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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. [ABSTRACT FROM AUTHOR]

Simple Summary: Cutaneous squamous cell carcinoma (cSCC) is a very common skin cancer with poor prognosis for patients with advanced disease. PI3K/AKT/mTOR and cell cycle signalling pathways are often dysregulated in mcSCC. A combination drug approach targeting both pathways concurrently has been theorised to overcome the underwhelming clinical performance of targeted inhibitors individually. This study investigates the potential of PI3K inhibitors (PI3Ki) and cell-cycle inhibitors (CDKi) as single agents and in combination against patient-derived mcSCC cell lines. Whilst PI3Ki and CDKi as single agents potently induced cancer cell death, PI3Ki synergistically enhanced the potential of dinaciclib to induce cell death in one mcSCC cell line, but not another. Interestingly, this pattern was reversed in more complex cell culture models. PI3Ki and CDKi effectively stopped the cell cycle and induced programmed cell death both individually and in combination. These findings suggest that personalised medicine approaches targeting PI3K and CDK pathways in combination may yield some benefit, although further investigation is required to address discrepancies between simple and more complex culture models. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Metastases from cutaneous squamous cell carcinoma (cSCC) occur in 2%–5% of cases. Surgery is the standard treatment, often combined with adjuvant radiotherapy. Concurrent carboplatin treatment with post-operative radiotherapy may be prescribed, although it has not shown benefit in recent clinical trials in high-risk cSCC patients. The novel high-Z nanoparticle thulium (III) oxide has been shown to enhance radiation dose delivery to brain tumors by specific uptake of these nanoparticles into the cancerous tissue. As the dose-enhancement capacity of thulium oxide nanoparticles following radiotherapy against metastatic cSCC cells is unknown, its efficacy as a radiosensitizer was evaluated, with and without carboplatin. Novel and validated human patient-derived cell lines of metastatic cSCC were used. The sensitivity of the cells to radiation was investigated using short-term proliferation assays as well as clonogenic survival as the radiobiological endpoint. Briefly, cells were irradiated with 125 kVp orthovoltage x-rays (0–6 Gy) with and without thulium oxide nanoparticles (99.9% trace metals basis; 50 µg ml−1) or low dose carboplatin pre-sensitization. Cellular uptake of the nanoparticles was first confirmed by microscopy and found to have no impact on short-term cell survival for the cSCC cells, highlighting the biocompatibility of thulium oxide nanoparticles. Clonogenic cell survival assays confirmed radio-sensitization when exposed to thulium nanoparticles, with the cell sensitivity increasing by a factor of 1.24 (calculated at the 10% survival fraction) for the irradiated cSCC cells. The combination of carboplatin with thulium oxide nanoparticles with irradiation did not result in significant further reductions in survival compared to nanoparticles alone. This is the first study to provide in vitro data demonstrating the independent radiosensitization effect of high-Z nanoparticles against metastatic cSCC with or without carboplatin. Further preclinical investigations with radiotherapy plus high-Z nanoparticles for the management of metastatic cSCC are warranted. [ABSTRACT FROM AUTHOR]

Cutaneous squamous cell carcinoma (SCC) is second only in incidence to basal cell carcinoma (BCC), effecting up to 500 000 people in the United States annually. Metastasis to regional lymph nodes occurs in approximately 5% of cases and imparts significant morbidity. Standard treatment in this group involves a combination of surgery and adjuvant radiation. Currently, there are no clinically useful biomarkers of metastatic potential in primary cutaneous SCC and histological predictors can be unreliable. The high level of mutational burden in normal UV-exposed skin has hampered the search for novel drivers of invasive disease, and indeed metastatic potential. This review outlines the clinical problems in high-risk and metastatic cutaneous SCCs, reviews the known genetic events and molecular mechanisms in high-risk primary cutaneous SCC and metastasis, and identifies avenues for further investigation and potential therapy. [ABSTRACT FROM AUTHOR]

