BACKGROUND & AIMS:Patients with cholangiocarcinoma (CCA) have poor prognosis. Current cisplatin-based first-line chemotherapy offers limited survival benefit. Cisplatin induces single-strand DNA breaks, activating DNA repair mechanisms that diminish its effectiveness. Here, we present the design, chemical synthesis, and therapeutic evaluation of a new generation of chemotherapeutic agents (Aurkines) with unique polyelectrophilic properties. These agents cause a high frequency of double-strand DNA breaks, bypassing DNA repair, and promoting cancer cell death. METHODS:Two novel compounds, Aurkine 16 and Aurkine 18, were designed and evaluated for their antitumor effects in both naïve and cisplatin-resistant CCA cells, cancer-associated fibroblasts, healthy cholangiocytes, and in vivo models. RESULTS:Aurkines effectively induced double-strand DNA breaks, leading to increased DNA damage and elevated levels of reactive oxygen species, resulting in greater cytotoxicity than cisplatin in CCA cells. Phosphoproteomic and molecular analysis revealed that cisplatin activates DNA repair pathways, while Aurkines primarily induce apoptosis. Importantly, Aurkines also triggered apoptosis in cisplatin-resistant CCA cells and cancer-associated fibroblasts without harming healthy cholangiocytes. Additionally, Aurkines demonstrated cytotoxicity in other cisplatin-resistant cancers, such as breast and ovarian cancer. This tumor selectivity results from reduced uptake, increased efflux, and compact chromatin structure in normal cells, limiting Aurkine-DNA interactions. In vivo, Aurkines inhibited the growth of subcutaneous naïve and cisplatin-resistant CCA tumors, as well as orthotopic tumors in immunocompetent mice, promoting antitumor immune cell recruitment without any adverse events. Transport studies revealed that Aurkines were selectively taken up by OCT1, OCT3, CTR1, and OATP1A2, whereas only CTR1 transported cisplatin. CONCLUSIONS:Aurkines represent promising therapeutic drugs for both naïve and cisplatin-resistant cancers due to their unique polyelectrophilic properties and selective targeting of malignant cells. IMPACT AND IMPLICATIONS:This study introduces a novel therapeutic strategy designed to induce frequent double-strand DNA breaks selectively in both naïve and cisplatin-resistant cancer cells, without evident toxic side effects at therapeutic doses. This approach may form the basis for new strategies to overcome the critical challenge of drug resistance in cancer treatment and has the potential to be a breakthrough not only for the treatment of biliary tumors but also for other cancers.

Objective Cytotoxic agents are the cornerstone of treatment for patients with advanced intrahepatic cholangiocarcinoma (iCCA), despite heterogeneous benefit. We hypothesised that the pretreatment molecular profiles of diagnostic biopsies can predict patient benefit from chemotherapy and define molecular bases of innate chemoresistance. Design We identified a cohort of advanced iCCA patients with comparable baseline characteristics who diverged as extreme outliers on chemotherapy (survival <6 m in rapid progressors, RP; survival >23 m in long survivors, LS). Diagnostic biopsies were characterised by digital pathology, then subjected to whole-transcriptome profiling of bulk and geospatially macrodissected tissue regions. Spatial transcriptomics of tumour-infiltrating myeloid cells was performed using targeted digital spatial profiling (GeoMx). Transcriptome signatures were evaluated in multiple cohorts of resected cancers. Signatures were also characterised using in vitro cell lines, in vivo mouse models and single cell RNA-sequencing data. Results Pretreatment transcriptome profiles differentiated patients who would become RPs or LSs on chemotherapy. Biologically, this signature originated from altered tumour-myeloid dynamics, implicating tumour-induced immune tolerogenicity with poor response to chemotherapy. The central role of the liver microenviroment was confrmed by the association of the RPLS transcriptome signature with clinical outcome in iCCA but not extrahepatic CCA, and in liver metastasis from colorectal cancer, but not in the matched primary bowel tumours. Conclusions The RPLS signature could be a novel metric of chemotherapy outcome in iCCA. Further development and validation of this transcriptomic signature is warranted to develop precision chemotherapy strategies in these settings.

