Ex vivo therapeutic screening of metastatic cSCC: A review of methodological considerations for clinical implementation.

Cutaneous squamous cell carcinoma (cSCC) is the second most common malignancy worldwide, with most deaths caused by locally advanced and metastatic disease. Treatment of resectable metastases is typically limited to invasive surgery with adjuvant radiotherapy; however, many patients fail to respond and there is minimal data to predict response or propose effective alternatives. Precision medicine could improve this, though genomic biomarkers remain elusive in the high mutational background and genomic complexity of cSCC. A phenotypic approach to precision medicine using patient-derived ex vivo tumour models is gaining favour for its capacity to directly assess biological responses to therapeutics as a functional, predictive biomarker. However, the use of ex vivo models for guiding therapeutic selection has yet to be employed for metastatic cSCC. This review will therefore evaluate the existing experimental models of metastatic cSCC and discuss how ex vivo methods could overcome the shortcomings of these existing models. Disease-specific considerations for a prospective methodological pipeline will also be discussed in the context of precision medicine.

PIK Your Poison: The Effects of Combining PI3K and CDK Inhibitors against Metastatic Cutaneous Squamous Cell Carcinoma In Vitro.

Cutaneous squamous cell carcinoma (cSCC) is a very common skin malignancy with poor prognosis for patients with locally advanced or metastatic cSCC (mcSCC). PI3K/AKT/mTOR and cell cycle signalling pathways are often dysregulated in mcSCC. A combination drug approach has been theorised to overcome the underwhelming clinical performance of targeted inhibitors as single agents. This study investigates the potential of targeted inhibition of the p110α-subunit of PI3K with PIK-75 or BGT226 (P13Ki), and of CDK1/2/5/9 with dinaciclib (CDKi) as single agents and in combination. The patient-derived mcSCC cell lines, UW-CSCC1 and UW-CSCC2, were used to assess cell viability, migration, cell signalling, cell cycle distribution, and apoptosis. PIK-75, BGT226, and dinaciclib exhibited strong cytotoxic potency as single agents. Notably, the non-malignant HaCaT cell line was unaffected. In 2D cultures, PIK-75 synergistically enhanced the cytotoxic effects of dinaciclib in UW-CSCC2, but not UW-CSCC1. Interestingly, this pattern was reversed in 3D spheroid models. Despite the combination of PIK-75 and dinaciclib resulting in an increase in cell cycle arrest and apoptosis, and reduced cell motility, these differences were largely negligible compared to their single-agent counterpart. The differential responses between the cell lines correlated with driver gene mutation profiles. These findings suggest that personalised medicine approaches targeting PI3K and CDK pathways in combination may yield some benefit for mcSCC, and that more complex 3D models should be considered for drug responsiveness studies in this disease.

Ion Transport and Inhibitor Binding by Human NHE1: Insights from Molecular Dynamics Simulations and Free Energy Calculations.

The human Na+/H+ exchanger (NHE1) plays a crucial role in maintaining intracellular pH by regulating the electroneutral exchange of a single intracellular H+ for one extracellular Na+ across the plasma membrane. Understanding the molecular mechanisms governing ion transport and the binding of inhibitors is of importance in the development of anticancer therapeutics targeting NHE1. In this context, we performed molecular dynamics (MD) simulations based on the recent cryo-electron microscopy (cryo-EM) structures of outward- and inward-facing conformations of NHE1. These simulations allowed us to explore the dynamics of the protein, examine the ion-translocation pore, and confirm that Asp267 is the ion-binding residue. Our free energy calculations did not show a significant difference between Na+ and K+ binding at the ion-binding site. Consequently, Na+ over K+ selectivity cannot be solely explained by differences in ion binding. Our MD simulations involving NHE1 inhibitors (cariporide and amiloride analogues) maintained stable interactions with Asp267 and Glu346. Our study highlights the importance of the salt bridge between the positively charged acylguanidine moiety and Asp267, which appears to play a role in the competitive inhibitory mechanism for this class of inhibitors. Our computational study provides a detailed mechanistic interpretation of experimental data and serves the basis of future structure-based inhibitor design.

