Alessia Isabella Esposito

Direct inhibition of hexokinase activity by metformin at least partially impairs glucose metabolism and tumor growth in experimental breast cancer

Emerging evidence suggests that metformin, a widely used anti-diabetic drug, may be useful in the prevention and treatment of different cancers. In the present study, we demonstrate that metformin directly inhibits the enzymatic function of hexokinase (HK) I and II in a cell line of triple-negative breast cancer (MDA-MB-231). The inhibition is selective for these isoforms, as documented by experiments with purified HK I and II as well as with cell lysates. Measurements of 18F-fluoro-deoxyglycose uptake document that it is dose- and time-dependent and powerful enough to virtually abolish glucose consumption despite unchanged availability of membrane glucose transporters. The profound energetic imbalance activates phosphorylation and is subsequently followed by cell death. More importantly, the “in vivo” relevance of this effect is confirmed by studies of orthotopic xenografts of MDA-MB-231 cells in athymic (nu/nu) mice. Administration of high drug doses after tumor development caused an evident tumor necrosis in a time as short as 48 h. On the other hand, 1 mo metformin treatment markedly reduced cancer glucose consumption and growth. Taken together, our results strongly suggest that HK inhibition contributes to metformin therapeutic and preventive potential in breast cancer.

Paradoxic effects of metformin on endothelial cells and angiogenesis

The biguanide metformin is used in type 2 diabetes management and has gained significant attention as a potential cancer preventive agent. Angioprevention represents a mechanism of chemoprevention, yet conflicting data concerning the antiangiogenic action of metformin have emerged. Here, we clarify some of the contradictory effects of metformin on endothelial cells and angiogenesis, using in vitro and in vivo assays combined with transcriptomic and protein array approaches. Metformin inhibits formation of capillary-like networks by endothelial cells; this effect is partially dependent on the energy sensor adenosine-monophosphate-activated protein kinase (AMPK) as shown by small interfering RNA knockdown. Gene expression profiling of human umbilical vein endothelial cells revealed a paradoxical modulation of several angiogenesis-associated genes and proteins by metformin, with short-term induction of vascular endothelial growth factor (VEGF), cyclooxygenase 2 and CXC chemokine receptor 4 at the messenger RNA level and downregulation of ADAMTS1. Antibody array analysis shows an essentially opposite regulation of numerous angiogenesis-associated proteins in endothelial and breast cancer cells including interleukin-8, angiogenin and TIMP-1, as well as selective regulation of angiopioetin-1, -2, endoglin and others. Endothelial cell production of the cytochrome P450 member CYP1B1 is upregulated by tumor cell supernatants in an AMPK-dependent manner, metformin blocks this effect. Metformin inhibits VEGF-dependent activation of extracellular signal-regulated kinase 1/2, and the inhibition of AMPK activity abrogates this event. Metformin hinders angiogenesis in matrigel pellets in vivo, prevents the microvessel density increase observed in obese mice on a high-fat diet, downregulating the number of white adipose tissue endothelial precursor cells. Our data show that metformin has an antiangiogenic activity in vitro and in vivo associated with a contradictory short-term enhancement of pro-angiogenic mediators, as well as with a differential regulation in endothelial and breast cancer cells.

Reference Profile Correlation Reveals Estrogen-like Trancriptional Activity of Curcumin

Background: Several secondary metabolites from herbal nutrient products act as weak estrogens (phytoestrogens), competing with endogenous estrogen for binding to the estrogen receptors and inhibiting steroid converting enzymes. However, it is still unclear whether these compounds elicit estrogen dependent transcription of genes at physiological concentrations. Methods: We compare the effects of physiological concentrations (100 nM) of the two phytoestrogens Enterolactone and Quercetin and the suspected phytoestrogen Curcumin on gene expression in the breast cancer cell line MCF7 with the effects elicited by 17-β-estradiol (E2). Results: All three phytocompounds have weak effects on gene transcription; most of the E2 genes respond to the phytoestrogens in the same direction though to a much lesser extent and in the order Curcumin > Quercetin > Enterolactone. Gene regulation induced by these compounds was low for genes strongly induced by E2 and similar to the latter for genes only weakly regulated by the classic estrogen. Of interest with regard to the treatment of menopausal symptoms, the survival factor Birc5/survivin and the oncogene MYBL1 are strongly induced by E2 but only marginally by phytoestrogens. Conclusion: This approach demonstrates estrogenic effects of putative phytoestrogens at physiological concentrations and shows, for the first time, estrogenic effects of Curcumin.

