ODZ1 allows glioblastoma to sustain invasiveness through a Myc-dependent transcriptional upregulation of RhoA.

Long-term survival remains low for most patients with glioblastoma (GBM), which reveals the need for markers of disease outcome and novel therapeutic targets. We describe that ODZ1 (also known as TENM1), a type II transmembrane protein involved in fetal brain development, plays a crucial role in the invasion of GBM cells. Differentiation of glioblastoma stem-like cells drives the nuclear translocation of an intracellular fragment of ODZ1 through proteolytic cleavage by signal peptide peptidase-like 2a. The intracellular fragment of ODZ1 promotes cytoskeletal remodelling of GBM cells and invasion of the surrounding environment both in vitro and in vivo. Absence of ODZ1 by gene deletion or downregulation of ODZ1 by small interfering RNAs drastically reduces the invasive capacity of GBM cells. This activity is mediated by an ODZ1-triggered transcriptional pathway, through the E-box binding Myc protein, that promotes the expression and activation of Ras homolog family member A (RhoA) and subsequent activation of Rho-associated, coiled-coil containing protein kinase (ROCK). Overexpression of ODZ1 in GBM cells reduced survival of xenografted mice. Consistently, analysis of 122 GBM tumour samples revealed that the number of ODZ1-positive cells inversely correlated with overall and progression-free survival. Our findings establish a novel marker of invading GBM cells and consequently a potential marker of disease progression and a therapeutic target in GBM.

Breast cancer dissemination promoted by a neuregulin-collagenase 3 signalling node.

Advances in the treatment of breast cancer have resulted in increased survival. However, in the metastatic setting, the disease remains incurable. Therefore, understanding of the mechanisms that promote dissemination of breast cancer cells may favor the development of novel therapeutic strategies to fight those tumors. Here, we show that the ErbB ligands, Neuregulins (NRGs), promote metastatic dissemination of breast cancer cells by switching on a kinase-metalloproteinase network. Clinicopathological analyses demonstrated that NRG expression in breast tumors associated to lymph node invasion and poor patient outcome. Preclinical in vivo analyses showed that NRG expression favored in situ tumor growth, local spreading and metastatic dissemination. Genomic, biochemical and functional studies identified matrix metalloproteinases, particularly stromelysin 2 and collagenase 3, as key mediators of the NRG-induced dissemination properties of breast cancer cells. Mechanistic analyses demonstrated that NRG augmented metalloproteinase expression through a route controlled by ERK1/2 kinases. ERK1/2 increased collagenase 3 expression by controlling the activity of an SBF1-related transcription factor. In conclusion, we describe a pathway linked to breast cancer dissemination. The clinical availability of agents that target some of the components of this signalling pathway suggests that patients with tumors fed by NRGs or other factors able to activate the ERK-Collagenase 3 route may benefit from agents that act on that signalling axis.

In vivo murine model of acquired resistance in myeloma reveals differential mechanisms for lenalidomide and pomalidomide in combination with dexamethasone.

The development of resistance to therapy is unavoidable in the history of multiple myeloma patients. Therefore, the study of its characteristics and mechanisms is critical in the search for novel therapeutic approaches to overcome it. This effort is hampered by the absence of appropriate preclinical models, especially those mimicking acquired resistance. Here we present an in vivo model of acquired resistance based on the continuous treatment of mice bearing subcutaneous MM1S plasmacytomas. Xenografts acquired resistance to two generations of immunomodulatory drugs (IMiDs; lenalidomide and pomalidomide) in combination with dexamethasone, that was reversible after a wash-out period. Furthermore, lenalidomide-dexamethasone (LD) or pomalidomide-dexamethasone (PD) did not display cross-resistance, which could be due to the differential requirements of the key target Cereblon and its substrates Aiolos and Ikaros observed in cells resistant to each combination. Differential gene expression profiles of LD and PD could also explain the absence of cross-resistance. Onset of resistance to both combinations was accompanied by upregulation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway and addition of selumetinib, a small-molecule MEK inhibitor, could resensitize resistant cells. Our results provide insights into the mechanisms of acquired resistance to LD and PD combinations and offer possible therapeutic approaches to addressing IMiD resistance in the clinic.