Abstract: Up-regulation of the urokinase plasminogen activation (uPA) system leads to increased cancer invasion and metastasis. Plasminogen activator inhibitors type-1 (PAI-1/SERPINE1) and type-2 (PAI-2/SERPINB2) have similar uPA inhibitory properties yet PAI-1 promotes cell invasion by modulating cell adhesion and migration. High tumour PAI-2 levels are associated with improved prognoses. Herein we show that PAI-2 is capable of inhibiting uPA in the presence of PAI-1, particularly on adherent cells in the presence of vitronectin. This suggests that elevated levels of PAI-2 in the tumour microenvironment could outcompete PAI-1 for uPA inhibition through its inhibitory serpin function. However, PAI-1 modulated cell adhesion in a largely uPA-independent manner consequently PAI-2 could not counteract the effects of PAI-1 on adhesion/migration. Thus studies aimed at further characterising the interplay between PAI-1 and PAI-2 on uPA-dependent pro-invasive processes are warranted. [Copyright &y& Elsevier]

Tumour expression of the urokinase plasminogen activator correlates with invasive capacity. Consequently, inhibition of this serine protease by physiological inhibitors should decrease invasion and metastasis. However, of the two main urokinase inhibitors, high tumour levels of the type 1 inhibitor actually promote tumour progression, whereas high levels of the type 2 inhibitor decrease tumour growth and metastasis. We propose that the basis of this apparently paradoxical action of two similar serine protease inhibitors lies in key structural differences controlling interactions with components of the extracellular matrix and endocytosis–signalling co-receptors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Prostate cancer is a leading cause of cancer-associated deaths in men over 60 years of age. Most patients are killed by tumor metastasis. Recent evidence has implicated a role of the tumor microenvironment and urokinase plasminogen activator (uPA) in cancer cell migration, invasion, and metastasis. Here, we examine the role of the Na+/H+ exchanger isoform 1 (NHE1) and uPA in DU 145 prostate cancer cell migration and colony formation. Knockout of NHE1 reduced cell migration. The effects of a series of novel NHE1/uPA hexamethylene-amiloride-based inhibitors with varying efficacy towards NHE1 and uPA were examined on prostate cancer cells. Inhibition of NHE1—alone, or with inhibitors combining NHE1 or uPA inhibition—generally did not prevent prostate cancer cell migration. However, uPA inhibition—but not NHE1 inhibition—prevented anchorage-dependent colony formation. Application of inhibitors at concentrations that only saturate uPA inhibition decreased tumor invasion in vivo. The results suggest that while knockout of NHE1 affects cell migration, these effects are not due to NHE1-dependent proton translocation. Additionally, while neither NHE1 nor uPA activity was critical in cell migration, only uPA activity appeared to be critical in anchorage-dependent colony formation of DU 145 prostate cancer cells and invasion in vivo. [ABSTRACT FROM AUTHOR]

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer. Most patients who develop metastases (2–5%) present with advanced disease that requires a combination of radical surgery and adjuvant radiation therapy. There are few effective therapies for refractory disease. In this study, we describe novel patient-derived cell lines from cSCC metastases of the head and neck (designated UW-CSCC1 and UW-CSCC2). The cell lines genotypically and phenotypically resembled the original patient tumor and were tumorogenic in mice. Differences in cancer-related gene expression between the tumor and cell lines after various culturing conditions could be largely reversed by xenografting and reculturing. The novel drug susceptibilities of UW-CSCC1 and an irradiated subclone UW-CSCC1-R to drugs targeting cell cycle, PI3K/AKT/mTOR, and DNA damage pathways were observed using high-throughput anti-cancer and kinase-inhibitor compound libraries, which correlate with either copy number variations, targetable mutations and/or the upregulation of gene expression. A secondary screen of top hits in all three cell lines including PIK3CA-targeting drugs supports the utility of targeting the PI3K/AKT/mTOR pathway in this disease. UW-CSCC cell lines are thus useful preclinical models for determining targetable pathways and candidate therapeutics. [ABSTRACT FROM AUTHOR]

