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Background: Invasive lobular carcinoma (ILC) is the second most common type of breast cancer. As few tools exist to study ILC metastasis, we isolated ILC cells with increased invasive properties to establish a spontaneously metastasising xenograft model.Methods: MDA-MB-134VI ILC cells were placed in transwells for 7 days. Migrated cells were isolated and expanded to create the VIVA1 cell line. VIVA1 cells were compared to parental MDA-MB-134VI cells in vitro for ILC marker expression and relative proliferative and invasive ability. An intraductally injected orthotopic xenograft model was used to assess primary and metastatic tumour growth in vivo.Results: Similar to MDA-MB-134VI, VIVA1 cells retained expression of oestrogen receptor (ER) and lacked expression of E-cadherin, however showed increased invasion in vitro. Following intraductal injection, VIVA1 and MDA-MB-134VI cells had similar primary tumour growth and survival kinetics. However, macrometastases were apparent in 7/10 VIVA1-injected animals. Cells from a primary orthotopic tumour (VIVA-LIG43) were isolated and showed similar proliferative rates but were also more invasive than parental cells. Upon re-injection intraductally, VIVA-LIG43 cells had more rapid tumour growth with similar metastatic incidence and location.Conclusions: We generated a new orthotopic spontaneously metastasising xenograft model for ER+ ILC amenable for the study of ILC metastasis. [ABSTRACT FROM AUTHOR]

Background: Small extracellular vesicles (sEVs) including exosomes, carrying the CD20, could be involved in immunotherapy resistance in diffuse large B cell lymphoma (DLBCL). We have reported endogenous brain-derived neurotrophic factor/TrkB (tropomyosin-related kinase B) survival axis in DLBCL. Here, we performed a comparative study of sEV production by germinal centre B cell (GCB) and activated B cell (ABC)-DLBCL cell lines, and analysed TrkB activation on this process.Methods: GCB (SUDHL4 and SUDHL6) and ABC (OCI-LY3, OCI-LY10 and U2932) cell lines were used. sEVs were characterised using nanoparticle tracking analysis technology and western blot. CD20 content was also analysed by enzyme-linked immunoassay, and complement-dependent cytotoxicity of rituximab was investigated. 7,8-Dihydroxyflavone (7,8-DHF) was used as a TrkB agonist. In vivo role of sEVs was evaluated in a xenograft model.Results: sEVs production varied significantly between DLBCL cells, independently of subtype. CD20 level was consistent with that of parental cells. Higher CD20 expression was found in sEVs after TrkB activation, with a trend in increasing their concentration. sEVs determined in vitro and in vivo protection from rituximab, which seemed CD20 level-dependent; the protection was enhanced when sEVs were produced by 7,8-DHF-treated cells.Conclusions: DLBCL-derived sEVs have the differential capacity to interfere with immunotherapy, which could be enhanced by growth factors like neurotrophins. Evaluating the sEV CD20 level could be useful for disease monitoring. [ABSTRACT FROM AUTHOR]

BackgroundBreast cancer, a heterogeneous disease has been broadly classified into oestrogen receptor positive (ER+) or oestrogen receptor negative (ER-) tumour types. Each of these tumours is dependent on specific signalling pathways for their progression. While high levels of survivin, an anti-apoptotic protein, increases aggressive behaviour in ER- breast tumours, oxidative stress (OS) promotes the progression of ER+ breast tumours. Mechanisms and molecular targets by which OS promotes tumourigenesis remain poorly understood.ResultsDETA-NONOate, a nitric oxide (NO)-donor induces OS in breast cancer cell lines by early re-localisation and downregulation of cellular survivin. Using in vivo models of HMLE(HRAS) xenografts and E2-induced breast tumours in ACI rats, we demonstrate that high OS downregulates survivin during initiation of tumourigenesis. Overexpression of survivin in HMLE(HRAS) cells led to a significant delay in tumour initiation and tumour volume in nude mice. This inverse relationship between survivin and OS was also observed in ER+ human breast tumours. We also demonstrate an upregulation of NADPH oxidase-1 (NOX1) and its activating protein p67, which are novel markers of OS in E2-induced tumours in ACI rats and as well as in ER+ human breast tumours.ConclusionOur data, therefore, suggest that downregulation of survivin could be an important early event by which OS initiates breast tumour formation.

Background: Cyclin-dependent kinase 9 (CDK9) is a promising prognostic marker and therapeutic target in cancers. Bufalin is an effective anti-tumour agent; however, the clinical application of bufalin is limited due to its high toxicity. Acetyl-bufalin, the bufalin prodrug, was designed and synthesised with higher efficiency and lower toxicity.Methods: Three non-small-cell lung cancer (NSCLC) cell lines, a xenograft model and a patient-derived xenograft (PDX) model were used to examine the effects of acetyl-bufalin. CDK9/STAT3 involvement was investigated by knockdown with siRNA, proteome microarray assay, western blot analysis and co-immunoprecipitation experiments. Acute toxicity test and pharmacokinetics (PK) study were conducted to assess the safety and PK. The human NSCLC tissues were analysed to verify high CDK9 expression.Results: We showed that CDK9 induced NSCLC cell proliferation and that this effect was associated with STAT3 activation, specifically an increase in STAT3 phosphorylation and transcription factor activity. Acetyl-bufalin is an effective and safety inhibitor of the CDK9/STAT3 pathway, leading to the impediment of various oncogenic processes in NSCLC. Molecular docking and high-throughput proteomics platform analysis uncovered acetyl-bufalin directly binds to CDK9. Consequently, acetyl-bufalin impaired the complex formation of CDK9 and STAT3, decreased the expressions of P-STAT3, and transcribed target genes such as cyclin B1, CDC2, MCL-1, Survivin, VEGF, BCL2, and it upregulated the expression levels of BAX and caspase-3 activity. Acetyl-bufalin inhibited tumour growth in NSCLC xenograft and PDX models.Conclusions: Acetyl-bufalin is a novel blocker of the CDK9/STAT3 pathway thus may have potential in therapy of NSCLC and other cancers. [ABSTRACT FROM AUTHOR]

Background: Our previous study demonstrated that lysine demethylase 2A (KDM2A) enhances stemness in breast cancer cells. This demethylase is also highly expressed in cancer-associated fibroblasts (CAFs). However, its clinical significance is unclear.Methods: The expression of KDM2A in CAFs was studied using immunohistochemical staining and its association with clinicopathological features and patient's survival was tested. Overexpression and knockdown strategies were used to investigate KDM2A-regulated genes in fibroblasts. Senescent cells were detected by using β-galactosidase staining. The in vivo tumour-promoting activity of stromal KDM2A was confirmed by animal study.Results: Increase of stromal KDM2A is associated with advanced tumour stage and poor clinical outcome in breast cancer patients. Cancer-derived cytokines stimulated KDM2A expression in normal fibroblasts and transformed them into CAFs. Upregulation of KDM2A induced p53-dependent senescence in fibroblasts and enhanced the release of cytokines, which reciprocally promoted cancer cell proliferation. Additionally, KDM2A upregulated programmed death-ligand 1 (PD-L1) expression via transcriptional activation in fibroblasts. Knockdown of KDM2A completely abolished the tumour-promoting activity of CAFs on breast tumour growth in vivo and diminished PD-L1 expression in the stroma of tumour tissues.Conclusions: Stromal KDM2A plays an oncogenic role in breast cancer and inhibition of KDM2A reduces fibroblast senescence and suppresses tumour growth. [ABSTRACT FROM AUTHOR]

Background: A large proportion of gastric cancer patients are susceptible to chemoresistance, while the underlying mechanism remains obscure. Stress granules (SGs) play a self-defence role for tumour cells in inhibiting chemotherapy-induced apoptosis. As an SG assembly effector, G3BP1 (Ras-GTPase-activating protein SH3 domain-binding protein) has been reported to be overexpressed in gastric cancer; thus, here we aim to explore its potent roles in gastric cancer chemoresistance.Methods: Kaplan-Meier analysis was used to compare survival rates in gastric cancer patients with different G3BP1 expression. The influence of G3BP1 on gastric cancer cell chemoresistance and apoptosis were evaluated by in vitro and in vivo approaches. The interaction between G3BP1 and YWHAZ was assessed by immunohistochemistry, immunoprecipitation and immunofluorescence.Results: G3BP1 was associated with the poor outcome of gastric cancer patients who received adjuvant chemotherapy. G3BP1 knockdown significantly increased the sensitivity of gastric cancer cells to chemotherapy drugs. Mechanically, cell apoptosis and pro-apoptotic-associated molecules were significantly elevated upon G3BP1 depletion. Gene co-expression network analyses identified YWHAZ as the critical interlayer of G3BP1; as a result, G3BP1 interacted with YWHAZ to sequester Bax into the cytoplasm. Clinically, G3BP1highYWHAZhigh gastric cancer patients displayed the worst outcome compared with other patients after chemotherapy.Conclusions: The expression of G3BP1 and YWHAZ could predict the adjuvant chemotherapy benefit in gastric cancer patients. [ABSTRACT FROM AUTHOR]