Cholangiocarcinomas (CCAs) are aggressive tumors arising along the biliary tract epithelium, whose incidence and mortality are increasing. CCAs are highly desmoplastic cancers characterized by a dense tumor microenvironment (TME), in which each single component plays a fundamental role in shaping CCA initiation, progression and resistance to therapies. The crosstalk between cancer cells and TME can affect the recruitment, infiltration and differentiation of immune cells. According to the stage of the disease and to intra- and inter-patient heterogeneity, TME may contribute to either protumoral or antitumoral activities. Therefore, a better understanding of the effect of each immune cell subtype may open the path to new personalized immune therapeutic strategies for the management of CCA. In this review, we describe the role of immune cells in CCA initiation and progression, and their crosstalk with both cancer-associated fibroblasts (CAFs) and the cancer-stem-cell-like (CSC) niche.

Targeting the human epidermal growth factor receptor 2 (HER2) has emerged as a promising therapeutic strategy for advanced biliary tract cancer (BTC). Javle et al. have recently reported a sub-analysis of the MyPathway trial on the efficacy of dual HER2 inhibition with pertuzumab plus trastuzumab in advanced BTC with ERBB2 amplification. After a median follow up of 8.1 months, 23% of patients achieved partial response, with a median duration of response of 10.8 months, median progression-free survival (PFS) of 4.0 months, and median overall survival of 10.9 months.[1]Javle M. Borad M.J. Azad N.S. Kurzrock R. Abou-Alfa G.K. George B. et al.Pertuzumab and trastuzumab for HER2-positive, metastatic biliary tract cancer (MyPathway): a multicentre, open-label, phase 2a, multiple basket study.Lancet Oncol. 2021; 22: 1290-1300Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar Whilst promising, these results indicate that resistance to anti-HER2 agents is still a significant challenge. Several molecular mechanisms causing resistance to anti-HER2 blockade have been described in breast cancer,[2]Rimawi M.F. De Angelis C. Schiff R. Resistance to anti-HER2 therapies in breast cancer.Am Soc Clin Oncol Educ Book. 2015; : e157-e164Crossref PubMed Scopus (16) Google Scholar while in BTC they are still poorly understood as the investigation of this therapeutic strategy is still in its infancy.[1]Javle M. Borad M.J. Azad N.S. Kurzrock R. Abou-Alfa G.K. George B. et al.Pertuzumab and trastuzumab for HER2-positive, metastatic biliary tract cancer (MyPathway): a multicentre, open-label, phase 2a, multiple basket study.Lancet Oncol. 2021; 22: 1290-1300Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar,3Javle M. Churi C. Kang H.C. Shroff R. Janku F. Surapaneni R. et al.HER2/neu-directed therapy for biliary tract cancer.J Hematol Oncol. 2015; Crossref PubMed Scopus (146) Google Scholar, 4Sorscher S. Marked radiographic response of a HER-2-overexpressing biliary cancer to trastuzumab.Cancer Manag Res. 2013; 9: 1-3Crossref PubMed Scopus (29) Google Scholar, 5Nam A.R. Kim J.W. Cha Y. Ha H. Park J.E. Bang J.H. et al.Therapeutic implication of HER2 in advanced biliary tract cancer.Oncotarget. 2016; 7: 58007-58021Crossref PubMed Scopus (40) Google Scholar, 6Hyman D.M. Piha-Paul S.A. Won H. Rodon J. Saura C. Shapiro G.I. et al.HER kinase inhibition in patients with HER2- and HER3-mutant cancers.Nature. 