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1.  Induction of BIM Is Essential for Apoptosis Triggered by EGFR Kinase Inhibitors in Mutant EGFR-Dependent Lung Adenocarcinomas 
PLoS Medicine  2007;4(10):e294.
Background
Mutations in the epidermal growth factor receptor (EGFR) gene are associated with increased sensitivity of lung cancers to kinase inhibitors like erlotinib. Mechanisms of cell death that occur after kinase inhibition in these oncogene-dependent tumors have not been well delineated. We sought to improve understanding of this process in order to provide insight into mechanisms of sensitivity and/or resistance to tyrosine kinase inhibitors and to uncover new targets for therapy.
Methods and Findings
Using a panel of human lung cancer cell lines that harbor EGFR mutations and a variety of biochemical, molecular, and cellular techniques, we show that EGFR kinase inhibition in drug-sensitive cells provokes apoptosis via the intrinsic pathway of caspase activation. The process requires induction of the proapoptotic BH3-only BCL2 family member BIM (i.e., BCL2-like 11, or BCL2L11); erlotinib dramatically induces BIM levels in sensitive but not in resistant cell lines, and knockdown of BIM expression by RNA interference virtually eliminates drug-induced cell killing in vitro. BIM status is regulated at both transcriptional and posttranscriptional levels and is influenced by the extracellular signal-regulated kinase (ERK) signaling cascade downstream of EGFR. Consistent with these findings, lung tumors and xenografts from mice bearing mutant EGFR-dependent lung adenocarcinomas display increased concentrations of Bim after erlotinib treatment. Moreover, an inhibitor of antiapoptotic proteins, ABT-737, enhances erlotinib-induced cell death in vitro.
Conclusions
In drug-sensitive EGFR mutant lung cancer cells, induction of BIM is essential for apoptosis triggered by EGFR kinase inhibitors. This finding implies that the intrinsic pathway of caspase activation may influence sensitivity and/or resistance of EGFR mutant lung tumor cells to EGFR kinase inhibition. Manipulation of the intrinsic pathway could be a therapeutic strategy to enhance further the clinical outcomes of patients with EGFR mutant lung tumors.
Using a panel of human drug-sensitive EGFR mutant lung cancer cells, William Pao and colleagues show that induction of BIM, a member of the BCL2 family, is essential for apoptosis triggered by EGFR kinase inhibitors.
Editors' Summary
Background.
Lung cancer, a common type of cancer, has a very low cure rate. Like all cancers, it occurs when cells begin to divide uncontrollably because of changes (mutations) in their genes. Chemotherapy drugs kill these rapidly dividing cells but, because some normal tissues are sensitive to these agents, it is hard to destroy the cancer without causing serious side effects. Recently, “targeted” therapies have brought new hope to some patients with cancer. These therapies attack the changes in cancer cells that allow them to divide uncontrollably but leave normal cells unscathed. One of the first molecules for which a targeted therapy was developed was the epidermal growth factor receptor (EGFR). In normal cells, messenger proteins bind to EGFR and activate its “tyrosine kinase,” an enzyme that sticks phosphate groups on tyrosine (an amino acid) in other proteins. These proteins then tell the cell to divide. Alterations to this signaling system drive uncontrolled cell division in some cancers so blocking the EGFR signaling pathway should stop these cancers growing. Indeed, some lung cancers with mutations in the tyrosine kinase of EGFR shrink dramatically when treated with gefitinib or erlotinib, two tyrosine kinase inhibitors (TKIs).
Why Was This Study Done?
TKI-sensitive lung cancers shrink when treated with TKIs because of drug-induced cell death, but what are the molecular mechanisms underlying this death? A better understanding of how TKIs kill cancer cells might provide new insights into why not all cancer cells with mutations in EGFR (the gene from which EGFR is made) are sensitive to TKIs. It might also uncover new targets for therapy. TKIs do not completely kill lung cancers, but if the mechanism of TKI-induced cell death were understood, it might be possible to enhance their effects. In this study, the researchers have investigated how cell death occurs after kinase inhibition in a panel of human lung cancer cell lines (cells isolated from human tumors that grow indefinitely in dishes) that carry EGFR mutations.
What Did the Researchers Do and Find?