RESCUE OF THE MUTANT CFTR CHLORIDE CHANNEL BY PHARMACOLOGICAL CORRECTORS AND LOW TEMPERATURE ANALYZED BY GENE EXPRESSION PROFILING

The F508del mutation, the most frequent in cystic fibrosis (CF), impairs the maturation of the CFTR chloride channel. The F508del defect can be partially overcome at low temperature (27°C) or with pharmacological correctors. However, the efficacy of correctors on the mutant protein appears to be dependent on the cell expression system. We have used a bronchial epithelial cell line, CFBE41o-, to determine the efficacy of various known treatments and to discover new correctors. Compared with other cell types, CFBE41o- shows the largest response to low temperature and the lowest one to correctors such as corr-4a and VRT-325. A screening of a small-molecule library identified 9-aminoacridine and ciclopirox, which were significantly more effective than corr-4a and VRT-325. Analysis with microarrays revealed that 9-aminoacridine, ciclopirox, and low temperature, in contrast to corr-4a, cause a profound change in cell transcriptome. These data suggest that 9-aminoacridine and ciclopirox act on F508del-CFTR maturation as proteostasis regulators, a mechanism already proposed for the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). However, we found that 9-aminoacridine, ciclopirox, and SAHA, in contrast to corr-4a, VRT-325, and low temperature, do not increase chloride secretion in primary bronchial epithelial cells from CF patients. These conflicting data appeared to be correlated with different gene expression signatures generated by these treatments in the cell line and in primary bronchial epithelial cells. Our results suggest that F508del-CFTR correctors acting by altering the cell transcriptome may be particularly active in heterologous expression systems but markedly less effective in native epithelial cells.

A prognostic multigene classifier for squamous cell carcinomas of the larynx.

Survival after diagnosis of laryngeal cancer has not improved over the last 20 years. Selection of patients for radio- and chemotherapy or surgery or follow-up strategies based on a prognostic classifier could improve survival without unduly extending radical surgery. We performed microarray gene expression analysis and developed a four-gene classifier for laryngeal cancer using Prediction Analysis of Microarray and leave-one-out cross validation. A four-gene classifier containing the non-coding gene H19, the histone HIST1H3F and the two small nucleolar RNAs, SNORA16A and SNORD14C was developed that assigns cases to low and high risk classes. The high risk class has a relative risk of 6.5 (CI=1.817-23.258, Fisher exact test p<0.0001). The maternally imprinted gene H19 is the top classifier gene.

Down-regulation of the PTTG1 proto-oncogene contributes to the melanoma suppressive effects of the cyclin-dependent kinase inhibitor PHA-848125

We previously demonstrated that PHA-848125, a cyclin-dependent kinase inhibitor presently under Phase II clinical investigation, impairs melanoma cell growth. In this study, gene expression profiling showed that PHA-848125 significantly modulated the expression of 128 genes, predominantly involved in cell cycle control, in the highly drug-sensitive GL-Mel (p53 wild-type) melanoma cells. Up-regulation of 4 selected genes (PDCD4, SESN2, DDIT4, DEPDC6), and down-regulation of 6 selected genes (PTTG1, CDC25A, AURKA, AURKB, PLK1, BIRC5) was confirmed at protein levels. The same protein analysis performed in PHA-848125-treated M10 melanoma cells - p53 mutated and less sensitive to the drug than GL-Mel cells - revealed no DEPDC6 expression and no changes of PTTG1, PDCD4 and BIRC5 levels. Upon PHA-848125 treatment, a marked PTTG1 down-modulation was also observed in A375 cells (p53 wild-type) but not in CN-Mel cells (p53 mutated). PTTG1 silencing significantly inhibited melanoma cell proliferation and induced senescence, with effects less pronounced in p53 mutated cells. PTTG1 silencing increased PHA-848125 sensitivity of p53 mutated cells but not that of A375 or GL-Mel cells. Accordingly, in M10 but not in A375 cells a higher level of senescence was detected in PHA-848125-treated/PTTG1-silenced cells with respect to PHA-848125-treated controls. In A375 and GL-Mel cells, TP53 silencing attenuated PHA-848125-induced down-modulation of PTTG1 and decreased cell sensitivity to the drug. These findings indicate that PHA-848125-induced down-regulation of PTTG1 depends, at least in part, on p53 function and contributes to the antiproliferative activity of the drug. Our study provides further molecular insight into the antitumor mechanism of PHA-848125.

