molecular mechanisms underlying cancer development and evolution have evolved rapidly over recent years, and the variation from one patient to another is now widely recognized. Consequently, one-size-fits-all approaches to the treatment of cancer have been superseded by precision medicines that target specific disease characteristics, promising maximum clinical efficacy, minimal safety concerns, and reduced economic burden. While precision oncology has been very successful in the treatment of some tumors with specific characteristics, a large number of patients do not yet have access to precision medicines for their disease. The success of next-generation precision oncology depends on the discovery of new actionable disease characteristics, rapid, accurate, and comprehensive diagnosis of complex phenotypes within each patient, novel clinical trial designs with improved response rates, and worldwide access to novel targeted anticancer therapies for all patients. This review outlines some of the current technological trends, and highlights some of the complex multidisciplinary efforts that are underway to ensure that many more patients with cancer will be able to benefit from precision oncology in the near future.

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.

Pancreatic Cancer Organotypic Models.

Pancreatic cancer has a dismal prognosis for patients due to late diagnosis and ineffective treatment options. There is a desperate need for more accurate disease models to enable improved therapies and diagnostic tests to reach the clinic. Pancreatic tumours have a high content of desmoplastic stroma, which forms a stiff, hypoxic tumour mass and contributes significantly to tumour development and metastatic spread. Therefore, 2D cell culture is not sufficient for understanding the complex biology of this disease. 3D in vitro models offer a more representative method of culturing cells for research in the laboratory. There are many different 3D models that can be used in research, organoids formed from patient or murine tumours are grown embedded in collagen or matrigel matrices, giving the potential for screening treatment options and personalised therapy in the future. Also, organotypic models using pancreatic cancer cell lines and stromal cells can be easily manipulated to study different aspects of pancreatic cancer and new therapeutic options in the laboratory. There are new emerging pancreatic cancer 3D models being developed, including microchip technology or synthetic scaffolds instead collagen and matrigel. All of these 3D culturing methods give an advantage over traditional 2D cell culture and could lead to improved understanding of this disease, translating to a better prognosis for patients in the clinic.

Dysregulated FGF signalling in neoplastic disorders.

The fibroblast growth factor (FGF) signalling pathway contributes to the regulation of a multitude of cellular functions, impacting on proliferation, survival, differentiation and migration. This biological importance is reflected by its prominent role in carcinogenesis; often being hijacked by cancer cells to offer growth or survival advantage. FGF signalling can contribute a driving force in the malignancy of different cancer types; through alterations in ligands, receptors or regulatory molecules. The dramatic advances in genomics technologies have highlighted how mutation, amplification, translocation or loss of elements in the FGF signalling network can contribute to cancer. Added to this are the stromal influences of FGF signalling. Dissection of the mechanisms that underlie the pro-tumourigenic effects resulting from perturbations to the FGF signalling network will be of utmost importance to the development of therapeutic approaches to treat FGF receptor (FGFR)-driven cancers. In this review, we will focus on the mechanisms of FGF deregulation, the prevalence of aberrations in different cancer types, and how we are progressing in the development of targeted therapies.

A 3D in vitro model of the human breast duct: a method to unravel myoepithelial-luminal interactions in the progression of breast cancer.

BACKGROUND: 3D modelling fulfils a critical role in research, allowing for complex cell behaviour and interactions to be studied in physiomimetic conditions. With tissue banks becoming established for a number of cancers, researchers now have access to primary patient cells, providing the perfect building blocks to recreate and interrogate intricate cellular systems in the laboratory. The ducts of the human breast are composed of an inner layer of luminal cells supported by an outer layer of myoepithelial cells. In early-stage ductal carcinoma in situ, cancerous luminal cells are confined to the ductal space by an intact myoepithelial layer. Understanding the relationship between myoepithelial and luminal cells in the development of cancer is critical for the development of new therapies and prognostic markers. This requires the generation of new models that allows for the manipulation of these two cell types in a physiological setting. METHODS: Using access to the Breast Cancer Now Tissue Bank, we isolated pure populations of myoepithelial and luminal cells from human reduction mammoplasty specimens and placed them into 2D culture. These cells were infected with lentiviral particles encoding either fluorescent proteins, to facilitate cell tracking, or an inducible human epidermal growth factor receptor 2 (HER2) expression construct. Myoepithelial and luminal cells were then recombined in collagen gels, and the resulting cellular structures were analysed by confocal microscopy. RESULTS: Myoepithelial and luminal cells isolated from reduction mammoplasty specimens can be grown separately in 2D culture and retain their differentiated state. When recombined in collagen gels, these cells reform into physiologically reflective bilayer structures. Inducible expression of HER2 in the luminal compartment, once the bilayer has formed, leads to robust luminal filling, recapitulating ductal carcinoma in situ, and can be blocked with anti-HER2 therapies. CONCLUSIONS: This model allows for the interaction between myoepithelial and luminal cells to be investigated in an in-vitro environment and paves the way to study early events in breast cancer development with the potential to act as a powerful drug discovery platform.

