Teresa Puig

Brain metastases from lung cancer responding to erlotinib: the importance of EGFR mutation

Median survival of patients with brain metastases from nonsmall cell lung cancer (NSCLC) is poor and more effective treatments are urgently needed. We have evaluated the efficacy of erlotinib in this setting and its association with activating mutations in the epidermal growth factor receptor (EGFR) gene. We retrospectively identified patients with NSCLC and brain metastases treated with erlotinib. EGFR mutations in exons 19 and 21 were analysed by direct sequencing. Efficacy and tolerability were compared according to EGFR mutational status. 69 NSCLC patients with brain metastases were identified, 17 of whom harboured EGFR mutations. Objective response rate in patients with EGFR mutations was 82.4%; no responses were observed in unselected patients (p<0.001). Median (95% CI) time to progression within the brain for patients harbouring EGFR mutations was 11.7 (7.9–15.5) months, compared to 5.8 (5.2–6.4) months for control patients whose EGFR mutational status had not been assessed (p<0.05). Overall survival was 12.9 (6.2–19.7) months and 3.1 (2.5–3.9) months (p<0.001), respectively. The toxicity of erlotinib was as expected and no differences between cohorts were observed. Erlotinib is active in brain metastases from NSCLC; this clinical benefit is related to the presence of activating mutations in exons 19 or 21 of the EGFR gene.

Fatty acid metabolism in breast cancer cells: differential inhibitory effects of epigallocatechin gallate (EGCG) and C75

Endogenous fatty acid metabolism is crucial to maintain the cancer cell malignant phenotype. Lipogenesis is regulated by the enzyme fatty acid synthase (FASN); and breakdown of fatty acids is regulated by carnitine palmitoyltransferase-1 (CPT-I). FASN is highly expressed in breast cancer and most common human carcinomas. Several compounds can inhibit FASN, although the degree of specificity of this inhibition has not been addressed. We have tested the effects of C75 and (-)-epigallocatechin-3-gallate (EGCG) on fatty acid metabolism pathways, cellular proliferation, induction of apoptosis and cell signalling in human breast cancer cells. Our results show that C75 and EGCG had comparable effects in blocking FASN activity. Treating cancer cells with EGCG or C75 induced apoptosis and caused a decrease in the active forms of oncoprotein HER2, AKT and ERK1/2 to a similar degree. We observed, in contrast, marked differential effects between C75 and EGCG on the fatty acid oxidation pathway. While EGCG had either no effect or a moderate reduction in CPT-I activity, C75 stimulated CPT-I activity (up to 129%), even in presence of inhibitory levels of malonyl-CoA, a potent inhibitor of the CPT-I enzyme. Taken together, these findings indicate that pharmacological inhibition of FASN occurs uncoupled from the stimulation of CPT-I with EGCG but not with C75, suggesting that EGCG might be free of the CPT-I related in vivo weight-loss that has been associated with C75. Our results establish EGCG as a potent and specific inhibitor of fatty acid synthesis (FASN), which may hold promise as a target-directed anti-cancer drug.

Novel Inhibitors of Fatty Acid Synthase with Anticancer Activity

Purpose: Fatty acid synthase (FASN) is overexpressed in human breast carcinoma. The natural polyphenol (−)-epigallocatechin-3-gallate blocks in vitro FASN activity and leads to apoptosis in breast cancer cells without any effects on carnitine palmitoyltransferase-1 (CPT-1) activity, and in vivo, does not decrease body weight. We synthesized a panel of new polyphenolic compounds and tested their effects on breast cancer models. Experimental Design: We evaluated the in vitro effects of the compounds on breast cancer cell growth (SK-Br3, MCF-7, and MDA-MB-231), apoptosis [as assessed by cleavage of poly(ADP-ribose) polymerase], cell signaling (HER2, ERK1/2, and AKT), and fatty acid metabolism enzymes (FASN and CPT-1). In vivo, we have evaluated their antitumor activity and their effect on body weight in a mice model of BT474 breast cancer cells. Results: Two compounds potently inhibited FASN activity and showed high cytotoxicity. Moreover, the compounds induced apoptosis and caused a marked decrease in the active forms of HER2, AKT, and ERK1/2 proteins. Interestingly, the compounds did not stimulate CPT-1 activity in vitro. We show evidence that one of the FASN inhibitors blocked the growth of BT474 breast cancer xenografts and did not induce weight loss in vivo. Conclusions: The synthesized polyphenolic compounds represent a novel class of FASN inhibitors, with in vitro and in vivo anticancer activity, that do not exhibit cross-activation of β-oxidation and do not induce weight loss in animals. One of the compounds blocked the growth of breast cancer xenografts. These FASN inhibitors may represent new agents for breast cancer treatment. (Clin Cancer Res 2009;15(24):7608–15)

A novel inhibitor of fatty acid synthase shows activity against HER2+ breast cancer xenografts and is active in anti-HER2 drug-resistant cell lines.