PAI-1 Deficiency Drives Pulmonary Vascular Smooth Muscle Remodeling and Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive and potentially a rapidly fatal disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PA) leading to increased pulmonary vascular resistance and right heart failure. Central to the remodeling process is a switch of the smooth muscle cells in small PAs (PASMC) to a proliferative, apoptosis-resistant phenotype. There is reason to suspect that the plasminogen activator system may play an important role in the remodeling program in PAH based on its roles in vascular post-injury restenosis, fibrosis, angiogenesis and tumorigenesis. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of the plasminogen activators - urokinase-type and tissue-type (uPA and tPA, respectively). Immunohisto- chemical and immunoblot analyses revealed that PAI-1 was deficient in smooth muscle areas of small remodeled PAs and early-passage PASMC from subjects with PAH compared to non-PAH controls. PAI1-/- male and female mice developed spontaneous pulmonary vascular remodeling and pulmonary hypertension (PH) as evidenced by significant increase in PA medial thickness, systolic right ventricular pressure, and right ventricular hypertrophy. Lastly, the uPA inhibitors upamostat (WX-671) and amiloride analog BB2-30F down-regulated mTORC1 and SMAD3, restored PAI-1 levels, reduced proliferation, and induced apoptosis in human PAH PASMC. We examined the effect of inhibition of uPA catalytic activity by BB2-30F on the development of SU5416/Hypoxia (SuHx)-induced PH in mice. Vehicletreated SuHx-exposed mice had up-regulated mTORC1 in small PAs, developed pulmonary vascular remodeling and PH, as evidenced by significant increase of PA MT, sRVP, RV hypertrophy, and a significant decrease in the pulmonary artery acceleration time/pulmonary ejection time (PAAT/PET) ratio compared to age- and sex-matched normoxia controls, whereas BB2-30F-treated group was protected from all these pathological changes. Taken together, our data strongly suggest that PAI-1 down- regulation in PASMC from human PAH lungs promotes PASMC hyper-proliferation, remodeling, and spontaneous PH due to unopposed uPA activation. Further studies are needed to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate the progression and/or reverse pulmonary vascular remodeling and PH.

Sex as a Predictor of Response to Immunotherapy in Advanced Cutaneous Squamous Cell Carcinoma.

Approximately 3-5% of patients with cutaneous squamous cell carcinoma (CSCC) develop advanced disease, accounting for roughly 1% of all cancer deaths in Australia. Immunotherapy has demonstrated significant clinical benefit in advanced CSCC in several key phase II studies; however, there are limited data for patients treated outside of clinical trials. This is particularly relevant in advanced CSCC, which is most often seen in elderly patients with significant comorbidities. Thus, we aim to describe our experience with immunotherapy in a cohort of patients with advanced CSCC in Australia. We retrospectively reviewed all advanced CSCC patients treated with immunotherapy within the Illawarra and Shoalhaven Local Health District. Among the 51 patients treated with immunotherapy, there was an objective response rate (ORR) of 53% and disease control rate (DCR) of 67%. Our most significant predictor of response was sex, with male patients more likely to have better responses compared to female patients (DCR 85% vs. 41%, p < 0.0001), as well as improved progression-free survival (HR 4.6, 95%CI 1.9-10.8, p = 0.0007) and overall survival (HR 3.0, 95%CI 1.3-7.1, p = 0.006). Differential expression analysis of 770 immune-related genes demonstrated an impaired CD8 T-cell response in female patients. Our observed ORR of 53% is similar to that described in current literature with durable responses seen in the majority of patients.

Protocol for the generation and automated confocal imaging of whole multi-cellular tumor spheroids.

Multi-cellular tumor spheroids (MCTS) have found widespread use in pre-clinical research. However, their complex three-dimensional structure makes immunofluorescent staining and imaging challenging. Here, we present a protocol for whole spheroid staining and automated imaging using laser-scanning confocal microscopy. We describe steps for cell culture, seeding of spheroids and transfer of MCTS, and adhesion to Ibidi chamber slides. We then detail fixation, immunofluorescent staining based on optimized reagent concentrations and incubation times, and confocal imaging facilitated by glycerol-based optical clearing.

Automated Patch Clamp Screening of Amiloride and 5-N,N-Hexamethyleneamiloride Analogs Identifies 6-Iodoamiloride as a Potent Acid-Sensing Ion Channel Inhibitor.

