The role of FGFR3 mutation in tumour initiation, progression and invasion of urothelial cell carcinoma in mice

Bladder cancer is the 5th most common and the 9th most lethal cancer in the UK. Based on histopathological and genomic analysis, a model of two independent pathogenesis pathways has been suggested, resulting in either non-invasive superficial or invasive urothelial tumours with potential to metastasise. Prominently, the fibroblast growth factor receptor 3 (FGFR3) is found mutated in up to 84% of non-invasive superficial tumours. Alterations in FGFR3 such as mutation or wild type receptor overexpression are also found in 54% of muscle-invasive tumours. FGFR3 is a tyrosine kinase receptor for fibroblast growth factors (FGFs), which stimulates both the RAS/MAPK and the PI3K/AKT pathways and regulates a range of cellular processes such as cell growth and division during development. In this study we examined the role of FGFR3 in bladder cancer by using mice as a model organism. Firstly, we addressed whether combination of Fgfr3 and Pten mutation, UroIICre Fgfr3+/K644E Ptenflox/flox, is able to drive non-invasive superficial bladder cancer. We observed that the thickness of the double mutant urothelium was significantly increased compared to singly mutated Fgfr3 or Pten, UroIICre Fgfr3+/K644E and UroIICre Ptenflox/flox. Moreover, several cellular abnormalities were detected that were accompanied by differential expression of layer-specific markers, which strongly suggested that they were caused cooperatively by Fgfr3 mutation and Pten deletion. The results supported the hypothesis that FGFR3 activation can play a causative role in urothelial pathogenesis of non-invasive superficial bladder cancer together with upregulated PI3K-AKT signalling. Secondly, we aimed to identify mutations that cooperate with Fgfr3 and with other common bladder cancer mutations such as Pten and Ras, in promoting urothelial tumourigenesis by Sleeping Beauty (SB) insertional mutagenesis in mice. The SB system may constitute an inefficient tool in the bladder to induce urothelial tumourigenesis, since it failed to produce bladder tumours in Fgfr3 as well as in Hras mutant mice. In mice with Pten deletion, one tumour was generated and general hypertrophy with cellular abnormalities was observed in all samples. No direct association between Fgfr3 and Pten mutations was found; however, SB mutagenesis supported that Fgfr3 and Pten cooperation may merge at the signalling downstream. Thirdly, we examined the role of the most common mutation in FGFR3, S249C, in the urothelium and in tumour progression and invasion by subjecting Fgfr3 mutant mice to a bladder-specific carcinogen, N-butyl-N-(hydroxybutyl)-nitrosamine (OH-BBN). We showed that FGFR3 S249C mutation by itself does not lead to urothelial abnormalities. However, in OH-BBN-induced tumours the presence of S249C increased the number of animals that formed bladder tumours by 4.4-fold. Our results present for the first time an effect of FGFR3 S249C mutation in invasive bladder cancer. Lastly, we sought to establish methods to generate and assess invasive bladder tumours using in vivo and in vitro techniques. First we examined the effectiveness of a Cre-expressing adenovirus (AdenoCre) to generate mouse models of bladder cancer with different combinations of genetic mutations. p53 deletion or mutation together with Pten loss led to formation of aggressive bladder tumours; however the origin of these tumours was likely to be the bladder muscle. Hras activation in combination with Pten deletion did not produce tumours or any cellular abnormalities by 8 months. AdenoCre-mediated tumour induction was successful in the presence of β-catenin and Hras mutation. However, an issue of AdenoCre transduction was the frequent observation of tumours in various other tissues such as the pelvic soft tissue, liver, pancreas and lung. Using an optimised AdenoCre procedure, the technique would allow lineage tracing of cancer stem cells in a developing bladder tumour and potentially during metastatic spread. Secondly, we tested imaging techniques in the living animals and validated ultrasound as a functional method to detect bladder wall thickening, as well as to monitor tumour growth in vivo. Thirdly, with the aim to assess cell transformation, migration and response to drug treatment, we tested essential ex vivo techniques and assays such as 3D sphere culture, organotypic slice culture as well as a Collagen-I invasion assay. The 3D tumour sphere culture was successful with murine Wnt-activated tumours as well as with invasive human cell lines. The organotypic slice culture was assessed as a system to test the effect of therapeutic drugs on the tumour cells; however, an issue of tissue disintegration has yet to be overcome. The Collagen-I assay successfully recapitulated invasion of a human bladder cancer cell line; however, the system needs to be adapted to murine bladder tumours. Taken together, this study presents for the first time evidence that support the functional role of FGFR3 signalling in the early stages of non-invasive urothelial carcinoma as well as in tumour progression of established neoplasms in mice. Given the wide availability of inhibitors specific to FGF signalling, our FGFR3 mouse models in conjunction with optimised ex vivo assays and imaging systems may open the avenue for FGFR3-targeted translation in urothelial disease

