A Unique Panel of Patient-Derived Cutaneous Squamous Cell Carcinoma Cell Lines Provides a Preclinical Pathway for Therapeutic Testing.

Background: Cutaneous squamous cell carcinoma (cSCC) incidence continues to rise with increasing morbidity and mortality, with limited treatment options for advanced disease. Future improvements in targeted therapy will rely on advances in genomic/transcriptomic understanding and the use of model systems for basic research. We describe here the panel of 16 primary and metastatic cSCC cell lines developed and characterised over the past three decades in our laboratory in order to provide such a resource for future preclinical research and drug screening.

Identification of Rigosertib for the Treatment of Recessive Dystrophic Epidermolysis Bullosa-Associated Squamous Cell Carcinoma.

Purpose: Squamous cell carcinoma (SCC) of the skin is the leading cause of death in patients with the severe generalized form of the genetic disease recessive dystrophic epidermolysis bullosa (RDEB). Although emerging data are identifying why patients suffer this fatal complication, therapies for treatment of RDEB SCC are in urgent need. We previously identified polo-like kinase 1 (PLK1) as a therapeutic target in skin SCC, including RDEB SCC.

Pre-operative stromal stiffness measured by shear wave elastography is independently associated with breast cancer-specific survival.

Introduction: With the increased use of neoadjuvant therapy for breast cancer, there is a need for pre-operative prediction of prognosis. We aimed to assess the prognostic value of tumour stiffness measured by ultrasound shear wave elastography (SWE).

Methods: A consecutive cohort of patients with invasive breast cancer underwent breast ultrasound (US) including SWE.

Large Intragenic Deletion in DSTYK Underlies Autosomal-Recessive Complicated Spastic Paraparesis, SPG23.

SPG23 is an autosomal-recessive neurodegenerative subtype of lower limb spastic paraparesis with additional diffuse skin and hair dyspigmentation at birth followed by further patchy pigment loss during childhood. Previously, genome-wide linkage in an Arab-Israeli pedigree mapped the gene to an approximately 25 cM locus on chromosome 1q24-q32. By using whole-exome sequencing in a further Palestinian-Jordanian SPG23 pedigree, we identified a complex homozygous 4-kb deletion/20-bp insertion in DSTYK (dual serine-threonine and tyrosine protein kinase) in all four affected family members.

Inactivation of TGFβ receptors in stem cells drives cutaneous squamous cell carcinoma.

Melanoma patients treated with oncogenic BRAF inhibitors can develop cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, driven by paradoxical RAS/RAF/MAPK pathway activation. Here we identify frequent TGFBR1 and TGFBR2 mutations in human vemurafenib-induced skin lesions and in sporadic cSCC. Functional analysis reveals these mutations ablate canonical TGFβ Smad signalling, which is localized to bulge stem cells in both normal human and murine skin.

Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin.

Mutations in GRHL2 result in an autosomal-recessive ectodermal Dysplasia syndrome.

Grainyhead-like 2, encoded by GRHL2, is a member of a highly conserved family of transcription factors that play essential roles during epithelial development. Haploinsufficiency for GRHL2 has been implicated in autosomal-dominant deafness, but mutations have not yet been associated with any skin pathology. We investigated two unrelated Kuwaiti families in which a total of six individuals have had lifelong ectodermal defects.

High levels of type VII collagen expression in recessive dystrophic epidermolysis bullosa cutaneous squamous cell carcinoma keratinocytes increases PI3K and MAPK signalling, cell migration and invasion.

Background: Epidermolysis bullosa is a group of inherited skin fragility diseases varying in severity from mild scarring to infant mortality. Great efforts are being undertaken to develop therapeutic strategies to treat the more pernicious forms of this disease, particularly those associated with recessive, loss-of-function mutations. In such cases significant effort is directed toward delivering recombinant protein at levels sufficient to demonstrate clinical benefit.

Type VII collagen regulates expression of OATP1B3, promotes front-to-rear polarity and increases structural organisation in 3D spheroid cultures of RDEB tumour keratinocytes.