Acid-sensing ion channels (ASICs) are transmembrane sensors of extracellular acidosis and potential drug targets in several disease indications, including neuropathic pain and cancer metastasis. The K+-sparing diuretic amiloride is a moderate nonspecific inhibitor of ASICs and has been widely used as a probe for elucidating ASIC function. In this work, we screened a library of 6-substituted and 5,6-disubstituted amiloride analogs using a custom-developed automated patch clamp protocol and identified 6-iodoamiloride as a potent ASIC1 inhibitor. Follow-up IC50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 94 (hASIC1 94 IC50 = 88 nM, cf. amiloride 11 IC50 = 1.7 µM). A similar improvement in activity was observed in ASIC3-mediated currents from rat dorsal root ganglion neurons (rDRG single-concentration 94 IC50 = 230 nM, cf. 11 IC50 = 2.7 µM). 6-Iodoamiloride represents the amiloride analog of choice for studying the effects of ASIC inhibition on cell physiology.

Comparing Genomic Landscapes of Oral and Cutaneous Squamous Cell Carcinoma of the Head and Neck: Quest for Novel Diagnostic Markers.

Squamous cell carcinoma is the most common head and neck malignancy arising from the oral mucosa and the skin. The histologic and immunohistochemical features of oral squamous cell carcinoma (OSCC) and head and neck cutaneous squamous cell carcinoma (HNcSCC) are similar, making it difficult to identify the primary site in cases of metastases. With the advent of immunotherapy, reliable distinction of OSCC and HNcSCC at metastatic sites has important treatment and prognostic implications. Here, we investigate and compare the genomic landscape of OSCC and HNcSCC to identify diagnostically useful biomarkers. Whole-genome sequencing data from 57 OSCC and 41 HNcSCC patients were obtained for tumor and matched normal samples. Tumor mutation burden (TMB), Catalogue of Somatic Mutations in Cancer (COSMIC) mutational signatures, frequent chromosomal alterations, somatic single nucleotide, and copy number variations were analyzed. The median TMB of 3.75 in primary OSCC was significantly lower (P < .001) than that of 147.51 mutations/Mb in primary HNcSCC. The COSMIC mutation signatures were significantly different (P < .001) between OSCC and HNcSCC. OSCC showed COSMIC single-base substitution (SBS) mutation signature 1 and AID/APOBEC activity-associated signature 2 and/or 13. All except 1 HNcSCC from hair-bearing scalp showed UV damage-associated COSMIC SBS mutation signature 7. Both OSCC and HNcSCC demonstrated a predominance of tumor suppressor gene mutations, predominantly TP53. The most frequently mutated oncogenes were PIK3CA and MUC4 in OSCC and HNcSCC, respectively. The metastases of OSCC and HNcSCC demonstrated TMB and COSMIC SBS mutation signatures similar to their primary counterparts. The combination of high TMB and UV signature in a metastatic keratinizing squamous cell carcinoma suggests HNcSCC as the primary site and may also facilitate decisions regarding immunotherapy. HNcSCC and OSCC show distinct genomic profiles despite histologic and immunohistochemical similarities. Their genomic characteristics may underlie differences in behavior and guide treatment decisions in recurrent and metastatic settings.

Recent Advances in Targeting the Urokinase Plasminogen Activator with Nanotherapeutics.

The aberrant proteolytic landscape of the tumor microenvironment is a key contributor of cancer progression. Overexpression of urokinase plasminogen activator (uPA) and/or its associated cell-surface receptor (uPAR) in tumor versus normal tissue is significantly associated with worse clinicopathological features and poorer patient survival across multiple cancer types. This is linked to mechanisms that facilitate tumor cell invasion and migration, via direct and downstream activation of various proteolytic processes that degrade the extracellular matrix─ultimately leading to metastasis. Targeting uPA has thus long been considered an attractive anticancer strategy. However, poor bioavailability of several uPA-selective small-molecule inhibitors has limited early clinical progress. Nanodelivery systems have emerged as an exciting method to enhance the pharmacokinetic (PK) profile of existing chemotherapeutics, allowing increased circulation time, improved bioavailability, and targeted delivery to tumor tissue. Combining uPA inhibitors with nanoparticle-based delivery systems thus offers a remarkable opportunity to overcome existing PK challenges associated with conventional uPA inhibitors, while leveraging potent candidates into novel targeted nanotherapeutics for an improved anticancer response in uPA positive tumors.