2018; 554: 189-194Crossref PubMed Scopus (401) Google Scholar Herein, we report the clinical course and the integrated molecular analysis of tissue and longitudinal liquid biopsies in a patient with co-amplified ERBB2 and EGFR gallbladder cancer (GBC) treated with concurrent HER2 and EGFR inhibitors. Our data suggest that EGFR copy number gain might represent a mechanism of resistance to trastuzumab in GBC, providing useful insights for the application of HER2 inhibitors in clinical practice. A 58-year-old female patient was diagnosed with a stage IV (CK7+, CK19+) GBC with multiple liver metastases in February 2019 (Fig. 1A). After progression on first-line cisplatin+gemcitabine (May 2019), she received a targeted inhibitor of thymidylate synthase within a phase I trial, which was withdrawn because of hepatotoxicity. Third-line FOLFOX chemotherapy was administered between September 2019 and March 2020, when due to stabilization of disease and onset of the COVID-19 pandemic she was switched to active surveillance. At progression (August 2020) the patient was re-challenged with FOLFOX, but treatment was halted 2 months later due to radiological progression. She was then considered for an off-licence targeted therapy based on the next-generation sequencing (NGS) results of her diagnostic liver biopsy [FoundationOne cDx 324 gene panel], which documented double amplification of ERBB2 and EGFR, along with SMAD and TP53 mutations. In view of the double ERBB2-EGFR amplification, dual blockade with trastuzumab (HER2-directed antibody; loading dose 8 mg/kg followed by 6mg/kg q21) and lapatinib (reversible EGFR/HER2 kinase inhibitor; 1,250 mg/day continuously) was initiated in November 2020. Monthly circulating tumour DNA (ctDNA) and tumour markers were used to monitor response (Fig. 1B,C). Pre-treatment ctDNA analysis [Avenio ctDNA extended 77 gene assay] showed concordance with tissue data confirming the presence of EGFR and ERBB2 amplifications (copy number variation [CNV] scores 26.4 and 14.2, respectively) and p.Arg361His SMAD4 and p.Tyr220Cys TP53 mutations (variant allele frequency [VAF] 48.12% and 53.46%, respectively) (Fig. 1D). Liquid biopsy analysis after a month of treatment documented significant reduction in tumour markers and ctDNA load with normalisation of SMAD4 and TP53 mutations (VAF 0.32% and 0.36%, respectively) and undetectability of ERBB2 and EGFR CNV (Fig. 1C,E). Lapatinib was discontinued 2.1 months later because of grade 4 diarrhoea. As soon as EGFR inhibition was halted, tumour markers and ctDNA showed a steep increase. Trastuzumab was continued as a single agent until March 2021, when it was interrupted due to biochemical and radiological progression (progression-free survival 4.1 months). ctDNA at progression showed SMAD4 and TP53 mutations (VAF 54% and 63%, respectively) along with ERBB2 and EGFR amplifications (CNV scores 26.62 and 14.6, respectively). Of note, de novo mutations arose at 2.5 months (p.Glu545Lys PIK3CA) and 3.73 months (p.Val266Leu PDGFRA and p.Ile673Ile EGFR) (Fig. 1E). The patient died in June 2021. We report the clinical and molecular evolution of a patient whose GBC harbored a co-amplification of ERBB2 and EGFR: dual ERBB2 and EGFR inhibition was initially associated with clinical, biochemical and ctDNA signs of response. As soon as EGFR inhibition was lifted due to ongoing toxicity from the tyrosine-kinase inhibitor, the patient showed disease progression. These observations suggest two, non-mutually exclusive hypotheses. First, EGFR copy number gain might have been the driver event of progression in this patient, an assumption supported by the observation that EGFR-amplified cancers have a significantly poorer prognosis than ERBB2-amplified cancers in a pan-cancer analysis (Fig. S1). Second, similarly to other gastrointestinal cancers treated with BRAF, KRAS G12C and multi-tyrosine kinase inhibitors,7Prahallad A. Sun C. Huang S. Di Nicolantonio F. Salazar R. Zecchin D. et al.Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR.Nature. 2012; 483: 100-103Crossref PubMed Scopus (1423) Google Scholar, 8Amodio V. Yaeger R. Arcella P. Cancelliere C. Lamba S. Lorenzato A. et al.EGFR blockade reverts resistance to KRAS(G12C) inhibition in colorectal cancer.Cancer Discov. 2020; 10: 1129-1139Crossref PubMed Scopus (110) Google Scholar, 9Jin H. Shi Y. Lv Y. Yuan S. Ramirez C.F.A. Lieftink C. et al.EGFR activation limits the response of liver cancer to lenvatinib.Nature. 2021; 595: 730-734Crossref PubMed Scopus (41) Google Scholar EGFR signaling, especially in the context of an EGFR-amplification, might have represented a mechanism of bypass or rebound upon HER2 inhibition. Although co-amplification of receptor tyrosine kinases is relatively infrequent in BTC, our data suggest that testing for the presence of other receptor tyrosine kinase amplifications might be a sound approach to identify mechanisms of resistance and optimize combinatorial therapeutic strategies such as the use of a pan-HER inhibitor like afatinib in cases with EGFR and ERBB2 co-amplifications. Under these assumptions, as HER2 inhibition becomes a widely used therapeutic approach in BTC, single testing with HER2 fluorescence in situ hybridization appears to fall short of providing a comprehensive assessment of the molecular architecture of BTC – broader genomic analyses should be considered for more robust patient screening and selection. Finally, in our patient, we observed emergence of de novo mutations in PDGRA and PIK3CA upon progression, which have previously been associated with resistance to lapatinib[10]Siravegna G. Lazzari L. Crisafulli G. Sartore-Bianchi A. Mussolin B. Cassingena A. et al.Radiologic and genomic evolution of individual metastases during HER2 blockade in colorectal cancer.Cancer Cell. 2018; 34: 148-162 e147Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar in colon cancer, underlining the paramount importance of ctDNA in identifying sequential adaptive therapies in this group of patients. This study has been supported by the Lord Kelvin Adam Smith readership ( University of Glasgow ), the ISSF Excellence and Innovation Catalyst award ( Wellcome Trust ), and the COST-ACTION-18122 ( Euro-Cholangio-Net ). RC: collection of data and draft of the manuscript; FA: collection of data and draft of the manuscript; CR: collection of data; SP: collection of data; CB: ideation and design of project, analysis, revision of manuscript. CB receives honoraria from Incyte and Servier. Please refer to the accompanying ICMJE disclosure forms for further details. Collaborators: Tamsin Nash2,3, Martin MacLeod2,3, Paula Sanchon-Sanchez1,4, Patricia Roxburgh1,2,3, Jeff Evans1,2,3, Janet Graham2,3, Fraser Duthie2, Nicola Valeri5, Pawel Herzyk6,7 Julie Galbraith6,7 1Institute of Cancer Science, University of Glasgow, UK 2NHS Greater Glasgow and Clyde, Glasgow, UK 3Beatson West of Scotland Cancer Centre, Glasgow, UK 4Experimental Hepatology and Drug Targeting (HEVEPHARM), University of Salamanca, Institute for Biomedical Research of Salamanca (IBSAL), Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, ES 5Division of Surgery and Cancer, Imperial College London, UK 6Institute of Molecular Cell & Systems Biology, University of Glasgow, UK 7Glasgow Polyomics, University of Glasgow, UK The following are the supplementary data to this article: Download .pdf (1.74 MB) Help with pdf files Multimedia component 1 Download .pdf (.49 MB) Help with pdf files Multimedia component 2