Autophagy Inhibition in BRAF-Driven Cancers

Several BRAF-driven cancers, including advanced BRAFV600E/K-driven melanoma, non-small-cell lung carcinoma, and thyroid cancer, are currently treated using first-line inhibitor combinations of BRAFV600E plus MEK1/2. However, despite the success of this vertical inhibition strategy, the durability of patient response is often limited by the phenomenon of primary or acquired drug resistance. It has recently been shown that autophagy, a conserved cellular recycling process, is increased in BRAF-driven melanoma upon inhibition of BRAFV600E signaling. Autophagy is believed to promote tumor progression of established tumors and also to protect cancer cells from the cytotoxic effects of chemotherapy. To this end, BRAF inhibitor (BRAFi)-resistant cells often display increased autophagy compared to responsive lines. Several mechanisms have been proposed for BRAFi-induced autophagy, such as activation of the endoplasmic reticulum (ER) stress gatekeeper GRP78, AMP-activated protein kinase, and transcriptional regulation of the autophagy regulating transcription factors TFEB and TFE3 via ERK1/2 or mTOR inhibition. This review describes the relationship between BRAF-targeted therapy and autophagy regulation, and discusses possible future treatment strategies of combined inhibition of oncogenic signaling plus autophagy for BRAF-driven cancers

Loss of Cxcr2 in myeloid cells promotes tumour progression and T cell infiltration in invasive bladder cancer

BACKGROUND:CXCR2 is a chemokine receptor expressed in myeloid cells, including neutrophils and macrophages. Pharmacological inhibition of CXCR2 has been shown to sensitize tumours to immune checkpoint inhibitor immunotherapies in some cancer types. OBJECTIVE:To investigate the effects of CXCR2 loss in regulation of tumour-infiltrating myeloid cells and their relationship to lymphocytes during bladder tumorigenesis. METHODS:Urothelial pathogenesis and immune contexture was investigated in an OH-BBN model of invasive bladder cancer with Cxcr2 deleted in myeloid cells (LysMCre Cxcr2floxflox). CXCR2 gene alterations and expression in human muscle invasive bladder cancer were analysed in The Cancer Genome Atlas. RESULTS:Urothelial tumour pathogenesis was significantly increased upon Cxcr2 deletion compared to wildtype mice. This was associated with a suppression of myeloid cell infiltration in Cxcr2-deleted bladders shortly after the carcinogen induction. Interestingly, following a transient increase of macrophages at the outset of tumour formation, an increase in T cell infiltration was observed in Cxcr2-deleted tumours. The increased tumour burden in the Cxcr2-deleted bladder was largely independent of T cells and the status of immune suppression. The Cxcr2-deleted mouse model reflected the low CXCR2 mRNA range in human bladder cancer, which showed poor overall survival. CONCLUSIONS:In contrast to previous reports of increased CXCR2 signalling associated with disease progression and poor prognosis, CXCR2 was protective against bladder cancer during tumour initiation. This is likely due to a suppression of acute inflammation. The strategy for sensitizing checkpoint immunotherapy by CXCR2 inhibition in bladder cancer may benefit from an examination of immune suppressive status

Ein rechnergestuetztes Verfahren fuer den Generalplanentwurf von Schiffen

Available from TIB Hannover: RA 489(517) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Comprehensive DNA methylation study identifies novel progression-related and prognostic markers for cutaneous melanoma

Background: Cutaneous melanoma is the deadliest skin cancer, with an increasing incidence and mortality rate. Currently, staging of patients with primary melanoma is performed using histological biomarkers such as tumor thickness and ulceration. As disruption of the epigenomic landscape is recognized as a widespread feature inherent in tumor development and progression, we aimed to identify novel biomarkers providing additional clinical information over current factors using unbiased genome-wide DNA methylation analyses. Methods: We performed a comprehensive DNA methylation analysis during all progression stages of melanoma using Infinium HumanMethylation450 BeadChips on a discovery cohort of benign nevi (n = 14) and malignant melanoma from both primary (n = 33) and metastatic (n = 28) sites, integrating the DNA methylome with gene expression data. We validated the discovered biomarkers in three independent validation cohorts by pyrosequencing and immunohistochemistry. Results: We identified and validated biomarkers for, and pathways involved in, melanoma development (e.g., HOXA9 DNA methylation) and tumor progression (e.g., TBC1D16 DNA methylation). In addition, we determined a prognostic signature with potential clinical applicability and validated PON3 DNA methylation and OVOL1 protein expression as biomarkers with prognostic information independent of tumor thickness and ulceration. Conclusions: Our data underscores the importance of epigenomic regulation in triggering metastatic dissemination through the inactivation of central cancer-related pathways. Inactivation of cell-adhesion and differentiation unleashes dissemination, and subsequent activation of inflammatory and immune system programs impairs anti-tumoral defense pathways. Moreover, we identify several markers of tumor development and progression previously unrelated to melanoma, and determined a prognostic signature with potential clinical utility