HER2-Positive Gastroesophageal Cancers Are Associated with a Higher Risk of Brain Metastasis.

Brain metastasis from gastroesophageal adenocarcinomas (GOCs) is a rare but a devastating diagnosis. Human epidermal growth factor receptor 2 (HER2) is a prognostic and predictive biomarker in GOCs. The association of HER2 with GOC brain metastasis is not known. We performed a retrospective analysis of patients with GOCs with known HER2 status between January 2015 and November 2021. HER2 was assessed on either the primary tumour or metastasis by immunohistochemistry or in situ hybridization. The diagnosis of brain metastasis was made on standard imaging techniques in patients with symptoms or signs. HER2 results were available for 201 patients, with 34 patients (16.9%) HER2 positive. A total of 12 patients developed symptomatic brain metastasis from GOCs, of which 7 (58.3%) were HER2 positive. The development of symptomatic brain metastasis was significantly higher in the HER2-positive GOCs (OR8.26, 95%CI 2.09-35.60; p = 0.0009). There was no significant association of HER2 status and overall survival in patients with brain metastasis. Although the rate of brain metastasis remains low in GOCs, the incidence of symptomatic brain metastasis was significantly higher in patients with HER2-positive tumours.

High-Dimensional and Spatial Analysis Reveals Immune Landscape-Dependent Progression in Cutaneous Squamous Cell Carcinoma.

PURPOSE: The tumor immune microenvironment impacts the biological behavior of the tumor, but its effect on clinical outcomes in head and neck cutaneous squamous cell carcinomas (HNcSCC) is largely unknown. EXPERIMENTAL DESIGN: We compared the immune milieu of high-risk HNcSCC that never progressed to metastasis with those that metastasized using multiparameter imaging mass cytometry. The cohort included both immunosuppressed patients (IS) and patients with an absence of clinical immune-suppression (ACIS). Spatial analyses were used to identify cellular interactions that were associated with tumor behavior. RESULTS: Nonprogressing primary HNcSCC were characterized by higher CD8+ and CD4+ T-cell responses, including numerically increased regulatory T cells. In contrast, primary lesions from HNcSCC patients who progressed were largely devoid of T cells with lower numbers of innate immune cells and increased expression of checkpoint receptors and in the metastatic lesions were characterized by an accumulation of B cells. Spatial analysis reveals multiple cellular interactions associated with nonprogressing primary tumors that were distinct in primary tumors of disease-progressing patients. Cellular regional analysis of the tumor microenvironment also shows squamous cell-enriched tumor regions associated with primary nonprogressing tumors. CONCLUSIONS: Effective responses from both CD8+ and CD4+ T cells in the tumor microenvironment are essential for immune control of primary HNcSCC. Our findings indicate that the early events that shape the immune responses in primary tumors dictate progression and disease outcomes in HNcSCC.

Whole genome analysis reveals the genomic complexity in metastatic cutaneous squamous cell carcinoma.

Metastatic cutaneous squamous cell carcinoma (CSCC) is a highly morbid disease requiring radical surgery and adjuvant therapy, which is associated with a poor prognosis. Yet, compared to other advanced malignancies, relatively little is known of the genomic landscape of metastatic CSCC. We have previously reported the mutational signatures and mutational patterns of CCCTC-binding factor (CTCF) regions in metastatic CSCC. However, many other genomic components (indel signatures, non-coding drivers, and structural variants) of metastatic CSCC have not been reported. To this end, we performed whole genome sequencing on lymph node metastases and blood DNA from 25 CSCC patients with regional metastases of the head and neck. We designed a multifaceted computational analysis at the whole genome level to provide a more comprehensive perspective of the genomic landscape of metastatic CSCC. In the non-coding genome, 3' untranslated region (3'UTR) regions of EVC (48% of specimens), PPP1R1A (48% of specimens), and ABCA4 (20% of specimens) along with the tumor-suppressing long non-coding RNA (lncRNA) LINC01003 (64% of specimens) were significantly functionally altered (Q-value < 0.05) and represent potential non-coding biomarkers of CSCC. Recurrent copy number loss in the tumor suppressor gene PTPRD was observed. Gene amplification was much less frequent, and few genes were recurrently amplified. Single nucleotide variants driver analyses from three tools confirmed TP53 and CDKN2A as recurrently mutated genes but also identified C9 as a potential novel driver in this disease. Furthermore, indel signature analysis highlighted the dominance of ID signature 13 (ID13) followed by ID8 and ID9. ID9 has previously been shown to have no association with skin melanoma, unlike ID13 and ID8, suggesting a novel pattern of indel variation in metastatic CSCC. The enrichment analysis of various genetically altered candidates shows enrichment of "TGF-beta regulation of extracellular matrix" and "cell cycle G1 to S check points." These enriched terms are associated with genetic instability, cell proliferation, and migration as mechanisms of genomic drivers of metastatic CSCC.

