Elisabet Ametller

Anticachectic effects of formoterol: a drug for potential treatment of muscle wasting.

In cancer cachexia both cardiac and skeletal muscle suffer an important protein mobilization as a result of increased proteolysis. Administration of the β2-agonist formoterol to both rats and mice bearing highly cachectic tumors resulted in an important reversal of the muscle-wasting process. The anti-wasting effects of the drug were based on both an activation of the rate of protein synthesis and an inhibition of the rate of muscle proteolysis. Northern blot analysis revealed that formoterol treatment resulted in a decrease in the mRNA content of ubiquitin and proteasome subunits in gastrocnemius muscles; this, together with the decreased proteasome activity observed, suggest that the main anti-proteolytic action of the drug may be based on an inhibition of the ATP-ubiquitin-dependent proteolytic system. Interestingly, the β2-agonist was also able to diminish the increased rate of muscle apoptosis (measured as DNA laddering as well as caspase-3 activity) present in tumor-bearing animals. The present results indicate that formoterol exerted a selective, powerful protective action on heart and skeletal muscle by antagonizing the enhanced protein degradation that characterizes cancer cachexia, and it could be revealed as a potential therapeutic tool in pathologic states wherein muscle protein hypercatabolism is a critical feature such as cancer cachexia or other wasting diseases.

Inference of Tumor Evolution during Chemotherapy by Computational Modeling and In Situ Analysis of Genetic and Phenotypic Cellular Diversity

Cancer therapy exerts a strong selection pressure that shapes tumor evolution, yet our knowledge of how tumors change during treatment is limited. Here, we report the analysis of cellular heterogeneity for genetic and phenotypic features and their spatial distribution in breast tumors pre- and post-neoadjuvant chemotherapy. We found that intratumor genetic diversity was tumor-subtype specific, and it did not change during treatment in tumors with partial or no response. However, lower pretreatment genetic diversity was significantly associated with pathologic complete response. In contrast, phenotypic diversity was different between pre- and posttreatment samples. We also observed significant changes in the spatial distribution of cells with distinct genetic and phenotypic features. We used these experimental data to develop a stochastic computational model to infer tumor growth patterns and evolutionary dynamics. Our results highlight the importance of integrated analysis of genotypes and phenotypes of single cells in intact tissues to predict tumor evolution.

Targeting of substance P induces cancer cell death and decreases the steady state of EGFR and Her2

NK1 is a tachykinin receptor highly relevant to tumorigenesis and metastasis development in breast cancer and other carcinomas. Despite the substantial efforts done to develop potent NK1 receptor antagonists, none of these antagonists had shown good antitumor activity in clinical trials. Now, we have tested the effect of inhibition of the neuropeptide Substance P (SP), a NK1 ligand, as a potential therapeutic approach in cancer. We found that the inhibition of SP with antibodies strongly inhibit cell growth and induce apoptosis in breast, colon, and prostate cancer cell lines. These effects were accompained by a decrease in the mitogen‐activated kinase singaling pathway. Interestingly, in some cell lines SP abrogation decreased the steady state of Her2 and EGFR, suggesting that SP‐mediated signaling is important for the basal activity of these ErbB receptors. In consequence, we observed a blockade of the cell cycle progression and the inhibition of several cell cycle‐related proteins including mTOR. SP inhibition also induced cell death in cell lines resistant to Lapatinib and Trastuzumab that have increased levels of active Her2, suggesting that this therapeutic approach could be also effective for those cancers resistant to current anti‐ErbB therapies. Thus, we propose a new therapeutic strategy for those cancers that express NK1 receptor and/or other tachykinin receptors, based in the immuno‐blockade of the neuropeptide SP. J. Cell. Physiol. 227: 1358–1366, 2012.

