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PDF file - 427K, Effects of the combination cetuximab plus fingolimod on tumor growth of mice xenografted with GEO-CR resistant tumors

PDF file - 5320K, Validation of antibodies for analysis of SphK1 expression on FFPE colorectal cancer tissues

PDF file - 503K, Effects of SphK1 modulation on cetuximab sensitivity in resistant colorectal cancer cell lines

PDF file - 1047K, Effects of the combination cetuximab plus fingolimod on apoptosis of GEO-CR tumor xenografts

PDF file - 2068K, Correlation of SphK1 expression with clinical data in colorectal cancer patients

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PDF file - 483K, Effects of SphK1 inhibition by DMS on cetuximab sensitivity in resistant colorectal cancer cell lines

PDF file - 357K, Sensitivity to cetuximab of a panel of human colorectal cancer cell lines

Purpose: Although the anti–EGF receptor (EGFR) monoclonal antibody cetuximab is an effective strategy in colorectal cancer therapy, its clinical use is limited by intrinsic or acquired resistance. Alterations in the “sphingolipid rheostat”—the balance between the proapoptotic molecule ceramide and the mitogenic factor sphingosine-1-phosphate (S1P)—due to sphingosine kinase 1 (SphK1) overactivation have been involved in resistance to anticancer-targeted agents. Moreover, cross-talks between SphK1 and EGFR-dependent signaling pathways have been described.Experimental design: We investigated SphK1 contribution to cetuximab resistance in colorectal cancer, in preclinical in vitro/in vivo models, and in tumor specimens from patients.Results: SphK1 was found overexpressed and overactivated in colorectal cancer cells with intrinsic or acquired resistance to cetuximab. SphK1 contribution to resistance was supported by the demonstration that SphK1 inhibition by N,N-dimethyl-sphingosine or silencing via siRNA in resistant cells restores sensitivity to cetuximab, whereas exogenous SphK1 overexpression in sensitive cells confers resistance to these agents. Moreover, treatment of resistant cells with fingolimod (FTY720), a S1P receptor (S1PR) antagonist, resulted in resensitization to cetuximab both in vitro and in vivo, with inhibition of tumor growth, interference with signal transduction, induction of cancer cells apoptosis, and prolongation of mice survival. Finally, a correlation between SphK1 expression and cetuximab response was found in colorectal cancer patients. Clin Cancer Res; 19(1); 138–47. ©2012 AACR.

PDF file - 908K, Multivariate analysis correlating the response to cetuximab-based regimens to different factors

ObjectiveTo evaluate whether eculizumab helps patients with anti–acetylcholine receptor–positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension.MethodsPatients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study.ResultsA total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1–4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected.ConclusionEculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population.ClinicalTrials.gov IdentifierREGAIN, NCT01997229; REGAIN open-label extension, NCT02301624.Classification of EvidenceThis study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo.

The terminal complement inhibitor eculizumab was shown to improve myasthenia gravis-related symptoms in the 26-week, phase 3, randomized, double-blind, placebo-controlled REGAIN study (NCT01997229). In this 52-week sub-analysis of the open-label extension of REGAIN (NCT02301624), eculizumab's efficacy and safety were assessed in 11 Japanese and 88 Caucasian patients with anti-acetylcholine receptor antibody-positive refractory generalized myasthenia gravis. For patients who had received placebo during REGAIN, treatment with open-label eculizumab resulted in generally similar outcomes in the Japanese and Caucasian populations. Rapid improvements were maintained for 52 weeks, assessed by change in score from open-label extension baseline to week 52 (mean [standard error]) using the following scales (in Japanese and Caucasian patients, respectively): Myasthenia Gravis Activities of Daily Living (-2.4 [1.34] and - 3.3 [0.65]); Quantitative Myasthenia Gravis (-2.9 [1.98] and - 4.3 [0.79]); Myasthenia Gravis Composite (-4.5 [2.63] and - 4.9 [1.19]); and Myasthenia Gravis Quality of Life 15-item questionnaire (-8.6 [5.68] and - 6.5 [1.93]). Overall, the safety of eculizumab was consistent with its known safety profile. In this interim sub-analysis, the efficacy and safety of eculizumab in Japanese and Caucasian patients were generally similar, and consistent with the overall REGAIN population. ispartof: JOURNAL OF THE NEUROLOGICAL SCIENCES vol:407 ispartof: location:Netherlands status: published

