Francesca Trapani

Hepatic mTORC2 Activates Glycolysis and Lipogenesis through Akt, Glucokinase, and SREBP1c

Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates and activates AGC kinase family members, including Akt, SGK1, and PKC, in response to insulin/IGF1. The liver is a key organ in insulin-mediated regulation of metabolism. To assess the role of hepatic mTORC2, we generated liver-specific rictor knockout (LiRiKO) mice. Fed LiRiKO mice displayed loss of Akt Ser473 phosphorylation and reduced glucokinase and SREBP1c activity in the liver, leading to constitutive gluconeogenesis, and impaired glycolysis and lipogenesis, suggesting that the mTORC2-deficient liver is unable to sense satiety. These liver-specific defects resulted in systemic hyperglycemia, hyperinsulinemia, and hypolipidemia. Expression of constitutively active Akt2 in mTORC2-deficient hepatocytes restored both glucose flux and lipogenesis, whereas glucokinase overexpression rescued glucose flux but not lipogenesis. Thus, mTORC2 regulates hepatic glucose and lipid metabolism via insulin-induced Akt signaling to control whole-body metabolic homeostasis. These findings have implications for emerging drug therapies that target mTORC2.

Activity of the Monocarboxylate Transporter 1 Inhibitor AZD3965 in Small Cell Lung Cancer

Purpose: The monocarboxylate transporter 1 (MCT1) inhibitor, AZD3965, is undergoing phase I evaluation in the United Kingdom. AZD3965 is proposed, via lactate transport modulation, to kill tumor cells reliant on glycolysis. We investigated the therapeutic potential of AZD3965 in small cell lung cancer (SCLC) seeking rationale for clinical testing in this disease and putative predictive biomarkers for trial use. Experimental Design: AZD3965 sensitivity was determined for seven SCLC cell lines, in normoxia and hypoxia, and for a tumor xenograft model. Proof of mechanism was sought via changes in intracellular/tumor lactate. Expression of MCT1 and related transporter MCT4 was assessed by Western blot analysis. Drug resistance was investigated via MCT4 siRNAi and overexpression. The expression and clinical significance of MCT1 and MCT4 were explored in a tissue microarray (TMA) from 78 patients with SCLC. Results: AZD3965 sensitivity varied in vitro and was highest in hypoxia. Resistance in hypoxia was associated with increased MCT4 expression. In vivo, AZD3965 reduced tumor growth and increased intratumor lactate. In the TMA, high MCT1 expression was associated with worse prognosis (P = 0.014). MCT1 and hypoxia marker CA IX expression in the absence of MCT4 was observed in 21% of SCLC tumors. Conclusions: This study provides a rationale to test AZD3965 in patients with SCLC. Our results suggest that patients with tumors expressing MCT1 and lacking in MCT4 are most likely to respond. Clin Cancer Res; 20(4); 926–37. ©2013 AACR.

Hepatic mTORC1 controls locomotor activity, body temperature, and lipid metabolism through FGF21.

Significance The mammalian target of rapamycin complex 1 (mTORC1) controls cell growth and metabolism in response to nutrients, growth factors, and cellular energy. Aberrant mTORC1 signaling is implicated in human diseases such as diabetes, obesity, and cancer. Our results reveal that ectopic mTORC1 activation in the liver controls the stress hormone fibroblast growth factor 21 (FGF21) in a peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α)–dependent manner via glutamine depletion, which in turn affects whole-body behavior and metabolism. mTORC1 signaling correlates with FGF21 expression in human liver tumors, suggesting that our findings in mice may have physiological relevance in glutamine-addicted human cancers. Thus, treatment with the anticancer drug rapamycin may have beneficial effects by blocking tumor growth and by preventing deregulation of whole-body physiology due to FGF21 expression. The liver is a key metabolic organ that controls whole-body physiology in response to nutrient availability. Mammalian target of rapamycin (mTOR) is a nutrient-activated kinase and central controller of growth and metabolism that is negatively regulated by the tumor suppressor tuberous sclerosis complex 1 (TSC1). To investigate the role of hepatic mTOR complex 1 (mTORC1) in whole-body physiology, we generated liver-specific Tsc1 (L-Tsc1 KO) knockout mice. L-Tsc1 KO mice displayed reduced locomotor activity, body temperature, and hepatic triglyceride content in a rapamycin-sensitive manner. Ectopic activation of mTORC1 also caused depletion of hepatic and plasma glutamine, leading to peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α)–dependent fibroblast growth factor 21 (FGF21) expression in the liver. Injection of glutamine or knockdown of PGC-1α or FGF21 in the liver suppressed the behavioral and metabolic defects due to mTORC1 activation. Thus, mTORC1 in the liver controls whole-body physiology through PGC-1α and FGF21. Finally, mTORC1 signaling correlated with FGF21 expression in human liver tumors, suggesting that treatment of glutamine-addicted cancers with mTOR inhibitors might have beneficial effects at both the tumor and whole-body level.

CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer.

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.

A caspase-3 ?death-switch? in colorectal cancer cells for induced and synchronous tumor apoptosis in vitro and in vivo facilitates the development of minimally invasive cell death biomarkers

Novel anticancer drugs targeting key apoptosis regulators have been developed and are undergoing clinical trials. Pharmacodynamic biomarkers to define the optimum dose of drug that provokes tumor apoptosis are in demand; acquisition of longitudinal tumor biopsies is a significant challenge and minimally invasive biomarkers are required. Considering this, we have developed and validated a preclinical ‘death-switch’ model for the discovery of secreted biomarkers of tumour apoptosis using in vitro proteomics and in vivo evaluation of the novel imaging probe [18F]ML-10 for non-invasive detection of apoptosis using positron emission tomography (PET). The ‘death-switch’ is a constitutively active mutant caspase-3 that is robustly induced by doxycycline to drive synchronous apoptosis in human colorectal cancer cells in vitro or grown as tumor xenografts. Death-switch induction caused caspase-dependent apoptosis between 3 and 24 hours in vitro and regression of ‘death-switched’ xenografts occurred within 24 h correlating with the percentage of apoptotic cells in tumor and levels of an established cell death biomarker (cleaved cytokeratin-18) in the blood. We sought to define secreted biomarkers of tumor apoptosis from cultured cells using Discovery Isobaric Tag proteomics, which may provide candidates to validate in blood. Early after caspase-3 activation, levels of normally secreted proteins were decreased (e.g. Gelsolin and Midkine) and proteins including CD44 and High Mobility Group protein B1 (HMGB1) that were released into cell culture media in vitro were also identified in the bloodstream of mice bearing death-switched tumors. We also exemplify the utility of the death-switch model for the validation of apoptotic imaging probes using [18F]ML-10, a PET tracer currently in clinical trials. Results showed increased tracer uptake of [18F]ML-10 in tumours undergoing apoptosis, compared with matched tumour controls imaged in the same animal. Overall, the death-switch model represents a robust and versatile tool for the discovery and validation of apoptosis biomarkers.

Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study

An explant model derived from EpCam negative mesenchymal non-small-cell lung (NSCLC) cancer circulating tumour cells (a ‘liquid biopsy’) recapitulates the histology of the donor patients diagnostic specimen and chemoresistance to cisplatin and pemetrexed. This proof-of-principal landmark model opens a new avenue for study of advanced NSCLC biology when tissue biopsies unavailable.

Clinical and histopathological correlations of fecal calprotectin release in colorectal carcinoma