Background: Recently, fusion variants of the breast cancer anti-oestrogen-resistance 4 (BCAR4) gene were recurrently discovered in lung adenocarcinoma from the genome-wide studies. However, the functional characterisation of BCAR4 fusion has not been investigated.Methods: Based on the analysis of RNA-sequencing data, we identified a fusion transcript of CD63-BCAR4 in a Korean patient with lung adenocarcinoma who did not harbour any known activating mutations in EGFR and KRAS genes. To investigate the oncogenic effect of CD63-BCAR4, in vitro and in vivo animal experiments were performed.Results: In vitro experiments showed strongly enhanced cell migration and proliferation by the exogenous expression of CD63-BCAR4 protein in bronchial epithelial cells. Cell migration was notably reduced after knockdown of BCAR4 fusion by small-interfering RNA. The tumorigenic and metastatic capability of the CD63-BCAR4 fusion was confirmed by using the mouse xenograft model. Fusion-overexpressed cells result in metastasis to the liver and lung as well as the primary tumours after subcutaneous injection into mice. Cyclin D1, MMP1, Slug and mesenchymal markers were significantly increased after CD63-BCAR4 overexpression in the in vitro and in vivo experiments.Conclusions: Taken together, our results suggest a newly identified fusion gene, CD63-BCAR4 as a potential novel oncogene in lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Background: Previously, we identified ITIH5 as a suppressor of pancreatic ductal adenocarcinoma (PDAC) metastasis in experimental models. Expression of ITIH5 correlated with decreased cell motility, invasion and metastasis without significant inhibition of primary tumour growth. Here, we tested whether secretion of ITIH5 is required to suppress liver metastasis and sought to understand the role of ITIH5 in human PDAC.Methods: We expressed mutant ITIH5 with deletion of the N-terminal secretion sequence (ITIH5Δs) in highly metastatic human PDAC cell lines. We used a human tissue microarray (TMA) to compare ITIH5 levels in uninvolved pancreas, primary and metastatic PDAC.Results: Secretion-deficient ITIH5Δs was sufficient to suppress liver metastasis. Similar to secreted ITIH5, expression of ITIH5Δs was associated with rounded cell morphology, reduced cell motility and reduction of liver metastasis. Expression of ITIH5 is low in both human primary PDAC and matched metastases.Conclusions: Metastasis suppression by ITIH5 may be mediated by an intracellular mechanism. In human PDAC, loss of ITIH5 may be an early event and ITIH5-low PDAC cells in primary tumours may be selected for liver metastasis. Further defining the ITIH5-mediated pathway in PDAC could establish future therapeutic exploitation of this biology and reduce morbidity and mortality associated with PDAC metastasis. [ABSTRACT FROM AUTHOR]

Background: Antioxidase alleviates the accumulation of radiation-induced reactive oxygen species (ROS) and therefore has strong connections with radioresistance. Isocitrate dehydrogenase 2 (IDH2) facilitates the turnover of antioxidase, but its role in radiotherapeutic efficiency in oesophageal squamous cell carcinoma (ESCC) still remains elusive.Methods: The involvement of IDH2 in radiotherapeutic efficacy in ESCC was investigated in vitro and vivo by IDH2 knockdown. IDH2 expression in biopsy specimens of 141 patients was identified to evaluate its clinical significance.Results: We found that Kyse510 and Kyse140 cells were more radioresistant and had higher IDH2 expression. In these two cell lines, IDH2 knockdown intensified the radiation-induced ROS overload and oxidative damage on lipid, protein, and nucleic acids. In addition, IDH2 silencing aggravated the radiation-induced mitochondrial dysfunction and cell apoptosis and ultimately promoted radiosensitisation via inhibiting AKT phosphorylation in a ROS-dependent manner. Furthermore, IDH2 depletion facilitated the radiation-induced growth inhibition and cell apoptosis in murine xenografts. Finally, IDH2 expression was correlated with definite chemoradiotherapy (dCRT) efficacy and served as an independent prognostic factor for survival of ESCC patients.Conclusions: IDH2 plays a key role in the radioresistance of ESCC. Targeting IDH2 could be a promising regimen to improve radiotherapeutic efficiency in ESCC patients. [ABSTRACT FROM AUTHOR]

Background: Stearoyl-CoA desaturase-1 (SCD1) is reported to play essential roles in cancer stemness among several cancers. Our previous research revealed significant overexpression of SCD1 in primary gastric cancer stem cells (GCSCs), with its functional role still unknown.Methods: We stably established three primary GCSCs by sphere-forming assays and flow cytometry. Protein quantification and bioinformatics analysis were performed to reveal the differential protein pattern. Lentivirus-based small-interfering RNA (siRNA) knockdown and pharmacological inhibition approaches were used to characterise the function and molecular mechanism role of SCD1 in the regulation of GC stemness and tumour metastasis capacity both in vitro and in vivo.Results: SCD1 was found to increase the population of GCSCs, whereas its suppression by an SCD1 inhibitor or knockdown by siRNA attenuated the stemness of GCSCs, including chemotherapy resistance and sphere-forming ability. Furthermore, SCD1 suppression reversed epithelial-to-mesenchymal transition and reduced the GC metastasis probability both in vitro and in vivo. Downregulation of SCD1 in GCSCs was associated with the expression of Yes-associated protein (YAP), a key protein in the Hippo pathway, and nuclear YAP translocation was also blocked by the SCD1 decrease.Conclusions: SCD1 promotes GCSC stemness through the Hippo/YAP pathway. Targeting SCD1 might be a novel therapeutic strategy, especially to suppress GC metastasis and sensitise chemotherapy. [ABSTRACT FROM AUTHOR]

Background: The small GTPase Ran is upregulated in multiple cancers and fundamental for cancer cell survival and progression, but its significance and molecular mechanisms in colorectal cancer (CRC) remain elusive.Methods: Ran expression was detected in CRC cell lines and tumour tissues. In vitro and in vivo functional assays were performed to examine the effects of Ran on cell proliferation and metastasis. The pathways and effectors regulated by Ran were explored by an unbiased screening. Bioinformatics prediction and experimental validation were used to identify the miRNA regulator for Ran.Results: Ran expression was frequently increased in metastatic CRC cells and tissues, especially in metastatic tissues. The upregulation of Ran correlated with poor CRC patient prognosis. Ran silencing reduced proliferation and metastasis of CRC cells both in vitro and in vivo. Ran regulated the expression of EGFR and activation of ERK and AKT signalling pathways. miR-802 was identified as an upstream regulator of Ran and miR-802 overexpression resulted in antiproliferative and antimetastatic activities.Conclusion: Our study demonstrates the oncogenic roles and underlying mechanisms of Ran in CRC and the novel miR-802/Ran/EGFR regulatory axis may provide potential biomarkers for the treatment of CRC. [ABSTRACT FROM AUTHOR]

Background: DAXX is a transcription repressor that has been implicated in several types of cancers, but its role in the development of gastric cancer remains unknown.Methods: We analysed the expression of DAXX in 83 pairs of gastric cancer samples, including neoplastic and adjacent tissues, and correlated the expression levels with clinical stages. We also investigated the molecular mechanisms by which DAXX downregulation promotes cancer growth using both in vitro and in vivo models.Results: DAXX was downregulated in advanced gastric cancer samples. The expression of DAXX inversely correlates with that of cancer stem cell markers CD44 and Oct4 in gastric cancer lines. DAXX overexpression in gastric cancer cells inhibited migration, invasion and epithelial- mesenchymal transition (EMT). The inhibition of EMT was achieved through the repression of SNAI3, a key inducer of EMT, by recruiting HDAC-1 into the nucleus. Using a xenograft mouse model, we demonstrated that the MKN45 cells formed smaller tumours when DAXX was overexpressed. Wild-type AGS cells were not able to form tumours in nude mice, but in contrast, formed visible tumours when DAXX was silenced in the cells.Conclusion: We for the first time demonstrated that DAXX functions as a tumour suppressor in gastric cancer by inhibiting stem cell growth and EMT. [ABSTRACT FROM AUTHOR]

Background: Epidemiological studies suggest that metformin may reduce the incidence of cancer in patients with diabetes and multiple late phase clinical trials assessing the potential of repurposing this drug are underway. Transcriptomic profiling of tumour samples is an excellent tool to understand drug bioactivity, identify candidate biomarkers and assess for mechanisms of resistance to therapy.Methods: Thirty-six patients with untreated primary breast cancer were recruited to a window study and transcriptomic profiling of tumour samples carried out before and after metformin treatment.Results: Multiple genes that regulate fatty acid oxidation were upregulated at the transcriptomic level and there was a differential change in expression between two previously identified cohorts of patients with distinct metabolic responses. Increase in expression of a mitochondrial fatty oxidation gene composite signature correlated with change in a proliferation gene signature. In vitro assays showed that, in contrast to previous studies in models of normal cells, metformin reduces fatty acid oxidation with a subsequent accumulation of intracellular triglyceride, independent of AMPK activation.Conclusions: We propose that metformin at clinical doses targets fatty acid oxidation in cancer cells with implications for patient selection and drug combinations.Clinical Trial Registration: NCT01266486. [ABSTRACT FROM AUTHOR]

