Phosphodiesterase type 5 inhibitors enhance chemotherapy in preclinical models of esophageal adenocarcinoma by targeting cancer-associated fibroblasts.

The chemotherapy resistance of esophageal adenocarcinomas (EACs) is underpinned by cancer cell extrinsic mechanisms of the tumor microenvironment (TME). We demonstrate that, by targeting the tumor-promoting functions of the predominant TME cell type, cancer-associated fibroblasts (CAFs) with phosphodiesterase type 5 inhibitors (PDE5i), we can enhance the efficacy of standard-of-care chemotherapy. In ex vivo conditions, PDE5i prevent the transdifferentiation of normal fibroblasts to CAF and abolish the tumor-promoting function of established EAC CAFs. Using shotgun proteomics and single-cell RNA-seq, we reveal PDE5i-specific regulation of pathways related to fibroblast activation and tumor promotion. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patient and in vivo PDX-based model systems. These findings demonstrate that CAFs drive chemotherapy resistance in EACs and can be targeted by repurposing PDE5i, a safe and well-tolerated class of drug administered to millions of patients world-wide to treat erectile dysfunction.

Author Correction: Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1.

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LSD1 inhibition attenuates androgen receptor V7 splice variant activation in castration resistant prostate cancer models.

BACKGROUND: Castrate resistant prostate cancer (CRPC) is often driven by constitutively active forms of the androgen receptor such as the V7 splice variant (AR-V7) and commonly becomes resistant to established hormonal therapy strategies such as enzalutamide as a result. The lysine demethylase LSD1 is a co-activator of the wild type androgen receptor and a potential therapeutic target in hormone sensitive prostate cancer. We evaluated whether LSD1 could also be therapeutically targeted in CRPC models driven by AR-V7. METHODS: We utilised cell line models of castrate resistant prostate cancer through over expression of AR-V7 to test the impact of chemical LSD1 inhibition on AR activation. We validated findings through depletion of LSD1 expression and in prostate cancer cell lines that express AR-V7. RESULTS: Chemical inhibition of LSD1 resulted in reduced activation of the androgen receptor through both the wild type and its AR-V7 splice variant forms. This was confirmed and validated in luciferase reporter assays, in LNCaP and 22Rv1 prostate cancer cell lines and in LSD1 depletion experiments. CONCLUSION: LSD1 contributes to activation of both the wild type and V7 splice variant forms of the androgen receptor and can be therapeutically targeted in models of CRPC. Further development of this approach is warranted.

Quantitative proteomic profiling of primary cancer-associated fibroblasts in oesophageal adenocarcinoma.

BACKGROUND: Cancer-associated fibroblasts (CAFs) form the major stromal component of the tumour microenvironment (TME). The present study aimed to examine the proteomic profiles of CAFs vs. normal fibroblasts (NOFs) from patients with oesophageal adenocarcinoma to gain insight into their pro-oncogenic phenotype. METHODS: CAFs/NOFs from four patients were sub-cultured and analysed using quantitative proteomics. Differentially expressed proteins (DEPs) were subjected to bioinformatics and compared with published proteomics and transcriptomics  datasets. RESULTS: Principal component analysis of all profiled proteins showed that CAFs had high heterogeneity and clustered separately from NOFs. Bioinformatics interrogation of the DEPs demonstrated inhibition of adhesion of epithelial cells, adhesion of connective tissue cells and cell death of fibroblast cell lines in CAFs vs. NOFs (p < 0.0001). KEGG pathway analysis showed a significant enrichment of the insulin-signalling pathway (p = 0.03). Gene ontology terms related with myofibroblast phenotype, metabolism, cell adhesion/migration, hypoxia/oxidative stress, angiogenesis, immune/inflammatory response were enriched in CAFs vs. NOFs. Nestin, a stem-cell marker up-regulated in CAFs vs. NOFs, was confirmed to be expressed in the TME with immunohistochemistry. CONCLUSIONS: The identified pathways and participating proteins may provide novel insight on the tumour-promoting properties of CAFs and unravel novel adjuvant therapeutic targets in the TME.

Epidermal Growth Factor Receptor Family Inhibition Identifies P38 Mitogen-activated Protein Kinase as a Potential Therapeutic Target in Bladder Cancer.

