Giorgia Nardo

Platelet‐derived growth factor‐D and Rho GTPases regulate recruitment of cancer‐associated fibroblasts in cholangiocarcinoma

Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer‐associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial‐mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)‐expressing human male CCA cell line (EGI‐1) after xenotransplantation into severe‐combined‐immunodeficient mice, (3) expression of platelet‐derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF‐D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF‐D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α‐SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α‐SMA‐expressing CAFs, which were negative for EGFP and the human Y‐probe, but positive for the murine Y‐probe. CCA cells were strongly immunoreactive for PDGF‐A and ‐D, whereas CAFs expressed PDGF receptor (PDGFR)β. PDGF‐D, a PDGFRβ agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF‐D and CCA conditioned medium and was significantly inhibited by PDGFRβ blockade with Imatinib and by silencing PDGF‐D expression in CCA cells. In fibroblasts, PDGF‐D activated the Rac1 and Cdc42 Rho GTPases and c‐Jun N‐terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF‐D‐induced fibroblast migration. Conclusion: CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF‐D, which stimulates fibroblast migration through PDGFRβ and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF‐D pathway may offer a novel therapeutic approach. (Hepatology 2013;53:1042–1053)

Nuclear Expression of S100A4 Calcium-Binding Protein Increases Cholangiocarcinoma Invasiveness and Metastasization

Cholangiocarcinoma (CCA) carries a severe prognosis because of its strong invasiveness and early metastasization. In several patients, otherwise eligible for surgical resection, micrometastasis are already present at the time of surgery. The mechanisms responsible for CCA invasiveness are unclear. S100A4, a member of the S100 family of small Ca2+‐binding proteins, is expressed in mesenchymal cells, regulates cell motility in several cell types, and is expressed in some epithelial cancers. Thus, we aimed to study the role of S100A4 in CCA invasiveness and metastasization. The expression of S100A4 was studied by immunohistochemistry in 93 human liver samples of CCA patients undergoing surgical resection and correlated with metastases development (67 cases) and patient survival following surgery using log rank tests and multivariate analysis. S100A4 expression was studied in EGI‐1 and TFK‐1, human CCA cell lines with and without nuclear S100A4 expression, respectively. Metastatic properties of CCA cells were assessed by xenotransplantation in severe combined immunodeficiency (SCID) mice after transduction with lentiviral vectors encoding firefly luciferase gene. Proliferation, motility (wound healing), invasiveness (Boyden chamber), and metalloproteinases (MMPs) secretion were studied in CCA cells, with or without lentiviral silencing of S100A4. Nuclear expression of S100A4 by neoplastic ducts was a strong predictor of metastasization and reduced survival after resection (P < 0.01). EGI‐1 CCA cells showed stronger metastatic properties than TFK‐1 when xenotransplanted in SCID mice. S100A4‐silenced EGI‐1 cells showed significantly reduced motility, invasiveness, and MMP‐9 secretion in vitro, without changes in cell proliferation. Conclusion: Nuclear S100A4 identifies a subset of CCA patients with a poor prognosis after surgical resection. Nuclear expression of S100A4 increases CCA cells invasiveness and metastasization, indicating that S100A4 may also represent a potential therapeutic target. (HEPATOLOGY 2011; 54:890–899)

GLYCOLYTIC PHENOTYPE AND AMP KINASE MODIFY THE PATHOLOGIC RESPONSE OF TUMOR XENOGRAFTS TO VEGF NEUTRALIZATION

VEGF antagonists are now widely used cancer therapeutics, but predictive biomarkers of response or toxicity remain unavailable. In this study, we analyzed the effects of anti-VEGF therapy on tumor metabolism and therapeutic response by using an integrated set of imaging techniques, including bioluminescence metabolic imaging, 18-fluorodeoxyglucose positron emission tomography, and MRI imaging and spectroscopy. Our results revealed that anti-VEGF therapy caused a dramatic depletion of glucose and an exhaustion of ATP levels in tumors, although glucose uptake was maintained. These metabolic changes selectively accompanied the presence of large necrotic areas and partial tumor regression in highly glycolytic tumors. In addition, we found that the central metabolic protein kinase AMP-activated protein kinase (AMPK)-a cellular sensor of ATP levels that supports cell viability in response to energy stress-was activated by anti-VEGF therapy in experimental tumors. AMPK-α2 attenuation increased glucose consumption, tumor cell sensitivity to glucose starvation, and tumor necrosis following anti-VEGF therapy. Taken together, our findings reveal functional links between the Warburg effect and the AMPK pathway with therapeutic responses to VEGF neutralization in tumor xenograft models.