Introduction: Several reports have suggested that a population of undifferentiated cells known as cancer stem cells (CSCs), is responsible for cancer formation and maintenance. In the last decade, the presence of CSCs in solid cancers have been reported. Areas covered: This review summarizes the main approaches for targeting CSCs drug resistance. It is indeed known that CSCs may contribute to resistance to conventional chemotherapy, radiotherapy and targeted agents. Among the mechanisms by which CSCs escape anticancer therapies, removal of therapeutic agents by drug efflux pumps, enhanced DNA damage repair, activation of mitogenic/anti-apoptotic pathways; the main features of CSCs, stemness and EMT, are involved, as well as the capability to evade immune response. Expert opinion: Different approaches are suitable to target CSCs mediated drug resistance. Some of them are currently under clinical evaluation in different cancer types. A better understanding of CSC biology, as well as more accurate study design, may maximize the therapeutic effects of these agents. In this respect, it is important to establish: (i) which molecules should be targeted; (ii) what drug combinations may be suitable; (iii) which patient settings will CSC targeting offer the highest clinical benefit; and (iv) how to integrate therapeutic approaches targeting CSCs with standard cancer therapy.

In several tumors the PI3K/AKT/mTOR pathway is frequently disrupted, an event that results in uncontrolled cell proliferation and tumor growth. Through the years, several compounds have been developed to inhibit the pathway at different steps: the mammalian target of rapamycin (mTOR) seemed to be the most qualified target. However, this kinase has such a key role in cell survival that mechanisms of resistance are rapidly developed. Nevertheless, clinical results obtained with mTOR inhibitors in breast cancer, renal cell carcinoma, neuroendocrine tumors and mantle cell lymphoma push oncologists to actively further develop these drugs, maybe by better selecting the population to which they are offered, through the research of predictive factors of responsiveness. In this review, we aim to describe mechanisms of resistance to mTOR inhibitors, from preclinical and clinical perspectives.

HER2 transmembrane receptor is an important target in immunotherapy treatment of breast and gastroesophageal cancer. Molecular imaging of HER2 expression may provide essential prognostic and predictive information concerning disseminated cancer and aid in selection of an optimal therapy. Radiolabeled low molecular weight peptide ligands are particularly attractive as probes for molecular imaging, since they reach and bind to the target and clear from non-target organs and blood stream faster than bulky antibodies. In this study, we evaluated a potential HER2-imaging probe, an A9 nonapeptide, derived from the trastuzumab-Fab portion. Its cellular uptake was investigated by mass spectrometry analysis of the cytoplasmic cellular extracts. Moreover, based on in-silico modeling, DTPA chelator was conjugated to N-terminus of A9. 111In-labeled A9 demonstrated nanomolar affinity to HER2-expressing BT474 cells and favorable biodistribution profile in NMRI mice. This study suggests that the peptide A9 represents a good lead candidate for development of molecular probe, to be used for imaging purposes and for the delivery of cytotoxic agents.

The urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored cell membrane receptor that focuses urokinase (uPA) proteolytic activity on the cell surface. Its expression is increased in many human cancers, including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and correlates with a poor prognosis and early invasion and metastasis. uPAR is able to control, through a cross-talk with tyrosine kinase receptors, the shift between tumor dormancy and proliferation, that usually precedes metastasis formation. Therefore, we investigated the role of uPAR expression in RAS mutated NSCLC and CRC cells. In this study we provided evidence, for the first time, that RAS mutational condition is functionally correlated to uPAR overexpression in NSCLC and CRC cancer cell lines and patient-derived tissue samples. Moreover, oncogenic features related to uPAR overexpression in RAS mutated NSCLC and CRC, such as adhesion, migration and metastatic process may be targeted, in vitro and in vivo, by new anti-uPAR small molecules, specific inhibitors of uPAR-vitronectin interaction. Therefore, anti-uPAR drugs could represent an effective pharmacological strategy for NSCLC and CRC patients carrying RAS mutations.

Background:Cetuximab is the only targeted agent approved for the treatment of head and neck squamous cell carcinomas (HNSCC), but low response rates and disease progression are frequently reported. As the phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) pathways have an important role in the pathogenesis of HNSCC, we investigated their involvement in cetuximab resistance.Methods:Different human squamous cancer cell lines sensitive or resistant to cetuximab were tested for the dual PI3K/mTOR inhibitor PF-05212384 (PKI-587), alone and in combination, both in vitro and in vivo.Results:Treatment with PKI-587 enhances sensitivity to cetuximab in vitro, even in the condition of epidermal growth factor receptor (EGFR) resistance. The combination of the two drugs inhibits cells survival, impairs the activation of signalling pathways and induces apoptosis. Interestingly, although significant inhibition of proliferation is observed in all cell lines treated with PKI-587 in combination with cetuximab, activation of apoptosis is evident in sensitive but not in resistant cell lines, in which autophagy is pre-eminent. In nude mice xenografted with resistant Kyse30 cells, the combined treatment significantly reduces tumour growth and prolongs mice survival.Conclusions:Phosphoinositide 3-kinase/mammalian target of rapamycin inhibition has an important role in the rescue of cetuximab resistance. Different mechanisms of cell death are induced by combined treatment depending on basal anti-EGFR responsiveness. © 2014 Cancer Research UK.