Background: Poly-ADP ribose polymerase (PARP) inhibitor-based cancer therapy selectively targets cells with deficient homologous recombination repair. Considering their long-term use in maintenance treatment, any potential mutagenic effect of PARP inhibitor treatment could accelerate the development of resistance or harm non-malignant somatic cells.Methods: We tested the mutagenicity of long-term treatment with the PARP inhibitor niraparib using whole-genome sequencing of cultured cell clones and whole-exome sequencing of patient-derived breast cancer xenografts.Results: We observed no significant increase in the number and alteration in the spectrum of base substitutions, short insertions and deletions and genomic rearrangements upon niraparib treatment of human DLD-1 colon adenocarcinoma cells, wild-type and BRCA1 mutant chicken DT40 lymphoblastoma cells and BRCA1-defective SUM149PT breast carcinoma cells, except for a minor increase in specific deletion classes. We also did not detect any contribution of in vivo niraparib treatment to subclonal mutations arising in breast cancer-derived xenografts.Conclusions: The results suggest that long-term inhibition of DNA repair with PARP inhibitors has no or only limited mutagenic effect. Mutagenesis due to prolonged use of PARP inhibitors in cancer treatment is therefore not expected to contribute to the genetic evolution of resistance, generate significant immunogenic neoepitopes or induce secondary malignancies. [ABSTRACT FROM AUTHOR]

Background: Hypoxia is negatively associated with glioblastoma (GBM) patient survival and contributes to tumour resistance. Anti-angiogenic therapy in GBM further increases hypoxia and activates survival pathways. The aim of this study was to determine the role of hypoxia-induced autophagy in GBM.Methods: Pharmacological inhibition of autophagy was applied in combination with bevacizumab in GBM patient-derived xenografts (PDXs). Sensitivity towards inhibitors was further tested in vitro under normoxia and hypoxia, followed by transcriptomic analysis. Genetic interference was done using ATG9A-depleted cells.Results: We find that GBM cells activate autophagy as a survival mechanism to hypoxia, although basic autophagy appears active under normoxic conditions. Although single agent chloroquine treatment in vivo significantly increased survival of PDXs, the combination with bevacizumab resulted in a synergistic effect at low non-effective chloroquine dose. ATG9A was consistently induced by hypoxia, and silencing of ATG9A led to decreased proliferation in vitro and delayed tumour growth in vivo. Hypoxia-induced activation of autophagy was compromised upon ATG9A depletion.Conclusions: This work shows that inhibition of autophagy is a promising strategy against GBM and identifies ATG9 as a novel target in hypoxia-induced autophagy. Combination with hypoxia-inducing agents may provide benefit by allowing to decrease the effective dose of autophagy inhibitors. [ABSTRACT FROM AUTHOR]

Background: The Hedgehog (Hh) pathway is upregulated in cervical cancer and associated with poor outcome. We explored the effects of Hh pathway inhibition in combination with RTCT in a patient derived orthotopic cervical cancer xenograft model (OCICx).Methods: 5E1, a monoclonal antibody for SHH, or Sonidegib (LDE225), a clinical SMO inhibitor (Novartis) were added to RTCT. We investigated tumour growth delay, metastasis and GI toxicity using orthotopic cervical cancer xenografts models. The xenografts were treated with radiotherapy (15 × 2 Gy daily fractions over 3 weeks) and weekly cisplatin 4 mg kg-1 concurrently, with or without 5E1 or Sonidegib (LDE225). The Hh inhibitors were administered by subcutaneous injection (5E1; 20 mg kg-1 weekly for 3 weeks), or by oral gavage (Sonidegib; 60 mg kg-1 daily for 3 weeks).Results: We observed that both Hh inhibitors administered with RTCT were well tolerated and showed increased tumour growth delay, and reduced metastasis, with no increase in acute GI-toxicity relative to RTCT alone.Conclusions: Our data suggest Hh can be a valid therapeutic target in cervical cancer and supports data suggesting a potential therapeutic role for targeting Hh in patients undergoing RTCT. This warrants further investigation in clinical trials. [ABSTRACT FROM AUTHOR]

Background: Clinical options for patients harbouring advanced/recurrent uterine serous carcinoma (USC), an aggressive variant of endometrial tumour, are very limited. Next-generation sequencing (NGS) data recently demonstrated that cyclin E1 (CCNE1) gene amplification and pik3ca driver mutations are common in USC and may therefore represent ideal therapeutic targets.Methods: Cyclin E1 expression was evaluated by immunohistochemistry (IHC) on 95 USCs. The efficacy of the cyclin-dependent kinase 2/9 inhibitor CYC065 was assessed on multiple primary USC cell lines with or without CCNE1 amplification. Cell-cycle analyses and knockdown experiments were performed to assess CYC065 targeting specificity. Finally, the in vitro and in vivo activity of CYC065, Taselisib (a PIK3CA inhibitor) and their combinations was tested on USC xenografts derived from CCNE1-amplified/pik3ca-mutated USCs.Results: We found that 89.5% of the USCs expressed CCNE1. CYC065 blocked cells in the G1 phase of the cell cycle and inhibited cell growth specifically in CCNE1-overexpressing USCs. Cyclin E1 knockdown conferred increased resistance to CYC065, whereas CYC065 treatment of xenografts derived from CCNE1-amplified USCs significantly reduced tumour growth. The combination of CYC065 and Taselisib demonstrated synergistic effect in vitro and was significantly more effective than single-agent treatment in decreasing tumour growth in xenografts of CCNE1-amplified/pik3ca-mutated USCs.Conclusions: Dual CCNE1/PIK3CA blockade may represent a novel therapeutic option for USC patients harbouring recurrent CCNE1-amplified/pi3kca-mutated tumours. [ABSTRACT FROM AUTHOR]

Background: Copy-number gain of the oncostatin-M receptor (OSMR) occurs frequently in cervical squamous cell carcinoma (SCC) and is associated with adverse clinical outcome. We previously showed that OSMR overexpression renders cervical SCC cells more sensitive to the major ligand oncostatin-M (OSM), which increases migration and invasion in vitro. We hypothesised that a major contribution to this phenotype would come from epithelial-mesenchymal transition (EMT).Methods: We performed a comprehensive integrated study, involving in vitro cell line studies, in vivo animal models and numerous clinical samples from a variety of anatomical sites.Results: In independent sets of cervical, head/neck and lung SCC tissues, OSMR expression levels correlated with multiple EMT-associated phenotypic markers and transcription factors. OSM treatment of OSMR overexpressing cervical SCC cells produced consistent EMT changes and increased tumour sphere formation in suspension culture. In a mouse model, OSMR overexpressing SCC cells treated with OSM showed significant increases in lung colonisation. The biological effects of exogenous OSM were mirrored by highly significant adverse overall survival in cervical SCCs with OSMR overexpression (N=251).Conclusions: OSM:OSMR interactions are able to induce EMT, increased cancer stem cell-like properties and enhanced lung colonisation in SCC cells. These changes are likely to contribute to the highly significant adverse outcome associated with OSMR overexpression in cervical SCCs. [ABSTRACT FROM AUTHOR]

Background: Focal adhesion kinase (FAK) is overexpressed in many types of tumours, including lung cancer. Y15, a small molecule which inhibits Y397 FAK autophosphorylation, decreases growth of human neuroblastoma, breast and pancreatic cancers. In this study, we investigated the in vitro and in vivo effects of Y15, and the underlying mechanism on non-small cell lung cancer cells.Methods: The cytotoxic effects of Y15 targeting FAK signalling were evaluated. Gene-knockdown experiments were performed to determine the anti-cancer mechanism. Xenografts with RAS or EGFR mutations were selected for in vivo Y15 treatment.Results: Y15 blocked autophosphorylation of FAK in a time- and dose-dependent manner. It caused dose-dependent decrease of lung cancer cell viability and clonogenicity. Y15 inhibited tumour growth of RAS-mutant (A549 with KRAS mutation and H1299 with NRAS mutation), as well as epidermal growth factor receptor (EGFR) mutant (H1650 and H1975) lung cancer xenografts. JNK activation is a mechanism underlying Y15-induced Bcl-2 and Mcl-1 downregulation. Moreover, knockdown of Bcl-2 or Bcl-xL potentiated the effects of Y15. The combination of various inhibitors of the Bcl-2 family of proteins with FAK inhibitors demonstrated synergy in multiple lung cancer cell lines in vitro.Conclusions: FAK inhibition demonstrated efficacy both in vitro and in vivo in lung cancers with either oncogenic RAS or EGFR mutations. In addition, FAK inhibition in combination with inhibitors of Bcl-2 family of anti-apoptotic proteins has synergistic activity in these MAPK-activated non-small cell lung cancer cell line models. [ABSTRACT FROM AUTHOR]