A tEMTing target? Clinical and experimental evidence for epithelial-mesenchymal transition in the progression of cutaneous squamous cell carcinoma (a scoping systematic review).

Cutaneous squamous cell carcinoma (cSCC) is a disease with globally rising incidence and poor prognosis for patients with advanced or metastatic disease. Epithelial-mesenchymal transition (EMT) is a driver of metastasis in many carcinomas, and cSCC is no exception. We aimed to provide a systematic overview of the clinical and experimental evidence for EMT in cSCC, with critical appraisal of type and quality of the methodology used. We then used this information as rationale for potential drug targets against advanced and metastatic cSCC. All primary literature encompassing clinical and cell-based or xenograft experimental studies reporting on the role of EMT markers or related signalling pathways in the progression of cSCC were considered. A screen of 3443 search results yielded 86 eligible studies comprising 44 experimental studies, 22 clinical studies, and 20 studies integrating both. From the clinical studies a timeline illustrating the alteration of EMT markers and related signalling was evident based on clinical progression of the disease. The experimental studies reveal connections of EMT with a multitude of factors such as genetic disorders, cancer-associated fibroblasts, and matrix remodelling via matrix metalloproteinases and urokinase plasminogen activator. Additionally, EMT was found to be closely tied to environmental factors as well as to stemness in cSCC via NFκB and ß-catenin. We conclude that the canonical EGFR, canonical TGF-ßR, PI3K/AKT and NFκB signalling are the four signalling pillars that induce EMT in cSCC and could be valuable therapeutic targets. Despite the complexity, EMT markers and pathways are desirable biomarkers and drug targets for the treatment of advanced or metastatic cSCC.

A Novel Role for Plasminogen Activator Inhibitor Type-2 as a Hypochlorite-Resistant Serine Protease Inhibitor and Holdase Chaperone.

Plasminogen activator inhibitor type-2 (PAI-2), a member of the serpin family, is dramatically upregulated during pregnancy and in response to inflammation. Although PAI-2 exists in glycosylated and non-glycosylated forms in vivo, the majority of in vitro studies of PAI-2 have exclusively involved the intracellular non-glycosylated form. This study shows that exposure to inflammation-associated hypochlorite induces the oligomerisation of PAI-2 via a mechanism involving dityrosine formation. Compared to plasminogen activator inhibitor type-1 (PAI-1), both forms of PAI-2 are more resistant to hypochlorite-induced inactivation of its protease inhibitory activity. Holdase-type extracellular chaperone activity plays a putative non-canonical role for PAI-2. Our data demonstrate that glycosylated PAI-2 more efficiently inhibits the aggregation of Alzheimer's disease and preeclampsia-associated amyloid beta peptide (Aß), compared to non-glycosylated PAI-2 in vitro. However, hypochlorite-induced modification of non-glycosylated PAI-2 dramatically enhances its holdase activity by promoting the formation of very high-molecular-mass chaperone-active PAI-2 oligomers. Both PAI-2 forms protect against Aß-induced cytotoxicity in the SH-SY5Y neuroblastoma cell line in vitro. In the villous placenta, PAI-2 is localised primarily to syncytiotrophoblast with wide interpersonal variation in women with preeclampsia and in gestational-age-matched controls. Although intracellular PAI-2 and Aß staining localised to different placental cell types, some PAI-2 co-localised with Aß in the extracellular plaque-like aggregated deposits abundant in preeclamptic placenta. Thus, PAI-2 potentially contributes to controlling aberrant fibrinolysis and the accumulation of misfolded proteins in states characterised by oxidative and proteostasis stress, such as in Alzheimer's disease and preeclampsia.