Metabolic reprogramming is a hallmark of cancer and allows tumour cells to meet the increased energy demands required for rapid proliferation, invasion, and metastasis. Indeed, many tumour cells acquire distinctive metabolic and bioenergetic features that enable them to survive in resource-limited conditions, mainly by harnessing alternative nutrients. Several recent studies have explored the metabolic plasticity of cancer cells with the aim of identifying new druggable targets, while therapeutic strategies to limit the access to nutrients have been successfully applied to the treatment of some tumours. Cholangiocarcinoma (CCA), a highly heterogeneous tumour, is the second most common form of primary liver cancer. It is characterised by resistance to chemotherapy and poor prognosis, with 5-year survival rates of below 20%. Deregulation of metabolic pathways have been described during the onset and progression of CCA. Increased aerobic glycolysis and glutamine anaplerosis provide CCA cells with the ability to generate biosynthetic intermediates. Other metabolic alterations involving carbohydrates, amino acids and lipids have been shown to sustain cancer cell growth and dissemination. In this review, we discuss the complex metabolic rewiring that occurs during CCA development and leads to unique nutrient addiction. The possible role of therapeutic interventions based on metabolic changes is also thoroughly discussed.

Background Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA. Methods Cholangiocarcinogenesis was induced in rats (TAA) and mice ( Jnk Δhepa + CCl 4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRAS G12D cells. Cell signaling, growth, gene regulation and [U- 13 C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model. Results Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRAS G12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRAS G12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRAS G12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression. Conclusions In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA.

In the search for the ideal model of tumours, the use of three-dimensional in vitro models is advancing rapidly. These are intended to mimic the in vivo properties of the tumours which affect cancer development, progression and drug sensitivity, and take into account cell–cell interactions, adhesion and invasiveness. Importantly, it is hoped that successful recapitulation of the structure and function of the tissue will predict patient response, permitting the development of personalized therapy in a timely manner applicable to the clinic. Furthermore, the use of co-culture systems will allow the role of the tumour microenvironment and tissue–tissue interactions to be taken into account and should lead to more accurate predictions of tumour development and responses to drugs. In this review, the relative merits and limitations of patient-derived organoids will be discussed compared to other in vitro and ex vivo cancer models. We will focus on their use as models for drug testing and personalized therapy and how these may be improved. Developments in technology will also be considered, including the use of microfluidics, 3D bioprinting, cryopreservation and circulating tumour cell-derived organoids. These have the potential to enhance the consistency, accessibility and availability of these models.

Background Neuroblastoma accounts for 7% of paediatric malignancies but is responsible for 15% of all childhood cancer deaths. Despite rigorous treatment involving chemotherapy, surgery, radiotherapy and immunotherapy, the 5-year overall survival rate of high-risk disease remains < 40%, highlighting the need for improved therapy. Since neuroblastoma cells exhibit aberrant metabolism, we determined whether their sensitivity to radiotherapy could be enhanced by drugs affecting cancer cell metabolism. Methods Using a panel of neuroblastoma and glioma cells, we determined the radiosensitising effects of inhibitors of glycolysis (2-DG) and mitochondrial function (metformin). Mechanisms underlying radiosensitisation were determined by metabolomic and bioenergetic profiling, flow cytometry and live cell imaging and by evaluating different treatment schedules. Results The radiosensitising effects of 2-DG were greatly enhanced by combination with the antidiabetic biguanide, metformin. Metabolomic analysis and cellular bioenergetic profiling revealed this combination to elicit severe disruption of key glycolytic and mitochondrial metabolites, causing significant reductions in ATP generation and enhancing radiosensitivity. Combination treatment induced G(2)/M arrest that persisted for at least 24 h post-irradiation, promoting apoptotic cell death in a large proportion of cells. Conclusion Our findings demonstrate that the radiosensitising effect of 2-DG was significantly enhanced by its combination with metformin. This clearly demonstrates that dual metabolic targeting has potential to improve clinical outcomes in children with high-risk neuroblastoma by overcoming radioresistance.

FOLFIRINOX, a combination of chemotherapy drugs (Fluorouracil, Oxaliplatin, Irinotecan -FOI), provides the best clinical benefit in pancreatic ductal adenocarcinoma (PDAC) patients. In this study we explore the role of miRNAs (MIR) as modulators of chemosensitivity to identify potential biomarkers of response. We find that 41 and 84 microRNA inhibitors enhance the sensitivity of Capan1 and MiaPaCa2 PDAC cells respectively. These include a MIR1307-inhibitor that we validate in further PDAC cell lines. Chemotherapy-induced apoptosis and DNA damage accumulation are higher in MIR1307 knock-out (MIR1307KO) versus control PDAC cells, while re-expression of MIR1307 in MIR1307KO cells rescues these effects. We identify binding of MIR1307 to CLIC5 mRNA through covalent ligation of endogenous Argonaute-bound RNAs cross-linking immunoprecipitation assay. We validate these findings in an in vivo model with MIR1307 disruption. In a pilot cohort of PDAC patients undergoing FOLFIRONX chemotherapy, circulating MIR1307 correlates with clinical outcome.