Background: Development of targeted therapies for high-grade serous ovarian cancer (HGSC) remains challenging, as contributing molecular pathways are poorly defined or expressed heterogeneously. CUB-domain containing protein 1 (CDCP1) is a cell-surface protein elevated in lung, colorectal, pancreas, renal and clear cell ovarian cancer. Methods: CUB-domain containing protein 1 was examined by immunohistochemistry in HGSC and fallopian tube. The impact of targeting CDCP1 on cell growth and migration in vitro, and intraperitoneal xenograft growth in mice was examined. Three patient-derived xenograft (PDX) mouse models were developed and characterised for CDCP1 expression. The effect of a monoclonal anti-CDCP1 antibody on PDX growth was examined. Src activation was assessed by western blot analysis. Results: Elevated CDCP1 was observed in 77% of HGSC cases. Silencing of CDCP1 reduced migration and non-adherent cell growth in vitro and tumour burden in vivo. Expression of CDCP1 in patient samples was maintained in PDX models. Antibody blockade of CDCP1 significantly reduced growth of an HGSC PDX. The CDCP1-mediated activation of Src was observed in cultured cells and mouse xenografts. Conclusions: CUB-domain containing protein 1 is over-expressed by the majority of HGSCs. In vitro and mouse model data indicate that CDCP1 has a role in HGSC and that it can be targeted to inhibit progression of this cancer. [ABSTRACT FROM AUTHOR]

Background: FAT4, a cadherin-related protein, was shown to function as a tumour suppressor; however, its role in human gastric cancer remains largely unknown. Here, we investigated the role of FAT4 in gastric cancer and examined the underlying molecular mechanisms.Methods: The expression of FAT4 was evaluated by immunohistochemistry, western blotting, and qRT-PCR in relation to the clinicopathological characteristics of gastric cancer patients. The effects of FAT4 silencing on cell proliferation, migration, and invasion were assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay, and migration and invasion assays in gastric cancer cell lines in vitro and in a mouse xenograft model in vivo.Results: Downregulation of FAT4 expression in gastric cancer tissues compared with adjacent normal tissues was correlated with lymph-node metastasis and poor survival. Knockdown of FAT4 promoted the growth and invasion of gastric cancer cells via the activation of Wnt/β-catenin signalling, and induced epithelial-to-mesenchymal transition (EMT) in gastric cancer cells, as demonstrated by the upregulation and downregulation of mesenchymal and epithelial markers. Silencing of FAT4 promoted tumour growth and metastasis in a gastric cancer xenograft model in vivo.Conclusions: FAT4 has a tumour suppressor role mediated by the modulation of Wnt/β-catenin signalling, providing potential novel targets for the treatment of gastric cancer. [ABSTRACT FROM AUTHOR]

Background: MYB encodes for a transcription factor regulating the expression of a wide array of genes involved in cellular functions. It is reported to be amplified in a sub-set of pancreatic cancer (PC) cases; however, its pathobiological association has remained unclear thus far.Methods: Expression of MYB and other cellular proteins was analysed by immunoblot or qRT-PCR analyses. MYB was stably overexpressed in non-expressing (BxPC3) and silenced in highly expressing (MiaPaCa and Panc1) PC cells. Effect on growth was analysed by automated cell counting at 24-h interval. Cell-cycle progression and apoptotic indices of PC cells with altered MYB expression were measured through flow cytometry upon staining with respective biomarkers. Cell motility/invasion was examined in a Boyden's chamber assay using non-coated or Matrigel-coated membranes. Effect on tumorigenicity and metastatic potential was examined by non-invasive imaging and through end-point measurements of luciferase-tagged MYB-altered PC implanted in the pancreas of nude mice.Results: MYB was aberrantly expressed in all malignant cases of pancreas, whereas remained undetectable in normal pancreas. All the tested established PC cell lines except BxPC3 also exhibited MYB expression. Forced expression of MYB in BxPC3 cells promoted their growth, cell-cycle progression, survival and malignant behaviour, whereas its silencing in MiaPaCa and Panc1 cells produced converse effects. More importantly, ectopic MYB expression was sufficient to confer tumorigenic and metastatic capabilities to non-tumorigenic BxPC3 cells, while its silencing resulted in significant loss of the same in MYB-overexpressing cells as demonstrated in orthotopic mouse model. We also identified several MYB-regulated genes in PC cells that might potentially mediate its effect on tumour growth and metastasis.Conclusions: MYB is aberrantly overexpressed in PC cells and acts as a key determinant of pancreatic tumour growth and metastasis. [ABSTRACT FROM AUTHOR]

Background: Despite good initial response to chemotherapy, 30% of Ewing's sarcoma (EWS) patients with localised tumours develop recurrent disease, associated with poor prognosis. The aim of this study was to address this challenge by conducting preclinical evaluation of a death receptor targeted agent in vitro and in vivo, and by identifying predictive biomarkers.Methods: Cell viability assays, drug dose responses, immunoblots, rescue with gene transfer, mice tumour models, and statistical comparisons of tumour growth and Kaplan-Meier survival curves.Results: This study shows that many EWS cell lines are selectively sensitive to a death receptor DR5 antibody and are more resistant to a DR4 antibody. Preclinical evaluation of these cell lines indicates their sensitivity to human DR5 agonist antibody conatumumab in vitro, which induces rapid activation of caspase-8 and apoptosis. We also found that sensitivity to conatumumab correlates with expression of caspase-8. Furthermore, the catalytic activity of caspase-8 is both necessary and sufficient to confer this sensitivity. In vivo, conatumumab is active against an EWS cell line and a patient-derived xenograft with higher caspase-8 expression, but is not effective against another with lower caspase-8 expression.Conclusions: These studies suggest the potential of conatumumab as a therapeutic agent against EWS and caspase-8 as a predictive biomarker for sensitivity. [ABSTRACT FROM AUTHOR]

Background: Fibulin-3 (FBLN3) was recently presented as a promising novel biomarker for malignant pleural mesothelioma (MPM), warranting independent validation studies.Methods: ELISA was used to measure cellular and secreted FBLN3 in cell lines, in plasma of xenograft tumour-bearing mice, in plasma from two independent series of MPM and non-MPM patients and in pleural fluid from a third series. Diagnostic and prognostic potential of FBLN3 was assessed by receiver operating characteristics curve analysis and Kaplan-Meier method, respectively.Results: FBLN3 was expressed in all MPM and benign mesothelial cell lines tested, and a correlation was observed between cellular protein expression and secreted levels. Human FBLN3 was detectable in plasma of tumour-bearing mice, suggesting that MPM cells contribute to levels of circulating FBLN3. Plasma FBLN3 was significantly elevated in MPM patients from the Sydney cohort, but not the Vienna cohort, but the diagnostic accuracy was low (63%, (95% CI: 50.1-76.4) and 56% (95% CI: 41.5-71.0), respectively). Although FBLN3 levels in pleural effusions were not significantly different between cases and controls, FBLN3 levels in pleural effusion fluid were found to be independently associated with prognosis (hazard ratio of 9.92 (95% CI: 2.14-45.93)).Conclusions: These data confirm the potential prognostic value of pleural effusion FBLN3, but question the diagnostic value of this protein in MPM patients. [ABSTRACT FROM AUTHOR]

Background:Axl plays multiple roles in tumourigenesis in several cancers. Here we evaluated the expression and biological function of Axl in renal cell carcinoma (RCC).Methods:Axl expression was analysed in a tissue microarray of 174 RCC samples by immunostaining and a panel of 11 normal tumour pairs of human RCC tissues by western blot, as well as in RCC cell lines by both western blot and quantitative PCR. The effects of Axl knockdown in RCC cells on cell growth and signalling were investigated. The efficacy of a humanised Axl targeting monoclonal antibody hMAb173 was tested in histoculture and tumour xenograft.Results:We have determined by immunohistochemistry (IHC) that Axl is expressed in 59% of RCC array samples with moderate to high in 20% but not expressed in normal kidney tissue. Western blot analysis of 11 pairs of tumour and adjacent normal tissue show high Axl expression in 73% of the tumours but not normal tissue. Axl is also expressed in RCC cell lines in which Axl knockdown reduces cell viability and PI3K/Akt signalling. The Axl antibody hMAb173 significantly induced RCC cell apoptosis in histoculture and inhibited the growth of RCC tumour in vivo by 78%. The hMAb173-treated tumours also had significantly reduced Axl protein levels, inhibited PI3K signalling, decreased proliferation, and induced apoptosis.Conclusions:Axl is highly expressed in RCC and critical for RCC cell survival. Targeting Axl is a potential approach for RCC treatment. [ABSTRACT FROM AUTHOR]