Active kinase profiling, genetic and pharmacological data define mTOR as an important common target in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer. Despite response to chemotherapy, relapses are frequent and resistance to available treatments is often seen in the metastatic setting. Therefore, identification of new therapeutic targets is required. With this aim, we have profiled the activation status of 44 receptor tyrosine kinases (RTKs) and their major signaling pathways in patient-derived TNBC tumors. Frequent co-activation of several RTKs as well as the extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) and mammalian target of rapamycin (mTOR) routes was found. Pharmacologic targeting of the activated kinases indicated that agents that attack the mTOR route are more potent and efficient antitumoral treatments than agents targeting RTKs. mTOR signals through two multiprotein complexes, mTORC1 and mTORC2. We used a genetic approach to explore the contribution of each of the two mTOR branches to the regulation of cell number of TNBC cells. RNA interference experiments indicated that mTORC1 predominated over mTORC2 in the control of TNBC cell proliferation. Moreover, RNA interference of mTOR had a superior antiproliferative action than separately acting on mTORC1 or mTORC2. To analyze the relevance of mTOR targeting in vivo, we used mice with TNBC. Treatment of these mice with BEZ235, a drug that targets mTOR, slowed tumor growth. Mechanistically, BEZ235 delayed cell cycle progression without affecting cell viability. Our results show that TNBCs are particularly sensitive to inhibition of the mTOR pathway, and indicate that mTOR targeting may be a more efficient anti-TNBC therapy than exclusively acting on the mTORC1 branch of the pathway. This is relevant as most mTOR inhibitors used in the clinic act on mTORC1. Collectively with the fact that BEZ235 synergized with drugs commonly used in the treatment of TNBC, our data support the clinical development of agents that act on mTOR as a therapy for this disease.

ErbBs inhibition by lapatinib blocks tumor growth in an orthotopic model of human testicular germ cell tumor.

In this work, we have analyzed the expression of different members of the ErbB family in human samples of testicular germ cell tumors (GCTs). We observed expression of ErbB1 or ErbB2 in different tumor subtypes, but we also found high expression of ErbB3 in all GCTs tested. This pattern of expression was maintained when primary tumors were orthotopically implanted in nude mice. We have chosen a choriocarcinoma model characterized by high levels of ErbB1, but also of ErbB2 and ErbB3, to assay the in vivo effect of ErbB inhibitors on tumoral growth. Our results showed a complete lack of effect (refractoriness) to the pure ErbB1 receptor inhibitors cetuximab and gefitinib. While these inhibitors blocked ErbB1 phosphorylation, ErbB2 phosphorylation was not affected, suggesting an ErbB1-independent activation of this receptor. To confirm the importance of ErbB2 activation, animals were treated with lapatinib, a dual ErbB1 and ErbB2 inhibitor. Lapatinib treatment caused a 50% inhibition in tumor growth, an effect correlated with a blockade of both ErbB1 and ErbB2 phosphorylation levels, and of downstream signaling pathways (Akt, ERKs and Stat3). ErbB2 activation could still occur due to the formation of ErbB2/ErbB3 heterodimers, and ErbB3 activation was completely inhibited by lapatinib. Finally, combined inhibition of ErbB1 (gefitinib) and ErbB3 activities (knockdown expression by shRNA) inhibited tumoral testicular cells proliferation in a similar way to lapatinib. Our results explain why lapatinib but not anti-ErbB1 agents might be effective for treatment of testicular GCT patients.

A dominant-negative N-terminal fragment of HER2 frequently expressed in breast cancers.