Hypoxia Inducible Factor-1α Inactivation Unveils a Link between Tumor Cell Metabolism and Hypoxia-Induced Cell Death

Hypoxia and the acquisition of a glycolytic phenotype are intrinsic features of the tumor microenvironment. The hypoxia inducible factor-1alpha (HIF-1alpha) pathway is activated under hypoxic conditions and orchestrates a complex transcriptional program that enhances cell survival. Although the consequences of HIF-1alpha inactivation in cancer cells have been widely investigated, only a few studies have addressed the role of HIF-1alpha in the survival of cancer cells endowed with different glycolytic capacities. In this study, we investigated this aspect in ovarian cancer cells. Hypoxia-induced toxicity was increased in highly glycolytic cells compared with poorly glycolytic cells; it was also associated with a sharp decrease in intracellular ATP levels and was prevented by glucose supplementation. Stable HIF-1alpha silencing enhanced hypoxia-induced cell death in vitro due to a lack of cell cycle arrest. Tumors bearing attenuated HIF-1alpha levels had similar growth rates and vascularization as did controls, but tumors showed higher proliferation levels and increased necrosis. Moreover, tumors formed by HIF-1alpha deficient cells had higher levels of lactate and lower ATP concentrations than controls as shown by metabolic imaging. The findings that such metabolic properties can affect the survival of cancer cells under hypoxic conditions and that these properties contribute to the determination of the consequences of HIF-1alpha inactivation could have important implications on the understanding of the effects of anti-angiogenic and HIF-1alpha-targeting drugs in cancer.

VEGF-Targeted Therapy Stably Modulates the Glycolytic Phenotype of Tumor Cells

Anti-VEGF therapy perturbs tumor metabolism, severely impairing oxygen, glucose, and ATP levels. In this study, we investigated the effects of anti-VEGF therapy in multiple experimental tumor models that differ in their glycolytic phenotypes to gain insights into optimal modulation of the metabolic features of this therapy. Prolonged treatments induced vascular regression and necrosis in tumor xenograft models, with highly glycolytic tumors becoming treatment resistant more rapidly than poorly glycolytic tumors. By PET imaging, prolonged treatments yielded an increase in both hypoxic and proliferative regions of tumors. A selection for highly glycolytic cells was noted and this metabolic shift was stable and associated with increased tumor aggressiveness and resistance to VEGF blockade in serially transplanted mice. Our results support the hypothesis that the highly glycolytic phenotype of tumor cells studied in xenograft models, either primary or secondary, is a cell-autonomous trait conferring resistance to VEGF blockade. The finding that metabolic traits of tumors can be selected by antiangiogenic therapy suggests insights into the evolutionary dynamics of tumor metabolism.

Long Pentraxin-3 Inhibits FGF8b-Dependent Angiogenesis and Growth of Steroid Hormone–Regulated Tumors

Fibroblast growth factor-8b (FGF8b) exerts nonredundant autocrine/paracrine functions in steroid hormone–regulated tumors. Previous observations had shown that the soluble pattern recognition receptor long pentraxin-3 (PTX3) is a natural selective antagonist for a restricted number of FGF family members, inhibiting FGF2 but not FGF1 and FGF4 activity. Here, we assessed the capacity of PTX3 to antagonize FGF8b and to inhibit the vascularization and growth of steroid hormone–regulated tumors. Surface plasmon resonance analysis shows that PTX3 binds FGF8b with high affinity (Kd = 30–90 nmol/L). As a consequence, PTX3 prevents the binding of FGF8b to its receptors, inhibits FGF8b-driven ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells, and suppresses FGF8b-induced neovascularization in vivo. Also, PTX3 inhibits dihydrotestosterone (DHT)- and FGF8b-driven proliferation of androgen-regulated Shionogi 115 (S115) mouse breast tumor cells. Furthermore, DHT-treated, PTX3 overexpressing hPTX3_S115 cell transfectants show a reduced proliferation rate in vitro and a limited angiogenic activity in the chick embryo chorioallantoic membrane and murine s.c. Matrigel plug assays. Accordingly, hPTX3_S115 cells show a dramatic decrease of their tumorigenic activity when grafted in immunodeficient male mice. These results identify PTX3 as a novel FGF8b antagonist endowed with antiangiogenic and antineoplastic activity with possible implications for the therapy of hormonal tumors. Mol Cancer Ther; 10(9); 1600–10. ©2011 AACR.

Low dose paclitaxel reduces S100A4 nuclear import to inhibit invasion and hematogenous metastasis of cholangiocarcinoma