Background:Acquired resistance to molecularly targeted therapeutics is a key challenge in personalised cancer medicine, highlighting the need for identifying the underlying mechanisms and early biomarkers of relapse, in order to guide subsequent patient management.Methods:Here we use human head and neck squamous cell carcinoma (HNSCC) models and nuclear magnetic resonance (NMR) spectroscopy to assess the metabolic changes that follow acquired resistance to EGFR tyrosine kinase inhibitors (TKIs), and which could serve as potential metabolic biomarkers of drug resistance.Results:Comparison of NMR metabolite profiles obtained from control (CALS) and EGFR TKI-resistant (CALR) cells grown as 2D monolayers, 3D spheroids or xenograft tumours in athymic mice revealed a number of differences between the sensitive and drug-resistant models. In particular, we observed elevated levels of glycerophosphocholine (GPC) in CALR relative to CALS monolayers, spheroids and tumours, independent of the growth rate or environment. In addition, there was an increase in alanine, aspartate and creatine+phosphocreatine in resistant spheroids and xenografts, and increased levels of lactate, branched-chain amino acids and a fall in phosphoethanolamine only in xenografts. The xenograft lactate build-up was associated with an increased expression of the glucose transporter GLUT-1, whereas the rise in GPC was attributed to inhibition of GPC phosphodiesterase. Reduced glycerophosphocholine (GPC) and phosphocholine were observed in a second HNSCC model probably indicative of a different drug resistance mechanism.Conclusions:Our studies reveal metabolic signatures associated not only with acquired EGFR TKI resistance but also growth pattern, microenvironment and contributing mechanisms in HNSCC models. These findings warrant further investigation as metabolic biomarkers of disease relapse in the clinic. [ABSTRACT FROM AUTHOR]

Background:Autophagy allows recycling of cellular components and may facilitate cell survival after chemotherapy. Pantoprazole inhibits proton pumps and is reported to inhibit autophagy. Here we evaluate the effects of pantoprazole to modify cytotoxicity of the anticancer drug docetaxel, and underlying mechanisms.Methods:Effects of docetaxel±pantoprazole were studied against wild-type and autophagy-deficient PC3 cells and against four human xenografts. Effects of pantoprazole on autophagy were evaluated by quantifying LC3-I, LC3-II and p62 proteins in western blots, and by fluorescent microscopy of cells transfected with RFP-GFP-LC3. The distribution of drug effects and of autophagy was quantified in tumour sections in relation to blood vessels and hypoxia by immunohistochemistry using γH2AX, cleaved caspase-3, Ki67 and LC3/ p62.Results:Pantoprazole increased the toxicity of docetaxel in vitro, increased docetaxel-induced expression of γH2AX and cleaved caspase-3, and decreased Ki67 in tumour sections. Pantoprazole increased growth delay of four human xenografts of low, moderate and high sensitivity to docetaxel, with minimal increase in toxicity. Docetaxel led to increased autophagy throughout tumour sections. Pantoprazole inhibited autophagy, and effects of pantoprazole were reduced against genetically modified cells with decreased ability to undergo autophagy.Conclusions:Autophagy is a mechanism of resistance to docetaxel chemotherapy that may be modified by pantoprazole to improve therapeutic index. [ABSTRACT FROM AUTHOR]

Background:The phosphoinositide-3 kinase (PI3K) pathway is an attractive therapeutic target. However, difficulty in predicting therapeutic response limits the clinical implementation of PI3K inhibitors. This study evaluates the utility of clinically relevant magnetic resonance imaging (MRI) biomarkers for noninvasively assessing the in vivo response to the dual PI3K/mTOR inhibitor BEZ235 in two ovarian cancer models with differential PI3K pathway activity.Methods:The PI3K signalling activity of TOV-21G and TOV-112D human ovarian cancer cells was investigated in vitro. Cellular and vascular response of the xenografts to BEZ235 treatment (65 mg kg−1, 3 days) was assessed in vivo using diffusion-weighted (DW) and dynamic contrast-enhanced (DCE)-MRI. Micro-computed tomography was performed to investigate changes in vascular morphology.Results:The TOV-21G cells showed higher PI3K signalling activity than TOV-112D cells in vitro and in vivo. Treated TOV-21G xenografts decreased in volume and DW-MRI revealed an increased water diffusivity that was not found in TOV-112D xenografts. Treatment-induced improvement in vascular functionality was detected with DCE-MRI in both models. Changes in vascular morphology were not found.Conclusions:Our results suggest that DW- and DCE-MRI can detect cellular and vascular response to PI3K/mTOR inhibition in vivo. However, only DW-MRI could discriminate between a strong and weak response to BEZ235. [ABSTRACT FROM AUTHOR]

Background:Effective vascular normalisation following vascular endothelial growth factor (VEGF) inhibition is associated with endothelial cell regression leaving empty basement membrane sleeves (BMS). These long-lived BMS permit the rapid regrowth of tumour vasculature upon treatment cessation and promote resistance to VEGF-targeting drugs. Previous attempts at removing BMS have failed. Angiopoietin-2 (Ang2) is a vascular destabilizing factor that antagonises normalisation. We hypothesised that Ang2 inhibition could permit vascular normalisation at significantly reduced doses of VEGF inhibition, avoiding excessive vessel regression and the formation of empty BMS.Methods:Mice xenografted with human colorectal cancer cells (LS174T) were treated with low (0.5 mg kg−1) or high (5 mg kg−1) doses of the VEGF-targeting antibody bevacizumab with or without an Ang2 blocking peptibody L1-10. Tumour growth, BMS formation and normalisation parameters were examined including vessel density, pericyte coverage, adherence junctions, leakiness, perfusion, hypoxia and proliferation.Results:Dual targeting of VEGF and Ang2 achieved effective normalisation at only one-tenth of the dose required with bevacizumab alone. Pericyte coverage, vascular integrity, adherence junctions and perfusion as prerequisites for improved access of chemotherapy were improved without inducing empty BMS that facilitate rapid vascular regrowth.Conclusions:Dual targeting of VEGF and Ang2 can potentiate the effectiveness of VEGF inhibitors and avoid the formation of empty BMS. [ABSTRACT FROM AUTHOR]

Background:Isolation of colorectal cancer (CRC) cell populations enriched for cancer stem cells (CSCs) may facilitate target identification. There is no consensus regarding the best methods for isolating CRC stem cells (CRC-SCs). We determined the suitability of various cellular models and various stem cell markers for the isolation of CRC-SCs.Methods:Established human CRC cell lines, established CRC cell lines passaged through mice, patient-derived xenograft (PDX)-derived cells, early passage/newly established cell lines, and cells directly from clinical specimens were studied. Cells were FAC-sorted for the CRC-SC markers CD44, CD133, and aldehyde dehydrogenase (ALDH). Sphere formation and in vivo tumorigenicity studies were used to validate CRC-SC enrichment.Results:None of the markers studied in established cell lines, grown either in vitro or in vivo, consistently enriched for CRC-SCs. In the three other cellular models, CD44 and CD133 did not reliably enrich for stemness. In contrast, freshly isolated PDX-derived cells or early passage/newly established CRC cell lines with high ALDH activity formed spheres in vitro and enhanced tumorigenicity in vivo, whereas cells with low ALDH activity did not.Conclusions:PDX-derived cells, early passages/newly established CRC cell lines and cells from clinical specimen with high ALDH activity can be used to identify CRC-SC-enriched populations. Established CRC cell lines should not be used to isolate CSCs. [ABSTRACT FROM AUTHOR]

Background:Sunitinib (VEGFR/PDGFR inhibitor) and everolimus (mTOR inhibitor) are both approved for advanced renal cell carcinoma (RCC) as first-line and second-line therapy, respectively. In the clinics, sunitinib treatment is limited by the emergence of acquired resistance, leading to a switch to second-line treatment at progression, often based on everolimus. No data have been yet generated on programmed alternating sequential strategies combining alternative use of sunitinib and everolimus before progression. Such strategy is expected to delay the emergence of acquired resistance and improve tumour control. The aim of our study was to assess the changes in tumours induced by three different sequences administration of sunitinib and everolimus.Methods:In human Caki-1 RCC xenograft model, sunitinib was alternated with everolimus every week, every 2 weeks, or every 3 weeks. Effects on necrosis, hypoxia, angiogenesis, and EMT status were assessed by immunohisochemistry and immunofluorescence.Results:Sunitinib and everolimus programmed sequential regimens before progression yielded longer median time to tumour progression than sunitinib and everolimus monotherapies. In each group of treatment, tumour growth control was associated with inhibition of mTOR pathway and changes from a mesenchymal towards an epithelial phenotype, with a decrease in vimentin and an increase in E-cadherin expression. The sequential combinations of these two agents in a RCC mouse clinical trial induced antiangiogenic effects, leading to tumour necrosis.Conclusions:In summary, our study showed that alternate sequence of sunitinib and everolimus mitigated the development of mesenchymal phenotype compared with sunitinib as single agent. [ABSTRACT FROM AUTHOR]