Several evidences suggest a marked angiogenic dependency in triple-negative breast cancer (TNBC) tumorigenesis and a potential sensitivity to anti-angiogenic agents. Herein, the putative role of Hedgehog (Hh) pathway in regulating TNBC-dependent angiogenesis was investigated. Background: Several evidences suggest a marked angiogenic dependency in triple-negative breast cancer (TNBC) tumorigenesis and a potential sensitivity to anti-angiogenic agents. Herein, the putative role of Hedgehog (Hh) pathway in regulating TNBC-dependent angiogenesis was investigated.Methods: Expression and regulation of the Hh pathway transcription factor glioma-associated oncogene homolog1 protein (GLI1) were studied on the endothelial compartment and on TNBC-initiated angiogenesis. To evaluate the translational relevance of our findings, the combination of paclitaxel with the Smo inhibitor NVP-LDE225 was tested in TNBC xenografted mice.Results: Tissue microarray analysis on 200 TNBC patients showed GLI1 overexpression paired with vascular endothelial growth factor receptor 2 (VEGFR2) expression. In vitro, Hh pathway promotes TNBC progression in an autocrine manner, regulating the VEGF/VEGFR2 loop on cancer cell surface, and in a paracrine manner, orchestrating tumour vascularisation. These effects were counteracted by Smo pharmacological inhibition. In TNBC xenografted mice, scheduling NVP-LDE225 rather than bevacizumab provided a better sustained inhibition of TNBC cells proliferation and endothelial cells organisation.Conclusions: This study identifies the Hh pathway as one of the main regulators of tumour angiogenesis in TNBC, thus suggesting Hh inhibition as a potential new anti-angiogenic therapeutic option to be clinically investigated in GLI1 overexpressing TNBC patients.

Background Airborne allergens can induce an immunological chronic disease characterized by airway hyper responsiveness and inflammation, mediated by exaggerated Th2 immune response. Allergen-specific immunotherapy (AIT) is effective for treating this condition because it is able to modify its natural course by opposing the underlying pathogenic mechanisms and determining immune suppression, immune deviation and tolerance. The rational for the present study was to investigate the possibility of improving allergoid-based IT in terms of efficacy and safety. Recently, 1α,25-dihydroxyvitamin D3 (VD3), the active metabolite of vitamin D3, was described to be a potent inducer of T regulatory cells and to be a good adjuvant in AIT settings. Methods We investigated whether the co-administration of VD3 could potentiate the effect of AIT even when added to a low dose of chemically-modified monomeric allergoid of Der p 2 (d2-OID), in a Derp p 2 (d2)-sensitized BALB/c mice model. Control groups where treated with sham, VD3 alone or d2-OID only. Results The d2-OID alone was not fully successful, as expected for a low dose. VD3 administration was associated with some valuable, although limited, changes in the immunological parameters in the lung. On the contrary, the VD3 adjuvated allergoid vaccine induced the most prominent reduction of airway eosinophilia and Th2 cytokines and concomitant increase of T regulatory cells and IL-10 in the lung and Der p 2-specific IgG2a in the serum. Conclusions The addition of VD3 to a conventional AIT protocol would allow the reduction of allergoid dose needed and therefore, the production costs. Moreover, beneficial immunomodulatory effects have been achieved by the oral administration which might favour the management of the therapy by the patients and their adherence, possibly enhancing the efficacy of the treatment. Electronic supplementary material The online version of this article (doi:10.1186/s12948-016-0044-1) contains supplementary material, which is available to authorized users.

Aberrant motility and invasive ability are relevant hallmarks of malignant tumor cells. Pathways regulating the movement of cancer cells from the site of primary tumor toward adjacent and/or distant tissues are not entirely defined. By using a model of malignant transformation induced by Ras, we identified Wnt4 as an early target of Ras oncogenic signaling. Here we show that Wnt4 is repressed by Ras and that forced Wnt4 expression inhibits Ras-induced cell motility. Accordingly, we found that Wnt4 is downregulated in human anaplastic thyroid carcinomas, the most malignant and metastatic thyroid cancer histotype. Wnt4 interferes with Ras-induced actin cytoskeleton reorganization through non-canonical pathways, by altering the balance between the activation of different Rho-family small guanosine triphosphatases (GTPases). Finally, we demonstrate that Wnt4 is post-transcriptionally repressed by miR-24, a Ras-induced micro RNA (miRNA) targeting the 3'-untranslated region (UTR) of Wnt4. Taken together our data highlight a novel Ras-regulated miRNA-dependent circuitry regulating the motile phenotype of cancer cells.Oncogene advance online publication, 1 October 2012; doi:10.1038/onc.2012.419.