The researchers show, first, that erlotinib induces a type of cell death called apoptosis in erlotinib-sensitive cell lines but not in resistant cell lines. Apoptosis can be activated by two major pathways. In this instance, the researchers report, the so-called “intrinsic” pathway activates apoptosis. This pathway is stimulated by proapoptotic members of the BCL2 family of proteins and is blocked by antiapoptotic members, so the researchers examined the effect of erlotinib treatment on the expression of BCL2 family members in the EGFR mutant cell lines. Erlotinib treatment increased the expression of the proapoptotic protein BIM in sensitive but not in resistant cell lines. It also removed phosphate groups from BIM—dephosphorylated BIM is a more potent proapoptotic protein. Conversely, blocking BIM expression using a technique called RNA interference virtually eliminated the ability of erlotinib to kill EGFR mutant cell lines. The researchers also report that erlotinib treatment increased BIM expression in erlotinib-sensitive lung tumors growing in mice and that an inhibitor of the anti-apoptotic protein BCL2 enhanced erlotinib-induced death in drug-sensitive cells growing in dishes.
What Do These Findings Mean?
These findings indicate that BIM activity is essential for the apoptosis triggered by TKIs in drug-sensitive lung cancer cells that carry EGFR mutations, and that treatment of these cells with TKIs induces both the expression and dephosphorylation of BIM. The finding that the intrinsic pathway of apoptosis activation is involved in TKI-induced cell death suggests that changes in this pathway (possibly mutations in some of its components) might influence the sensitivity of EGFR mutant lung cancers to TKIs. Finally, these findings suggest that giving drugs that affect the intrinsic pathway of apoptosis activation at the same time as TKIs might further improve the clinical outcome for patients with EGFR mutant tumors. Such combinations will have to be tested in clinical trials before being used routinely.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040294.
US National Cancer Institute information for patients and professionals on lung cancer (in English and Spanish)
Information for patients from Cancer Research UK on lung cancer including information on treatment with TKIs
Wikipedia pages on apoptosis, epidermal growth factor receptor, and BCL-2 proteins (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
Information for patients from Cancerbackup on erlotinib and gefitinib
doi:10.1371/journal.pmed.0040294
PMCID: PMC2001209  PMID: 17927446
2.  KRAS Testing for Anti-EGFR Therapy in Advanced Colorectal Cancer 
Executive Summary
In February 2010, the Medical Advisory Secretariat (MAS) began work on evidence-based reviews of the literature surrounding three pharmacogenomic tests. This project came about when Cancer Care Ontario (CCO) asked MAS to provide evidence-based analyses on the effectiveness and cost-effectiveness of three oncology pharmacogenomic tests currently in use in Ontario.
Evidence-based analyses have been prepared for each of these technologies. These have been completed in conjunction with internal and external stakeholders, including a Provincial Expert Panel on Pharmacogenomics (PEPP). Within the PEPP, subgroup committees were developed for each disease area. For each technology, an economic analysis was also completed by the Toronto Health Economics and Technology Assessment Collaborative (THETA) and is summarized within the reports.
The following reports can be publicly accessed at the MAS website at: www.health.gov.on.ca/mas or at www.health.gov.on.ca/english/providers/program/mas/mas_about.html
Gene Expression Profiling for Guiding Adjuvant Chemotherapy Decisions in Women with Early Breast Cancer: An Evidence-Based and Economic Analysis
Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: an Evidence-Based and Economic Analysis
K-RAS testing in Treatment Decisions for Advanced Colorectal Cancer: an Evidence-Based and Economic Analysis.
Objective
The objective of this systematic review is to determine the predictive value of KRAS testing in the treatment of metastatic colorectal cancer (mCRC) with two anti-EGFR agents, cetuximab and panitumumab. Economic analyses are also being conducted to evaluate the cost-effectiveness of KRAS testing.
Clinical Need: Condition and Target Population
Metastatic colorectal cancer (mCRC) is usually defined as stage IV disease according to the American Joint Committee on Cancer tumour node metastasis (TNM) system or stage D in the Duke’s classification system. Patients with advanced colorectal cancer (mCRC) either present with metastatic disease or develop it through disease progression.
KRAS (Kristen-RAS, a member of the rat sarcoma virus (ras) gene family of oncogenes) is frequently mutated in epithelial cancers such as colorectal cancer, with mutations occurring in mutational hotspots (codons 12 and 13) of the KRAS protein. Involved in EGFR-mediated signalling of cellular processes such as cell proliferation, resistance to apoptosis, enhanced cell motility and neoangiogenesis, a mutation in the KRAS gene is believed to be involved in cancer pathogenesis. Such a mutation is also hypothesized to be involved in resistance to targeted anti-EGFR (epidermal growth factor receptor with tyrosine kinase activity) treatments such as cetuximab and panitumumab, hence, the important in evaluating the evidence on the predictive value of KRAS testing in this context.
