ABSTRACT
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. DSTYK is located within the established linkage region and we also found the same mutation in the previously reported pedigree and another Israeli pedigree (total of ten affected individuals from three different families). The mutation removes the last two exons and part of the 3' UTR of DSTYK. Skin biopsies revealed reduced DSTYK protein levels along with focal loss of melanocytes. Ultrastructurally, swollen mitochondria and cytoplasmic vacuoles were also noted in remaining melanocytes and some keratinocytes and fibroblasts. Cultured keratinocytes and fibroblasts from an affected individual, as well as knockdown of Dstyk in mouse melanocytes, keratinocytes, and fibroblasts, were associated with increased cell death after ultraviolet irradiation. Keratinocytes from an affected individual showed loss of kinase activity upon stimulation with fibroblast growth factor. Previously, dominant mutations in DSTYK were implicated in congenital urological developmental disorders, but our study identifies different phenotypic consequences for a recurrent autosomal-recessive deletion mutation in revealing the genetic basis of SPG23.
Subject(s)
Pigmentation Disorders/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Sequence Deletion , Spastic Paraplegia, Hereditary/genetics , Vitiligo/genetics , Amino Acid Sequence , Animals , Apoptosis/genetics , Asian People/genetics , Chromosomes, Human, Pair 1/genetics , Exons , Facies , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Genetic Linkage , Genetic Loci , Genome-Wide Association Study , Homozygote , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Male , Melanocytes/cytology , Melanocytes/metabolism , Mice , NIH 3T3 Cells , Pedigree , Pigmentation Disorders/diagnosis , Spastic Paraplegia, Hereditary/diagnosis , Vitiligo/diagnosis , Young AdultABSTRACT
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. METHODS: Primary keratinocytes were isolated from cSCC tumours and metastases, and cell lines were established. These were characterised using short tandem repeat (STR) profiling and genotyped by whole exome sequencing. Multiple in vitro assays were performed to document their morphology, growth characteristics, migration and invasion characteristics, and in vivo xenograft growth. RESULTS: STR profiles of the cSCC lines allow the confirmation of their unique identity. Phylogenetic trees derived from exome sequence analysis of the matched primary and metastatic lines provide insight into the genetic basis of disease progression. The results of in vivo and in vitro analyses allow researchers to select suitable cell lines for specific experimentation. CONCLUSIONS: There are few well-characterised cSCC lines available for widespread preclinical experimentation and drug screening. The described cSCC cell line panel provides a critical tool for in vitro and in vivo experimentation.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Squamous Cell/pathology , Disease Models, Animal , Drug Evaluation, Preclinical/methods , Skin Neoplasms/pathology , Animals , Biomarkers, Tumor , Biopsy , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Cell Line, Tumor , Gene Expression Profiling , Humans , Immunohistochemistry , Keratinocytes/drug effects , Keratinocytes/metabolism , Male , Mutation , Neoplasm Metastasis , Neoplasm Staging , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Xenograft Model Antitumor AssaysABSTRACT
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. The following were recorded prospectively: US diameter, stiffness at SWE, presentation source, core biopsy grade, oestrogen receptor (ER) status and pre-operative nodal status. Breast cancer-specific survival (BCSS) was analysed with regard to US size and stiffness, tumour grade on core biopsy, ER status, presentation mode and pre-operative nodal status. Analysis used Cox proportional hazards regression. RESULTS: Of the 520 patients, 42 breast cancer and 53 non-breast cancer deaths were recorded at mean follow-up of 5.4 years. Hazard ratios (HR) for tertiles of stiffness were 1, 4.8 and 8.1 (P = 0.0001). HR for 2 groups based on US size < or ≥ 20 mm were 1 and 5.1 (P < 0.0001). HR for each unit increase in tumour grade on core biopsy was 3.9 (P < 0.0001). The HR for ER positivity compared to ER negativity was 0.21 (P < 0.001). BCSS was also associated with presentation mode and pre-operative nodal status. In a multivariable model, stiffness, US size and ER status were independently associated with BCSS. CONCLUSION: Multiple pre-operative factors including stromal stiffness at SWE have independent prognostic significance. A larger dataset with longer follow-up could be used in the future to construct a pre-operative prognostic model to guide treatment decisions.