Among peritumoral cells, cancer-associated fibroblasts (CAFs) are major facilitators of tumor progression. This study describes the effects of two urokinase-derived, novel decapeptides, denoted as Pep 1 and its cyclic derivative Pep 2. In a mouse model of tumor dissemination, using HT1080 fibrosarcoma cells, Pep 2 reduced the number and size of lung metastases. Specific binding of fluoresceinated Pep 2 to HT1080 and telomerase immortalised fibroblasts (TIF) cell surfaces was enhanced by αv overexpression or abolished by excess vitronectin, anti-αv antibodies or silencing of ITGAV αv gene, identifying αv-integrin as the Pep 2 molecular target. In 3D-organotypic assays, peptide-exposed TIFs and primary CAFs from breast carcinoma patients both exhibited a markedly reduced pro-invasive ability of either HT1080 fibrosarcoma or MDA-MB-231 mammary carcinoma cells, respectively. Furthermore, TIFs, either exposed to Pep 2, or silenced for αv integrin, were impaired in their ability to chemoattract cancer cells and to contract collagen matrices, exhibiting reduced α-smooth muscle actin (α-SMA) levels. Finally, peptide exposure of αv-expressing primary CAFs led to the downregulation of α-SMA protein and to a dramatic reduction of their pro-invasive capability. In conclusion, the ability of the novel decapeptides to interfere with tumor cell invasion directly and through the down-modulation of CAF phenotype suggests their use as lead compounds for co-targeting anti-cancer strategies. [ABSTRACT FROM AUTHOR]

[Display omitted] The K+-sparing diuretic amiloride elicits anticancer activities in multiple animal models. During our recent medicinal chemistry campaign aiming to identify amiloride analogs with improved properties for potential use in cancer, we discovered novel 6-(hetero)aryl-substituted amiloride and 5-(N , N -hexamethylene)amiloride (HMA) analogs with up to 100-fold higher potencies than the parent compounds against urokinase plasminogen activator (uPA), one of amiloride's putative anticancer targets, and no diuretic or antikaliuretic effects. Here, we report the systematic evaluation of structure–property relationships (lipophilicity, aqueous solubility and in vitro metabolic stability in human and mouse liver microsomes) in twelve matched pair analogs selected from our 6-substituted amiloride and HMA libraries. Mouse plasma stability, plasma protein binding, Caco-2 cell permeability, cardiac ion channel activity and pharmacokinetics in mice (PO and IV) and rats (IV) are described alongside amiloride and HMA comparators for a subset of the four most promising matched-pair analogs. The findings combined with earlier uPA activity/selectivity and other data ultimately drove selection of two analogs (AA1-39 and AA1-41) that showed efficacy in separate mouse cancer metastasis studies. [ABSTRACT FROM AUTHOR]

Sialic acid at the terminus of cell surface glycoconjugates is a critical element in cell-cell recognition, receptor binding and immune responses. Sialyltransferases (ST), the enzymes responsible for the biosynthesis of sialylated glycans are highly upregulated in cancer and the resulting hypersialylation of the tumour cell surface correlates strongly with tumour growth, metastasis and drug resistance. Inhibitors of human STs, in particular human ST6Gal I, are thus expected to be valuable chemical tools for the discovery of novel anticancer drugs. Herein, we report on the computationally-guided design and development of uridine-based inhibitors that replace the charged phosphodiester linker of known ST inhibitors with a neutral carbamate to improve pharmacokinetic properties and synthetic accessibility. A series of 24 carbamate-linked uridyl-based compounds were synthesised by coupling aryl and hetaryl α-hydroxyphosphonates with a 5′-amino-5′-deoxyuridine fragment. The inhibitory activities of the newly synthesised compounds against recombinant human ST6Gal I were determined using a luminescent microplate assay, and five promising inhibitors with K i 's ranging from 1 to 20 µM were identified. These results show that carbamate-linked uridyl-based compounds are a potential new class of readily accessible, non-cytotoxic ST inhibitors to be further explored. [ABSTRACT FROM AUTHOR]

The oral K+-sparing diuretic amiloride shows anti-cancer side-activities in multiple rodent models. These effects appear to arise, at least in part, through moderate inhibition of the urokinase-type plasminogen activator (uPA, K i = 2.4 µM), a pro-metastatic trypsin-like serine protease that is upregulated in many aggressive solid malignancies. In applying the selective optimization of side-activity (SOSA) approach, a focused library of twenty two 6-substituted amiloride derivatives were prepared, with multiple examples displaying uPA inhibitory potencies in the nM range. X-ray co-crystal structures revealed that the potency increases relative to amiloride arise from increased occupancy of uPA's S1β subsite by the appended 6-substituents. Leading compounds were shown to have high selectivity over related trypsin-like serine proteases and no diuretic or anti-kaliuretic effects in rats. Compound 15 showed anti-metastatic effects in a xenografted mouse model of late-stage lung metastasis. [ABSTRACT FROM AUTHOR]