Nuclear expression of the calcium-binding protein S100A4 is a biomarker of increased invasiveness in cholangiocarcinoma, a primary liver cancer with scarce treatment opportunities and dismal prognosis. In this study, we provide evidence that targeting S100A4 nuclear import by low-dose paclitaxel, a microtubule-stabilizing agent, inhibits cholangiocarcinoma invasiveness and metastatic spread. Administration of low-dose paclitaxel to established (EGI-1) and primary (CCA-TV3) cholangiocarcinoma cell lines expressing nuclear S100A4 triggered a marked reduction in nuclear expression of S100A4 without modifying its cytoplasmic levels, an effect associated with a significant decrease in cell migration and invasiveness. While low-dose paclitaxel did not affect cellular proliferation, apoptosis, or cytoskeletal integrity, it significantly reduced SUMOylation of S100A4, a critical posttranslational modification that directs its trafficking to the nucleus. This effect of low-dose paclitaxel was reproduced by ginkolic acid, a specific SUMOylation inhibitor. Downregulation of nuclear S100A4 by low-dose paclitaxel was associated with a strong reduction in RhoA and Cdc42 GTPase activity, MT1-MMP expression, and MMP-9 secretion. In an SCID mouse xenograft model, low-dose metronomic paclitaxel treatment decreased lung dissemination of EGI-1 cells without significantly affecting their local tumor growth. In the tumor mass, nuclear S100A4 expression by cholangiocarcinoma cells was significantly reduced, whereas rates of proliferation and apoptosis were unchanged. Overall, our findings highlight nuclear S100A4 as a candidate therapeutic target in cholangiocarcinoma and establish a mechanistic rationale for the use of low-dose paclitaxel in blocking metastatic progression of cholangiocarcinoma. Cancer Res; 76(16); 4775-84. ©2016 AACR.

Metabolic effects of anti-angiogenic therapy in tumors

Anti-angiogenic therapy has recently been added to the panel of cancer therapeutics, but predictive biomarkers of response are still not available. In animal models, anti-angiogenic therapy causes tumor starvation by increasing hypoxia and impairing nutrients supply. It is thus conceivable that angiogenesis inhibition causes remarkable metabolic perturbations in tumors, although they remain largely uncharted. We review here recent acquisitions about metabolic effects of angiogenesis blockade in tumors and discuss the possibility that some metabolic features of tumor cells - i.e. their dependency from glucose as primary energy substrate - might affect tumor responses to anti-VEGF treatment.

LKB1 Expression Correlates with Increased Survival in Patients with Advanced Non-Small Cell Lung Cancer Treated with Chemotherapy and Bevacizumab

Purpose: LKB1 is a key sensor of metabolic stress, including hypoxia and glucose deprivation, two features of the tumor microenvironment exacerbated by antiangiogenic therapy. We investigated the role of LKB1 as a potential predictive marker of sensitivity to bevacizumab in advanced non–small cell lung cancer (aNSCLC). Experimental design: We retrospectively analyzed LKB1 expression by IHC in 98 samples from 125 patients with aNSCLC, including 59 patients treated with chemotherapy and 39 treated with chemotherapy plus bevacizumab. IHC intensity was recoded in two classes (negative/weak vs. moderate/intense) and correlated with outcome according to treatment arm. Patient-derived tumor xenografts (PDXs) were used to investigate mechanisms involved in preclinical models. Results: In the whole study population (125), median OS and PFS were 11.7 [95% confidence interval (CI), 9.1–15.3] and 6.7 (95% CI, 5.7–7.2) months, respectively. Differential impact of the marker on outcome of the 98 patients was highlighted according to the treatment. Patients with negative/weak LKB1 status did not have a statistically significant benefit from bevacizumab added to chemotherapy (HR for patients treated with bevacizumab: 0.89; 95% CI, 0.51–1.56; P = 0.6803), whereas patients expressing moderate/intense LKB1 and receiving bevacizumab had significant lower risk of death compared with those not receiving bevacizumab (HR, 0.26; 95% CI, 0.10–0.64; P = 0.0035). Loss of LKB1 was associated with reduced AMPK activation in PDXs and increased tumor necrosis following bevacizumab administration, highlighting impaired control of the metabolic stress caused by this antiangiogenic drug. Conclusions: Our data hint at a possible predictive impact of LKB1 expression in patients with aNSCLC treated with chemotherapy plus bevacizumab. Clin Cancer Res; 23(13); 3316–24. ©2017 AACR.

Morphological and genetic heterogeneity in multifocal lung adenocarcinoma: The case of a never-smoker woman

Discrimination of multifocal primary lung cancers from lung metastases is crucial to allow for an appropriate clinical management. We report here a case of multifocal lung adenocarcinomas with different morphological and molecular patterns. Radical surgery of one lung nodule was performed at the time of diagnosis, and subsequently on two other lung nodules. At the time of distant relapse, biopsy was repeated for molecular characterization. The patient was treated with EGFR tyrosine kinase inhibitor according to the detection of EGFR exon 21 mutation in metastatic sample and in one of the three lung tumors, characterized by lower mutated allele frequency. The progression free survival was three months according to radiological criteria and the treatment was provided for six months, until clinical progression. Following the assessment of EGFR mutations by pyrosequencing, tumor samples were analyzed by a 30-gene next generation sequencing (NGS) panel, allowing to study intra- and inter-tumor heterogeneity and to confirm the three lung tumors as independent. Different molecular profiles of synchronous tumors and identical EGFR, PIK3CA and TP53 mutations in one of three primary lung tumors and the metachronous metastasis were identified. In conclusion, morphological and molecular characterization of multiple lung nodules by NGS may help to define synchronous and metachronous adenocarcinomas, thus affecting surgical indication and systemic treatment. Intratumor heterogeneity may be associated with differential sensitivity to targeted treatment.