The transmembrane tyrosine kinase HER2 (ErbB2, neu) is a prototypical biomarker for breast cancers and a therapeutic target. Although anti-HER2 therapies are remarkably effective, HER2-positive tumors are heterogeneous and some subtypes do not respond or develop resistance to these therapies. Here we show that H2NTF, a novel N-terminal fragment of HER2, is expressed at variable levels in 60% of the breast cancer samples analyzed. Characterization of H2NTF shows that it is devoid of the tyrosine kinase domain but it readily interacts with full-length HER2 and other HER receptors. As a consequence, H2NTF acts as a dominant-negative, attenuating the signaling triggered by full-length HER receptors. Expression of H2NTF results in resistance to the treatment with low concentrations of trastuzumab in vitro. However, cells expressing H2NTF and non-expressing cells have similar sensitivity to trastuzumab in vivo, indicating that H2NTF/trastuzumab complexes trigger antibody-dependent cell-mediated cytotoxicity.

The epoxyketone-based proteasome inhibitors carfilzomib and orally bioavailable oprozomib have anti-resorptive and bone-anabolic activity in addition to anti-myeloma effects.

Proteasome inhibitors (PIs), namely bortezomib, have become a cornerstone therapy for multiple myeloma (MM), potently reducing tumor burden and inhibiting pathologic bone destruction. In clinical trials, carfilzomib, a next generation epoxyketone-based irreversible PI, has exhibited potent anti-myeloma efficacy and decreased side effects compared with bortezomib. Carfilzomib and its orally bioavailable analog oprozomib, effectively decreased MM cell viability following continual or transient treatment mimicking in vivo pharmacokinetics. Interactions between myeloma cells and the bone marrow (BM) microenvironment augment the number and activity of bone-resorbing osteoclasts (OCs) while inhibiting bone-forming osteoblasts (OBs), resulting in increased tumor growth and osteolytic lesions. At clinically relevant concentrations, carfilzomib and oprozomib directly inhibited OC formation and bone resorption in vitro, while enhancing osteogenic differentiation and matrix mineralization. Accordingly, carfilzomib and oprozomib increased trabecular bone volume, decreased bone resorption and enhanced bone formation in non-tumor bearing mice. Finally, in mouse models of disseminated MM, the epoxyketone-based PIs decreased murine 5TGM1 and human RPMI-8226 tumor burden and prevented bone loss. These data demonstrate that, in addition to anti-myeloma properties, carfilzomib and oprozomib effectively shift the bone microenvironment from a catabolic to an anabolic state and, similar to bortezomib, may decrease skeletal complications of MM.

Clinical significance of CD81 expression by clonal plasma cells in high-risk smoldering and symptomatic multiple myeloma patients.

The presence of CD19 in myelomatous plasma cells (MM-PCs) correlates with adverse prognosis in multiple myeloma (MM). Although CD19 expression is upregulated by CD81, this marker has been poorly investigated and its prognostic value in MM remains unknown. We have analyzed CD81 expression by multiparameter flow cytometry in MM-PCs from 230 MM patients at diagnosis included in the Grupo Español de Mieloma (GEM)05>65 years trial as well as 56 high-risk smoldering MM (SMM). CD81 expression was detected in 45% (103/230) MM patients, and the detection of CD81(+) MM-PC was an independent prognostic factor for progression-free (hazard ratio=1.9; P=0.003) and overall survival (hazard ratio=2.0; P=0.02); this adverse impact was validated in an additional series of 325 transplant-candidate MM patients included in the GEM05 <65 years trial. Moreover, CD81(+) SMM (n=34/56, 57%) patients had a shorter time to progression to MM (P=0.02). Overall, our results show that CD81 may have a relevant role in MM pathogenesis and represent a novel adverse prognostic marker in myeloma.

Differential action of small molecule HER kinase inhibitors on receptor heterodimerization: therapeutic implications.