Prostate cancers, like many other types of cancer, express elevated levels of fatty acid synthase (FASN) to make more fatty acids, which are required for energy, signaling, and proliferation. Because inhibition of FASN has been shown to sensitize tumors to chemotherapy and radiation, we studied the effect of C75, a radiosensitizing FASN inhibitor, and compared its single agent and radiosensitizing activities in 2 prostate cancer cell lines, PC3 and LNCaP, with alternative FASN inhibitors that have progressed into clinical trials. We also investigated the effect of serum and fatty acid supplementation on responses to FASN inhibitors, probing expression of key proteins related to fatty acid uptake in response to FASN inhibition, irradiation, and serum lipid concentration and how this may be modulated to increase the potency of C75. We demonstrated that C75 was the only FASN inhibitor to sensitize cells to ionizing radiation; no sensitization was apparent with FASN inhibitors TVB-3166 or Orlistat. The prostate cancer cell lines were able to take up fatty acids from the culture medium, and the availability of fatty acids affected sensitivity of these cells to C75 but not the other FASN inhibitors tested. C75 also increased expression of fatty acid transporter proteins FATP1 and CD36. Furthermore, blocking CD36 with antibody increased the sensitivity of cells to C75. We suggest that the potency of C75 is affected by fatty acid availability and that the effectiveness of FASN inhibitors in combination with ionizing radiation can be further enhanced by regulating fatty acid uptake.

Cancer organoids are 3D phenotypic cultures that can be established from resected or biopsy tumour samples and can be grown as mini tumours in the dish. Flourishing evidence supports the feasibility of patient derived organoids (PDO) from a number of solid tumours. Evidence for cholangiocarcinoma (CCA) PDO is still sparse but growing. CCA PDO lines have been established from resected early stage disease, advanced cancers and highly chemorefractory tumours. Cancer PDO was shown to recapitulate the 3D morphology, genomic landscape and transcriptomic profile of the source counterpart. They proved to be a valued model for drug discovery and sensitivity testing, and they showed to mimic the drug response observed in vivo in the patients. However, PDO lack representation of the intratumour heterogeneity and the tumour-stroma interaction. The efficiency rate of CCA PDO within the three different subtypes, intrahepatic, perihilar and distal, is still to be explored. In this manuscript we will review evidence for CCA PDO highlighting advantages and limitations of this novel disease model.

Although radiotherapy is often used to treat localized disease and for palliative care in prostate cancer patients, novel methods are required to improve the sensitivity of aggressive disease to ionizing radiation. AMP-activated protein kinase (AMPK) is an energy sensor which regulates proliferation, aggressiveness and survival of cancer cells. We assessed the ability of the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) to sensitize prostate cancer cells to radiation. Prostate cancer cell lines LNCaP and PC3 were treated with X-rays and AICAR then assessed for clonogenic survival, spheroid growth delay, cell cycle progression, and AMPK and p53 activity. AICAR synergistically enhanced the clonogenic killing capacity, spheroid growth inhibition and pro-apoptotic effect of X-rays. The mechanism of radiosensitization appeared to involve cell cycle regulation, but not oxidative stress. Moreover, it was not dependent on p53 status. Treatment of PC3 cells with a fatty acid synthase inhibitor further enhanced clonogenic killing of the combination of X-rays and AICAR, whereas mTOR inhibition caused no additional enhancement. These results indicate that interference with metabolic signalling pathways which protect cells against irradiation have the potential to enhance radiotherapy. Activation of AMPK in combination with radiotherapy has the potential to target metabolically active and aggressive tumors which are currently untreatable.

