Loreta Pia Ciuffreda

Increased TGF-α as a Mechanism of Acquired Resistance to the Anti-EGFR Inhibitor Cetuximab through EGFR–MET Interaction and Activation of MET Signaling in Colon Cancer Cells

Purpose: Although cetuximab, an anti-EGF receptor (EGFR) monoclonal antibody, is an effective treatment for patients with KRAS wild-type metastatic colorectal cancer (mCRC), its clinical use is limited by onset of resistance. Experimental Design: We characterized two colorectal cancer models to study the mechanisms of acquired resistance to cetuximab. Results: Following chronic treatment of nude mice bearing cetuximab-sensitive human GEO colon xenografts, cetuximab-resistant GEO (GEO-CR) cells were obtained. In GEO-CR cells, proliferation and survival signals were constitutively active despite EGFR inhibition by cetuximab treatment. Whole gene expression profiling identified a series of genes involved in the hepatocyte growth factor (HGF)-MET–dependent pathways, which were upregulated in GEO-CR cells. Furthermore, activated, phosphorylated MET was detected in GEO-CR cells. A second colorectal cancer cell line with acquired resistance to cetuximab was obtained (SW48-CR). Inhibition of MET expression by siRNA restored cetuximab sensitivity in GEO-CR and SW48-CR cells, whereas exogenous activation of MET by HGF stimulation in cetuximab-sensitive GEO and SW48 cells induced resistance to cetuximab. Treatment of GEO-CR and SW48-CR cells with PHA665752, a selective MET inhibitor, inhibited cell growth, proliferation, and survival signals and impaired cancer cell migration. Overexpression of TGF-α, a specific EGFR ligand, was involved in the acquisition of cetuximab resistance in GEO-CR and SW48-CR cells. In fact, TGF-α overexpression induced the EGFR–MET interaction, with subsequent MET phosphorylation and activation of MET downstream effectors in GEO-CR and SW48-CR cells. Conclusions: These results suggest that overexpression of TGF-α through induction of EGFR–MET interaction contributes to cetuximab resistance in colorectal cancer cells. The combined inhibition of EGFR and MET receptor could represent a strategy for preventing and/or overcoming cetuximab resistance in patients with colorectal cancer. Clin Cancer Res; 19(24); 6751–65. ©2013 AACR.

Antitumor activity of pimasertib, a selective MEK 1/2 inhibitor, in combination with PI3K/mTOR inhibitors or with multi‐targeted kinase inhibitors in pimasertib‐resistant human lung and colorectal cancer cells

The RAS/RAF/MEK/MAPK and the PTEN/PI3K/AKT/mTOR pathways are key regulators of proliferation and survival in human cancer cells. Selective inhibitors of different transducer molecules in these pathways have been developed as molecular targeted anti‐cancer therapies. The in vitro and in vivo anti‐tumor activity of pimasertib, a selective MEK 1/2 inhibitor, alone or in combination with a PI3K inhibitor (PI3Ki), a mTOR inhibitor (everolimus), or with multi‐targeted kinase inhibitors (sorafenib and regorafenib), that block also BRAF and CRAF, were tested in a panel of eight human lung and colon cancer cell lines. Following pimasertib treatment, cancer cell lines were classified as pimasertib‐sensitive (IC50 for cell growth inhibition of 0.001 µM) or pimasertib‐resistant. Evaluation of basal gene expression profiles by microarrays identified several genes that were up‐regulated in pimasertib‐resistant cancer cells and that were involved in both RAS/RAF/MEK/MAPK and PTEN/PI3K/AKT/mTOR pathways. Therefore, a series of combination experiments with pimasertib and either PI3Ki, everolimus, sorafenib or regorafenib were conducted, demonstrating a synergistic effect in cell growth inhibition and induction of apoptosis with sustained blockade in MAPK‐ and AKT‐dependent signaling pathways in pimasertib‐resistant human colon carcinoma (HCT15) and lung adenocarcinoma (H1975) cells. Finally, in nude mice bearing established HCT15 and H1975 subcutaneous tumor xenografts, the combined treatment with pimasertib and BEZ235 (a dual PI3K/mTOR inhibitor) or with sorafenib caused significant tumor growth delays and increase in mice survival as compared to single agent treatment. These results suggest that dual blockade of MAPK and PI3K pathways could overcome intrinsic resistance to MEK inhibition.