OBJECTIVE: To investigate perturbations in downstream signaling pathway activation and potential resistance mechanisms to epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER2) inhibition in cell line models of bladder cancer. METHODS: We undertook a structured screening approach by phosphokinase array, followed by validation steps, to detect activated downstream signaling pathway nodes after therapeutic inhibition of EGFR or HER2 in bladder cancer cell lines. RESULTS: Erlotinib treatment of RT112 cells induced phosphorylation of 9 activated phosphoprotein targets (p38 mitogen-activated protein kinase [MAPK] [Thr180/Tyr182], GSK-3α/ß [Ser21/9], MEK1/2 [Ser218/222, Ser222/226], Akt (protein kinase B) [Ser473], TOR [target of rapamycin] [Ser2448], Src [Tyr419], p27 [Thr198], p27 [Thr157], and PLCγ-1 [Tyr783]), whereas STAT4 (signal transducer and activator of transcription 4) (Tyr693) phosphorylation was reduced. Of these, p38 MAPK phosphorylation was confirmed to occur in response to inhibition of either EGFR or HER2 signaling through multiple validation steps, including differing bladder cancer cell lines (RT112, UM-UC-3, and T24) and methods of receptor pathway inhibition (erlotinib, lapatinib, and siRNA depletion of EGFR or HER2). Chemical inhibition of p38 MAPK with SB203580 led to inhibition of proliferation in RT112, UM-UC-3, and T24 cell lines (IC50 20.85, 76.78, and 79.12 µM, respectively). Fractional effect analyses indicated a synergistic interaction for inhibition of cell proliferation when combining SB203580 with lapatinib. CONCLUSION: p38 MAPK is a potential therapeutic target in bladder cancer and this strategy warrants further development in this disease. It may also allow combination therapy strategies to be developed in conjunction with EGFR or HER2 inhibition.

Fibroblasts derived from oesophageal adenocarcinoma differ in DNA methylation profile from normal oesophageal fibroblasts.

Oesophageal adenocarcinoma (OAC) is increasing in incidence and has a poor prognosis. Tumour derived fibroblasts (TDFs) differ functionally from normal fibroblasts (NDFs), and play a pivotal role in cancer. Many of the differences persist through subculture. We measured the DNA methylation profiles of 10 TDFs from OAC with 12 NDF from normal oesophageal mucosa using Infinium HumanMethylation450 Beadchips and found they differed in multidimensional scaling analysis. We identified 4,856 differentially methylated CpGs (DMCs, adjusted p < 0.01 and absolute difference in average ß-value > 0.15), of which 3,243 (66.8%) were hypomethylated in TDFs compared to NDFs. Hypermethylated DMCs were enriched at transcription start sites (TSSs) and in CpG islands, and depleted in transcriptional enhancers. Gene ontology analysis of genes with DMCs at TSSs revealed an enrichment of genes involved in development, morphogenesis, migration, adhesion, regulation of processes and response to stimuli. Alpha-smooth muscle actin (α-SMA) is a marker of activated fibroblasts and a poor prognostic indicator in OAC. Hypomethylated DMCs were observed at the TSS of transcript variant 2 of α-SMA, which correlated with an increase in α-SMA protein expression. These data suggest that DNA methylation may contribute to the maintenance of the TDF phenotype.

Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1.

New biological tools are required to understand the functional significance of genetic events revealed by whole genome sequencing (WGS) studies in oesophageal adenocarcinoma (OAC). The MFD-1 cell line was isolated from a 55-year-old male with OAC without recombinant-DNA transformation. Somatic genetic variations from MFD-1, tumour, normal oesophagus, and leucocytes were analysed with SNP6. WGS was performed in tumour and leucocytes. RNAseq was performed in MFD-1, and two classic OAC cell lines FLO1 and OE33. Transposase-accessible chromatin sequencing (ATAC-seq) was performed in MFD-1, OE33, and non-neoplastic HET1A cells. Functional studies were performed. MFD-1 had a high SNP genotype concordance with matched germline/tumour. Parental tumour and MFD-1 carried four somatically acquired mutations in three recurrent mutated genes in OAC: TP53, ABCB1 and SEMA5A, not present in FLO-1 or OE33. MFD-1 displayed high expression of epithelial and glandular markers and a unique fingerprint of open chromatin. MFD-1 was tumorigenic in SCID mouse and proliferative and invasive in 3D cultures. The clinical utility of whole genome sequencing projects will be delivered using accurate model systems to develop molecular-phenotype therapeutics. We have described the first such system to arise from the oesophageal International Cancer Genome Consortium project.

A subset of myofibroblastic cancer-associated fibroblasts regulate collagen fiber elongation, which is prognostic in multiple cancers.