Cancer Progression Gene Expression Profiling Identifies the Urokinase Plasminogen Activator Receptor as a Biomarker of Metastasis in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) of the head and neck region is the second most prevalent skin cancer, with metastases to regional lymph nodes occurring in 2%-5% of cases. To further our understanding of the molecular events characterizing cSCC invasion and metastasis, we conducted targeted cancer progression gene expression and pathway analysis in non-metastasizing (PRI-) and metastasizing primary (PRI+) cSCC tumors of the head and neck region, cognate lymph node metastases (MET), and matched sun-exposed skin (SES). The highest differentially expressed genes in metastatic (MET and PRI+) versus non-metastatic tumors (PRI-) and SES included PLAU, PLAUR, MMP1, MMP10, MMP13, ITGA5, VEGFA, and various inflammatory cytokine genes. Pathway enrichment analyses implicated these genes in cellular pathways and functions promoting matrix remodeling, cell survival and migration, and epithelial to mesenchymal transition, which were all significantly activated in metastatic compared to non-metastatic tumors (PRI-) and SES. We validated the overexpression of urokinase plasminogen activator receptor (uPAR, encoded by PLAUR) in an extended patient cohort by demonstrating higher uPAR staining intensity in metastasizing tumors. As pathway analyses identified epidermal growth factor (EGF) as a potential upstream regulator of PLAUR, the effect of EGF on uPAR expression levels and cell motility was functionally validated in human metastatic cSCC cells. In conclusion, we propose that uPAR is an important driver of metastasis in cSCC and represents a potential therapeutic target in this disease.

Urokinase plasminogen activator as an anti-metastasis target: inhibitor design principles, recent amiloride derivatives, and issues with human/mouse species selectivity.

The urokinase plasminogen activator (uPA) is a widely studied anticancer drug target with multiple classes of inhibitors reported to date. Many of these inhibitors contain amidine or guanidine groups, while others lacking these groups show improved oral bioavailability. Most of the X-ray co-crystal structures of small molecule uPA inhibitors show a key salt bridge with the side chain carboxylate of Asp189 in the S1 pocket of uPA. This review summarises the different classes of uPA inhibitors, their binding interactions and experimentally measured inhibitory potencies and highlights species selectivity issues with attention to recently described 6-substituted amiloride and 5­N,N-(hexamethylene)amiloride (HMA) derivatives.

The Urokinase Plasminogen Activation System in Pancreatic Cancer: Prospective Diagnostic and Therapeutic Targets.

Pancreatic cancer is a highly aggressive malignancy that features high recurrence rates and the poorest prognosis of all solid cancers. The urokinase plasminogen activation system (uPAS) is strongly implicated in the pathophysiology and clinical outcomes of patients with pancreatic ductal adenocarcinoma (PDAC), which accounts for more than 90% of all pancreatic cancers. Overexpression of the urokinase-type plasminogen activator (uPA) or its cell surface receptor uPAR is a key step in the acquisition of a metastatic phenotype via multiple mechanisms, including the increased activation of cell surface localised plasminogen which generates the serine protease plasmin. This triggers multiple downstream processes that promote tumour cell migration and invasion. Increasing clinical evidence shows that the overexpression of uPA, uPAR, or of both is strongly associated with worse clinicopathological features and poor prognosis in PDAC patients. This review provides an overview of the current understanding of the uPAS in the pathogenesis and progression of pancreatic cancer, with a focus on PDAC, and summarises the substantial body of evidence that supports the role of uPAS components, including plasminogen receptors, in this disease. The review further outlines the clinical utility of uPAS components as prospective diagnostic and prognostic biomarkers for PDAC, as well as a rationale for the development of novel uPAS-targeted therapeutics.

Metastatic phenotype and immunosuppressive tumour microenvironment in pancreatic ductal adenocarcinoma: Key role of the urokinase plasminogen activator (PLAU).