KRAS Mutation Testing in Advanced Colorectal Cancer
Both cetuximab and panitumumab are indicated by Health Canada in the treatment of patients with metastatic colorectal cancer whose tumours are WT for the KRAS gene. Cetuximab may be offered as monotherapy in patients intolerant to irinotecan-based chemotherapy or in patients who have failed both irinotecan and oxaliplatin-based regimens and who received a fluoropyrimidine. It can also be administered in combination with irinotecan in patients refractory to other irinotecan-based chemotherapy regimens. Panitumumab is only indicated as a single agent after failure of fluoropyrimidine-, oxaliplatin-, and irinotecan-containing chemotherapy regimens.
In Ontario, patients with advanced colorectal cancer who are refractory to chemotherapy may be offered the targeted anti-EGFR treatments cetuximab or panitumumab. Eligibility for these treatments is based on the KRAS status of their tumour, derived from tissue collected from surgical or biopsy specimens. It is believed that KRAS status is not affected by treatments, therefore, for patients for whom surgical tissue is available for KRAS testing, additional biopsies prior to treatment with these targeted agents is not necessary. For patients that have not undergone surgery or for whom surgical tissue is not available, a biopsy of either the primary or metastatic site is required to determine their KRAS status. This is possible as status at the metastatic and primary tumour sites is considered to be similar.
Research Question
To determine if there is predictive value of KRAS testing in guiding treatment decisions with anti-EGFR targeted therapies in advanced colorectal cancer patients refractory to chemotherapy.
Research Methods
Literature Search
The Medical Advisory Secretariat followed its standard procedures and on May 18, 2010, searched the following electronic databases: Ovid MEDLINE, EMBASE, Ovid MEDLINE In-Process & Other Non-Indexed Citations, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews and The International Network of Agencies for Health Technology Assessment database.
The subject headings and keywords searched included colorectal cancer, cetuximab, panitumumab, and KRAS testing. The search was further restricted to English-language articles published between January 1, 2009 and May 18, 2010 resulting in 1335 articles for review. Excluded were case reports, comments, editorials, nonsystematic reviews, and letters. Studies published from January 1, 2005 to December 31, 2008 were identified in a health technology assessment conducted by the Agency for Healthcare Research and Quality (AHRQ), published in 2010. In total, 14 observational studies were identified for inclusion in this EBA: 4 for cetuximab monotherapy, 7 for the cetuximab-irinotecan combination therapy, and 3 to be included in the review for panitumumab monotherapy
Inclusion Criteria
English-language articles, and English or French-language HTAs published from January 2005 to May 2010, inclusive.
Randomized controlled trials (RCTs) or observational studies, including single arm treatment studies that include KRAS testing.
Studies with data on main outcomes of interest, overall and progression-free survival.
Studies of third line treatment with cetuximab or panitumumab in patients with advanced colorectal cancer refractory to chemotherapy.
For the cetuximab-irinotecan evaluation, studies in which at least 70% of patients in the study received this combination therapy.
Exclusion Criteria
Studies whose entire sample was included in subsequent publications which have been included in this EBA.
Studies in pediatric populations.
Case reports, comments, editorials, or letters.
Outcomes of Interest
Overall survival (OS), median
Progression-free-survival (PFS), median.
Response rates.
Adverse event rates.
Quality of life (QOL).
Summary of Findings of Systematic Review
Cetuximab or Panitumumab Monotherapy
Based on moderate GRADE observational evidence, there is improvement in PFS and OS favouring patients without the KRAS mutation (KRAS wildtype, or KRAS WT) compared to those with the mutation.
Cetuximab-Irinotecan Combination Therapy
There is low GRADE evidence that testing for KRAS may optimize survival benefits in patients without the KRAS mutation (KRAS wildtype, or KRAS WT) compared to those with the mutation.
However, cetuximab-irinotecan combination treatments based on KRAS status discount any effect of cetuximab in possibly reversing resistance to irinotecan in patients with the mutation, as observed effects were lower than for patients without the mutation. Clinical experts have raised concerns about the biological plausibility of this observation and this conclusion would, therefore, be regarded as hypothesis generating.
Economic Analysis
Cost-effectiveness and budget impact analyses were conducted incorporating estimates of effectiveness from this systematic review. Evaluation of relative cost-effectiveness, based on a decision-analytic cost-utility analysis, assessed testing for KRAS genetic mutations versus no testing in the context of treatment with cetuximab monotherapy, panitumumab monotherapy, cetuximab in combination with irinotecan, and best supportive care.
Of importance to note is that the cost-effectiveness analysis focused on the impact of testing for KRAS mutations compared to no testing in the context of different treatment options, and does not assess the cost-effectiveness of the drug treatments alone.
Conclusions
KRAS status is predictive of outcomes in cetuximab and panitumumab monotherapy, and in cetuximab-irinotecan combination therapy.
