Overcoming resistance to rapalogs in gliomas by combinatory therapies

Glioblastoma is the most common and aggressive brain tumor type, with a mean patient survival of approximately 1 year. Many previous analyses of the glioma kinome have identified key deregulated pathways that converge and activate mammalian target of rapamycin (mTOR). Following the identification and characterization of mTOR-promoting activity in gliomagenesis, data from preclinical studies suggested the targeting of mTOR by rapamycin or its analogs (rapalogs) as a promising therapeutic approach. However, clinical trials with rapalogs have shown very limited efficacy on glioma due to the development of resistance mechanisms. Analysis of rapalog-insensitive glioma cells has revealed increased activity of growth and survival pathways compensating for mTOR inhibition by rapalogs that are suitable for therapeutic intervention. In addition, recently developed mTOR inhibitors show high anti-glioma activity. In this review, we recapitulate the regulation of mTOR signaling and its involvement in gliomagenesis, discuss mechanisms resulting in resistance to rapalogs, and speculate on strategies to overcome resistance. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Strategies to target energy metabolism in consensus molecular subtype 3 along with Kirsten rat sarcoma viral oncogene homolog mutations for colorectal cancer therapy

Alterations in cellular energy metabolism play a critical role in colorectal cancer (CRC), which has been identified as the definition of consensus molecular subtypes (CMSs), and CMS3 tumors exhibit energy metabolism signatures along with Kirsten rat sarcoma viral oncogene homolog (KRAS)-activating mutations. This review summarizes the relationship between CMS3 tumors associated with mutated KRAS and energy metabolism in CRC, especially for the dysregulated energy metabolism that affects tumor cell proliferation, invasion, and migration. Furthermore, this review concentrates on the role of metabolic genes and factors and signaling pathways, which coupled with a primary energy source connected with the CMS3 associated with mutated KRAS, induce metabolic alterations. The strategies to target energy metabolism for the metabolic alterations in mutated KRAS CRC are also introduced. In conclusion, dysregulated energy metabolism has a close relationship with mutated KRAS in CMS3 tumors. Therefore, selective inhibitors or agents against metabolic targets or KRAS signaling may be clinically useful for CMS3 tumor treatment through a personalized approach for patients with cancer.     

The contribution of tumor and host tissue factor expression to oncogene-driven gliomagenesis

Glioblastoma multiforme (GBM) is an aggressive form of glial brain tumors, associated with angiogenesis, thrombosis, and upregulation of tissue factor (TF), the key cellular trigger of coagulation and signaling. Since TF is upregulated by oncogenic mutations occurring in different subsets of human brain tumors we investigated whether TF contributes to tumourigenesis driven by oncogenic activation of EGFR (EGFRvIII) and RAS pathways in the brain. Here we show that TF expression correlates with poor prognosis in glioma, but not in GBM. In situ, the TF protein expression is heterogeneously expressed in adult and pediatric gliomas. GBM cells harboring EGFRvIII (U373vIII) grow aggressively as xenografts in SCID mice and their progression is delayed by administration of monoclonal antibodies blocking coagulant (CNTO 859) and signaling (10H10) effects of TF in vivo. Mice in which TF gene is disrupted in the neuroectodermal lineage exhibit delayed progression of spontaneous brain tumors driven by oncogenic N-ras and SV40 large T antigen (SV40LT) expressed under the control of sleeping beauty transposase. Reduced host TF levels in low-TF/SCID hypomorphic mice mitigated growth of glioma subcutaneously but not in the brain. Thus, we suggest that tumor-associated TF may serve as therapeutic target in the context of oncogene-driven disease progression in a subset of glioma.     

Combination therapy with KRAS siRNA and EGFR inhibitor AZD8931 suppresses lung cancer cell growth in vitro