Targeted therapies have been approved for various malignancies but the acquisition of resistance remains a substantial challenge in the clinical management of advanced cancers. Twenty-five per cent of breast cancers overexpress ErbB2/HER2, which confers a more aggressive phenotype and is associated with a poor prognosis. HER2-targeting therapies (trastuzumab, pertuzumab, TDM1 and lapatinib) are available, but a significant fraction of HER2-positive breast cancers eventually relapse or progress. This suggests that acquired or intrinsic resistance enables escape from HER2 inhibition. This review focuses on mechanisms of intrinsic/acquired resistance to lapatinib identified in preclinical and clinical studies. A better understanding of these mechanisms could lead to novel predictive markers of lapatinib response and to novel therapeutic strategies for breast cancer patients.

Introduction: Src tyrosine kinase overactivation has been correlated with a poor response to human epidermal growth factor receptor 2 (HER2) inhibitors in breast cancer. To identify the mechanism by which Src overexpression sustains this resistance, we tested a panel of breast cancer cell lines either sensitive or resistant to lapatinib.Methods: To determine the role of Src in lapatinib resistance, we evaluated the effects of Src inhibition/silencing in vitro on survival, migration, and invasion of lapatinib-resistant cells. In vivo experiments were performed in JIMT-1 lapatinib-resistant cells orthotopically implanted in nude mice. We used artificial metastasis assays to evaluate the effect of Src inhibition on the invasiveness of lapatinib-resistant cells. Src-dependent signal transduction was investigated with Western blot and ELISA analyses.Results: Src activation was higher in lapatinib-resistant than in lapatinib-sensitive cells. The selective small-molecule Src inhibitor saracatinib combined with lapatinib synergistically inhibited the proliferation, migration, and invasion of lapatinib-resistant cells. Saracatinib combined with lapatinib significantly prolonged survival of JIMT-1-xenografted mice compared with saracatinib alone, and impaired the formation of lung metastases. Unexpectedly, in lapatinib-resistant cells, Src preferentially interacted with epidermal growth factor receptor (EGFR) rather than with HER2. Moreover, EGFR targeting and lapatinib synergistically inhibited survival, migration, and invasion of resistant cells, thereby counteracting Src-mediated resistance. These findings demonstrate that Src activation in lapatinib-resistant cells depends on EGFR-dependent rather than on HER2-dependent signaling.Conclusions: Complete pharmacologic EGFR/HER2 inhibition is required to reverse Src-dependent resistance to lapatinib in breast cancer. © 2014 Formisano et al.; licensee BioMed Central Ltd.

Background The EGFR tyrosine kinase inhibitors gefitinib and erlotinib are first-line therapy for NSCLCs harbouring EGFR-activating mutations. The intrinsic and acquired resistance to these agents is a relevant clinical issue. Although Src tyrosine kinase has been involved in such resistance in preclinical models, clinical development of these agents has been so far limited. Methods We used a panel of human NSCLC cell lines: PC9 and HCC827 (EGFR-activating mutation; highly sensitive to erlotinib), Calu3 (EGFR/Ras wild-type, wt; moderately sensitive), Calu3-ER (with acquired resistance), H1299 and A549 (Ras mutant; resistant), H1975 (EGFR T790M mutant; resistant). In these models, we tested three different Src inhibitors (saracatinib, dasatinib, and bosutinib), both in vitro and in vivo. Findings NSCLC cell lines showed different activation of EGFR-dependent and Src-dependent pathways and variable sensitivity to Src inhibitors. A kinase assay demonstrated that all the compounds are able to directly inhibit EGFR tyrosine kinase variants. In cell lysates only saracatinib and bosutinib efficiently reduced EGFR activation, while dasatinib was the more effective agent in inhibiting Src tyrosine kinase. Consistently, in EGFR-activating mutant, erlotinib sensitive cells, saracatinib and bosutinib showed anti-proliferative effects related to simultaneous EGFR/Src inhibition. In EGFR wt/Ras mutant cells Src inhibition by dasatinib interfered with cell proliferation and signal transduction. Since Src inhibitors had only moderate effects as single agents we tested the combination of saracatinib with EGFR inhibitors (erlotinib or cetuximab) in EGFR-addicted cells, and of dasatanib with MEK inhibitors (selumetinib) in Ras mutant, erlotinib resistant models. These combinations were effective both in vitro and in nude mice, inhibiting tumour growth, prolonging mice survival, and interfering with signal transduction. Importantly, the combination of saracatanib and cetuximab was effective also in the erlotinib resistant, EGFR T790M mutant model. Interpretation Src inhibitors may act with different mechanisms in NSCLC cell lines, depending on EGFR/Ras mutational profile. Integration of anti-Src agents with EGFR or MEK inhibitors could represent effective therapeutic options for different cohorts of NSCLC patients.