Subject(s)
Breast Neoplasms/diagnostic imaging , Breast Neoplasms/mortality , Elasticity Imaging Techniques , Elasticity , Preoperative Period , Tumor Microenvironment , Adult , Aged , Aged, 80 and over , Biomarkers , Breast Neoplasms/surgery , Female , Humans , Kaplan-Meier Estimate , Middle Aged , Neoplasm Grading , Neoplasm Staging , PrognosisABSTRACT
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. The clinical features comprised nail dystrophy or nail loss, marginal palmoplantar keratoderma, hypodontia, enamel hypoplasia, oral hyperpigmentation, and dysphagia. In addition, three individuals had sensorineural deafness, and three had bronchial asthma. Taken together, the features were consistent with an unusual autosomal-recessive ectodermal dysplasia syndrome. Because of consanguinity in both families, we used whole-exome sequencing to search for novel homozygous DNA variants and found GRHL2 mutations common to both families: affected subjects in one family were homozygous for c.1192T>C (p.Tyr398His) in exon 9, and subjects in the other family were homozygous for c.1445T>A (p.Ile482Lys) in exon 11. Immortalized keratinocytes (p.Ile482Lys) showed altered cell morphology, impaired tight junctions, adhesion defects, and cytoplasmic translocation of GRHL2. Whole-skin transcriptomic analysis (p.Ile482Lys) disclosed changes in genes implicated in networks of cell-cell and cell-matrix adhesion. Our clinical findings of an autosomal-recessive ectodermal dysplasia syndrome provide insight into the role of GRHL2 in skin development, homeostasis, and human disease.
Subject(s)
Cleft Palate/genetics , DNA-Binding Proteins/genetics , Ectodermal Dysplasia/genetics , Genes, Recessive/genetics , Intellectual Disability/genetics , Mutation/genetics , Skin/pathology , Syndactyly/genetics , Transcription Factors/genetics , Blotting, Western , Child , DNA-Binding Proteins/metabolism , Exons/genetics , Female , Humans , Male , Pedigree , Phenotype , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Skin/metabolism , Syndrome , Transcription Factors/metabolismABSTRACT
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. OATP1B3 expression is limited to the liver in healthy tissues, but is frequently detected in multiple cancer types and is reported to be associated with differing clinical outcome. The mechanism and functional significance of tumour-specific expression of OATP1B3 has yet to be determined. Here, we identify SLCO1B3 expression in tumour keratinocytes isolated from RDEB and UV-induced cSCC and demonstrate that SLCO1B3 expression and promoter activity are modulated by type VII collagen. We show that reduction of SLCO1B3 expression upon expression of full-length type VII collagen in RDEB cSCC coincides with acquisition of front-to-rear polarity and increased organisation of 3D spheroid cultures. In addition, we show that type VII collagen positively regulates the abundance of markers implicated in cellular polarity, namely ELMO2, PAR3, E-cadherin, B-catenin, ITGA6 and Ln332.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Cell Polarity , Collagen Type VII/physiology , Epidermolysis Bullosa Dystrophica/metabolism , Organic Anion Transporters, Sodium-Independent/metabolism , Skin Neoplasms/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antigens, CD , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cadherins/genetics , Cadherins/metabolism , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Coculture Techniques , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Epidermolysis Bullosa Dystrophica/genetics , Epidermolysis Bullosa Dystrophica/pathology , Gene Expression Regulation, Neoplastic , Humans , Integrin alpha6/genetics , Integrin alpha6/metabolism , Keratinocytes , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Neoplasm Transplantation , Organic Anion Transporters, Sodium-Independent/genetics , Promoter Regions, Genetic , Protein Transport , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Solute Carrier Organic Anion Transporter Family Member 1B3 , Transcription, Genetic , Tumor Cells, Cultured , beta Catenin/genetics , beta Catenin/metabolism , KalininABSTRACT