The Role of MMP7 and Its Cross-Talk with the FAS/FASL System during the Acquisition of Chemoresistance to Oxaliplatin

Background The efficacy of oxaliplatin in cancer chemotherapy is limited by the development of drug resistance. MMP7 has been related to the loss of tumor cell response to cytotoxic agents although the exact mechanism is not fully understood. Moreover, MMP7 is an independent prognosis factor for survival in patients with colorectal cancer. The aim of the present study was to analyze the role of MMP7 and its cross-talk with the Fas/FasL system during the acquisition of oxaliplatin resistance in colon cancer cells. Principal Findings For this purpose we have developed three different oxaliplatin-resistant cell lines (RHT29, RHCT116 p53+/+, RHCT116 p53−/−) from the parental HT29, HCT116 p53+/+ and HCT116 p53−/− colon cancer cells. MMP7 basal expression was higher in the resistant compared to the parental cell lines. MMP7 was also upregulated by oxaliplatin in both HT29 (p53 mutant) and RHCT116 p53−/− but not in the RHCT116 p53+/+. Inhibition of MMP by 1,10-phenantroline monohydrate or siRNA of MMP7 restores cell sensitivity to oxaliplatin-induced apoptosis in both HT29 and RHCT116 p53−/− but not in the RHCT116 p53+/+. Some of these effects are caused by alterations in Fas receptor. Fas is upregulated by oxaliplatin in colon cancer cells, however the RHT29 cells treated with oxaliplatin showed a 3.8-fold lower Fas expression at the cell surface than the HT29 cells. Decrease of Fas at the plasma membrane seems to be caused by MMP7 since its inhibition restores Fas levels. Moreover, functional analysis of Fas demonstrates that this receptor was less potent in inducing apoptosis in RHT29 cells and that its activation induces MAPK signaling in resistant cells. Conclusions Taking together, these results suggest that MMP7 is related to the acquisition of oxaliplatin-resistance and that its inhibition restores drug sensitivity by increasing Fas receptor. Furthermore, Fas undergoes a change in its functionality in oxaliplatin-resistant cells inducing survival pathways instead of apoptotic signals.

Substance P autocrine signaling contributes to persistent HER2 activation that drives malignant progression and drug resistance in breast cancer

ERBB receptor transmodulation by heterologous G-protein-coupled receptors (GPCR) generates functional diversity in signal transduction. Tachykinins are neuropeptides and proinflammatory cytokines that promote cell survival and cancer progression by activating several GPCRs. In this work, we found that the pain-associated tachykinin Substance P (SP) contributes to persistent transmodulation of the ERBB receptors, EGFR and HER2, in breast cancer, acting to enhance malignancy and therapeutic resistance. SP and its high-affinity receptor NK-1R were highly expressed in HER2(+) primary breast tumors (relative to the luminal and triple-negative subtypes) and were overall correlated with poor prognosis factors. In breast cancer cell lines and primary cultures derived from breast cancer samples, we found that SP could activate HER2. Conversely, RNA interference-mediated attenuation of NK-1R, or its chemical inhibition, or suppression of overall GPCR-mediated signaling, all strongly decreased steady-state expression of EGFR and HER2, establishing that their basal activity relied upon transdirectional activation by GPCR. Thus, SP exposure affected cellular responses to anti-ERBB therapies. Our work reveals an important oncogenic cooperation between NK-1R and HER2, thereby adding a novel link between inflammation and cancer progression that may be targetable by SP antagonists that have been clinically explored.

Are Peroxisome Proliferator-Activated Receptors Involved in Skeletal Muscle Wasting during Experimental Cancer Cachexia? Role of β2-Adrenergic Agonists

Implantation of the Yoshida AH-130 ascites hepatoma to rats resulted in a decrease in muscle weight 7 days after the inoculation of the tumor. These changes were associated with increases in the mRNA content for both peroxisome proliferator-activated receptor (PPAR) gamma and PPAR delta in skeletal muscle. The increase in gene expression for these transcription factors was related to increases in the expression of several genes involved in fatty acid transport, activation, and oxidation. Tumor burden also resulted in increases in PPAR gamma coactivator-1 alpha gene expression and pyruvate dehydrogenase kinase 4. All these changes in lipid metabolism genes suggest that a metabolic shift occurs in skeletal muscle of tumor-bearing rats toward a more oxidative phenotype. Formoterol treatment to tumor-bearing rats resulted in an amelioration of all the changes observed as a result of tumor burden. Administration of this beta(2)-adrenergic agonist also resulted in a decrease in mRNA content of muscle PPAR alpha, PPAR delta, and PPAR gamma, as well as in mRNA levels of many of the genes involved in both lipid and mitochondrial metabolism. All these results suggest an involvement of the different PPARs as transcription factors related with muscle wasting and also indicate that a possible mode of action of the anticachectic compound formoterol may involve a normalization of the levels of these transcription factors.