Deregulation of ErbB/HER receptor tyrosine kinases has been linked to several types of cancer. The mechanism of activation of these receptors includes establishment of receptor dimers. Here, we have analyzed the action of different small molecule HER tyrosine kinase inhibitors (TKIs) on HER receptor dimerization. Breast cancer cell lines were treated with distinct TKIs and the formation of HER2-HER3 dimers was analyzed by coimmunoprecipitation and western blot or by Förster resonance energy transfer assays. Antibody-dependent cellular cytotoxicity was analyzed by measuring the release of lactate dehydrogenase and cell viability. Lapatinib and neratinib interfered with ligand-induced dimerization of HER receptors; while pelitinib, gefitinib, canertinib or erlotinib did not. Moreover, lapatinib and neratinib were able to disrupt previously formed receptor dimers. Structural analyses allowed the elucidation of the mechanism by which some TKIs prevent the formation of HER receptor dimers, while others do not. Experiments aimed at defining the functional importance of dimerization indicated that TKIs that impeded dimerization prevented down-regulation of HER2 receptors, and favored the action of trastuzumab. We postulate that TKIs that prevent dimerization and down-regulation of HER2 may augment the antitumoral action of trastuzumab, and this mechanism of action should be considered in the treatment of HER2 positive tumors which combine TKIs with antireceptor antibodies.

Sox2 expression in breast tumours and activation in breast cancer stem cells.

The cancer stem cell (CSC) model does not imply that tumours are generated from transformed tissue stem cells. The target of transformation could be a tissue stem cell, a progenitor cell, or a differentiated cell that acquires self-renewal ability. The observation that induced pluripotency reprogramming and cancer are related has lead to the speculation that CSCs may arise through a reprogramming-like mechanism. Expression of pluripotency genes (Oct4, Nanog and Sox2) was tested in breast tumours by immunohistochemistry and it was found that Sox2 is expressed in early stage breast tumours. However, expression of Oct4 or Nanog was not found. Mammosphere formation in culture was used to reveal stem cell properties, where expression of Sox2, but not Oct4 or Nanog, was induced. Over-expression of Sox2 increased mammosphere formation, effect dependent on continuous Sox2 expression; furthermore, Sox2 knockdown prevented mammosphere formation and delayed tumour formation in xenograft tumour initiation models. Induction of Sox2 expression was achieved through activation of the distal enhancer of Sox2 promoter upon sphere formation, the same element that controls Sox2 transcription in pluripotent stem cells. These findings suggest that reactivation of Sox2 represents an early step in breast tumour initiation, explaining tumour heterogeneity by placing the tumour-initiating event in any cell along the axis of mammary differentiation.

P-Rex1 participates in Neuregulin-ErbB signal transduction and its expression correlates with patient outcome in breast cancer.

The Neuregulins and their receptors, the ErbB/HER subfamily of receptor tyrosine kinases, have critical roles in animal physiology, and their deregulation is frequent in cancer. Here we report the identification of the guanine nucleotide exchange factor, phosphatidylinositol 3,4,5-triphosphate-dependent Rac exchanger 1 (P-Rex1), as a novel mediator in signalling by ErbB/HER receptors. P-Rex1 was formerly described as a phosphoinositide 3-kinase and Gßγ activated protein that regulates Rac function. We define how ErbB/HER receptors regulate P-Rex1 function, which involves dephosphorylation of inhibitory residues, and phosphorylation of activating residues of P-Rex. The net balance resulting from activation of this phosphorylation/dephosphorylation cycle of P-Rex1 favours Rac activation. Molecular and biological studies indicated that P-Rex1 phosphorylation regulated the proliferation of breast cancer cells, and P-Rex1 knockdown affected their migration or invasiveness, as well as their in vivo tumourigenic potential. Moreover, as we found correlation between high P-Rex1 expression and poor patient outcome in breast cancer, P-Rex1 targeting may be therapeutically relevant in cancer.

The synergy of panobinostat plus doxorubicin in acute myeloid leukemia suggests a role for HDAC inhibitors in the control of DNA repair.