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.5786delC (p.Pro1929Leufs(∗)8) in EXPH5, which truncates the 1,989 amino acid Slac2-b protein by 52 residues. The clinical features comprised generalized scale-crusts and occasional blisters, mostly induced by trauma, as well as mild diffuse pigmentary mottling on the trunk and proximal limbs. There was no increased bleeding tendency, no neurologic abnormalities, and no increased incidence of infection. Analysis of an affected person's skin showed loss of Slac2-b immunostaining (C-terminal antibody), disruption of keratinocyte adhesion within the lower epidermis, and an increased number of perinuclear vesicles. A role for Slac2-b in keratinocyte biology was supported by findings of cytoskeletal disruption (mainly keratin intermediate filaments) and decreased keratinocyte adhesion in both keratinocytes from an affected subject and after shRNA knockdown of Slac2-b in normal keratinocytes. Slac2-b was also shown to colocalize with Rab27B and ß4 integrin to early adhesion initiation sites in spreading normal keratinocytes. Collectively, our findings identify an unexpected role for Slac2-b in inherited skin fragility and expand the clinical spectrum of human disorders of GTPase effector proteins.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Germ-Line Mutation , Hair Diseases/congenital , Hair Diseases/genetics , Adaptor Proteins, Signal Transducing/metabolism , Base Sequence , Female , Hair Diseases/diagnosis , Humans , Keratinocytes/metabolism , Keratinocytes/pathology , Male , Pedigree , Skin/pathology , Skin/ultrastructure , rab GTP-Binding Proteins/metabolismABSTRACT
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. RESULTS: 10(-8)M octreotide markedly inhibited both basal and VEGF-stimulated HUVEC proliferation, had no effect on endothelial cell migration, but inhibited endothelial tubule formation. HUVEC cocultured with the somatostatin- and VEGF-producing STC-1 cells presented a markedly decreased proliferation, a slightly increased motility and an increased capacity of tubule formation; in this system, the inhibition of endothelial cell proliferation was abolished by neutralizing anti-somatostatin but was restored in the presence of anti-VEGF antibodies. This suggests that somatostatin is able to antagonize the effects of VEGF on endothelial cell proliferation but not on endothelial cell sprouting. Finally, no significant effect of octreotide on tumor growth and intratumoral microvascular density was detected in an experimental model of intrahepatic dissemination of STC-1 cells. CONCLUSION: The in vitro antiangiogenic effects of somatostatin and its analogues are likely to be efficiently counterbalanced in the tumor microenvironment by the concomitant release of proangiogenic factors like VEGF.
Subject(s)
Cell Communication/drug effects , Enteroendocrine Cells/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Octreotide/pharmacology , Somatostatin/pharmacology , Animals , Antineoplastic Agents/pharmacology , Carcinoma/pathology , Cell Communication/physiology , Cells, Cultured , Enteroendocrine Cells/metabolism , Enteroendocrine Cells/pathology , Female , Gastrointestinal Neoplasms/pathology , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Mice , Mice, Nude , Mice, Transgenic , Neoplasm Invasiveness , Pancreatic Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
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. Tumor cells were grafted in the adventitial layer of the caecal wall of nude mice, sacrificed after 8 wk. RESULTS: At 8 wk, "primary" tumors were present in all animals. STC-1 derived tumors were morphologically moderately differentiated, with high proliferative and apoptotic activities; in contrast, INS-r3 derived tumors were well differentiated, with low proliferative and apoptotic activities. VEGF was expressed in <50% grafted STC-1 cells but in >90% of grafted INS-r3 cells. Microvascular density was significantly higher in INS-r3 derived tumors than in STC-1 derived tumors. All STC-1 derived tumors (n = 8) have invaded the mucosa, in contrast to none of the INS-r3 derived tumors (n = 8); liver metastases were detected in 7/8 animals bearing STC-1 derived tumors and in 0/8 animals with INS-r3 derived tumors, despite the presence of lymph node metastases. CONCLUSIONS: Our experimental data concur with clinical findings to suggest that in well differentiated digestive neuroendocrine tumors angiogenesis is disconnected from tumor progression: the development of a highly vascular tumor microenvironment is correlated with VEGF secretion but is not associated with invasive and metastatic properties; it must therefore be regarded as an indirect marker of differentiation.