While KRAS testing is cost-effective for all strategies considered, it is not equally cost-effective for all treatment options.
PMCID: PMC3377508  PMID: 23074403
3.  Luteolin Suppresses Cancer Cell Proliferation by Targeting Vaccinia-Related Kinase 1 
PLoS ONE  2014;9(10):e109655.
Uncontrolled proliferation, a major feature of cancer cells, is often triggered by the malfunction of cell cycle regulators such as protein kinases. Recently, cell cycle-related protein kinases have become attractive targets for anti-cancer therapy, because they play fundamental roles in cellular proliferation. However, the protein kinase-targeted drugs that have been developed so far do not show impressive clinical results and also display severe side effects; therefore, there is undoubtedly a need to investigate new drugs targeting other protein kinases that are critical in cell cycle progression. Vaccinia-related kinase 1 (VRK1) is a mitotic kinase that functions in cell cycle regulation by phosphorylating cell cycle-related substrates such as barrier-to-autointegration factor (BAF), histone H3, and the cAMP response element (CRE)-binding protein (CREB). In our study, we identified luteolin as the inhibitor of VRK1 by screening a small-molecule natural compound library. Here, we evaluated the efficacy of luteolin as a VRK1-targeted inhibitor for developing an effective anti-cancer strategy. We confirmed that luteolin significantly reduces VRK1-mediated phosphorylation of the cell cycle-related substrates BAF and histone H3, and directly interacts with the catalytic domain of VRK1. In addition, luteolin regulates cell cycle progression by modulating VRK1 activity, leading to the suppression of cancer cell proliferation and the induction of apoptosis. Therefore, our study suggests that luteolin-induced VRK1 inhibition may contribute to establish a novel cell cycle-targeted strategy for anti-cancer therapy.
doi:10.1371/journal.pone.0109655
PMCID: PMC4195671  PMID: 25310002
4.  Update on Aurora Kinase Targeted Therapeutics in Oncology 
Introduction
Mammalian cells contain three distinct serine/threonine protein kinases with highly conserved catalytic domains, including aurora A and B kinases that are essential regulators of mitotic entry and progression. Overexpression of aurora A and/or B kinase is associated with high proliferation rates and poor prognosis, making them ideal targets for anti-cancer therapy. Disruption of mitotic machinery is a proven anti-cancer strategy employed by multiple chemotherapeutic agents. Numerous small molecule inhibitors of the aurora kinases have been discovered and tested in vivo and in vitro, with a few currently in phase II testing.
Areas covered
This review provides the reader with updated results from both preclinical and human studies for each of the aurora kinase inhibitors (AKI) that are currently being investigated. The paper also covers in detail the late breaking and phase I data presented for AKIs thereby allowing the reader to compare and contrast individual and class-related effects of AKIs.
Expert opinion
While the successful development and approval of an AKI for anti-cancer therapy remains unresolved, pre-clinical identification of resistant mechanisms would help design better early phase clinical trials where relevant combinations may be evaluated prior to phase II testing. The authors believe that aurora kinases are important anti-cancer targets that operate in collaboration with other oncogenes intimately involved in uncontrolled tumor proliferation and by providing a unique, targeted and complimentary anti-cancer mechanism, expand the available armamentarium against cancer.
PMCID: PMC3039426  PMID: 18991785
Mitosis; Centrosome assembly; Aurora kinase inhibitor (AKI); Small molecule inhibitors; Targeted cancer therapy; Spindle assembly checkpoint; Aneuploidy
5.  Update on Aurora Kinase Targeted Therapeutics in Oncology 
Expert opinion on drug discovery  2011;6(3):291-307.
Introduction
Mammalian cells contain three distinct serine/threonine protein kinases with highly conserved catalytic domains, including aurora A and B kinases that are essential regulators of mitotic entry and progression. Overexpression of aurora A and/or B kinase is associated with high proliferation rates and poor prognosis, making them ideal targets for anti-cancer therapy. Disruption of mitotic machinery is a proven anti-cancer strategy employed by multiple chemotherapeutic agents. Numerous small molecule inhibitors of the aurora kinases have been discovered and tested in vivo and in vitro, with a few currently in phase II testing.
Areas covered
This review provides the reader with updated results from both preclinical and human studies for each of the aurora kinase inhibitors (AKI) that are currently being investigated. The paper also covers in detail the late breaking and phase I data presented for AKIs thereby allowing the reader to compare and contrast individual and classrelated effects of AKIs.