Intrinsic resistance to selumetinib, a selective inhibitor of MEK1/2, by cAMP-dependent protein kinase A activation in human lung and colorectal cancer cells

Background:MEK is activated in ∼40% colorectal cancer (CRC) and 20–30% non-small cell lung cancer (NSCLC). Selumetinib is a selective inhibitor of MEK1/2, which is currently in clinical development.Methods:We evaluated the effects of selumetinib in vitro and in vivo in CRC and NSCLC cell lines to identify cancer cell characteristics correlating with sensitivity to MEK inhibition.Results:Five NSCLC and six CRC cell lines were treated with selumetinib and classified according to the median inhibitory concentration (IC50) values as sensitive (⩽1 μM) or resistant (>1 μM). In selumetinib-sensitive cancer cell lines, selumetinib treatment induced G1 cell-cycle arrest and apoptosis and suppression of tumour growth as xenografts in immunodeficient mice. Evaluation of intracellular effector proteins and analysis of gene mutations showed no correlation with selumetinib sensitivity. Microarray gene expression profiles revealed that the activation of cAMP-dependent protein kinase A (PKA) was associated with MEK inhibitor resistance. Combined targeting of both MEK and PKA resulted in cancer cell growth inhibition of MEK inhibitor-resistant cancer cell lines in vitro and in vivo.Conclusion:This study provides molecular insights to explain resistance to an MEK inhibitor in human cancer cell lines.

SIRT1 activation attenuates diastolic dysfunction by reducing cardiac fibrosis in a model of anthracycline cardiomyopathy

BACKGROUND Doxorubicin (DOXO) is an effective anti-neoplastic drug but its clinical benefits are hampered by cardiotoxicity. Oxidative stress, apoptosis and myocardial fibrosis mediate the anthracycline cardiomyopathy. ROS trigger TGF-β pathway that activates cardiac fibroblasts promoting fibrosis. Myocardial stiffness contributes to diastolic dysfunction, less studied aspect of anthracycline cardiomyopathy. Considering the role of SIRT1 in the inhibition of the TGF-β/SMAD3 pathway, resveratrol (RES), a SIRT1 activator, might improve cardiac function by interfering with the development of cardiac fibrosis in a model of DOXO-induced cardiomyopathy. METHODS F344 rats received a cumulative dose of 15 mg/kg of DOXO in 2 weeks or DOXO+RES (DOXO and RES, 2.5mg/kg/day, concomitantly for 2 weeks and then RES alone for 1 more week). The effects of RES on cardiac fibroblasts were also tested in vitro. RESULTS Along with systolic dysfunction, DOXO was also responsible of diastolic abnormalities. Myocardial stiffness correlated with fibroblast activation and collagen deposition. DOXO+RES co-treatment significantly improved ± dP/dt and, more interestingly, ameliorated end-diastolic pressure/volume relationship. Treatment with RES resulted in reduced fibrosis and fibroblast activation and, most importantly, the mortality rate was significantly reduced in DOXO+RES group. Fibroblasts isolated from DOXO+RES-treated rats, in which SIRT1 was upregulated, showed decreased levels of TGF-β and pSMAD3/SMAD3 when compared to cells isolated from DOXO-exposed hearts. CONCLUSIONS Our findings reveal a key role of SIRT1 in supporting animal survival and functional parameters of the heart. SIRT1 activation by interfering with fibrogenesis can improve relaxation properties of myocardium and attenuate myocardial remodeling related to chemotherapy.

SIRT1 activation rescues doxorubicin-induced loss of functional competence of human cardiac progenitor cells

BACKGROUND The search for compounds able to counteract chemotherapy-induced heart failure is extremely important at the age of global cancer epidemic. The role of SIRT1 in the maintenance of progenitor cell homeostasis may contribute to its cardioprotective effects. SIRT1 activators, by preserving progenitor cells, could have a clinical relevance for the prevention of doxorubicin (DOXO)-cardiotoxicity. METHODS To determine whether SIRT1 activator, resveratrol (RES), interferes with adverse effects of DOXO on cardiac progenitor cells (CPCs): 1) human CPCs (hCPCs) were exposed in vitro to DOXO or DOXO+RES and their regenerative potential was tested in vivo in an animal model of DOXO-induced heart failure; 2) the in vivo effects of DOXO+RES co-treatment on CPCs were studied in a rat model. RESULTS In contrast to healthy cells, DOXO-exposed hCPCs were ineffective in a model of anthracycline cardiomyopathy. The in vitro activation of SIRT1 decreased p53 acetylation, overcame suppression of the IGF-1/Akt pro-survival and anti-apoptotic signaling, enhanced oxidative stress defense and prevented senescence and growth arrest of hCPCs. Priming with RES counterbalanced the onset of dysfunctional phenotype in DOXO-exposed hCPCs, partly restoring their ability to repair the damage with improvement in cardiac function and animal survival. The in vivo co-treatment DOXO+RES prevented the anthracycline-induced alterations in CPCs, partly preserving cardiac function. CONCLUSION SIRT1 activation protects DOXO-exposed CPCs and re-establishes their proper function. Pharmacological intervention at the level of tissue-specific progenitor cells may provide cardiac benefits for the growing population of long-term cancer survivors that are at risk of chemotherapy-induced cardiovascular toxicity.