IntroductionInhibiting the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of breast carcinoma cells, and this is linked to human epidermal growth factor receptor 2 (HER2) signaling pathways in models of simultaneous expression of FASN and HER2.MethodsIn a xenograft model of breast carcinoma cells that are FASN+ and HER2+, we have characterised the anticancer activity and the toxicity profile of G28UCM, the lead compound of a novel family of synthetic FASN inhibitors. In vitro, we analysed the cellular and molecular interactions of combining G28UCM with anti-HER drugs. Finally, we tested the cytotoxic ability of G28UCM on breast cancer cells resistant to trastuzumab or lapatinib, that we developed in our laboratory.ResultsIn vivo, G28UCM reduced the size of 5 out of 14 established xenografts. In the responding tumours, we observed inhibition of FASN activity, cleavage of poly-ADPribose polymerase (PARP) and a decrease of p-HER2, p- protein kinase B (AKT) and p-ERK1/2, which were not observed in the nonresponding tumours. In the G28UCM-treated animals, no significant toxicities occurred, and weight loss was not observed. In vitro, G28UCM showed marked synergistic interactions with trastuzumab, lapatinib, erlotinib or gefitinib (but not with cetuximab), which correlated with increases in apoptosis and with decreases in the activation of HER2, extracellular signal-regulated kinase (ERK)1/2 and AKT. In trastuzumab-resistant and in lapatinib-resistant breast cancer cells, in which trastuzumab and lapatinib were not effective, G28UCM retained the anticancer activity observed in the parental cells.ConclusionsG28UCM inhibits fatty acid synthase (FASN) activity and the growth of breast carcinoma xenografts in vivo, and is active in cells with acquired resistance to anti-HER2 drugs, which make it a candidate for further pre-clinical development.

Different fatty acid metabolism effects of (−)-Epigallocatechin-3-Gallate and C75 in Adenocarcinoma lung cancer

BackgroundFatty acid synthase (FASN) is overexpressed and hyperactivated in several human carcinomas, including lung cancer. We characterize and compare the anti-cancer effects of the FASN inhibitors C75 and (−)-epigallocatechin-3-gallate (EGCG) in a lung cancer model.MethodsWe evaluated in vitro the effects of C75 and EGCG on fatty acid metabolism (FASN and CPT enzymes), cellular proliferation, apoptosis and cell signaling (EGFR, ERK1/2, AKT and mTOR) in human A549 lung carcinoma cells. In vivo, we evaluated their anti-tumour activity and their effect on body weight in a mice model of human adenocarcinoma xenograft.ResultsC75 and EGCG had comparable effects in blocking FASN activity (96,9% and 89,3% of inhibition, respectively). In contrast, EGCG had either no significant effect in CPT activity, the rate-limiting enzyme of fatty acid β-oxidation, while C75 stimulated CPT up to 130%. Treating lung cancer cells with EGCG or C75 induced apoptosis and affected EGFR-signaling. While EGCG abolished p-EGFR, p-AKT, p-ERK1/2 and p-mTOR, C75 was less active in decreasing the levels of EGFR and p-AKT. In vivo, EGCG and C75 blocked the growth of lung cancer xenografts but C75 treatment, not EGCG, caused a marked animal weight loss.ConclusionsIn lung cancer, inhibition of FASN using EGCG can be achieved without parallel stimulation of fatty acid oxidation and this effect is related mainly to EGFR signaling pathway. EGCG reduce the growth of adenocarcinoma human lung cancer xenografts without inducing body weight loss. Taken together, EGCG may be a candidate for future pre-clinical development.

New Synthetic Inhibitors of Fatty Acid Synthase with Anticancer Activity

Fatty acid synthase (FASN) is a lipogenic enzyme that is highly expressed in different human cancers. Here we report the development of a new series of polyphenolic compounds 5-30 that have been evaluated for their cytotoxic capacity in SK-Br3 cells, a human breast cancer cell line with high FASN expression. The compounds with an IC(50) < 50 μM have been tested for their ability to inhibit FASN activity. Among them, derivative 30 blocks the 90% of FASN activity at low concentration (4 μM), is highly cytotoxic in a broad panel of tumor cells, induces apoptosis, and blocks the activation of HER2, AKT, and ERK pathways. Remarkably, 30 does not activate carnitine palmitoyltransferase-1 (CPT-1) nor induces in mice weight loss, which are the main drawbacks of other previously described FASN inhibitors. Thus, FASN inhibitor 30 may aid the validation of this enzyme as a therapeutic target for the treatment of cancer.