Acute myeloid leukemia (AML) is a clonal disorder characterized by the accumulation of myeloid blasts in the bone marrow. Here, we report the effects of the novel histone deacetylase inhibitor panobinostat (LBH589) in combination with doxorubicin on AML cells. Panobinostat exhibited potent anti-AML activity in all AML cell lines tested and in primary AML cells from patients (IC(50)<20 nM). In addition, panobinostat potentiated the action of several standard-of-care anti-AML compounds, particularly, doxorubicin. The molecular effects induced by panobinostat and doxorubicin treatment were investigated by analyzing gene expression, cell cycle, apoptosis and signaling pathways. Analyses of gene expression profiles identified 588 genes whose expression was exclusively affected by the combination of panobinostat and doxorubicin. The combination induced AML cell death by an increase in the mitochondrial outer membrane permeability and release of cytochrome c from the mitochondria, resulting in caspase-dependent apoptosis and accompanied by the upregulation of Bax, Bak and, particularly, Bad. The drug combination provoked a strong activation of a DNA damage response, indicating that this combination may trigger cell death by a mechanism that induced DNA double-strand breaks. These data indicate that the combination of panobinostat and doxorubicin may be an effective therapy for the treatment of AML.

Mesenchymal stem cells from multiple myeloma patients display distinct genomic profile as compared with those from normal donors.

It is an open question whether in multiple myeloma (MM) bone marrow stromal cells contain genomic alterations, which may contribute to the pathogenesis of the disease. We conducted an array-based comparative genomic hybridization (array-CGH) analysis to compare the extent of unbalanced genomic alterations in mesenchymal stem cells from 21 myeloma patients (MM-MSCs) and 12 normal donors (ND-MSCs) after in vitro culture expansion. Whereas ND-MSCs were devoid of genomic imbalances, several non-recurrent chromosomal gains and losses (>1 Mb size) were detected in MM-MSCs. Using real-time reverse transcription PCR, we found correlative deregulated expression for five genes encoded in regions for which genomic imbalances were detected using array-CGH. In addition, only MM-MSCs showed a specific pattern of 'hot-spot' regions with discrete (<1 Mb) genomic alterations, some of which were confirmed using fluorescence in situ hybridization (FISH). Within MM-MSC samples, unsupervised cluster analysis did not correlate with particular clinicobiological features of MM patients. We also explored whether cytogenetic abnormalities present in myelomatous plasma cells (PCs) were shared by matching MSCs from the same patients using FISH. All MM-MSCs were cytogenetically normal for the tested genomic alterations. Therefore we cannot support a common progenitor for myeloma PCs and MSCs.

Synergic antitumoral effect of an IGF-IR inhibitor and trastuzumab on HER2-overexpressing breast cancer cells.

BACKGROUND: Receptor tyrosine kinases play an important role in breast cancer. One of them, the type I insulin-like growth factor, has been linked to resistance to trastuzumab (Herceptin), an agent that targets human epidermal growth factor receptor 2. Here, we show that the insulin-like growth factor-I receptor (IGF-IR) antagonist NVP-AEW541 inhibits proliferation of breast cancer cells and synergizes with trastuzumab. PATIENTS AND METHODS: Patient samples and breast cancer cell lines were evaluated for IGF-IR expression or activation by western blotting. 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) uptake assays and Annexin V staining were used for the analyses of cell proliferation/apoptosis. Biochemical and genomic studies were carried out to gain insights into the mechanism of action of NVP-AEW541. RESULTS: The IGF-IR was expressed above normal levels in a number of breast cancer samples. Activation of this receptor was inhibited by NVP-AEW541 that also decreased cell proliferation and increased apoptosis. NVP-AEW541 decreased the amount of pAkt and increased the level of p27. Combination studies with several drugs used in the breast cancer clinic showed that NVP-AEW541 synergistically increased the action of trastuzumab. CONCLUSIONS: Our results show the anti-breast cancer action of NVP-AEW541 and support the clinical development of anti-IGF-IR agents, especially in combination with trastuzumab.

Pemetrexed acts as an antimyeloma agent by provoking cell cycle blockade and apoptosis.