Doxorubicin cardiotoxicity and target cells: a broader perspective

The cardiotoxicity of doxorubicin is becoming an interdisciplinary point of interest given a growing population of cancer survivors. The complex and not completely understood pathogenesis of this complication makes difficult to design successful preventive or curative measures. Although cardiomyocyte has been considered a classical cellular target, other cells including various types of undifferentiated cells are involved in myocardial homeostasis. Such perspective may shed light on previously unrecognized aspects of cardiotoxicity and promote new experimental and clinical cardioprotective strategies. In this review, different cellular targets of doxorubicin are discussed with the focus on cardiac progenitor cells, oxidative stress, DNA damage, senescence and apoptosis all of which contribute to their compromised functional properties.

Effects of ranolazine in a model of doxorubicin-induced left ventricle diastolic dysfunction

Doxorubicin is a highly effective anticancer drug, but its clinical application is hampered by cardiotoxicity. Asymptomatic diastolic dysfunction can be the earliest manifestation of doxorubicin cardiotoxicity. Therefore, a search for therapeutic intervention that can interfere with early manifestations and possibly prevent later development of cardiotoxicity is warranted. Increased doxorubicin‐dependent ROS may explain, in part, Ca2+ and Na+ overload that contributes to diastolic dysfunction and development of heart failure. Therefore, we tested whether the administration of ranolazine, a selective blocker of late Na+ current, immediately after completing doxorubicin therapy, could affect diastolic dysfunction and interfere with the progression of functional decline.

Sitagliptin reduces inflammation, fibrosis and preserves diastolic function in a rat model of heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a systemic syndrome driven by co‐morbidities, and its pathophysiology is poorly understood. Several studies suggesting that dipeptidyl peptidase 4 (DPP4) might be involved in the pathophysiology of heart failure have prompted experimental and clinical investigations of DPP4 inhibitors in the cardiovascular system. Here we have investigated whether the DPP4 inhibitor sitagliptin affected the progression of HFpEF independently of its effects on glycaemia.

Long-term administration of ranolazine attenuates diastolic dysfunction and adverse myocardial remodeling in a model of heart failure with preserved ejection fraction

BACKGROUND To investigate the effects of chronic administration of ranolazine (RAN) on experimental model of heart failure with preserved ejection fraction. METHODS Seven-weeks old Dahl salt-sensitive rats were fed a high salt diet for 5weeks to induce hypertension. Afterwards, rats continued with a high salt diet and were administered either with vehicle or RAN (20mg/kg/die, ip) for the following 8weeks. Control rats were maintained on a low salt diet. RESULTS While systolic parameters were not altered, diastolic parameters were changed in high salt animals. Hemodynamic analysis showed a decreased dP/dt min, increased LVEDP, longer time constant and steeper slope of the end-diastolic pressure-volume relationship. Treatment with RAN attenuated these alterations and determined a reduction in mortality. Additionally, the magnitude of myocardial hypertrophy and activation of PI3K/Akt pathway were reduced. Alteration in diastolic compliance as a consequence of elevated myocardial stiffness was confirmed by an increase of collagen deposition and activation of pro-fibrotic TGF-β/SMAD3/CTGF signaling. These effects were counteracted by RAN. High salt rats had a decrease in SERCA2 and an increase in Na(+)/Ca(2+) exchanger (NCX). Treatment with RAN reduced NCX expression and determined an increment of SERCA2. Moreover, the levels of nitrotyrosine and oxidized dyhydroethidium were higher in high salt rats. RAN induced a decrement of oxidative stress, supporting the concept that reduction in ROS may mediate beneficial effects. CONCLUSIONS Our findings support the possibility that diastolic dysfunction can be attenuated by RAN, indicating its ability to affect active relaxation and passive diastolic compliance.

Dipeptidyl peptidase 4 inhibitor sitagliptin reduces inflammation, fibrosis and preserves diastolic function in a rat model of heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a systemic syndrome driven by co‐morbidities, and its pathophysiology is poorly understood. Several studies suggesting that dipeptidyl peptidase 4 (DPP4) might be involved in the pathophysiology of heart failure have prompted experimental and clinical investigations of DPP4 inhibitors in the cardiovascular system. Here we have investigated whether the DPP4 inhibitor sitagliptin affected the progression of HFpEF independently of its effects on glycaemia.