Subject(s)
Carcinoma, Neuroendocrine/pathology , Digestive System Neoplasms/pathology , Intestinal Neoplasms/pathology , Neovascularization, Pathologic/pathology , Animals , Antigens, Viral, Tumor/genetics , Carcinoma, Neuroendocrine/blood supply , Cell Line, Tumor , Digestive System Neoplasms/blood supply , Disease Progression , Glucagon/genetics , Insulin/genetics , Intestinal Neoplasms/blood supply , Lymphatic Metastasis/pathology , Mice , Mice, Nude , Microcirculation , Promoter Regions, Genetic , Rats , Simian virus 40/immunology , Transplantation, Heterologous , Vascular Endothelial Growth Factor A/geneticsABSTRACT
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.Experimental Design: We previously identified polo-like kinase 1 (PLK1) as a therapeutic target in skin SCC, including RDEB SCC. Here, we undertake a screen of 6 compounds originally designated as PLK1 inhibitors, and detail the efficacy of the lead compound, the multipathway allosteric inhibitor ON-01910, for targeting RDEB SCC in vitro and in vivo. RESULTS: ON-01910 (or rigosertib) exhibited significant specificity for RDEB SCC: in culture rigosertib induced apoptosis in 10 of 10 RDEB SCC keratinocyte populations while only slowing the growth of normal primary skin cells at doses 2 orders of magnitude higher. Furthermore, rigosertib significantly inhibited the growth of two RDEB SCC in murine xenograft studies with no apparent toxicity. Mechanistically, rigosertib has been shown to inhibit multiple signaling pathways. Comparison of PLK1 siRNA with MEK inhibition, AKT inhibition, and the microtubule-disrupting agent vinblastine in RDEB SCC shows that only PLK1 reduction exhibits a similar sensitivity profile to rigosertib. CONCLUSIONS: These data support a "first in RDEB" phase II clinical trial of rigosertib to assess tumor targeting in patients with late stage, metastatic, and/or unresectable SCC.
Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/etiology , Epidermolysis Bullosa Dystrophica/complications , Epidermolysis Bullosa Dystrophica/genetics , Glycine/analogs & derivatives , Skin Neoplasms/drug therapy , Skin Neoplasms/etiology , Sulfones/therapeutic use , Antineoplastic Agents/pharmacology , Apoptosis , Carcinoma, Squamous Cell/diagnosis , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Gene Knockdown Techniques , Genes, Recessive , Glycine/pharmacology , Glycine/therapeutic use , Humans , Keratinocytes/drug effects , Keratinocytes/metabolism , Molecular Targeted Therapy , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA, Messenger , RNA, Small Interfering , Skin Neoplasms/diagnosis , Sulfones/pharmacology , Polo-Like Kinase 1ABSTRACT
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 TGFß Smad signalling, which is localized to bulge stem cells in both normal human and murine skin. MAPK pathway hyperactivation (through Braf(V600E) or Kras(G12D) knockin) and TGFß signalling ablation (through Tgfbr1 deletion) in LGR5(+ve) stem cells enables rapid cSCC development in the mouse. Mutation of Tp53 (which is commonly mutated in sporadic cSCC) coupled with Tgfbr1 deletion in LGR5(+ve) cells also results in cSCC development. These findings indicate that LGR5(+ve) stem cells may act as cells of origin for cSCC, and that RAS/RAF/MAPK pathway hyperactivation or Tp53 mutation, coupled with loss of TGFß signalling, are driving events of skin tumorigenesis.