Lung cancer is a leading cause of cancer-related deaths worldwide, with less than a 5-year survival rate for both men and women. Epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma oncogene (KRAS) signaling pathways play a critical role in the proliferation and progression of various cancers, including lung cancer. Genetic studies have shown that amplification, over-expression, or mutation of EGFR is an early and major molecular event in many human tumors. KRAS mutation is a negative factor in various cancer, including non-small-cell lung cancer, and complicates therapeutic approaches with adjuvant chemotherapy and anti-EGFR directed therapies. This article is dedicated to evaluating the synergistic effect of a novel EGFR inhibitor AZD8931 and KRAS small interfering RNA (siRNA) on the proliferation and apoptosis of lung adenocarcinoma cancer cells. A549 lung cancer cells were treated with KRAS siRNA and the EGFR inhibitor alone or in combination. The cytotoxic effects of KRAS siRNA and te EGFR inhibitor were determined usingMTT assay, and induction of apoptosis was determined by FACS analysis. Suppression of KRAS, Her-2, and EGFR expression by treatments was measured by qRT-PCR and western blotting. KRAS siRNA and the EGFR inhibitor significantly reduced the proliferation of A549 cells as well as KRAS and EGFR mRNA levels 24 hr after treatment. The results also indicated that the silencing of KRAS and EGFR has synergistic effects on the induction of apoptosis on the A549 cells. These results indicated that KRAS and EGFR might play important roles in the progression of lung cancer and could be potential therapeutic targets for treatment of lung cancer.     

LKB1 in lung cancerigenesis: a serine/threonine kinase as tumor suppressor

Lung cancer is featured with high mortality, with a 15% five-year survival rate worldwide. Genetic alterations, such as loss of function of tumor suppressor genes, frequently contribute to lung cancer initiation, progression and metastasis. Liver kinase B1 (LKB1), as a serine/threonine kinase and tumor suppressor, is frequently mutated and inactivated in non-small cell lung cancer (NSCLC). Recent studies have provided strong evidences that LKB1 loss promotes lung cancerigenesis process, especially lung cancer progression and metastasis. This review will summarize recent progress on how LKB1 modulates the process of lung cancerigenesis, emphasizing on LKB1 downstream signaling pathways and biological functions. We will further discuss the potential development of prognostic biomarkers or therapeutic targets in lung cancer clinic based on the molecular alteration associated with deregulated LKB1 signaling.     

Targeted therapy for gastric cancer: Molecular pathways and ongoing investigations

Gastric cancer is currently the second leading cause of worldwide cancer mortality. Ongoing collaborative sequencing efforts have highlighted recurrent somatic genomic aberrations in gastric cancer, however, despite advances in characterizing the genomic landscape, there have been few advances in patient outcomes. Prognosis remains poor with a median overall survival of 12 months for advanced disease. The improved survival with trastuzumab, and more recently ramucirumab, underscore the promise of targeted and biologic therapies and the importance of molecular tumor characterization in gastric cancer. Here we review the most frequent actionable alterations in gastric cancer and highlight ongoing clinical investigations attempting to translate biologic understanding into improved clinical outcomes.     

Antagonistic functional duality of cancer genes

Cancer evolution is a stochastic process both at the genome and gene levels. Most of tumors contain multiple genetic subclones, evolving in either succession or in parallel, either in a linear or branching manner, with heterogeneous genome and gene alterations, extensively rewired signaling networks, and addicted to multiple oncogenes easily switching with each other during cancer progression and medical intervention. Hundreds of discovered cancer genes are classified according to whether they function in a dominant (oncogenes) or recessive (tumor suppressor genes) manner in a cancer cell. However, there are many cancer “gene-chameleons”, which behave distinctly in opposite way in the different experimental settings showing antagonistic duality. In contrast to the widely accepted view that mutant NADP⁺-dependent isocitrate dehydrogenases 1/2 (IDH1/2) and associated metabolite 2-hydroxyglutarate (R)-enantiomer are intrinsically “the drivers” of tumourigenesis, mutant IDH1/2 inhibited, promoted or had no effect on cell proliferation, growth and tumorigenicity in diverse experiments. Similar behavior was evidenced for dozens of cancer genes. Gene function is dependent on genetic network, which is defined by the genome context. The overall changes in karyotype can result in alterations of the role and function of the same genes and pathways. The diverse cell lines and tumor samples have been used in experiments for proving gene tumor promoting/suppressive activity. They all display heterogeneous individual karyotypes and disturbed signaling networks. Consequently, the effect and function of gene under investigation can be opposite and versatile in cells with different genomes that may explain antagonistic duality of cancer genes and the cell type- or the cellular genetic/context-dependent response to the same protein. Antagonistic duality of cancer genes might contribute to failure of chemotherapy. Instructive examples of unexpected activity of cancer genes and “paradoxical” effects of different anticancer drugs depending on the cellular genetic context/signaling network are discussed.     

mTOR/S6K1 and MAPK/RSK signaling pathways coordinately regulate estrogen receptor α serine 167 phosphorylation

Resistance to anti-estrogen therapy is a major clinical concern in treatment of breast cancer. Estrogen-independent phosphorylation of estrogen receptor α, specifically on Ser167, is one of the contributing causes to development of resistance, and a prognostic marker for the disease. Here, we dissect the signaling pathways responsible for Ser167 phosphorylation. We report that the mTOR/S6K1 and MAPK/RSK contribute non-overlapping inputs into ERα activation via Ser167 phosphorylation. This cooperation may be targeted in breast cancer treatment by a combination of mTOR and MAPK inhibitors.     