Anti-stromal treatment together with chemotherapy targets multiple signalling pathways in pancreatic adenocarcinoma.

Stromal targeting for pancreatic ductal adenocarcinoma (PDAC) is rapidly becoming an attractive option, due to the lack of efficacy of standard chemotherapy and increased knowledge about PDAC stroma. We postulated that the addition of stromal therapy may enhance the anti-tumour efficacy of chemotherapy. Gemcitabine and all-trans retinoic acid (ATRA) were combined in a clinically applicable regimen, to target cancer cells and pancreatic stellate cells (PSCs) respectively, in 3D organotypic culture models and genetically engineered mice (LSL-Kras(G12D) (/+) ;LSL-Trp53(R172H) (/+) ;Pdx-1-Cre: KPC mice) representing the spectrum of PDAC. In two distinct sets of organotypic models as well as KPC mice, we demonstrate a reduction in cancer cell proliferation and invasion together with enhanced cancer cell apoptosis when ATRA is combined with gemcitabine, compared to vehicle or either agent alone. Simultaneously, PSC activity (as measured by deposition of extracellular matrix proteins such as collagen and fibronectin) and PSC invasive ability were both diminished in response to combination therapy. These effects were mediated through a range of signalling cascades (Wnt, hedgehog, retinoid, and FGF) in cancer as well as stellate cells, affecting epithelial cellular functions such as epithelial-mesenchymal transition, cellular polarity, and lumen formation. At the tissue level, this resulted in enhanced tumour necrosis, increased vascularity, and diminished hypoxia. Consequently, there was an overall reduction in tumour size. The enhanced effect of stromal co-targeting (ATRA) alongside chemotherapy (gemcitabine) appears to be mediated by dampening multiple signalling cascades in the tumour-stroma cross-talk, rather than ablating stroma or targeting a single pathway. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Identification of ZDHHC14 as a novel human tumour suppressor gene.

Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase-dependent apoptotic pathway and heterozygous knockout of ZDHHC14 increased [CORRECTED] cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down-regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer.

FGF receptors 1 and 2 are key regulators of keratinocyte migration in vitro and in wounded skin.

Efficient wound repair is essential for the maintenance of the integrity of the skin. The repair process is controlled by a variety of growth factors and cytokines, and their abnormal expression or activity can cause healing disorders. Here, we show that wound repair is severely delayed in mice lacking fibroblast growth factor receptors (FGFR) 1 and 2 in keratinocytes. As the underlying mechanism, we identified impaired wound contraction and a delay in re-epithelialization that resulted from impaired keratinocyte migration at the wound edge. Scratch wounding and transwell assays demonstrated that FGFR1/2-deficient keratinocytes had a reduced migration velocity and impaired directional persistence owing to inefficient formation and turnover of focal adhesions. Underlying this defect, we identified a significant reduction in the expression of major focal adhesion components in the absence of FGFR signaling, resulting in a general migratory deficiency. These results identify FGFs as key regulators of keratinocyte migration in wounded skin.

The ins and outs of fibroblast growth factor receptor signalling.

FGFR (fibroblast growth factor receptor) signalling plays critical roles in embryogensis, adult physiology, tissue repair and many pathologies. Of particular interest over recent years, it has been implicated in a wide range of cancers, and concerted efforts are underway to target different aspects of FGFR signalling networks. A major focus has been identifying the canonical downstream signalling pathways in cancer cells, and these are now relatively well understood. In the present review, we focus on two distinct but emerging hot topics in FGF biology: its role in stromal cross-talk during cancer progression and the potential roles of FGFR signalling in the nucleus. These neglected areas are proving to be of great interest clinically and are intimately linked, at least in pancreatic cancer. The importance of the stroma in cancer is well accepted, both as a conduit/barrier for treatment and as a target in its own right. Nuclear receptors are less acknowledged as targets, largely due to historical scepticism as to their existence or importance. However, increasing evidence from across the receptor tyrosine kinase field is now strong enough to make the study of nuclear growth factor receptors a major area of interest.