Subject(s)
Antineoplastic Agents/adverse effects , Carcinoma, Squamous Cell/chemically induced , Carcinoma, Squamous Cell/genetics , Melanoma/drug therapy , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Receptors, Transforming Growth Factor beta/genetics , Skin Neoplasms/chemically induced , Skin Neoplasms/genetics , Animals , Biopsy , Carcinogenesis/genetics , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , DNA Mutational Analysis/methods , Female , Humans , Indoles/adverse effects , Male , Mice , Mice, Inbred Strains , Mutation , Neoplasms, Experimental/chemically induced , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Receptor, Transforming Growth Factor-beta Type I , Receptor, Transforming Growth Factor-beta Type II , Signal Transduction/drug effects , Skin Neoplasms/pathology , Stem Cells , Sulfonamides/adverse effects , Transforming Growth Factor beta/metabolism , Tumor Suppressor Protein p53/genetics , Vemurafenib , Exome SequencingABSTRACT
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. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention.
Subject(s)
Basement Membrane/enzymology , Basement Membrane/pathology , Epidermolysis Bullosa Dystrophica/enzymology , Epidermolysis Bullosa Dystrophica/pathology , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/analysis , Basement Membrane/metabolism , Bone Marrow Transplantation , Cells, Cultured , Collagen Type VII/analysis , Collagen Type VII/metabolism , Epidermolysis Bullosa Dystrophica/metabolism , Epidermolysis Bullosa Dystrophica/therapy , Humans , Keratinocytes/enzymology , Keratinocytes/metabolism , Keratinocytes/pathology , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/metabolism , Protein Interaction Maps , Skin/enzymology , Skin/metabolism , Skin/pathologyABSTRACT
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. As expected, frequent NICD1 staining was observed in T lymphoblastic leukemia/lymphoma, a tumor in which activating NOTCH1 mutations are common. However, when IHC was used to gauge NOTCH1 activation in other human cancers, several unexpected findings emerged. Among B cell tumors, NICD1 staining was much more frequent in chronic lymphocytic leukemia than would be predicted based on the frequency of NOTCH1 mutations, while mantle cell lymphoma and diffuse large B cell lymphoma showed no evidence of NOTCH1 activation. NICD1 was also detected in 38% of peripheral T cell lymphomas. Of interest, NICD1 staining in chronic lymphocytic leukemia cells and in angioimmunoblastic lymphoma was consistently more pronounced in lymph nodes than in surrounding soft tissues, implicating factors in the nodal microenvironment in NOTCH1 activation in these diseases. Among carcinomas, diffuse strong NICD1 staining was observed in 3.8% of cases of triple negative breast cancer (3 of 78 tumors), but was absent from 151 non-small cell lung carcinomas and 147 ovarian carcinomas. Frequent staining of normal endothelium was also observed; in line with this observation, strong NICD1 staining was also seen in 77% of angiosarcomas. These findings complement insights from genomic sequencing studies and suggest that IHC staining is a valuable experimental tool that may be useful in selection of patients for clinical trials.
Subject(s)
Receptor, Notch1/metabolism , Animals , Cell Line, Tumor , Heterografts , Humans , Immunohistochemistry , Mice , Mutation , Receptor, Notch1/geneticsABSTRACT
Wnt5a is one of the so-called non-canonical Wnt ligands which do not act through ß-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. Dysregulated Wnt5a signalling facilitates invasion of multiple tumor types into adjacent tissue. However, the expression and distribution of Wnt5a in cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), as well as the effect of Wnt5a on keratinocyte migration has not been studied in detail to date. We here report that Wnt5a is upregulated in SCC and BCC and localised to the leading edge of tumors, as well as tumor-associated fibroblasts. The Wnt5a-triggered bundling of its receptor Fzd3 provides evidence of Wnt5a concentration gradients projecting into the tumor. In vitro migration assays show that Wnt5a concentration gradients determine its effect on keratinoctye migration: While chemotactic migration is inhibited by Wnt5a present in homogenous concentrations, it is enhanced in the presence of a Wnt5a gradient. Expression profiling of the Wnt pathway shows that the upregulation of Wnt5a in SCC is coupled to repression of canonical Wnt signalling. This is confirmed by immunohistochemistry showing lack of nuclear ß-catenin, as well as absent accumulation of Axin2. Since both types of Wnt signalling act mutually antogonistically at multiple levels, the concurrent repression of canonical Wnt signalling suggests hyper-active Wnt5a signal transduction. Significantly, this combination of gene dysregulation is not observed in the benign hyperproliferative inflammatory skin disease psoriasis. Collectively, our data strongly suggest that Wnt5a signalling contributes to tissue invasion by non-melanoma skin cancer.