Crosstalk of the EphA2 receptor with a serine/threonine phosphatase suppresses the Akt-mTORC1 pathway in cancer cells

Receptor tyrosine kinases of the Eph family play multiple roles in the physiological regulation of tissue homeostasis and in the pathogenesis of various diseases, including cancer. The EphA2 receptor is highly expressed in most cancer cell types, where it has disparate activities that are not well understood. It has been reported that interplay of EphA2 with oncogenic signaling pathways promotes cancer cell malignancy independently of ephrin ligand binding and receptor kinase activity. In contrast, stimulation of EphA2 signaling with ephrin-A ligands can suppress malignancy by inhibiting the Ras-MAP kinase pathway, integrin-mediated adhesion, and epithelial to mesenchymal transition. Here we show that ephrin-A1 ligand-dependent activation of EphA2 decreases the growth of PC3 prostate cancer cells and profoundly inhibits the Akt-mTORC1 pathway, which is hyperactivated due to loss of the PTEN tumor suppressor. Our results do not implicate changes in the activity of Akt upstream regulators (such as Ras family GTPases, PI3 kinase, integrins, or the Ship2 lipid phosphatase) in the observed loss of Akt T308 and S473 phosphorylation downstream of EphA2. Indeed, EphA2 can inhibit Akt phosphorylation induced by oncogenic mutations of not only PTEN but also PI3 kinase. Furthermore, it can decrease the hyperphosphorylation induced by constitutive membrane-targeting of Akt. Our data suggest a novel signaling mechanism whereby EphA2 inactivates the Akt-mTORC1 oncogenic pathway through Akt dephosphorylation mediated by a serine/threonine phosphatase. Ephrin-A1-induced Akt dephosphorylation was observed not only in PC3 prostate cancer cells but also in other cancer cell types. Thus, activation of EphA2 signaling represents a possible new avenue for anti-cancer therapies that exploit the remarkable ability of this receptor to counteract multiple oncogenic signaling pathways.     

Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display

There is an ever-increasing demand to select specific, high-affinity binding molecules against targets of biomedical interest. The success of such selections depends strongly on the design and functional diversity of the library of binding molecules employed, and on the performance of the selection strategy. We recently developed SRP phage display that employs the cotranslational signal recognition particle (SRP) pathway for the translocation of proteins to the periplasm. This system allows efficient filamentous phage display of highly stable and fast-folding proteins, such as designed ankyrin repeat proteins (DARPins) that are virtually refractory to conventional phage display employing the post-translational Sec pathway. DARPins comprise a novel class of binding molecules suitable to complement or even replace antibodies in many biotechnological or biomedical applications. So far, all DARPins have been selected by ribosome display. Here, we harnessed SRP phage display to generate a phage DARPin library containing more than 10¹⁰ individual members. We were able to select well behaved and highly specific DARPins against a broad range of target proteins having affinities as low as 100 pM directly from this library, without affinity maturation. We describe efficient selection on the Fc domain of human IgG, TNFα, ErbB1 (EGFR), ErbB2 (HER2) and ErbB4 (HER4) as examples. Thus, SRP phage display makes filamentous phage display accessible for DARPins, allowing, for example, selection under harsh conditions or on whole cells. We envision that the use of SRP phage display will be beneficial for other libraries of stable and fast-folding proteins.     