Glyceraldehyde-3-phosphate dehydrogenase gene over expression correlates with poor prognosis in non small cell lung cancer patients.

BackgroundGlycolysis in presence of oxygen with high glucose consumption is known to be the metabolism of choice in many tumors. In lung cancer this phenomenon is routinely exploited in diagnostic PET imaging of fluorodeoxyglucose uptake, but not much is known about the prognostic capabilities of glycolysis level assessment in resected lung tumor samples.MethodsIn this retrospective study, we used real time polymerase chain reaction(RQ-PCR) to assess the expression level of the gene for Glyceraldehyde 3-phosphate dehydrogenase(GAPDH), key enzyme for glucose breakdown, in tumor samples from 82 consecutive early stages resected non small cell lung cancer(NSCLC) patients. We then compared our results in six large publicly available NSCLC microarray datasets collecting data from over 1250 total patients.ResultsIn our study GAPDH gene over expression was found to be an adverse prognostic factor in early stages NSCLC (n = 82 HR = 1.30 p = 0.050). This result was confirmed in 5 of 6 public datasets analyzed: Shedden et al. 2008: n = 442 HR = 1.54 p < 0.0001; Lee et al. 2008: n = 138 HR = 1.31 p = 0.043; Tomida et al. 2009: n = 117 HR = 1.59 p = 0.004; Roepman et al. 2009: n = 172 (TPI1 gene) HR = 1.51 p = 0.009; Okayama et al. 2012: n = 226 HR = 3.19 p < 0.0001; Botling et al. 2013: n = 196 HR = 1.00 p = 0.97). Furthermore, in the large and clinically well annotated Shedden et al. microarray dataset, GAPDH hazard ratio did not change whether calculated for the whole dataset or for the subgroup of adjuvant naive patients only (n = 330 HR = 1.49 p < 0.0001).ConclusionGAPDH gene over expression in resected tumor samples is an adverse prognostic factor in NSCLC. Our results confirm the prognostic value of glucose metabolism assessment in NSCLC.

A highly invasive subpopulation of MDA-MB-231 breast cancer cells shows accelerated growth, differential chemoresistance, features of apocrine tumors and reduced tumorigenicity in vivo

The acquisition of an invasive phenotype is a prerequisite for metastasization, yet it is not clear whether or to which extent the invasive phenotype is linked to other features characteristic of metastatic cells. We selected an invasive subpopulation from the triple negative breast cancer cell line MDA-MB-231, performing repeated cycles of preparative assays of invasion through Matrigel covered membranes. The invasive sub-population of MDA-MB-231 cells exhibits stronger migratory capacity as compared to parental cells confirming the highly invasive potential of the selected cell line. Prolonged cultivation of these cells did not abolish the invasive phenotype. ArrayCGH, DNA index quantification and karyotype analyses confirmed a common genetic origin of the parental and invasive subpopulations and revealed discrete structural differences of the invasive subpopulation including increased ploidy and the absence of a characteristic amplification of chromosome 5p14.1-15.33. Gene expression analyses showed a drastically altered expression profile including features of apocrine breast cancers and of invasion related matrix-metalloproteases and cytokines. The invasive cells showed accelerated proliferation, increased apoptosis, and an altered pattern of chemo-sensitivity with lower IC50 values for drugs affecting the mitotic apparatus. However, the invasive cell population is significantly less tumorigenic in orthotopic mouse xenografts suggesting that the acquisition of the invasive capacity and the achievement of metastatic growth potential are distinct events.