Subject(s)
Carcinoma, Basal Cell/metabolism , Carcinoma, Squamous Cell/metabolism , Frizzled Receptors/biosynthesis , Gene Expression Regulation, Neoplastic , Proto-Oncogene Proteins/biosynthesis , Skin Neoplasms/metabolism , Wnt Proteins/metabolism , Biopsy , Cell Line, Tumor , Cell Movement , Gene Expression Regulation , Humans , Immunohistochemistry/methods , Keratinocytes/cytology , Models, Biological , Protein Isoforms , Signal Transduction , Wnt Proteins/biosynthesis , Wnt-5a ProteinABSTRACT
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. Although gene expression of RDEB normal skin fibroblasts resembled that of cancer-associated fibroblasts, RDEB cancer-associated fibroblasts exhibited a distinct and divergent gene expression profile, with a large proportion of the differentially expressed genes involved in matrix and cell adhesion. RDEB cancer-associated fibroblasts conferred increased adhesion and invasion to tumor and nontumor keratinocytes. Reduction of COL7A1, the defective gene in RDEB, in normal dermal fibroblasts led to increased type XII collagen, thrombospondin-1, and Wnt-5A, while reexpression of wild type COL7A1 in RDEB fibroblasts decreased type XII collagen, thrombospondin-1, and Wnt-5A expression, reduced tumor cell invasion in organotypic culture, and restricted tumor growth in vivo. Overall, our findings show that matrix composition in patients with RDEB is a permissive environment for tumor development, and type VII collagen directly regulates the composition of matrix proteins secreted by dermal and cancer-associated fibroblasts.
Subject(s)
Carcinoma, Squamous Cell/genetics , Collagen Type VII/genetics , Epidermolysis Bullosa Dystrophica/genetics , Cell Proliferation , Cells, Cultured , Collagen Type VII/biosynthesis , Epidermolysis Bullosa Dystrophica/pathology , Fibroblasts/metabolism , Gene Expression Profiling , Humans , Neoplasm Invasiveness , Proto-Oncogene Proteins/biosynthesis , RNA, Small Interfering , Skin/cytology , Skin/metabolism , Thrombospondin 1/biosynthesis , Wnt Proteins/biosynthesis , Wnt-5a ProteinABSTRACT
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. SCC keratinocytes readily form tumours in vivo, which retain histological features of the parental tumour, making them an excellent model for the study and development of cancer therapies.
Subject(s)
Carcinoma, Squamous Cell/pathology , Cell Culture Techniques/methods , Cell Line, Tumor/pathology , Cell Separation/methods , Skin Neoplasms/pathology , Culture Media , DNA/genetics , DNA/isolation & purification , Humans , Keratinocytes/cytology , Keratinocytes/pathology , Polymorphism, Single Nucleotide/geneticsABSTRACT
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.