Background: hERG1 channels are aberrantly expressed in human cancers. The expression, functional role and clinical significance of hERG1 channels in pancreatic ductal adenocarcinoma (PDAC) is lacking. Methods: hERG1 expression was tested in PDAC primary samples assembled as tissue microarray by immunohistochemistry using an anti-hERG1 monoclonal antibody (α-hERG1-MoAb). The functional role of hERG1 was studied in PDAC cell lines and primary cultures. ERG1 expression during PDAC progression was studied in Pdx-1-Cre,LSL-Kras(G12D/+),LSL-Trp53(R175H/+) transgenic (KPC) mice. ERG1 expression in vivo was determined by optical imaging using Alexa-680-labelled α-hERG1-MoAb. Results: (i) hERG1 was expressed at high levels in 59% of primary PDAC; (ii) hERG1 blockade decreased PDAC cell growth and migration; (iii) hERG1 was physically and functionally linked to the Epidermal Growth Factor-Receptor pathway; (iv) in transgenic mice, ERG1 was expressed in PanIN lesions, reaching high expression levels in PDAC; (v) PDAC patients whose primary tumour showed high hERG1 expression had a worse prognosis; (vi) the α-hERG1-MoAb could detect PDAC in vivo. Conclusions: hERG1 regulates PDAC malignancy and its expression, once validated in a larger cohort also comprising of late-stage, non-surgically resected cases, may be exploited for diagnostic and prognostic purposes in PDAC either ex vivo or in vivo. [ABSTRACT FROM AUTHOR]

Background:AT7519 is a small-molecular inhibitor of multiple cyclin-dependent kinases (CDKs). It shows encouraging anti-cancer activity against multiple cell lines and in tumour xenografts. This phase I study was conducted to evaluate the safety and tolerability of AT7519 given as 1-h intravenous infusion on days 1, 4, 8 and 11 every 3 weeks.Methods:Patients with advanced refractory solid tumours or non-Hodgkin's lymphoma were enroled. Dose escalation occurred in a 3+3 manner based on toxicity assessment. Pharmacokinetic samples were collected after first AT7519 infusion, whereas pharmacodynamics (PD) samples were obtained in selected patients.Results:Thirty-four patients were enroled, and 32 received study treatments over 4 dose levels. Dose-limiting toxicities included mucositis, febrile neutropenia, rash, fatigue and hypokalemia. The recommended phase II dose (RP2D) was 27.0 mg m−2. Ten of 19 patients evaluable for efficacy had stable disease as the best response (median duration: 3.3 months; range: 2.5 to 11.1 months). There was no clinically significant QTc prolongation. There was an apparent dose proportional increase in AT7519 exposure. The PD studies showed reduction in markers of CDK activity in selected patients' skin biopsies post treatment.Conclusions:AT7519, when administered as an intravenous infusion on days 1, 4, 8 and 11, was well tolerated. The RP2D is 27.0 mg m−2. At this dose level, plasma AT7519 concentrations were above the biologically active concentrations, and preliminary anti-cancer activity was observed in patients. This dosing schedule is being further evaluated in multiple phase II studies. [ABSTRACT FROM AUTHOR]

Background:Glioblastoma (GBM), being a highly vascularised and locally invasive tumour, is an attractive target for anti-angiogenic and anti-invasive therapies. The GBM/endothelial cell response to gossypol/temozolomide (TMZ) treatment was investigated with a particular aim to assess treatment effects on cancer hallmarks.Methods:Cell viability, endothelial tube formation and GBM tumour cell invasion were variously assessed following combined treatment in vitro. The U87MG-luc2 subcutaneous xenograft model was used to investigate therapeutic response in vivo. Viable tumour response to treatment was interrogated using immunohistochemistry. Combined treatment protocols were also tested in primary GBM patient-derived cultures.Results:An endothelial/GBM cell viability inhibitory effect, as well as an anti-angiogenic and anti-invasive response, to combined treatment have been demonstrated in vitro. A significantly greater anti-proliferative (P=0.020, P=0.030), anti-angiogenic (P=0.040, P<0.0001) and pro-apoptotic (P=0.0083, P=0.0149) response was observed when combined treatment was compared with single gossypol/TMZ treatment response, respectively. GBM cell line and patient-specific response to gossypol/TMZ treatment was observed.Conclusions:Our results indicate that response to a combined gossypol/TMZ treatment is related to inhibition of tumour-associated angiogenesis, invasion and proliferation and warrants further investigation as a novel targeted GBM treatment strategy. [ABSTRACT FROM AUTHOR]

Background:Cetuximab is often combined with radiotherapy in advanced SCCHN. Alternative routes bypassing inhibition of EGFR with cetuximab may overshadow the efficacy of this combination. We undertook this study to investigate a possible role of dasatinib in this scenario.Methods:The SCC5, SCC25, SCC29, FaDu and A431 cell lines were assessed in vitro for cell proliferation under cetuximab and dasatinib treatments. In FaDu and A431 cells, dasatinib plus cetuximab resulted in higher proliferation than cetuximab alone. Then, FaDu and A431 cells were implanted into subcutaneous tissue of athymic mice that were irradiated with 30 Gy in 10 fractions over 2 weeks, and treated with cetuximab and dasatinib. Tumour growth, DNA synthesis and angiogenesis were determined. The EGFR, RAS-GTP activity, phosphorylated AKT, ERK1/2, SRC protein levels and VEGF secretion were determined in vitro.Results:The addition of dasatinib to cetuximab and radiotherapy increased tumour growth, DNA synthesis and angiogenesis that were associated with RAS, AKT and ERK1/2 activation, and SRC inhibition in FaDu and A431 cells.Conclusions:In xenografts derived from these two cell lines, dasatinib did not improve the efficacy of cetuximab combined with radiotherapy. On the contrary, it worsened tumour control achieved by the combination of these two treatments. [ABSTRACT FROM AUTHOR]

Background:A subset of human hepatocellular carcinomas (HCC) exhibit mutations of β-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO).Methods:Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1.Results:Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro.Conclusions:The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth. [ABSTRACT FROM AUTHOR]

Background:We conducted a phase I study in patients with advanced solid tumours to identify the recommended dose, assess pharmacokinetics (PK), pharmacodynamic activity and preclinical antitumour efficacy of the combination of sirolimus and gemcitabine.Methods:Nineteen patients were treated with sirolimus 2 or 5 mg daily and gemcitabine 800 or 1000 mg m−2 on days 1 and 8. Dose escalation depended on dose-limiting toxicity (DLT) rate during the first 3-week period. Paired skin biopsies were evaluated for phosphorylated S6 (pS6) as marker of mTOR (mammalian target of rapamycin) inhibition. Pharmacokinetics and preclinical evaluation of efficacy using two different sarcoma cell lines and leiomyosarcoma xenografts were also conducted.Results:Three DLTs were observed: grade 3 transaminitis, grade 3 thrombocytopenia and grade 4 thrombocytopenia. Common treatment-related adverse events included anaemia, neutropenia, thrombocytopenia and transaminitis. Pharmacodynamic analyses demonstrated mTOR inhibition with sirolimus 5 mg and PK showed no influence of sirolimus concentrations on gemcitabine clearance. In vitro and in vivo studies suggested mTOR pathway hyperactivation by gemcitabine that was reversed by sirolimus. Tumour growth in leiomyosarcoma xenografts was dramatically inhibited by the treatment.Conclusions:Recommended dose was sirolimus 5 mg per 24 h plus gemcitabine 800 mg m−2. Antitumour activity in preclinical sarcoma models and mTOR signalling inhibition were observed. A phase II study is currently ongoing. [ABSTRACT FROM AUTHOR]

Background:CD133 and CD44 are putative cancer stem cell (CSC) markers in colorectal cancer (CRC). However, their clinical significance is currently unclear. Here, we evaluated primary CRC cell isolates to determine the significance of several CSC markers, including CD133 and CD44, as predictors of tumourigenesis and prognosis.Methods:CD133- and CD44-positive cells from fresh clinical samples of 77 CRCs were selected by flow cytometric sorting and evaluated for tumourigenicity following subcutaneous transplantation into NOD/SCID mice. Cancer stem cell marker expression was examined in both xenografts and a complementary DNA library compiled from 167 CRC patient samples.Results:CD44+, CD133+ and CD133+CD44+ sub-populations were significantly more tumourigenic than the total cell population. The clinical samples expressed several transcript variants of CD44. Variant 2 was specifically overexpressed in both primary tumours and xenografts in comparison with the normal mucosa. A prognostic assay using qRT-PCR showed that the CD44v2high group (n=84, 5-year survival rate (5-OS): 0.74) had a significantly worse prognosis (P=0.041) than the CD44v2low group (n=83, 5-OS: 0.88).Conclusions:CD44 is an important CSC marker in CRC patients. Furthermore, CRC patients with high expression of CD44v2 have a poorer prognosis than patients with other CD44 variants. [ABSTRACT FROM AUTHOR]