Aim: Triple-negative breast cancer (TNBC) is a group of tumors that do not express HER2, estrogen and progesterone receptors. Due to reduced response to conventional antitumor therapies and poor prognosis, new targeted agents are needed for such aggressive sub-type of breast cancer. Multiple lines of evidence support the idea that deregulation of Hedgehog (Hh) signaling has a role in the pathogenesis of breast cancer, in part through the promotion of epithelial-stromal interactions. Therefore, the inhibition of the Hh pathway has been proposed as an interesting therapeutic approach Methods: The main objective of this study is to investigate the role of Hh signaling pathway in TNBC. To this aim, we used a panel of human breast cancer cell lines, including five cancer cells lines positive for ER, PR and HER2 expression (nTNBC) and five Triple Negative Breast Cancer cell lines (TNBC). The effects induced by the Smo-inhibitor NVP-LDE225 on proliferation, angiogenesis and signal transduction of breast cancer cells were investigated Results: GLI1, one of the major transcription factors induced by Hh signaling activation, is more expressed in TNBC than in nTNBC cell lines. Consistently, NVP-LDE225 treatment induced a more pronounced inhibitory effect on TNBC, in terms of tumor growth: while nTNBC cells display an IC50 of∼5mM, TNBC cell lines are more sensitive, with an average IC50 of∼2mM. In addition, Hh inhibition caused a robust impairment of TNBC cells invasion capabilities. These effects are coupled with a strong inhibition of VEGFA production by both tumor and stromal cells (human fibroblasts and HUVECs). Accordingly, NVP-LDE225 treatment interfered with HUVEC capillary tube formation, an effect even more evident than that observed with bevacizumab, the only targeted agent approved to date for TNBC patients Conclusions: Our results suggest that Hh has a specific role in breast epithelial-stromal interactions by regulation of angiogenesis. An orthotopic in vivo experiment in nude mice xenografted with TNBC cells, testing the combination of NVP-LDE225 with bevacizumab, is ongoing Disclosure: All authors have declared no conflicts of interest.

Background: Multiple lines of evidence support that the Hedgehog (Hh) signalling has a role in the maintenance and progression of different human cancers. Therefore, inhibition of the Hh pathway represents a valid anticancer therapeutic approach for renal cell carcinoma (RCC) patients. NVP-LDE225 is a Smoothened (Smo) antagonist that induces dose-related inhibition of Hh and Smo-dependent tumour growth. Methods: We assayed the effects of NVP-LDE225 alone or in combination with everolimus or sunitinib on the growth and invasion of human RCC models both in vitro and in vivo. To this aim, we used a panel of human RCC models, comprising cells with acquired resistance to sunitinib - a multiple tyrosine kinase inhibitor approved as a first-line treatment for RCC. Results: NVP-LDE225 cooperated with either everolimus or sunitinib to inhibit proliferation, migration, and invasion of RCC cells even in sunitinib-resistant (SuR) cells. Some major transducers involved in tumour cell motility, including paxillin, were also efficiently inhibited by the combination therapy, as demonstrated by western blot and confocal microscopy assays. Moreover, these combined treatments inhibited tumour growth and increased animal survival in nude mice xenografted with SuR RCC cells. Finally, lung micrometastasis formation was reduced when mice were treated with NVP-LDE225 plus everolimus or sunitinib, as evidenced by artificial metastatic assays. Conclusions: Hedgehog inhibition by NVP-LDE225 plus sunitinib or everolimus bolsters antitumour activity by interfering with tumour growth and metastatic spread, even in SuR cells. Thus, this new evidence puts forward a new promising therapeutic approach for RCC patients.