Subject(s)
Coculture Techniques/methods , 3T3 Cells , Animals , Cell Survival , Mice , MitosisABSTRACT
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). METHODS AND RESULTS: Leukocyte ABCA1, LXRα and PPARγ expression was measured by polymerase chain reaction in 63 men with varying degrees of glucose homeostasis. ABCA1 protein concentrations were measured in leukocytes. In a sub-group of 25 men, ABCA1 function was quantified as apolipoprotein-A1-mediated cholesterol efflux from 2-3 week cultured skin fibroblasts. Leukocyte ABCA1 expression correlated negatively with circulating HbA1c and glucose (rhoâ=â-0.41, p<0.001; rhoâ=â-0.34, pâ=â0.006 respectively) and was reduced in Type 2 diabetes (T2DM) (pâ=â0.03). Leukocyte ABCA1 protein was lower in T2DM (pâ=â0.03) and positively associated with plasma HDL cholesterol (HDL-C) (rhoâ=â0.34, pâ=â0.02). Apolipoprotein-A1-mediated cholesterol efflux correlated negatively with fasting glucose (rhoâ=â-0.50, pâ=â0.01) and positively with HDL-C (rhoâ=â0.41, pâ=â0.02). It was reduced in T2DM compared with controls (pâ=â0.04). These relationships were independent of LXRα and PPARγ expression. CONCLUSIONS: ABCA1 expression and protein concentrations in leukocytes, as well as function in cultured skin fibroblasts, are reduced in T2DM. ABCA1 protein concentration and function are associated with HDL-C levels. These findings indicate a glycaemia-related, persistent disruption of a key component of RCT.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Diabetes Mellitus, Type 2/genetics , Orphan Nuclear Receptors/genetics , PPAR gamma/genetics , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/metabolism , Case-Control Studies , Cells, Cultured , Cholesterol, HDL/metabolism , Diabetes Mellitus, Type 2/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Glycemic Index , Humans , Hyperglycemia/genetics , Hyperglycemia/metabolism , Liver X Receptors , Male , Middle Aged , Orphan Nuclear Receptors/metabolism , PPAR gamma/metabolism , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Skin/cytology , Skin/metabolismABSTRACT
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. We therefore designed an experimental model to study the intrahepatic growth of tumor endocrine cells; murine enteroendocrine STC-1 cells were injected into the spleen of nude mice to obtain their hepatic dissemination through the portal vein. Three stages of intrahepatic tumor growth were identified. Engraftment stage, until day 4 after intrasplenic injection of STC-1 cells, was avascular. Early growth, until day 17, resulted in small, infralobular nodules. Late growth, after day 17, was characterized by the development of large nodules associated with peritumoral vessels and containing abnormal intratumoral vessels. To test the effects of potentially anti-angiogenic agents on tumor growth, we then used STC-1 cells stably transfected with the endostatin-coding sequence. Intrahepatic tumor volume showed no significant change at days 4 and 8, but a dramatic decrease at day 28 (9.7 +/- 1.7% of liver tissue versus 25.2 +/- 2.4% in controls), because of a markedly lower number of large nodules (11 +/- 1.8% versus 42 +/- 5.8%) likely to result from an increased apoptotic index (39.4 +/- 5.6% versus 18.3 +/- 3.4). Our results suggest that active angiogenesis is not necessary for the engraftment and early growth of endocrine cells metastatic to the liver but is required at a later stage of progression.
Subject(s)
Enteroendocrine Cells/pathology , Intestinal Neoplasms/secondary , Liver Neoplasms, Experimental/secondary , Neovascularization, Pathologic/pathology , Animals , Apoptosis/physiology , Cell Division/physiology , Cell Line, Tumor , Cell Movement/physiology , Endostatins/genetics , Endothelial Cells/cytology , Enteroendocrine Cells/physiology , Female , Humans , Intestinal Neoplasms/physiopathology , Liver Neoplasms, Experimental/physiopathology , Mice , Mice, Nude , Neoplasm Transplantation , Neovascularization, Pathologic/physiopathology , Spleen , Transfection , Transplantation, Heterologous , Umbilical Veins/cytologyABSTRACT
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. Indirect immunofluorescent staining of SCC cryosections and Western blotting of cultured keratinocyte lysates identified two RDEB individuals who did not express detectable levels of type VII collagen. Mutation analysis revealed that these two patients harbor compound heterozygous nonsense mutations within the region of the COL7A1 gene encoding the NC1 domain. These data suggest that individuals with RDEB can develop SCC regardless of type VII collagen expression and that additional factors have a role in explaining the high incidence of tumors complicating this genodermatosis.