Targeted radiotherapy of metastatic neuroblastoma using the somatostatin receptor (SSTR)-targeted octreotide analogue DOTATATE radiolabelled with lutetium-177 (177Lu-DOTATATE) is a promising strategy. This study evaluates whether its effectiveness may be enhanced by combination with radiosensitising drugs. The growth rate of multicellular tumour spheroids, derived from the neuroblastoma cell lines SK-N-BE(2c), CHLA-15 and CHLA-20, was evaluated following treatment with 177Lu-DOTATATE, nutlin-3 and topotecan alone or in combination. Immunoblotting, immunostaining and flow cytometric analyses were used to determine activation of p53 signalling and cell death. Exposure to 177Lu-DOTATATE resulted in a significant growth delay in CHLA-15 and CHLA-20 spheroids, but not in SK-N-BE(2c) spheroids. Nutlin-3 enhanced the spheroid growth delay induced by topotecan in CHLA-15 and CHLA-20 spheroids, but not in SK-N-BE(2c) spheroids. Importantly, the combination of nutlin-3 with topotecan enhanced the spheroid growth delay induced by X-irradiation or by exposure to 177Lu-DOTATATE. The efficacy of the combination treatments was p53-dependent. These results indicate that targeted radiotherapy of high risk neuroblastoma with 177Lu-DOTATATE may be improved by combination with the radiosensitising drugs nutlin-3 and topotecan.

Prostate cancer is the most common malignancy of men. Treatment options include radiotherapy with or without hormonal manipulation and radical prostatectomy. However, there is no effective treatment for disseminated disease. A hallmark of malignancy is abnormal metabolism which also confers survival advantages and contributes to resistance to therapy. In response to exposure to ionizing radiation, metabolic pathways are activated which can protect the cell from irreversible injury. Tumor cell glycolytic activity is elevated and correlates with aggressiveness and radio resistance, indicating that targeting glucose metabolism may sensitize cancer cells to radiation. We have demonstrated that the clonogenic kill of PC3 cells induced by exposure to x-rays was enhanced by the glycolytic inhibitor 2-deoxyglucose (2DG). In contrast, treatment with 2DG failed to inhibit growth of multicellular spheroids derived from LNCaP cells. However, 2DG treatment, in the absence of irradiation, induced similar toxicity to PC3 and LNCaP cells cultured as monolayers. Radiation-induced cell cycle arrest was prevented by the simultaneous administration of 2DG in both cell lines, indicating a possible mechanism underlying sensitization. Therefore, we hypothesise that observed differences in cellular response to incubation with 2DG in the presence or absence of ionizing radiation resulted from variation in metabolic processes between tumor cell types. We conclude that inhibition of glucose metabolism by 2DG is an effective method for sensitizing prostate cancer cells to experimental radiotherapy and that this may occur by preventing DNA repair during radiation-induced cell cycle arrest.

The elevated activity of fatty acid synthase has been reported in a number of cancer types. Inhibition of this enzyme has been demonstrated to induce cancer cell death and reduce tumor growth. In addition, the fatty acid synthase inhibitor drug C75 has been reported to synergistically enhance the cancer-killing ability of ionizing radiation. However, clinical use of C75 has been limited due to its producing weight loss, believed to be caused by alterations in the activity of carnitine palmitoyltransferase-1. C75 is administered in the form of a racemic mixture of (-) and (+) enantiomers that may differ in their regulation of fatty acid synthase and carnitine palmitoyltransferase-1. Therefore, we assessed the relative cancer-killing potency of different enantiomeric forms of C75 in prostate cancer cells. These results suggest that (-)-C75 is the more cytotoxic enantiomer and has greater radiosensitizing capacity than (+)-C75. These observations will stimulate the development of fatty acid synthase inhibitors that are selective for cancer cells and enhance the tumor-killing activity of ionizing radiation, while minimizing weight loss in cancer patients.