The AP-1/CJUN signaling cascade is involved in muscle differentiation: Implications in muscle wasting during cancer cachexia

The aim of the present study was to investigate a possible role of the AP‐1 signaling cascade in the process of wasting associated with cancer cachexia at the level of skeletal muscle. The injection of virus containing the TAM67 protein (a blocker of the AP‐1 protein) to the gastrocnemius muscle of tumour‐bearing rats resulted in a significant recovery of the muscle mass (which is dramatically reduced as a result of tumour burden), therefore suggesting that AP‐1 is certainly involved in the signaling associated with muscle protein accretion. In conclusion, the gene therapy approach presented here clearly suggests an important role for AP‐1 in muscle signaling during catabolic states.

UCP3 overexpression neutralizes oxidative stress rather than nitrosative stress in mouse myotubes

The deleterious effects of oxidants on proteins may be modified by overexpression of uncoupling protein 3 (UCP3) in skeletal muscle cells exposed to hyperoxia or H2O2. UCP3 overexpression significantly attenuated the increase in protein carbonylation in response to hyperoxia and H2O2 exposures. However, antioxidant enzyme content and activity (superoxide dismutases, peroxiredoxins, glutathione peroxidase‐I, and catalase) were reduced or not modified in UCP3‐overexpressing myotubes exposed to oxidants. Protein nitration increased in UCP3‐overexpressing cells exposed to hyperoxia, but not to H2O2. We conclude that protein oxidation rather than nitration is neutralized by UPC3 overexpression in mouse myotubes exposed to abundant reactive oxygen species.

Effects of IL-15 on Rat Brown Adipose Tissue : Uncoupling Proteins and PPARs

Objectives: Interleukin‐15 (IL‐15) plays an important role in lipid metabolism as its administration to rats causes a marked depletion of white adipose tissue (WAT). This reduction in fat mass seems to be caused by and related to hipotriglyceridemia as a result of a lower hepatic lipogenesis and an increased fatty acid oxidation. We have previously observed that IL‐15 treatment induces the expression of uncoupling proteins (UCPs) in muscle. The aim of this study was to investigate the effects of IL‐15 on brown adipose tissue (BAT), and in particular on genes related to lipid metabolism in this tissue.

Sirtuin 1 in skeletal muscle of cachectic tumour-bearing rats: a role in impaired regeneration?

Background:In advanced malignant disease, cachexia and muscle wasting appear to be among the most common manifestations. This phenomenon is partially related with a decreased muscle regeneration capacity, as previously described in our laboratory.Methods and results:Rats bearing the Yoshida AH-130 ascites hepatoma were used in the experiments. The animals experienced a marked weight loss with decreases in skeletal muscle weights (13% gastrocnemius, 18% extensor digitorum longus, and 12% tibialis muscles). Muscle gene expression was measured using real-time polymerase chain reaction. Skeletal muscle from cachectic tumour-bearing rats is associated with a decreased expression of genes involved in regeneration such as Pax-7 (39%), myogenin (24%), and MyoD (17%). mRNA levels of Sirt1 increased (91%) in cachectic skeletal muscle. The Sirt1 gene has been shown to be associated with changes in muscle myoblast differentiation. Treatment of the tumour-bearing animals with formoterol—a beta2-agonist—normalizes the expression of genes involved in regeneration (i.e., increase of Pax7 (139%)), at the same time as it does with that of Sirt1 (42% decrease).Conclusions:It is suggested that the lack of muscle regeneration observed during muscle wasting in tumour-bearing animals is linked to the action of Sirt-1, possibly via PGC-1α. These factors may constitute possible targets of pharmacological treatment against muscle loss, thus potentially contributing to the understanding and mitigation of muscle atrophy associated with disease.