Type VII collagen is the main component of anchoring fibrils, structures that are integral to basement membrane homeostasis in skin. Mutations in the gene encoding type VII collagen COL7A1 cause recessive dystrophic epidermolysis bullosa (RDEB) an inherited skin blistering condition complicated by frequent aggressive cutaneous squamous cell carcinoma (cSCC). OATP1B3, which is encoded by the gene SLCO1B3, is a member of the OATP (organic anion transporting polypeptide) superfamily responsible for transporting a wide range of endogenous and xenobiotic compounds.

Infrared laser pulse triggers increased singlet oxygen production in tumour cells.

Photodynamic therapy (PDT) is a technique developed to treat the ever-increasing global incidence of cancer. This technique utilises singlet oxygen ((1)O2) generation via a laser excited photosensitiser (PS) to kill cancer cells. However, prolonged sensitivity to intensive light (6-8 weeks for lung cancer), relatively low tissue penetration by activating light (630 nm up to 4 mm), and the cost of PS administration can limit progressive PDT applications.

Gauging NOTCH1 Activation in Cancer Using Immunohistochemistry.

Fixed, paraffin-embedded (FPE) tissues are a potentially rich resource for studying the role of NOTCH1 in cancer and other pathologies, but tests that reliably detect activated NOTCH1 (NICD1) in FPE samples have been lacking. Here, we bridge this gap by developing an immunohistochemical (IHC) stain that detects a neoepitope created by the proteolytic cleavage event that activates NOTCH1. Following validation using xenografted cancers and normal tissues with known patterns of NOTCH1 activation, we applied this test to tumors linked to dysregulated Notch signaling by mutational studies.

Germline Mutation in EXPH5 Implicates the Rab27B Effector Protein Slac2-b in Inherited Skin Fragility.

The Rab GTPase Rab27B and one of its effector proteins, Slac2-b (also known as EXPH5, exophilin-5), have putative roles in intracellular vesicle trafficking but their relevance to human disease is not known. By using whole-exome sequencing, we identified a homozygous frameshift mutation in EXPH5 in three siblings with inherited skin fragility born to consanguineous Iraqi parents. All three individuals harbor the mutation c.

Fibroblast-derived dermal matrix drives development of aggressive cutaneous squamous cell carcinoma in patients with recessive dystrophic epidermolysis bullosa.

Patients with the genetic skin blistering disease recessive dystrophic epidermolysis bullosa (RDEB) develop aggressive cutaneous squamous cell carcinoma (cSCC). Metastasis leading to mortality is greater in RDEB than in other patient groups with cSCC. Here we investigate the dermal component in RDEB using mRNA expression profiling to compare cultured fibroblasts isolated from individuals without cSCC and directly from tumor matrix in RDEB and non-RDEB samples.

Wnt5a is strongly expressed at the leading edge in non-melanoma skin cancer, forming active gradients, while canonical Wnt signalling is repressed.

Wnt5a is one of the so-called non-canonical Wnt ligands which do not act through β-catenin. In normal development, Wnt5a is secreted and directs the migration of target cells along concentration gradients. The effect of Wnt5a on target cells is regulated by many factors, including the expression level of inhibitors and receptors.

Mutations in AEC syndrome skin reveal a role for p63 in basement membrane adhesion, skin barrier integrity and hair follicle biology.

Background: AEC (ankyloblepharon-ectodermal defects-clefting) syndrome is an autosomal dominant ectodermal dysplasia disorder caused by mutations in the transcription factor p63. Clinically, the skin is dry and often fragile; other features can include partial eyelid fusion (ankyloblepharon), hypodontia, orofacial clefting, sparse hair or alopecia, and nail dystrophy.

Objectives: To investigate how p63 gene mutations affect gene and protein expression in AEC syndrome skin.

Type 2 diabetes is associated with reduced ATP-binding cassette transporter A1 gene expression, protein and function.

Objective: Increasing plasma glucose levels are associated with increasing risk of vascular disease. We tested the hypothesis that there is a glycaemia-mediated impairment of reverse cholesterol transport (RCT). We studied the influence of plasma glucose on expression and function of a key mediator in RCT, the ATP binding cassette transporter-A1 (ABCA1) and expression of its regulators, liver X receptor-α (LXRα) and peroxisome proliferator-activated receptor-γ (PPARγ).

Effects of somatostatin and octreotide on the interactions between neoplastic gastroenteropancreatic endocrine cells and endothelial cells: a comparison between in vitro and in vivo properties.