Purpose: Although the anti–EGF receptor (EGFR) monoclonal antibody cetuximab is an effective strategy in colorectal cancer therapy, its clinical use is limited by intrinsic or acquired resistance. Alterations in the “sphingolipid rheostat”—the balance between the proapoptotic molecule ceramide and the mitogenic factor sphingosine-1-phosphate (S1P)—due to sphingosine kinase 1 (SphK1) overactivation have been involved in resistance to anticancer-targeted agents. Moreover, cross-talks between SphK1 and EGFR-dependent signaling pathways have been described. Experimental design: We investigated SphK1 contribution to cetuximab resistance in colorectal cancer, in preclinical in vitro/in vivo models, and in tumor specimens from patients. Results: SphK1 was found overexpressed and overactivated in colorectal cancer cells with intrinsic or acquired resistance to cetuximab. SphK1 contribution to resistance was supported by the demonstration that SphK1 inhibition by N,N-dimethyl-sphingosine or silencing via siRNA in resistant cells restores sensitivity to cetuximab, whereas exogenous SphK1 overexpression in sensitive cells confers resistance to these agents. Moreover, treatment of resistant cells with fingolimod (FTY720), a S1P receptor (S1PR) antagonist, resulted in resensitization to cetuximab both in vitro and in vivo, with inhibition of tumor growth, interference with signal transduction, induction of cancer cells apoptosis, and prolongation of mice survival. Finally, a correlation between SphK1 expression and cetuximab response was found in colorectal cancer patients. Clin Cancer Res; 19(1); 138–47. ©2012 AACR.

Nerve growth factor (NGF) induces terminal differentiation in PC12, a pheochromocytoma-derived cell line. NGF binds a specific receptor on the membrane and triggers the ERK1/2 cascade, which stimulates the transcription of neural genes. We report that NGF significantly affects mitochondrial metabolism by reducing mitochondrial-produced reactive oxygen species and stabilizing the electrochemical gradient. This is accomplished by stimulation of mitochondrial manganese superoxide dismutase (MnSOD) both transcriptionally and post-transcriptionally via Ki-Ras and ERK1/2. Activation of MnSOD is essential for completion of neuronal differentiation because 1) expression of MnSOD induces the transcription of a neuronal specific promoter and neurite outgrowth, 2) silencing of endogenous MnSOD by small interfering RNA significantly reduces transcription induced by NGF, and 3) a Ki-Ras mutant in the polylysine stretch at the COOH terminus, unable to stimulate MnSOD, fails to induce complete differentiation. Overexpression of MnSOD restores differentiation in cells expressing this mutant. ERK1/2 is also downstream of MnSOD, as a SOD mimetic drug stimulates ERK1/2 with the same kinetics of NGF and silencing of MnSOD reduces NGF-induced late ERK1/2. Long term activation of ERK1/2 by NGF requires SOD activation, low levels of hydrogen peroxide, and the integrity of the microtubular cytoskeleton. Confocal immunofluorescence shows that NGF stimulates the formation of a complex containing membrane-bound Ki-Ras, microtubules, and mitochondria. We propose that active NGF receptor induces association of mitochondria with plasma membrane. Local activation of ERK1/2 by Ki-Ras stimulates mitochondrial SOD, which reduces reactive oxygen species and produces H(2)O(2). Low and spatially restricted levels of H(2)O(2) induce and maintain long term ERK1/2 activity and ultimately differentiation of PC12 cells.

Aim: The EGFR TKIs gefitinib and erlotinib are the first-line therapy for NSCLCs harboring EGFR-activating mutations. The intrinsic resistance to these agents and the onset of acquired resistance in the responders is a relevant clinical issue and the aim of this work is to test the efficacy of three different Src inhibitors in these models of resistance. Methods: We used a panel of human NSCLC cell lines with different EGFR/Ras mutational profile and erlotinib sensitivity: PC9 and HCC827 (EGFR-activating mutation, highly sensitive), Calu3 (EGFR/Ras wt; moderately sensitive), Calu3-ER (with acquired resistance), H1299 and A549 (Ras mutant; resistant), H1975 (EGFR T790M mutant; resistant). In these models, we tested three different Src inhibitors (saracatinib, dasatinib and bosutinib), both in vitro and in vivo Results: NSCLC cell lines showed different activation of EGFR- and Src-dependent pathways and variable sensitivity to Src inhibitors. A kinase assay demonstrated that all the compounds are able to directly inhibit not only Src, but also EGFR TK variants. However, in cell lysates only saracatinib and bosutinib efficiently reduced EGFR activation, while dasatinib was the more effective agent in inhibiting Src TK. In EGFR-activating mutant, erlotinib sensitive cells, saracatinib and bosutinib showed anti-proliferative effects related to simultaneous EGFR/Src inhibition. In EGFR wt/Ras mutant cells Src inhibition by dasatinib interfered with cell proliferation and signal transduction. Since Src inhibitors had only moderate effects as single agents, both in vitro and in vivo, we tested the combination of saracatinib with EGFR inhibitors (erlotinib or cetuximab) in EGFR-addicted cells, and of dasatanib with MEK inhibitors (selumetinib) in Ras mutant, erlotinib resistant models. These combinations were effective both in vitro and in vivo, inhibiting tumor growth, prolonging mice survival and interfering with signal transduction. Importantly, the combination of saracatanib and cetuximab was effective also in the erlotinib resistant, EGFR T790M mutant model. Conclusions: Src inhibitors may act with different mechanisms in NSCLC cell lines, depending on EGFR/Ras mutational profile. Integration of anti-Src agents with EGFR or MEK inhibitors could represent effective therapeutic options for different cohorts of NSCLC patients. Disclosure: All authors have declared no conflicts of interest.