Collagen structure has been shown to influence tumor cell invasion, metastasis and clinical outcome in breast cancer. However, it remains unclear how it affects other solid cancers. Here we utilized multi-photon laser scanning microscopy and Second Harmonic Generation to identify alterations to collagen fiber structure within the tumor stroma of head & neck, esophageal and colorectal cancers. Image segmentation algorithms were then applied to quantitatively characterize these morphological changes, showing that elongated collagen fibers significantly correlated with poor clinical outcome (Log Rank p < 0.05). We used TGF-ß treatment to model fibroblast conversion to smooth muscle actin SMA-positive cancer associated fibroblasts (CAFs) and found that these cells induce the formation of elongated collagen fibers in vivo. However, proteomic/transcriptomic analysis of SMA-positive CAFs cultured ex-vivo showed significant heterogeneity in the expression of genes with collagen fibril organizing gene ontology. Notably, stratifying patients according to stromal SMA-positivity and collagen fiber elongation was found to provide a highly significant correlation with poor survival in all 3 cancer types (Log Rank p ≤ 0.003). In summary, we show that increased collagen fiber length correlates with poor patient survival in multiple tumor types and that only a sub-set of SMA-positive CAFs can mediate the formation of this collagen structure.

Cancer-associated fibroblasts predict poor outcome and promote periostin-dependent invasion in oesophageal adenocarcinoma.

Interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an important role in tumour development and progression. In this study we investigated the functional role of CAFs in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of 183 EAC patients for CAF markers related to disease mortality. We characterized CAFs and normal oesophageal fibroblasts (NOFs) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3D organotypic culture and xenograft models were used to examine the effects on EAC cell function and to dissect molecular mechanisms regulating invasion. Most EACs (93%) contained CAFs with a myofibroblastic (α-SMA-positive) phenotype, which correlated significantly with poor survival [p = 0.016; HR 7. 1 (1.7-29.4)]. Primary CAFs isolated from EACs have a contractile, myofibroblastic phenotype and promote EAC cell invasion in vitro (Transwell assays, p ≤ 0.05; organotypic culture, p < 0.001) and in vivo (p ≤ 0.05). In vitro, this pro-invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAFs and acts as a ligand for EAC cell integrins αvß3 and αvß5, promoting activation of the PI3kinase-Akt pathway. In patient samples, periostin expression at the tumour cell-stromal interface correlates with poor overall and disease-free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF-secreted periostin and EAC-expressed integrins results in PI3 kinase-Akt activation and increased tumour cell invasion. Most EACs contain a myofibroblastic CAF-rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patients.

The Nrf2 transcription factor contributes to resistance to cisplatin in bladder cancer.

OBJECTIVES: Cisplatin is the key systemic chemotherapeutic agent used for bladder cancer, but chemoresistance is a major clinical problem. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates various critical cellular processes, including cellular antioxidant response, cellular detoxification, and drug uptake/efflux. These processes, and the expression of multiple Nrf2 target genes, have been found to be associated with bladder cancer prognosis and chemotherapy resistance. We, therefore, investigated whether Nrf2 might regulate cisplatin resistance in bladder cancer. MATERIALS AND METHODS: We first used bladder cancer cell lines, including a cisplatin-resistant RT112 subline (RT112-CP), to investigate Nrf2 expression and activation and its association with cisplatin response. We then undertook immunohistochemical analysis of a tissue microarray of archival bladder cancer radical cystectomy specimens to test the relevance of clinical Nrf2 expression to outcomes following either neoadjuvant chemotherapy and cystectomy or cystectomy alone. RESULTS: Bladder cancer cell lines showed variable Nrf2 expression. Nrf2 expression was greater in RT112-CP cisplatin-resistant cells compared with that in parental RT112 cells. Nrf2 overexpression was functional in this model as it was associated with increased antioxidant response element reporter construct activity, Nrf2 target gene expression (metallothionein and glutathione reductase), and basal glutathione levels. Cisplatin resistance was associated with Nrf2 expression, and in RT112-CP cells, its depletion partially restored cisplatin sensitivity. We demonstrated increased cytoplasmic or nuclear Nrf2 expression or both in 32% of clinical bladder cancer samples compared with that in normal tissue samples. Expression of Nrf2 in bladder cancer following radical cystectomy was associated with unfavorable overall (median = 0.65 vs. 2.11 y, P = 0.045), bladder cancer-specific, and recurrence-free survival in those patients who also received neoadjuvant cisplatin-based chemotherapy but not in those treated with cystectomy alone. CONCLUSIONS: Nrf2 overexpression in bladder cancer is associated with clinically relevant cisplatin resistance that is reversible in experimental models and should now be tested in prospective studies.

Relaxin modulates human and rat hepatic myofibroblast function and ameliorates portal hypertension in vivo.