Background/aims: Experimental studies in vitro suggest that somatostatin and some of its analogues used in clinical practice, such as octreotide, may have potent antiangiogenic properties. However, the clinical transposition of these data is difficult.

Methods: To address this issue, we designed a comparative study of the effects of somatostatin and octreotide on the interactions between neoplastic endocrine cells and endothelial cells in several in vitro and in vivo experimental models, including primary cultures of human umbilical vein endothelial cells (HUVEC), indirect cocultures between HUVEC and the somatostatin-producing endocrine cell line STC-1, and an animal model of intrahepatic dissemination of STC-1 cells.

Integrative mRNA profiling comparing cultured primary cells with clinical samples reveals PLK1 and C20orf20 as therapeutic targets in cutaneous squamous cell carcinoma.

Identifying therapeutic targets for cancer treatment relies on consistent changes within particular types or sub-types of malignancy. The ability to define either consistent changes or sub-types of malignancy is often masked by tumor heterogeneity. To elucidate therapeutic targets in cutaneous squamous cell carcinoma (cSCC), the most frequent skin neoplasm with malignant potential, we have developed an integrated approach to gene expression profiling beginning with primary keratinocytes in culture.

Feeder layers: co-culture with nonneoplastic cells.

Maintenance of a mitotically inactive feeder layer which is able to provide extracellular matrix and growth factors can be critical in establishing and maintaining primary tumor cells. How feeder cells are handled and processed is crucial for providing trouble-free support for primary tumor cells and spontaneously immortalized lines.

Isolation and culture of squamous cell carcinoma lines.

Cutaneous squamous cell carcinoma (SCC) keratinocytes readily grow, expand in culture, and continuously passage, suggesting either spontaneous immortalisation at the early stage of culture or inherent proliferative capacity. One feature of SCC keratinocytes is genomic DNA rearrangement and single-nucleotide polymorphism studies of fresh frozen primary tumour, early and late passage SCC keratinocytes suggest that these rearrangements are stable in culture and retain the parental tumour lesions. SCC keratinocytes are isolated using standard primary culture techniques and become feeder cell independent with little or no observed "crisis" period.

Angiogenesis and tumor progression in neuroendocrine digestive tumors.

Background: Clinical observations suggest that in neuroendocrine digestive tumors a high intratumoral microvascular density is associated with good prognosis. We used an experimental orthotopic xenograft model to analyze the relations between angiogenic activity and tumor progression in this tumor subset.

Material And Methods: We compared 2 endocrine cell lines: STC-1, a low vascular endothelial growth factor (VEGF)-producing cell line, and INS-r3, a high VEGF-producing cell line.

Unusual molecular findings in Kindler syndrome.

Kindler syndrome (KS) is a rare inherited skin disorder with blistering and poikiloderma as its main clinical features. It is caused by loss-of-function mutations in the C20orf42 (KIND1) gene which encodes kindlin-1, an actin cytoskeleton-focal contact-associated protein which is predominantly expressed in keratinocytes. We investigated the molecular basis of KS in a 16-year-old Indian boy who had additional clinical findings, including scleroatrophic changes of the hands and feet, pseudoainhum and early onset of squamous cell carcinoma on his foot.

The role of angiogenesis in endocrine liver metastases: an experimental study.

Liver metastases are a major adverse event during the evolution of digestive endocrine tumors. However, little is known about their natural history and the determinants of their growth. In particular, whereas liver endocrine metastases, like their primary counterparts, are hypervascular, the role of tumor-associated angiogenesis has been little explored.

Patients with recessive dystrophic epidermolysis bullosa develop squamous-cell carcinoma regardless of type VII collagen expression.

Recent data suggest that individuals with recessive dystrophic epidermolysis bullosa (RDEB) only develop squamous-cell carcinoma (SCC) in the presence of the NC1 domain of type VII collagen. This conclusion was based on experimental work in which cryosections of SCCs from 10 people with RDEB all showed positive type VII collagen immunostaining and observations in a murine model of SCC development in which tumors only occurred using keratinocytes from RDEB subjects that expressed detectable levels of the NC1 domain of the type VII collagen protein. To assess whether the clinical interpretation was valid in another cohort of RDEB patients, we examined expression of type VII collagen in 17 SCC tumors excised from 11 patients.