Multiple myeloma (MM) is a malignancy of terminally differentiated B lymphocytes, characterized by accumulation of a monotypic plasma cell population in the bone marrow, monoclonal immunoglobulin in serum and/or urine and osteolytic lesions. Despite recent advances in the treatment, MM remains an incurable disease. This calls for an effort to develop novel therapeutics in order to eradicate the disease. Here we have evaluated the potential antimyeloma action of Pemetrexed, an antifolate drug that has shown promising results in other neoplastic diseases. Pemetrexed had a potent antimyeloma effect on cell lines sensitive and resistant to conventional therapeutic agents, and was also efficient on fresh cells from patients and in a murine MM model. Furthermore, Pemetrexed abrogated the protective action on MM cell death of several growth factors produced by the bone marrow microenvironment. Mechanistic studies indicated that Pemetrexed provoked this action by a combined effect that included cell cycle blockade, probably by p21 upregulation, and induction of apoptosis through caspase-dependent and -independent mechanisms. These data, together with the fact that Pemetrexed is already licensed for the therapy of other neoplastic diseases, opens the possibility for the inclusion of Pemetrexed in the therapeutic armamentarium to battle MM.

Targeting receptor tyrosine kinases and their signal transduction routes in head and neck cancer.

Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer in the world. At present several therapeutic approaches, including surgical removal, chemotherapy and radiotherapy, are used. Yet a significant number of patients relapse, often with metastases. In an attempt to improve treatment of SCCHN new targeted therapies are emerging. Among them special interest has been devoted to agents that act on the epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases, or the signal transduction routes used by these receptors to induce tumour cell proliferation. Such treatments include monoclonal antibodies and small molecule inhibitors of either the intracellular tyrosine kinase activity of these receptors or relevant signalling intermediates. Here we review the biological bases of these new targeted treatments, with special emphasis on the clinical results that point to an implementation of these drugs into the therapeutic armamentarium against SCCHN.

TGF-beta1 induces COX-2 expression and PGE2 synthesis through MAPK and PI3K pathways in human mesangial cells.

Transforming growth factor-beta1 (TGF-beta1) plays a fundamental role in the progression of renal diseases. Accumulating evidence has suggested that eicosanoids derived from cyclooxygenase-2 (COX-2) participate in a number of pathological processes in immune-mediated renal diseases. Mesangial cells (MC) play a major role in physiological and pathophysiological renal processes. MC express receptors for TGF-beta1, and COX-2 expression can be induced in MC. However, to date, there are no published data on the possible role of TGF-beta1 in COX-2 expression in human mesangial cells (HMC). We designed studies to determine (1) whether TGF-beta1 stimulates COX-2 expression in primary HMC, (2) whether mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) cascades are involved in TGF-beta1-induced COX-2 expression, and (3) whether prostaglandin (PG)E2 synthesis is affected by TGF-beta1 and MAP kinases and PI3K activation. Studies were performed in primary cultures of HMC and in an immortalized line of HMC. TGF-beta1 induces COX-2 promoter activity and COX-2 mRNA and protein expression in HMC. COX-2 induction is accompanied by increased PGE2 synthesis. Extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and PI3K pathway inhibition blunted TGF-beta1-induced COX-2 overexpression. We demonstrate that TGF-beta1 regulates COX-2 expression in HMC through the activation of ERK1/2, p38 MAPK, and PI3K. These results can help to elucidate the molecular mechanisms underlying the regulation of COX-2 and open up specific strategies for the treatment of glomerular disease.

Transforming growth factor-beta1 induces collagen synthesis and accumulation via p38 mitogen-activated protein kinase (MAPK) pathway in cultured L(6)E(9) myoblasts.

Transforming growth factor-beta (TGF-beta) plays a pivotal role in the extracellular matrix accumulation observed in chronic progressive tissue fibrosis, but the intracellular signaling mechanism by which TGF-beta stimulates this process remains poorly understood. We examined whether mitogen-activated protein kinase (MAPK) routes were involved in TGF-beta1-induced collagen expression in L(6)E(9) myoblasts. TGF-beta1 induced p38 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation whereas no effect on Jun N-terminal kinase phosphorylation was observed. Biochemical blockade of p38 but not of the ERK MAPK pathway abolished TGF-beta1-induced alpha(2)(I) collagen mRNA expression and accumulation. These data indicate that TGF-beta1-induced p38 activation is involved in TGF-beta1-stimulated collagen synthesis.