Background: Previous studies have revealed the role of dysregulated urokinase plasminogen activator (encoded by PLAU) expression and activity in several pathways associated with cancer progression. However, systematic investigation into the association of PLAU expression with factors that modulate PDAC (pancreatic ductal adenocarcinoma) progression is lacking, such as those affecting stromal (pancreatic stellate cell, PSC)-cancer cell interactions, tumour immunity, PDAC subtypes and clinical outcomes from potential PLAU inhibition. Methods: This study used an integrated bioinformatics approach to identify prognostic markers correlated with PLAU expression using different transcriptomics, proteomics, and clinical data sets. We then determined the association of dysregulated PLAU and correlated signatures with oncogenic pathways, metastatic phenotypes, stroma, immunosuppressive tumour microenvironment (TME) and clinical outcome. Finally, using an in vivo orthotopic model of pancreatic cancer, we confirmed the predicted effect of inhibiting PLAU on tumour growth and metastasis. Results: Our analyses revealed that PLAU upregulation is not only associated with numerous other prognostic markers but also associated with the activation of various oncogenic signalling pathways, aggressive phenotypes relevant to PDAC growth and metastasis, such as proliferation, epithelial-mesenchymal transition (EMT), stemness, hypoxia, extracellular cell matrix (ECM) degradation, upregulation of stromal signatures, and immune suppression in the tumour microenvironment (TME). Moreover, the upregulation of PLAU was directly connected with signalling pathways known to mediate PSC-cancer cell interactions. Furthermore, PLAU upregulation was associated with the aggressive basal/squamous phenotype of PDAC and significantly reduced overall survival, indicating that this subset of patients may benefit from therapeutic interventions to inhibit PLAU activity. Our studies with a clinically relevant orthotopic pancreatic model showed that even short-term PLAU inhibition is sufficient to significantly halt tumour growth and, importantly, eliminate visible metastasis. Conclusion: Elevated PLAU correlates with increased aggressive phenotypes, stromal score, and immune suppression in PDAC. PLAU upregulation is also closely associated with the basal subtype type of PDAC; patients with this subtype are at high risk of mortality from the disease and may benefit from therapeutic targeting of PLAU.

Disruption of Water Networks is the Cause of Human/Mouse Species Selectivity in Urokinase Plasminogen Activator (uPA) Inhibitors Derived from Hexamethylene Amiloride (HMA).

The urokinase plasminogen activator (uPA) plays a critical role in tumor cell invasion and migration and is a promising antimetastasis target. 6-Substituted analogues of 5-N,N-(hexamethylene)amiloride (HMA) are potent and selective uPA inhibitors that lack the diuretic and antikaliuretic properties of the parent drug amiloride. However, the compounds display pronounced selectivity for human over mouse uPA, thus confounding interpretation of data from human xenograft mouse models of cancer. Here, computational and experimental findings reveal that residue 99 is a key contributor to the observed species selectivity, whereby enthalpically unfavorable expulsion of a water molecule by the 5-N,N-hexamethylene ring occurs when residue 99 is Tyr (as in mouse uPA). Analogue 7 lacking the 5-N,N-hexamethylene ring maintained similar water networks when bound to human and mouse uPA and displayed reduced selectivity, thus supporting this conclusion. The study will guide further optimization of dual-potent human/mouse uPA inhibitors from the amiloride class as antimetastasis drugs.

Neuroserpin and transthyretin are extracellular chaperones that preferentially inhibit amyloid formation.

Neuroserpin is a secreted protease inhibitor known to inhibit amyloid formation by the Alzheimer's beta peptide (Aß). To test whether this effect was constrained to Aß, we used a range of in vitro assays to demonstrate that neuroserpin inhibits amyloid formation by several different proteins and protects against the associated cytotoxicity but, unlike other known chaperones, has a poor ability to inhibit amorphous protein aggregation. Collectively, these results suggest that neuroserpin has an unusual chaperone selectivity for intermediates on the amyloid-forming pathway. Bioinformatics analyses identified a highly conserved 14-residue region containing an α helix shared between neuroserpin and the thyroxine-transport protein transthyretin, and we subsequently demonstrated that transthyretin also preferentially inhibits amyloid formation. Last, we used rationally designed neuroserpin mutants to demonstrate a direct involvement of the conserved 14-mer region in its chaperone activity. Identification of this conserved region may prove useful in the future design of anti-amyloid reagents.