Towards defining biomarkers indicating resistances to targeted therapies

An impressive, but often short objective response was obtained in many tumor patients treated with different targeted therapies, but most of the patients develop resistances against these drugs. So far, a number of distinct mechanisms leading to intrinsic as well as acquired resistances have been identified in tumors of distinct origin. These can arise from genetic alterations, like mutations, truncations, and amplifications or due to deregulated expression of various proteins and signal transduction pathways, but also from cellular heterogeneity within tumors after an initial response. Therefore, biomarkers are urgently needed for cancer prognosis and personalized cancer medicine. The application of “ome”-based technologies including cancer (epi)genomics, next generation sequencing, cDNA microarrays and proteomics might led to the predictive or prognostic stratification of patients to categorize resistance mechanisms and to postulate combinations of treatment strategies. This review discusses the implementation of proteome-based analysis to identify markers of pathway (in)activation in tumors and the resistance mechanisms, which represent major clinical problems as a tool to optimize individually tailored therapies based on targeted drugs. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

p16INK4a/p19Arf与年龄相关性胰岛β细胞功能

2型糖尿病是一种与年龄密切相关的疾病,随着年龄增加,胰岛β细胞增殖能力和分泌能力下降,糖尿病患病率明显增加,但其机制尚不清楚. 最近研究发现,细胞周期蛋白依赖激酶(cyclin-dependent kinase, CDK)抑制剂p16INK4a是引起胰岛β细胞年龄相关性老化的重要调控因子.研究表明,通过p16INK4a介导的胰岛老化机制可能有如下两个：p38MAPK(mitogen-activated protein kinase)途径和PDGFR(platelet-derived growth factor receptor) 途径,两者均引起p16ink4a及p19ARF表达增加,而损害胰岛细胞增殖.本文综述年龄相关性胰岛β细胞功能下降的潜在机制,从而为改善胰岛β细胞功能提供新的分子靶点.     

Human Epidermal Growth Factor Receptor 2 (HER2) Impedes MLK3 Kinase Activity to Support Breast Cancer Cell Survival

Human epidermal growth factor receptor 2 (HER2) is amplified in ∼15–20% of human breast cancer and is important for tumor etiology and therapeutic options of breast cancer. Up-regulation of HER2 oncogene initiates cascades of events cumulating to the stimulation of transforming PI3K/AKT signaling, which also plays a dominant role in supporting cell survival and efficacy of HER2-directed therapies. Although investigating the underlying mechanisms by which HER2 promotes cell survival, we noticed a profound reduction in the kinase activity of a pro-apoptotic mixed lineage kinase 3 (MLK3) in HER2-positive (HER2+) but not in HER2-negative (HER2−) breast cancer tissues, whereas both HER2+ and HER2− tumors expressed a comparable level of MLK3 protein. Furthermore, the kinase activity of MLK3 was inversely correlated with HER2+ tumor grades. Moreover, HER2-directed drugs such as trastuzumab and lapatinib as well as depletion of HER2 or HER3 stimulated MLK3 kinase activity in HER2+ breast cancer cell lines. In addition, the noted inhibitory effect of HER2 on MLK3 kinase activity was mediated via its phosphorylation on Ser⁶⁷⁴ by AKT and that pharmacological inhibitors of PI3K/AKT prevented trastuzumab- and lapatinib-induced stimulation of MLK3 activity. Consistent with the pro-apoptotic function of MLK3, stable knockdown of MLK3 in the HER2+ cell line blunted the pro-apoptotic effects of trastuzumab and lapatinib. These findings suggest that HER2 activation inhibits the pro-apoptotic function of MLK3, which plays a mechanistic role in mediating anti-tumor activities of HER2-directed therapies. In brief, MLK3 represents a newly recognized integral component of HER2 biology in HER2+ breast tumors.     

Deconstructing breast cancer cell biology and the mechanisms of multidrug resistance

Cancer complexity constantly challenges the way that clinicians manage breast cancer therapy. Tumor heterogeneity and intratumoral stroma characteristics allow cells with different phenotypes and deregulated apoptotic, proliferative and migration abilities to co-exist contributing to a disappointing therapeutic response. While new approaches are being associated with conventional chemotherapy, such as hormonal therapy or target monoclonal antibodies, recurrence and metastasization are still observed. Membrane transporters are the cell's first line of contact with anticancer drugs having a major role in multidrug resistance events. This structural-based activity enables the cell to be drug-resistant by decreasing drug intracellular concentration through an efflux-transport mechanism, mainly associated with overexpression of ATP-binding cassette (ABC) proteins. This review focuses on some of the important structural and biological properties of the malignant cell and tumor microenvironment, addressing the role of the membrane ABC transporters in therapeutic outcomes, and highlighting related molecular pathways that may represent meaningful target therapies.     

Consensus Brain-derived Protein,Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.