AIM To determine calprotectin release before and after colorectal cancer operation and compare it to tumor and histopathological parameters. METHODS The study was performed on patients with diagnosed colorectal cancer admitted for operation. Calprotectin was measured in a single stool sample before and three months after the operation using an enzyme-linked immunosorbent assay (ELISA). Calprotectin levels greater than or equal to 50 μg/g were considered positive. The compliance for collecting stool samples was assessed and the value of calprotectin was correlated to tumor and histopathological parameters of intra- and peri-tumoral inflammation. Surgical specimens were fixed in neutral buffered formalin and stained with hematoxylin and eosin. Staging was performed according to the Dukes classification system and the 7(th) edition tumor node metastasis classification system. Intra- and peri-tumoral inflammation was graded according to the Klintrup criteria. Immunohistochemical quantification was performed for MPO, CD45R0, TIA-1, CD3, CD4, CD8, CD57, and granzyme B. Statistical significance was measured using Wilcoxon signed rank test, Kruskal Wallis test and Spearmans rank correlation coefficient as appropriate. RESULTS Between March 2009 and May 2011, 80 patients with colorectal cancer (46 men and 34 women, with mean age of 71 ± 11.7 years old) were enrolled in the study. Twenty-six patients had rectal carcinoma, 29 had left-side tumors, 23 had right-side tumors, and 2 had bilateral carcinoma. In total, 71.2% of the patients had increased levels of calprotectin before the operation (median 205 μg/g, range 50-2405 μg/g) and experienced a significant decrease three months after the operation (46 μg/g, range 10-384 μg/g, P < 0001). The compliance for collecting stool samples was 89.5%. Patients with T3 and T4 tumors had significantly higher values than those with T1 and T2 cancers (P = 0.022). For all other tumor parameters (N, M, G, L, V, Pn) and location, no significant difference in calprotectin concentration was found. Furthermore, the calprotectin levels and histological grading of both peri- and intra-tumoral inflammation was not correlated. Additional testing with specific markers for lymphocytes and neutrophils also revealed no statistically significant correlation. CONCLUSION Fecal calprotectin decreases significantly after colorectal cancer operation. Its value depends exclusively on the individual T-stage, but not on other tumor or histopathological parameters.

HMGA1 Expression in Human Hepatocellular Carcinoma Correlates with Poor Prognosis and Promotes Tumor Growth and Migration in in vitro Models

BACKGROUND: HMGA1 is a non-histone nuclear protein that regulates cellular proliferation, invasion and apoptosis and is overexpressed in many carcinomas. In this study we sought to explore the expression of HMGA1 in HCCs and cirrhotic tissues, and its effect in in vitro models. METHODS: We evaluated HMGA1 expression using gene expression microarrays (59 HCCs, of which 37 were matched with their corresponding cirrhotic tissue and 5 normal liver donors) and tissue microarray (192 HCCs, 108 cirrhotic tissues and 79 normal liver samples). HMGA1 expression was correlated with clinicopathologic features and patient outcome. Four liver cancer cell lines with stable induced or knockdown expression of HMGA1 were characterized using in vitro assays, including proliferation, migration and anchorage-independent growth. RESULTS: HMGA1 expression increased monotonically from normal liver tissues to cirrhotic tissue to HCC (P < .01) and was associated with Edmondson grade (P < .01). Overall, 51% and 42% of HCCs and cirrhotic tissues expressed HMGA1, respectively. Patients with HMGA1-positive HCCs had earlier disease progression and worse overall survival. Forced expression of HMGA1 in liver cancer models resulted in increased cell growth and migration, and vice versa. Soft agar assay showed that forced expression of HMGA1 led to increased foci formation, suggesting an oncogenic role of HMGA1 in hepatocarcinogenesis. CONCLUSIONS: HMGA1 is frequently expressed in cirrhotic tissues and HCCs and its expression is associated with high Edmondson grade and worse prognosis in HCC. Our results suggest that HMGA1 may act as oncogenic driver of progression, implicating it in tumor growth and migration potential in liver carcinogenesis.

The Combination of the PARP Inhibitor Olaparib and the WEE1 Inhibitor AZD1775 as a New Therapeutic Option for Small Cell Lung Cancer