Comprehensive DNA methylation study identifies novel progression-related and prognostic markers for cutaneous melanoma

Background: Cutaneous melanoma is the deadliest skin cancer, with an increasing incidence and mortality rate. Currently, staging of patients with primary melanoma is performed using histological biomarkers such as tumor thickness and ulceration. As disruption of the epigenomic landscape is recognized as a widespread feature inherent in tumor development and progression, we aimed to identify novel biomarkers providing additional clinical information over current factors using unbiased genome-wide DNA methylation analyses. Methods: We performed a comprehensive DNA methylation analysis during all progression stages of melanoma using Infinium HumanMethylation450 BeadChips on a discovery cohort of benign nevi (n = 14) and malignant melanoma from both primary (n = 33) and metastatic (n = 28) sites, integrating the DNA methylome with gene expression data. We validated the discovered biomarkers in three independent validation cohorts by pyrosequencing and immunohistochemistry. Results: We identified and validated biomarkers for, and pathways involved in, melanoma development (e.g., HOXA9 DNA methylation) and tumor progression (e.g., TBC1D16 DNA methylation). In addition, we determined a prognostic signature with potential clinical applicability and validated PON3 DNA methylation and OVOL1 protein expression as biomarkers with prognostic information independent of tumor thickness and ulceration. Conclusions: Our data underscores the importance of epigenomic regulation in triggering metastatic dissemination through the inactivation of central cancer-related pathways. Inactivation of cell-adhesion and differentiation unleashes dissemination, and subsequent activation of inflammatory and immune system programs impairs anti-tumoral defense pathways. Moreover, we identify several markers of tumor development and progression previously unrelated to melanoma, and determined a prognostic signature with potential clinical utility

Comprehensive DNA methylation study identifies novel progression-related and prognostic markers for cutaneous melanoma

Background: Cutaneous melanoma is the deadliest skin cancer, with an increasing incidence and mortality rate. Currently, staging of patients with primary melanoma is performed using histological biomarkers such as tumor thickness and ulceration. As disruption of the epigenomic landscape is recognized as a widespread feature inherent in tumor development and progression, we aimed to identify novel biomarkers providing additional clinical information over current factors using unbiased genome-wide DNA methylation analyses. Methods: We performed a comprehensive DNA methylation analysis during all progression stages of melanoma using Infinium HumanMethylation450 BeadChips on a discovery cohort of benign nevi (n = 14) and malignant melanoma from both primary (n = 33) and metastatic (n = 28) sites, integrating the DNA methylome with gene expression data. We validated the discovered biomarkers in three independent validation cohorts by pyrosequencing and immunohistochemistry. Results: We identified and validated biomarkers for, and pathways involved in, melanoma development (e.g., HOXA9 DNA methylation) and tumor progression (e.g., TBC1D16 DNA methylation). In addition, we determined a prognostic signature with potential clinical applicability and validated PON3 DNA methylation and OVOL1 protein expression as biomarkers with prognostic information independent of tumor thickness and ulceration. Conclusions: Our data underscores the importance of epigenomic regulation in triggering metastatic dissemination through the inactivation of central cancer-related pathways. Inactivation of cell-adhesion and differentiation unleashes dissemination, and subsequent activation of inflammatory and immune system programs impairs anti-tumoral defense pathways. Moreover, we identify several markers of tumor development and progression previously unrelated to melanoma, and determined a prognostic signature with potential clinical utility

Figure S2 from Genetic Silencing of AKT Induces Melanoma Cell Death via mTOR Suppression

Supplementary Figure S2 shows the classification of MTG001 and MTG004. Immunoblotting of MTG001 and MTG004 PDX-derived cell lines showed similar levels of P-AKT (Ser473), total AKT, P-ERK (Thr202/Tyr204), ERK1/2, P-Ribosomal S6 Kinase (Ser235/236), and S6 Kinase. GAPDH was used as a loading control. The MTG001 cells exhibited higher levels of total PTEN. Analysis of RNA sequencing data demonstrated that both MTG001 and MTG004 express wildtype PTEN.</p