Background. The dual EGFR/HER2 tyrosine kinase inhibitor (TKI) lapatinib is approved in metastatic breast cancer patients after trastuzumab failure. Unfortunately, several patients do not respond to treatment due to constitutive resistance, and even in responders the occurrence of disease progression is often observed. Recently, the activation of Src TK has been associated with resistance to anti-HER2 drugs, both trastuzumab and lapatinib, in HER2 overexpressing breast cancer cells. However, the molecular mechanisms by which Src activation may affect response to HER2 antagonists are still partially unclear. Methods. We used a panel of HER2 expressing human breast cancer cell lines with different degree of response to lapatinib: SKBR3, BT474 and MDA-MB-361 (high sensitivity), KPL-4 (moderate sensitivity), JIMT-1 (low sensitivity). Moreover, we generated a cell line with acquired resistance to lapatinib, MDA-MB-361-LR (Lapatinib Resistant). On these models, we investigated the effect of Src silencing via small interference RNA (siRNA) or pharmacological inhibition by saracatinib on lapatinib response, both in vitro and in vivo. Results. The combination of Src inhibition with lapatinib synergistically inhibited survival of human breast cancer cells with both intrinsic or acquired resistance to lapatinib, by interfering with signal transducers involved in cell proliferation and survival. Moreover, both saracatinib and Src specific siRNAs were able to reduce migration and invasion capabilities of breast cancer cells, by affecting Focal Adhesion Kinase (FAK) and paxillin activation. In nude mice xenografted with JIMT-1 cells, the combined treatment with saracatinib and lapatinib produced a cooperative antitumor effect with inhibition of tumor growth, interference with signal transduction and prolongation of mice survival. The combination strongly reduced the incidence of lung metastasis, as revealed through experimental metastasis assays. Interestingly, a significant reduction of EGFR/HER2 co-immunoprecipitation was found in resistant cells, thus suggesting that growth signals could be transduced by other type of TKRs dimers in these cells. Moreover, in breast cancer cell lines with both intrinsic or acquired resistance to lapatinib, Src interacted with EGFR rather than with HER2, in opposition to what was observed in sensitive cells. Consistently, EGFR inhibition through cetuximab or EGFR specific siRNAs was able to partially restore lapatinib sensitivity in resistant cells, by interfering with the reciprocal activation of EGFR and Src. Conclusions. Src plays an important role in the onset of resistance to lapatinib in breast cancer cell lines, and its interaction with EGFR may be involved in this event. Therefore, the combined inhibition of Src and EGFR/HER2 axis may represent a potentially effective therapeutic strategy in order to prevent or overcome resistance to lapatinib. Citation Format: Roberta Rosa, Lucia Nappi, Luigi Formisano, Claudia D'Amato, Valentina D'Amato, Vincenzo Damiano, Roberta Marciano, Ana Paula De Maio, Lucia Raimondo, Umberto Malapelle, Giancarlo Troncone, Sabino De Placido, Roberto Bianco. Src tyrosine kinase contributes to lapatinib resistance in human breast cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 950. doi:10.1158/1538-7445.AM2013-950 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Background HER2, a member of the HER family, is a transmembrane tyrosine kinase receptor overexpressed in almost 30% of breast cancer patients. Lapatinib is a small molecule HER2 inhibitor approved in metastatic breast cancer patients after trastuzumab failure. Unfortunately, the resistance against lapatinib is observed even in responding patients. Src is an intracellular kinase involved in tumor growth, angiogenesis and migration and it is related to trastuzumab resistance in human breast cancer cell lines. Materials and methods We used different HER2 expressing human breast cancer cell lines, such as MDA-MB-361, MDA-MB-231 and BT474 (sensitive to lapatinib) JIMT-1 and KPL-4 (low sensitivity against lapatinib), and MDA-MB-361-LR (acquired resistance to lapatinib). We performed survival, migration and invasion assays and WB analysis in all cell lines treated with lapatinib, saracatinib (a Src inhibitor) or the combination. We also used nude mice xenografted with JIMT-1 cells and in vivo artificial metastatic assay. Results We observed that the combination treatment of lapatinib plus saracatinib is able to inhibit survival, migration and invasion in vitro and to regulate several proteins such as Src, Akt, MAPK, paxillin and FAK in all cancer cell lines tested. In nude mice xenografted with JIMT-1 cells, the combined treatment reduced tumor growth, prolonged survival and strongly decreased the incidence of lung metastases. Interestingly, we observed that Src preferentially binds to HER2 in lapatinib sensitive cells and with EGFR in resistant ones. EGFR inhibition, through the use of cetuximab or siRNA silencing, strongly enhanced the effect of lapatinib on the growth of resistant cancer cells. The combined treatment of lapatinib and cetuximab, moreover, significantly reduced the activation of several intracellular transducers. Conclusions We demonstrated that Src plays an important role in the development of resistance to lapatinib in breast cancer cell lines, in particular affecting invasion and migration, and that EGFR may mediate its activation. These results may suggest the clinical development of lapatinib and cetuximab combination in patients resistant to lapatinib. Disclosure All authors have declared no conflicts of interest.