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.

Non-junctional human desmoglein 3 acts as an upstream regulator of Src in E-cadherin adhesion, a pathway possibly involved in the pathogenesis of pemphigus vulgaris.

E-cadherin, a classical cadherin, is an adhesion receptor in adherens junctions and has important functions in cell-cell adhesion and cell signalling. Recently we reported that a desmosomal cadherin, desmoglein 3 (Dsg3), an autoantigen in pemphigus vulgaris (PV), associates with E-cadherin and activates Src, which results in tyrosine phosphorylation of adherens junction proteins. However, the nature of such an interaction and its role in cell-cell adhesion remain unclear. In this report, we provide direct evidence that it is the detergent-soluble, non-desmosomal Dsg3 that regulates the activity of Src and its association with E-cadherin in adherens junction formation. Modulation of Dsg3 levels, either by Dsg3 silencing or over-expression, alters Src activity and its association with E-cadherin. Dsg3 silencing caused retardation of calcium-induced E-cadherin junction assembly and a reduction of desmosomal protein expression. Furthermore, we provide evidence that this signalling pathway is involved, at least in part, in the pathophysiology of pemphigus. Along with the reduced expression of Dsg3, loss and disruption of E-cadherin and a concomitant decreased pSrc signalling was identified in the basal keratinocytes surrounding the blisters in PV. These findings suggest a novel function for Dsg3 in the control of E-cadherin-Src signalling and cell-cell adhesion.

In silico targeting of colony-stimulating factor-1 receptor: delineating immunotherapy in cancer.

Aim: Delineate structure-based inhibition of colony-stimulating factor-1 receptor (CSF1R) by small molecule CSF1R inhibitors in clinical development for target identification and potential lead optimization in cancer therapeutics since CSF1R is a novel predictive biomarker for immunotherapy in cancer. Methods: Compounds were in silico modelled by induced fit docking protocol in a molecular operating environment (MOE, MOE.v.2015). The 3-dimensional (3D) X-ray crystallized structure of CSF1R kinase (Protein Databank, ID 4R7H) was obtained from Research Collaboratory for Structural Bioinformatics (RSCB) Protein Databank. The 3D conformers of edicotinib, DCC-3014, ARRY-382, BLZ-945, chiauranib, dovitinib, and sorafenib were obtained from PubChem Database. These structures were modelled in Amber10:EHT molecular force field, and quick prep application was used to correct and optimize the structures for missing residues, H-counts, termini capping, and alternates. The binding site was defined within the vicinity of the co-crystallized ligand of CSF1R kinase. The compounds were docked by the triangular matcher placement method and ranked by the London dG scoring function. The docked poses were further refined by the induced fit method. The pose with the lowest binding score (ΔG) was used to model the ligand interaction profile in Discovery Studio Visualizer v17.2. The co-crystallized ligand was docked in its apo conformation, and root-mean-square deviation was computed to validate the docking protocol. Results: All 7 CSF1R inhibitors interact with residue Met637 exhibiting selectivity except for edicotinib. The inhibitors maintain CSF1R in an auto-inhibitory conformation by interacting with Asp797 of the Asp-Phe-Gly (DFG) motif and/or hindering the conserved salt bridge formed between Glu633 and Lys616 thus stabilizing the activation loop, or interacting with tryptophan residue (Trp550) in the juxtamembrane domain. DCC-3014, ARRY-382, BLZ-945, and sorafenib bind with the lowest binding energy with CSF1R kinase. Conclusions: Pyrimidines are potent inhibitors that interact with CSF1R residues. DCC-3014 and ARRY-382 exhibit exceptional pharmaceutical potential exhibiting great structural stability and affinity.

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.

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.

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.

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.

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.

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.

Regulation of lymphatic-blood vessel separation by endothelial Rac1.

Sprouting angiogenesis and lymphatic-blood vessel segregation both involve the migration of endothelial cells, but the precise migratory molecules that govern the decision of blood vascular endothelial cells to segregate into lymphatic vasculature are unknown. Here, we deleted endothelial Rac1 in mice (Tie1-Cre(+);Rac1(fl/fl)) and revealed, unexpectedly, that whereas blood vessel morphology appeared normal, lymphatic-blood vessel separation was impaired, with corresponding edema, haemorrhage and embryonic lethality. Importantly, normal levels of Rac1 were essential for directed endothelial cell migratory responses to lymphatic-inductive signals. Our studies identify Rac1 as a crucial part of the migratory machinery required for endothelial cells to separate and form lymphatic vasculature.