Background:We investigated the biologic and pharmacologic activities of a chromosome region maintenance 1 (CRM1) inhibitor against human non-small cell lung cancer (NSCLC) cells both in vitro and in vivo.Methods:The in vitro and in vivo effects of a novel CRM1 inhibitor (KPT-330) for a large number of anticancer parameters were evaluated using a large panel of 11 NSCLC cell lines containing different key driver mutations. Mice bearing human NSCLC xenografts were treated with KPT-330, and tumour growth was assessed.Results:KPT-330 inhibited proliferation and induced cell cycle arrest and apoptosis-related proteins in 11 NSCLC cells lines. Moreover, the combination of KPT-330 with cisplatin synergistically enhanced the cell kill of the NSCLC cells in vitro. Human NSCLC tumours growing in immunodeficient mice were markedly inhibited by KPT-330. Also, KPT-330 was effective even against NSCLC cells with a transforming mutation of either exon 20 of EGFR, TP53, phosphatase and tensin homologue, RAS or PIK3CA, suggesting the drug might be effective against a variety of lung cancers irrespective of their driver mutation.Conclusions:Our results support clinical testing of KPT-330 as a novel therapeutic strategy for NSCLC. [ABSTRACT FROM AUTHOR]

Background:Dichloroacetate (DCA) has been found to have antitumour properties.Methods:We investigated the cellular and metabolic responses to DCA treatment and recovery in human colorectal (HT29, HCT116 WT and HCT116 Bax-ko), prostate carcinoma cells (PC3) and HT29 xenografts by flow cytometry, western blotting, electron microscopy, 1H and hyperpolarised 13C-magnetic resonance spectroscopy.Results:Increased expression of the autophagy markers LC3B II was observed following DCA treatment both in vitro and in vivo. We observed increased production of reactive oxygen species (ROS) and mTOR inhibition (decreased pS6 ribosomal protein and p4E-BP1 expression) as well as increased expression of MCT1 following DCA treatment. Steady-state lactate excretion and the apparent hyperpolarised [1-13C] pyruvate-to-lactate exchange rate (kPL) were decreased in DCA-treated cells, along with increased NAD+/NADH ratios and NAD+. Steady-state lactate excretion and kPL returned to, or exceeded, control levels in cells recovered from DCA treatment, accompanied by increased NAD+ and NADH. Reduced kPL with DCA treatment was found in HT29 tumour xenografts in vivo.Conclusions:DCA induces autophagy in cancer cells accompanied by ROS production and mTOR inhibition, reduced lactate excretion, reduced kPL and increased NAD+/NADH ratio. The observed cellular and metabolic changes recover on cessation of treatment. [ABSTRACT FROM AUTHOR]

Background:The NUT midline carcinoma (NMC) is a rare but fatal cancer for which systematic testing of therapy options has never been performed.Methods:On the basis of disease biology, we compared the efficacy of the CDK9 inhibitor flavopiridol (FP) with a panel of anticancer agents in NMC cell lines and mouse xenografts.Results:In vitro anthracyclines, topoisomerase inhibitors, and microtubule poisons were among the most cytotoxic drug classes for NMC cells, while efficacy of the bromodomain inhibitor JQ1 varied considerably between lines carrying different BRD4 (bromodomain-containing protein 4)-NUT (nuclear protein in testis) translocations. Efficacy of FP was comparable to vincristine and doxorubicin, drugs that have been previously used in NMC patients. All three compounds showed significantly better activity than etoposide and vorinostat, agents that have also been used in NMC patients. Statins and antimetabolites demonstrated intermediate single-agent efficacy. In vivo, vincristine significantly inhibited tumour growth in two different NMC xenografts. Flavopiridol in vivo was significantly effective in one of the two NMC xenograft lines, demonstrating the biological heterogeneity of this disease.Conclusions:These results demonstrate that FP may be of benefit to a subset of patients with NMC, and warrant a continued emphasis on microtubule inhibitors, anthracyclines, and topoisomerase inhibitors as effective drug classes in this disease. [ABSTRACT FROM AUTHOR]

Background:Hypoxia can activate autophagy, a self-digest adaptive process that maintains cell turnover. Mammalian target of rapamycin (mTOR) inhibitors are used to treat cancer but also stimulate autophagy.Methods:Human mammary cancer cells and derived xenografts were used to examine whether hypoxia could exacerbate autophagy-mediated resistance to the mTOR inhibitor rapamycin.Results:Rapamycin exerted potent antitumour effects in MCF-7 and MDA-MB-231 mammary tumours through a marked inhibition of angiogenesis, but the autophagy inhibitor chloroquine (CQ) failed to further sensitise tumours to mTOR inhibition. Rapamycin treatment actually led to tumour reoxygenation, thereby preventing the development of autophagy. Chloroquine alone, however, blocked the growth of MCF-7 tumours and in vitro blunted the hypoxia-induced component of autophagy in these cells. Finally, when initiating CQ treatment in large, hypoxic tumours, a robust antitumour effect could be observed, which also further increased the antiproliferative effects of rapamycin.Conclusion:The mTOR inhibitor rapamycin significantly contributes to tumour growth inhibition and normalisation of the tumour vasculature through potent antiangiogenic effects. The resulting reduction in hypoxia accounts for a lack of sensitisation by the autophagy inhibitor CQ, except if the tumours are already at an advanced stage, and thus largely hypoxic at the initiation of the combination of rapamycin and CQ treatment. [ABSTRACT FROM AUTHOR]

Background:Antibody drug conjugates (ADCs) and immunotoxins (ITs) are promising anticancer immunotherapeutics. Despite their encouraging performance in clinical trials, both ADCs and ITs often suffer from disadvantages such as stoichiometrically undefined chemical linkage of the cytotoxic payload (ADCs) and the potential immunogenicity of toxins derived from bacteria and plants (ITs).Methods:Human microtubule-associated protein tau (MAP) was cloned in-frame with human EGF, expressed in E. coli and purified by standard chromatographic methods. The in vitro activity was confirmed by flow cytometry, cell viability assays and tubulin polymerisation assay. The in vivo efficacy was demonstrated using noninvasive far-red in vivo imaging.Results:The EGF-MAP selectively induced apoptosis in EGFR-overexpressing proliferating cancer cells through stabilisation of microtubules. Nonproliferating cells were not affected, demonstrating superior selectivity of EGF-MAP for cancer cells. The EGF-MAP was well tolerated at high doses in mice compared with the ETA'-based control. The in vivo efficacy of EGF-MAP was demonstrated in a tumour xenograft mouse model.Conclusion:Our data indicate the general mechanism of action for a new class of human immunotherapeutic reagents suitable for the treatment of cancer. This approach combines the binding specificity of targeting ligands with the selective cytotoxicity of MAP towards proliferating cells. [ABSTRACT FROM AUTHOR]

Objective:This study aims to investigate the using of bone marrow mesenchymal stem cells (BMSCs) genetically engineered to produce interferon-β (IFN-β) as a gene delivery system to treat hepatocellular carcinoma (HCC) in vitro and in vivo.Methods:To measure the effects on tumour cell growth in vitro, IFN-β-producing BMSCs (BMSC/IFN-β) were co-cultured with the HCC cell line HepG2 and Huh7. Enzyme-linked immunosorbent assay (ELISA) was used to detect the IFN-β secretion in the BMSC culture condition medium (CM). The effect of BMSC/IFN-β on HCC cells proliferation was examined both in vitro and in vivo by using MTT, colony formation assay, BrdU staining, cell cycle analysis, and xenografted NOD/SCID mouse tumour model. To examine the impact of BMSC/IFN-β on the AKT/FOXO3a signalling, RT-PCR and western blotting were performed.Results:The BMSC/IFN-β cells can stably secrete high levels of IFN-β. Both MTT and colony forming assay showed that HCC cells had a lower growth rate when cultured in BMSC/IFN-β-CM as compared with that in BMSC/vector-CM or DMEM culture group. Co-culture with BMSC/IFN-β-CM dramatically decreased the percentages of cells with incorporated BrdUrd. In BMSC/IFN-β-CM-treated HCC cells, the proportion of G1-phase cells increased but it decreased in the S phase of the cell. The BMSC/IFN-β inhibited HCC growth in NOD/SCID mice and proved the survival period of these mice. Compared with the control group, p21 and p27 expression of hepatoma cells increased, whereas cyclin D1 and phosphorylation of Rb expression decreased when co-cultured with BMSC/IFN-β-CM. It was associated with suppression of Akt activity and enhanced transcriptional activity of FOXO3a.Conclusion:The IFN-β gene-modified BMSCs can effectively inhibit the proliferation of HCC cells in vitro and in vivo through inhibiting AKT/FOXO3a pathway. These results indicate that BMSC/IFN-β are a powerful anticancer cytotherapeutic tool for HCC. [ABSTRACT FROM AUTHOR]