Purpose: Introduced in 1987, platinum-based chemotherapy remains standard of care for small cell lung cancer (SCLC), a most aggressive, recalcitrant tumor. Prominent barriers to progress are paucity of tumor tissue to identify drug targets and patient-relevant models to interrogate novel therapies. Following our development of circulating tumor cell patient–derived explants (CDX) as models that faithfully mirror patient disease, here we exploit CDX to examine new therapeutic options for SCLC. Experimental Design: We investigated the efficacy of the PARP inhibitor olaparib alone or in combination with the WEE1 kinase inhibitor AZD1775 in 10 phenotypically distinct SCLC CDX in vivo and/or ex vivo. These CDX represent chemosensitive and chemorefractory disease including the first reported paired CDX generated longitudinally before treatment and upon disease progression. Results: There was a heterogeneous depth and duration of response to olaparib/AZD1775 that diminished when tested at disease progression. However, efficacy of this combination consistently exceeded that of cisplatin/etoposide, with cures in one CDX model. Genomic and protein analyses revealed defects in homologous recombination repair genes and oncogenes that induce replication stress (such as MYC family members), predisposed CDX to combined olaparib/AZD1775 sensitivity, although universal predictors of response were not noted. Conclusions: These preclinical data provide a strong rationale to trial this combination in the clinic informed by prevalent, readily accessed circulating tumor cell–based biomarkers. New therapies will be evaluated in SCLC patients after first-line chemotherapy, and our data suggest that the combination of olaparib/AZD1775 should be used as early as possible and before disease relapse. Clin Cancer Res; 24(20); 5153–64. ©2018 AACR.

Abstract 3371: Genetic, phenotypic and functional characterisation of vasculogenic mimicry in small-cell lung cancer

Background: Despite a good initial response to chemotherapy, most small cell lung cancer (SCLC) patients relapse with drug resistant disease. Targeting tumor vasculature in SCLC with anti-angiogenic drugs produced disappointing results, therefore angiogenesis-independent tumor vascularisation pathways warrant further investigation. Vasculogenic mimicry (VM) is the ability of tumor cells to mimic endothelial cells by trans-endothelial differentiation, characterised by increased expression of vascular markers including VE-Cadherin. We previously demonstrated that VM correlates with poor Overall Survival in Limited Stage SCLC patients and sought to phenotypically and genetically characterise VM vessels using SCLC Circulating Tumour Cells (CTCs) and CTC-Derived eXplant (CDX) models1 and to explore the functional significance of VE-Cadherin for VM formation in vitro and in vivo.Methods: VM was evaluated using CD31/periodic acid-Schiff (PAS) staining in tumors from 10 CDX models. Laser Capture Microdissection (LCM) and Copy Number Alteration (CNA) analysis was performed on CDX regions with high and low levels of VM. VE-Cadherin expression in SCLC CTCs was evaluated following ISET microfiltration of patients’ blood and Immunofluorescence staining for DAPI, CD45, pan Cytokeratin and VE-Cadherin. VE-Cadherin function was evaluated in vitro by Matrigel network assay using H446 SCLC cells and H446 shRNA VE-Cadherin knockdown (KD) cell lines, and in vivo by growth as xenografts, further treated with Cisplatin. Results: VM was present in CDX models and LCM followed by CNA analysis of VM vessels confirmed their SCLC origin. ISET microfiltration and immunofluorescent staining of CTCs from 37/38 SCLC patients revealed VE-Cadherin as a putative VM biomarker in SCLC CTCs. VE-Cadherin shRNA KD in VM competent H446 SCLC cells abrogated their ability to form VM networks in vitro and in vivo. Cisplatin treatment of mice bearing H446 VE-Cadherin KD tumors resulted in reduced cisplatin binding compared to parental H446 tumors. Conclusions: VM is present in CDX models and co-localises with VE-Cadherin expression. CNA confirms that VM vessels originate from tumour, and these VM-enriched regions bear a unique chromosomal signature compared to the low-VM regions. VE-Cadherin is required for network formation in vitro and VE-Cadherin levels and VM vessel numbers are positively correlated in vivo. Moreover, levels of VM in tumors had significant impact on both tumour growth kinetics and cisplatin delivery which has implications for drug senstivity. Our future research will interrogate the VM signaling pathway in SCLC and its role in chemosensitivity and establish feasibility for therapeutic targeting. Citation Format: Francesca Trapani, Stuart Williamson, Robert L. Metcalf, Hui Sun Leong, Benjamin Abbott, Jenny Antonello, Cassandra Hodgkinson, Lynsey Franklin, Mary J. Hendrix, Richard E b Seftor, Elisabeth Seftor, Dominic Rothwell, Ged Brady, Crispin Miller, Fiona H. Blackhall, Kathryn L. Simpson, Caroline Dive. Genetic, phenotypic and functional characterisation of vasculogenic mimicry in small-cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3371.