Resistance to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, often related to Ras or secondary EGFR mutations, is a relevant clinical issue in Non- Small Cell Lung Cancer (NSCLC). Although Src TK has been involved in such resistance, clinical development of its inhibitors has been so far limited. To better define the molecular targets of the Src TKIs saracatinib, dasatinib and bosutinib, we used a variety of in vitro/in vivo studies. Kinase assays supported by docking analysis demonstrated that all the compounds directly inhibit EGFR TK variants. However, in live cells only saracatinib efficiently reduced EGFR activation, while dasatinib was the most effective agent in inhibiting Src TK. Consistently, a pronounced anti-proliferative effect was achieved with saracatinib, in EGFR mutant cells, or with dasatinib, in wt EGFR/Ras mutant cells, poorly dependent on EGFR and erlotinib-resistant. We then identified the most effective drug combinations to overcome resistance to EGFR inhibitors, both in vitro and in nude mice: in T790M EGFR erlotinib-resistant cells, saracatinib with the anti-EGFR mAb cetuximab; in Ras mutant erlotinib-resistant models, dasatinib with the MEK inhibitor selumetinib. Src inhibitors may act with different mechanisms in NSCLCs, depending on EGFR/Ras mutational profile, and may be integrated with EGFR or MEK inhibitors for different cohorts of NSCLCs.

Almost all people in developed countries are exposed to metal nanoparticles (MeNPs) that are used in a large number of applications including medical (for diagnostic and therapeutic purposes). Once inside the body, absorbed by inhalation, contact, ingestion and injection, MeNPs can translocate to tissues and, as any foreign substance, are likely to encounter the innate immunity system that represent a non-specific first line of defense against potential threats to the host. In this review, we will discuss the possible effects of MeNPs on various components of the innate immunity (both specific cells and barriers). Most important is that there are no reports of immune diseases induced by MeNPs exposure: we are operating in a safe area. However, in vitro assays show that MeNPs have some effects on innate immunity, the main being toxicity (both cyto- and genotoxicity) and interference with the activity of various cells through modification of membrane receptors, gene expression and cytokine production. Such effects can have both negative and positive relevant impacts on humans. On the one hand, people exposed to high levels of MeNPs, as workers of industries producing or applying MeNPs, should be monitored for possible health effects. On the other hand, understanding the modality of the effects on immune responses is essential to develop medical applications for MeNPs. Indeed, those MeNPs that are able to stimulate immune cells could be used to develop of new vaccines, promote immunity against tumors and suppress autoimmunity.

Inhibition of the mechanistic target of rapamycin (mTOR) is a promising treatment strategy for several cancer types. Rapamycin derivatives such as everolimus are allosteric mTOR inhibitors acting through interaction with the intracellular immunophilin FKBP12, a prolyl isomerase with different cellular functions. Although mTOR inhibitors have significantly improved survival of different cancer patients, resistance and lack of predictive factors of response remain unsolved issues. To elucidate the mechanisms of resistance to everolimus, we evaluated Met activation in everolimus-sensitive/resistant human cancer cells, in vitro and in vivo. Biochemical and computational analyses were performed. Everolimus-resistant cells were xenografted into mice (10/group) and studied for their response to everolimus and Met inhibitors. The statistical significance of the in vitro results was evaluated by Student's t test. Everolimus reduced Met phosphorylation in everolimus-sensitive cells. This event was mediated by the formation of a Met-FKBP12 complex, which in turn is disrupted by everolimus. Aberrant Met activation in everolimus-resistant cells and overexpression of wild-type/mutant Met caused everolimus resistance. Pharmacological inhibition and RNA silencing of Met are effective in condition of everolimus resistance (P