Breast Cancer Genomics: From Portraits to Landscapes

Breast cancer is the most frequent female cancer and still one of the major causes of death although early diagnosis and improved therapies have had a great impact on survival after breast cancer diagnosis. However, there are still many unresolved problems in breast cancer such as the fraction of breast cancers that do not respond to current therapies and considerable overtreatment due to imperfect prognostication. The application of genomics to breast cancer has led to the identification of clinically relevant molecular subtypes, especially the distinction of luminal A and luminal B subtypes within the class of hormone receptor positive cancers. Many prognostic signatures have been developed and two of them are being applied in oncologic decision making yet their utility most likely does not go beyond the distinction of luminal A and B subtypes that show a highly different proliferative potential. Integration of copy number variation has identified even more subclasses with distinct clinical characteristics. Genome wide association studies have identified many single nucleotide polymorphisms that are associated with breast cancer risk and several of them resist in validation studies. Their application for the design of risk based preventive strategies has been proposed. Next generation sequencing shows a wide variation of driver mutations in breast cancer, most of them within interrelated signaling pathways. Several genes such as TP53 or PIK3CA show frequent mutations but many mutations are almost private. Sequencing also identified several actionable mutations, among which those that occur in genes more frequently involved in other cancers that could indicate specific treatments. Better prognostication and response prediction by means of genomic analyses and mutation screening will almost certainly contribute to the improvement of therapy and to the reduction of unnecessary toxicity. Breast cancer genomics has also led to a conceptual shift in our understanding of the process of metastasis that seems to be determined from very early stages of the disease although additional mutations occur at later stages.

Abstract 1182: Metformin affects breast cancer cell growth and disturbs an IGF1/insulin related gene network that correlates with breast cancer progression

Background Obesity and the insulin resistance syndrome are risk factors for breast cancer and might also affect breast cancer progression. The anti-diabetic drug Metformin (METF) reduces the breast cancer risk in diabetic women. Insulin like growth factor 1 (IGF1) and insulin are involved in breast cancer tumorigenesis and progression. We tested the effect of METF on the IGF1/insulin pathway and its involvement in breast cancer progression. Methods We developed a prognostic signature based on IGF1/insulin pathway genes using the Stockholm breast cancer microarray dataset of 149 cases for training and primary validation and the Uppsala dataset of 249 for external validation. The effect of METF on the prognostic gene set identified was tested in vitro on a panel of breast cancer cell lines. METF effects on proliferation and glucose metabolism were analyzed in vitro and in vivo. The insulin receptor substrate 2 (IRS2) was silenced by transfection with shRNA-lentiviral vectors. Xenograft growth, in the presence and absence of METF, was studied and 18FDG-uptake was measured in vitro and in vivo. Results A 15-gene signature (Insulin sensitivity score, ISS) was developed and predicted breast cancer metastasis with an accuracy similar to the Recurrence Score. ISS genes were expressed at variable levels in a breast cancer cell line panel and showed variable responsiveness to METF. The high expression correlation among the ISS genes observed in untreated breast cancer cell lines was lost upon treatment with METF. METF reduced breast cancer cell growth in vitro with IC50 values ranging from 1mM to 25mM. Growth of MDA-MB-231 cells and hyper-invasive subpopulations derived therefrom was reduced in vivo by oral administration of METF to xenografted nude mice. Response to METF in terms of IC50 values correlated with basal expression of the 15 ISS genes with the strongest inverse correlation observed for IRS2. Stable silencing of IRS2 reduced the MDA-231 cell responsiveness to METF in vitro. Discussion METF acts on the insulin/IGF1 axis by disturbing a network of breast cancer progression related genes and appears to depend in its action on the expression of IRS2 that inversely correlates with the sensitivity of cell lines to the drug. The disruption of the ISS gene network is expected to correlate with an effect on breast cancer growth and progression and in fact, mouse xenografts show reduced growth upon treatment with METF. IRS2 appears to be a major mediator of METF effects. Citation Format: Alessia I. Esposito, Adriana Amaro, Giovanna Angelini, Laura Emionite, Alessandra Gennari, Stefano Indraccolo, Davide Maggi, Cecilia Marini, Barabara Salani, Gianmario Sambuceti, Maria Pia Sormani, Ulrich Pfeffer. Metformin affects breast cancer cell growth and disturbs an IGF1/insulin related gene network that correlates with breast cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1182. doi:10.1158/1538-7445.AM2015-1182