Background:Sorafenib is a potent inhibitor against Raf kinase and several receptor tyrosine kinases that has been approved for the clinical treatment of advanced renal and liver cancer. Combining sorafenib with other agents has been shown to improve its antitumour efficacy by not only reducing the toxic side effects but also preventing primary and acquired resistance to sorafenib. We have previously observed that tetrandrine exhibits potent antitumour effects in human hepatocellular carcinoma. In this study, we investigated the synergistic antitumour activity of sorafenib in combination with tetrandrine.Methods:This was a two-part investigation that included the in vitro effects of sorafenib in combination with tetrandrine on cancer cells and the in vivo antitumour efficacy of this drug combination on tumour xenografts in nude mice.Results:Combined treatment showed a good synergistic antitumour effect yet spared nontumourigenic cells. The potential molecular mechanism may be mainly that it activated mitochondrial death pathway and induced caspase-dependent apoptosis in the cancer cells. Accumulation of intracellular reactive oxygen species (ROS) and subsequent activation of Akt may also be involved in apoptosis induction.Conclusion:The antitumour activity of sorafenib plus tetrandrine may be attributed to the induction of the intrinsic apoptosis pathway through ROS/Akt signaling. This finding provides a novel approach that may broaden the clinical application of sorafenib. [ABSTRACT FROM AUTHOR]

Background:Bcl-xL has an important role in the control of cell death through its inhibition of apoptosis. The aim of this study was to investigate the clinicopathological significance of Bcl-xL in upper urinary tract urothelial carcinoma (UTUC) and the therapeutic effect of targeting Bcl-xL protein in urothelial carcinoma (UC) cells.Methods:We evaluated the immunohistochemical expression of Bcl-xL in 175 UTUC patients to determine the clinical role of Bcl-xL expression in clinical outcome. We used bafilomycin A1 (BMA) as a specific inhibitor of Bcl-xL to examine the biological effects in UC cells in vitro and in vivo.Results:Immunohistochemical analysis of Bcl-xL expression revealed that patients with a high Bcl-xL score had a significantly lower 5-year cancer-specific survival (CSS) rate (53.2%) than those with a low Bcl-xL score (77.2%) (P=0.0011). Multivariate analysis indicated that a high Bcl-xL score was an independent prognostic factor of CSS (P=0.023). BMA inhibited UMUC-3 cell proliferation in vitro by induction of apoptosis. Treatment with BMA significantly inhibited tumour growth in UMUC-3 tumours in this mouse xenograft model accompanied by an elevated apoptosis induction.Conclusion:Bcl-xL appears to be a significant molecular marker for the prognosis of UTUCs. Targeting Bcl-xL may be a promising therapeutic strategy for patients with UC. [ABSTRACT FROM AUTHOR]

Background:Ex vivo colospheres have been previously characterised as a colorectal cancer (CRC) well-rounded multicellular model, exclusively formed by carcinoma cells, and derived from fresh CRC tissue after mechanical dissociation. The ability to form colospheres was correlated with tumour aggressiveness. Their three-dimensional conformation prompted us to further investigate their potential interest as a preclinical cancer tool.Methods:Patient-derived CRC xenografts were used to produce numerous colospheres. Mechanism of formation was elucidated by confocal microscopy. Expression analysis of a panel of 64 selected cancer-related genes by real-time qRT-PCR and hierarchical clustering allowed comparison of colospheres with parent xenografts. In vitro and in vivo assays were performed for migration and chemosensitivity studies.Results:Colospheres, formed by tissue remodelling and compaction, remained viable several weeks in floating conditions, escaping anoikis through their strong cell-cell interactions. Colospheres matched the gene expression profile of the parent xenograft tissue. Colosphere-forming cells migrated in collagen I matrix and metastasised when subrenally implanted in nude mice. Besides, the colosphere responses to 5-fluorouracil and irinotecan, two standard drugs in CRC, reproduced those of the in vivo original xenografts.Conclusion:Colospheres closely mimic biological characteristics of in vivo CRC tumours. Consequently, they would be relevant ex vivo CRC models. [ABSTRACT FROM AUTHOR]

Background:High-grade astrocytomas are malignant brain tumours that infiltrate the surrounding brain tissue and have a poor prognosis. Activation of formyl peptide receptor (FPR1) on the human astrocytoma cell line U87 promotes cell motility, growth and angiogenesis. We therefore investigated the FPR1 inhibitor, Chemotaxis Inhibitory Protein of S. aureus (CHIPS), as a potential anti-astrocytoma drug.Methods and results:FPR1 expression was studied immunohistochemically in astrocytomas WHO grades I-IV. With intracellular calcium mobilisation and migration assays, human ligands were tested for their ability to activate FPR1 on U87 cells and on a cell line derived from primary astrocytoma grade IV patient material. Thereafter, we selectively inhibited these ligand-induced responses of FPR1 with an anti-inflammatory compound called Chemotaxis Inhibitory Protein of S. aureus (CHIPS). U87 xenografts in NOD-SCID mice served to investigate the effects of CHIPS in vivo. FPR1 was expressed in 29 out of 32 (90%) of all grades of astrocytomas. Two human mitochondrial-derived formylated peptides, formyl-methionil-leucine-lysine-isoleucine-valine (fMLKLIV) and formyl-methionil-methionil-tyrosine-alanine-leucine-phenylalanine (fMMYALF), were potent activators of FPR1 on tumour cells. Ligand-induced responses of FPR1-expressing tumour cells could be inhibited with FPR1 inhibitor CHIPS. Treatment of tumour-bearing mice with CHIPS slightly reduced tumour growth and improved survival as compared to non-treated animals (P=0.0019).Conclusion:Targeting FPR1 with CHIPS reduces cell motility and tumour cell activation, and prolongs the survival of tumour-bearing mice. This strategy could be explored in future research to improve treatment results for astrocytoma patients. [ABSTRACT FROM AUTHOR]

Background:Sorafenib has recently been shown to reduce tumour growth in hepatoblastoma (HB) xenografts. The effect of a combined administration with cytostatic agents was now investigated.Methods:Cell viability after treatment with sorafenib and different cytostatic agents was evaluated in two HB cell lines (HUH6 and HepT1) using MTT assay. ERK signalling was investigated by western blot, NOXA expression by rt-PCR, and formation of DNA adducts using immunocytology. NMRI mice bearing subcutaneous HUH6-derived tumours were treated with sorafenib alone or in combination with cisplatin. Tumour progression, viability, apoptosis, and vascularisation were monitored by tumour volume, AFP levels, TUNEL assay, and CD31 immunostaining, respectively.Results:The combination of sorafenib and cisplatin led to a remarkable decrease in cell viability. The cisplatin-induced enhanced ERK1/2 activation, but not NOXA expression and the formation of DNA adducts was partly abrogated by sorafenib. In HB xenografts, both, sorafenib and alternated application of sorafenib and cisplatin significantly reduced tumour growth (P<0.05). Levels of AFP were lower in both treated groups (P=0.08). Relative apoptotic areas were increased (P=0.003). Mean vascular density was the lowest in the sorafenib/CDDP group (P=0.02).Conclusion:The combination of sorafenib with cisplatin might be a promising treatment option for high risk or recurrent HB. [ABSTRACT FROM AUTHOR]

Background:Sunitinib (Su), a tyrosine kinase inhibitor of VEGFR, is effective at producing tumour response in clear cell renal cell carcinoma (cRCC), but resistance to therapy is inevitable. As COX-2 is a known mediator of tumour growth, we explored the potential benefit of COX-2 inhibition in combination with VEGFR inhibition in attempts at delaying tumour progression on Su.Methods:COX-2 expression was compared with areas of hypoxia in tumours that progressed on Su vs untreated tumours. Mice bearing human cRCC xenografts were treated with Su and the COX-2 inhibitor, celecoxib, and the effects on tumour growth were assessed. Sequential vs concurrent regimens were compared.Results:COX-2 expression was increased in cRCC xenografts in areas of tumour hypoxia. The combination of Su and celecoxib achieved longer times to tumour progression compared to treatment with either agent alone or to untreated control animals in four models. This effect was seen with concurrent but not with sequential therapy.Conclusion:COX-2 inhibition can extend the effectiveness of VEGFR inhibition. This effect is dependent on the timing of therapy. Clinical trials combining Su and COX-2 inhibitors should be considered as a means delaying time to progression on sunitinib in patients with metastatic cRCC. [ABSTRACT FROM AUTHOR]