A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity.

Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human pancreatic ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human pancreatic ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5-15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization signal. In these experiments, PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. Although the Arg introduction modified the net charge of the protein and somehow impaired recognition by the cytosolic inhibitor, control variants, which had the same number of charges or were not recognized by the inhibitor, were not toxic. We concluded that targeting a ribonuclease to the nucleus results in cytotoxicity. This effect is probably due to ribonuclease interference with rRNA processing and ribosome assembly within the nucleolus.

Androgen-independent prostate cancer cells circumvent EGFR inhibition by overexpression of alternative HER receptors and ligands

The deregulation of the epidermal growth factor receptor (EGFR) pathway plays a major role in the pathogenesis of prostate cancer (PCa). However, therapies targeting EGFR have demonstrated limited effectiveness in PCa. A potential mechanism to overcome EGFR blockade in cancer cells is the autocrine activation of alternative receptors of the human EGFR (HER) family through the overexpression of the HER receptors and ligands. In the present study, we were interested in analyzing if this intrinsic resistance mechanism might contribute to the inefficacy of EGFR inhibitors in PCa. To this end, we selected two androgen-independent human prostate carcinoma cell lines (DU145 and PC3) and established DU145 erlotinib-resistant cells (DUErR). Cells were treated with three EGFR inhibitors (cetuximab, gefinitib and erlotinib) and the sensitivity to each treatment was assessed. The gene expression of the four EGFR/HER receptors and seven ligands of the HER family was analyzed by real-time PCR prior to and after each treatment. The receptors expression and activation were further characterized by flow cytometry and western blot analysis. EGFR inhibition rapidly induced enhanced gene expression of the EGF, betacellulin and neuregulin-1 ligands along with HER2, HER3 and HER4 receptors in the DU145 cells. In contrast, slight changes were observed in the PC3 cells, which are defective in the phosphatase and tensin homolog (PTEN) tumor suppressor gene. In the erlotinib-resistant DUErR cells, the expression of HER2 and HER3 was increased at mRNA and protein levels together with neuregulin-1, leading to enhanced HER3 phosphorylation and the activation of the downstream PI3K/Akt survival pathway. HER3 blockage by a monoclonal antibody restored the cytostatic activity of erlotinib in DUErR cells. Our results confirm that the overexpression and autocrine activation of HER3 play a key role in mediating the resistance to EGFR inhibitors in androgen-independent PCa cells.

Antimicrobial cyclic decapeptides with anticancer activity.

Antimicrobial peptides have been considered as potential candidates for cancer therapy. We report here the cytotoxicity of a library of 66 antibacterial cyclodecapeptides on human carcinoma cell lines, and their effects on apoptosis [as assessed by cleavage of poly(ADP-ribose) polymerase (PARP)] and cell signaling proteins (p53 and ERK1/2) in cultured human cervical carcinoma cells. A design of experiments approach permitted to analyze the results of a subset of 16 peptides and define rules for high anticancer activity against MDA-MB-231 breast carcinoma cells. Eight peptides were identified with IC(50) values ranging from 18.5 to 57.5 μM against the five cell lines tested, being HeLa cells the most sensitive. Among these sequences, BPC88, BPC96, BPC98, and BPC194 displayed specificity and high cytotoxicity against HeLa cells (IC(50) of 22.5-38.5 μM), showed low hemolytic activity and low cytotoxicity to non-malignant fibroblasts, and were stable to proteases in human serum. Induction of apoptosis by these peptides was observed and the apoptotic effect of BPC88 and BPC96 caused a marked decrease on the activated form of ERK1/2 kinase and an induction of p53. We further showed that BPC96 at low doses synergized the cytotoxic effect of cisplatin. These findings suggest that cyclic decapeptides may represent novel anticancer agents providing a new strategy in cancer therapy.

Novel anti-fatty acid synthase compounds with anti-cancer activity in HER2+ breast cancer.

Fatty acid synthase (FASN) expression and activity has emerged as a common phenotype in most human carcinomas, including breast cancer, and its expression is tightly linked to HER2 signaling pathways. The development of inhibitors of FASN activity has consequently appeared as a novel antitarget modality for treating cancer. However, the clinical use of FASN inhibitors, such as cerulenin, C75, and epigallocatechin 3‐gallate (EGCG), is limited by anorexia and induced body weight loss or by its low in vivo potency and stability. Here, we summarize the design and development of G28UCM, the lead‐compound of a novel family of synthetic FASN inhibitors, with both in vitro and in vivo activity in a human breast cancer model of FASN+ and HER2+.