UNLABELLED: Active myofibroblast (MF) contraction contributes significantly to the increased intrahepatic vascular resistance that is the primary cause of portal hypertension (PHT) in cirrhosis. We sought proof of concept for direct therapeutic targeting of the dynamic component of PHT and markers of MF activation using short-term administration of the peptide hormone relaxin (RLN). We defined the portal hypotensive effect in rat models of sinusoidal PHT and the expression, activity, and function of the RLN-receptor signaling axis in human liver MFs. The effects of RLN were studied after 8 and 16 weeks carbon tetrachloride intoxication, following bile duct ligation, and in tissue culture models. Hemodynamic changes were analyzed by direct cannulation, perivascular flowprobe, indocyanine green imaging, and functional magnetic resonance imaging. Serum and hepatic nitric oxide (NO) levels were determined by immunoassay. Hepatic inflammation was assessed by histology and serum markers and fibrosis by collagen proportionate area. Gene expression was analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting and hepatic stellate cell (HSC)-MF contractility by gel contraction assay. Increased expression of RLN receptor (RXFP1) was shown in HSC-MFs and fibrotic liver diseases in both rats and humans. RLN induced a selective and significant reduction in portal pressure in pathologically distinct PHT models, through augmentation of intrahepatic NO signaling and a dramatic reduction in contractile filament expression in HSC-MFs. Critical for translation, RLN did not induce systemic hypotension even in advanced cirrhosis models. Portal blood flow and hepatic oxygenation were increased by RLN in early cirrhosis. Treatment of human HSC-MFs with RLN inhibited contractility and induced an antifibrogenic phenotype in an RXFP1-dependent manner. CONCLUSION: We identified RXFP1 as a potential new therapeutic target for PHT and MF activation status.

Enantioselective synthesis of tranylcypromine analogues as lysine demethylase (LSD1) inhibitors.

Asymmetric cyclopropanation of styrenes by tert-butyl diazoacetate followed by ester hydrolysis and Curtius rearrangement gave a series of tranylcypromine analogues as single enantiomers. The o,- m- and p-bromo analogues were all more active than tranylcypromine in a LSD1 enzyme assay. The m- and p-bromo analogues were micromolar growth inhibitors of the LNCaP prostate cancer cell line as were the corresponding biphenyl analogues prepared from the bromide by Suzuki crosscoupling.

Total synthesis of largazole and analogues: HDAC inhibition, antiproliferative activity and metabolic stability.

The total synthesis of largazole and four analogues is reported. All analogues were nanomolar HDAC inhibitors. The antiproliferative activity is driven by lipophilicity and cell permeability. In murine liver homogenates, largazole is rapidly metabolized (half-life ≤5 min) to the thiol which has a half-life of 51 min.

S-adenosylhomocysteine hydrolase inhibition by 3-deazaneplanocin A analogues induces anti-cancer effects in breast cancer cell lines and synergy with both histone deacetylase and HER2 inhibition.

Epigenetic abnormalities including abnormal histone methyltransferase activity contribute to breast cancer pathogenesis. An example is over expression of the polycomb repressive complex (PRC) 2 member enhancer of zeste homolog 2 (EZH2) which is linked to epigenetic silencing and poor prognosis. Recent evidence shows that S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors (AHI) such as 3-deazaneplanocin A (DZNep) modulate chromatin through indirect inhibition of histone methyltransferases including EZH2. We investigated the biological effects of AdoHcy hydrolase inhibition using DZNep and its structural analogues 3-deazaadenosine (DZA) and neplanocin A (Nep A) in breast cancer cells. EZH2 protein expression was decreased and dose dependent growth inhibition occurred with variable potencies in MCF7, MDA-MB-231 and SKBr3 breast cancer cells. Cellular proliferation was inhibited through G(2)/M cell cycle arrest and apoptosis. In addition breast cancer cells accumulated cytoplasmic lipid droplets in response to AdoHcy hydrolase inhibition consistent with a differentiating effect. Each analogue induced a similar pattern of biological activity against breast cancer cells but with differences in potency (DZA > DZNep > Nep A). Co-administration with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) induced synergistic inhibition of breast cancer cell proliferation. Furthermore, the relatively AHI resistant human epidermal growth factor receptor 2 (HER2) positive cell line SKBr3 underwent synergistic growth inhibition in response to co-treatment with the HER2 directed therapeutic antibody trastuzumab. In conclusion, AHI induce growth inhibition, cell cycle arrest, apoptosis and differentiation in breast cancer cells and synergise with HDAC and HER2 inhibition. Targeting histone methyltransferase activity might be of therapeutic value in breast cancer.