Expert opinion
While the successful development and approval of an AKI for anti-cancer therapy remains unresolved, pre-clinical identification of resistant mechanisms would help design better early phase clinical trials where relevant combinations may be evaluated prior to phase II testing. The authors believe that aurora kinases are important anti-cancer targets that operate in collaboration with other oncogenes intimately involved in uncontrolled tumor proliferation and by providing a unique, targeted and complimentary anti-cancer mechanism, expand the available armamentarium against cancer.
doi:10.1517/17460441.2011.555395
PMCID: PMC3088914  PMID: 21556291
Mitosis; Centrosome assembly; Aurora kinase inhibitor (AKI); Small molecule inhibitors; Targeted cancer therapy; Spindle assembly checkpoint; Aneuploidy
6.  A Novel Tumor-Promoting Function Residing in the 5′ Non-coding Region of vascular endothelial growth factor mRNA 
PLoS Medicine  2008;5(5):e94.
Background
Vascular endothelial growth factor-A (VEGF) is one of the key regulators of tumor development, hence it is considered to be an important therapeutic target for cancer treatment. However, clinical trials have suggested that anti-VEGF monotherapy was less effective than standard chemotherapy. On the basis of the evidence, we hypothesized that vegf mRNA may have unrecognized function(s) in cancer cells.
Methods and Findings
Knockdown of VEGF with vegf-targeting small-interfering (si) RNAs increased susceptibility of human colon cancer cell line (HCT116) to apoptosis caused with 5-fluorouracil, etoposide, or doxorubicin. Recombinant human VEGF165 did not completely inhibit this apoptosis. Conversely, overexpression of VEGF165 increased resistance to anti-cancer drug-induced apoptosis, while an anti-VEGF165-neutralizing antibody did not completely block the resistance. We prepared plasmids encoding full-length vegf mRNA with mutation of signal sequence, vegf mRNAs lacking untranslated regions (UTRs), or mutated 5′UTRs. Using these plasmids, we revealed that the 5′UTR of vegf mRNA possessed anti-apoptotic activity. The 5′UTR-mediated activity was not affected by a protein synthesis inhibitor, cycloheximide. We established HCT116 clones stably expressing either the vegf 5′UTR or the mutated 5′UTR. The clones expressing the 5′UTR, but not the mutated one, showed increased anchorage-independent growth in vitro and formed progressive tumors when implanted in athymic nude mice. Microarray and quantitative real-time PCR analyses indicated that the vegf 5′UTR-expressing tumors had up-regulated anti-apoptotic genes, multidrug-resistant genes, and growth-promoting genes, while pro-apoptotic genes were down-regulated. Notably, expression of signal transducers and activators of transcription 1 (STAT1) was markedly repressed in the 5′UTR-expressing tumors, resulting in down-regulation of a STAT1-responsive cluster of genes (43 genes). As a result, the tumors did not respond to interferon (IFN)α therapy at all. We showed that stable silencing of endogenous vegf mRNA in HCT116 cells enhanced both STAT1 expression and IFNα responses.
Conclusions
These findings suggest that cancer cells have a survival system that is regulated by vegf mRNA and imply that both vegf mRNA and its protein may synergistically promote the malignancy of tumor cells. Therefore, combination of anti-vegf transcript strategies, such as siRNA-based gene silencing, with anti-VEGF antibody treatment may improve anti-cancer therapies that target VEGF.
Shigetada Teshima-Kondo and colleagues find that cancer cells have a survival system that is regulated by vegf mRNA and that vegf mRNA and its protein may synergistically promote the malignancy of tumor cells.
Editors' Summary
Background
Normally, throughout life, cell division (which produces new cells) and cell death are carefully balanced to keep the body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—disorganized masses of cells. When a cancer is small, it uses the body's existing blood supply to get the oxygen and nutrients it needs for its growth and survival. But, when it gets bigger, it has to develop its own blood supply. This process is called angiogenesis. It involves the release by the cancer cells of proteins called growth factors that bind to other proteins (receptors) on the surface of endothelial cells (the cells lining blood vessels). The receptors then send signals into the endothelial cells that tell them to make new blood vessels. One important angiogenic growth factor is “vascular endothelial growth factor” (VEGF). Tumors that make large amounts of VEGF tend to be more abnormal and more aggressive than those that make less VEGF. In addition, high levels of VEGF in the blood are often associated with poor responses to chemotherapy, drug regimens designed to kill cancer cells.
Why Was This Study Done?
Because VEGF is a key regulator of tumor development, several anti-VEGF therapies—drugs that target VEGF and its receptors—have been developed. These therapies strongly suppress the growth of tumor cells in the laboratory and in animals but, when used alone, are no better at increasing the survival times of patients with cancer than standard chemotherapy. Scientists are now looking for an explanation for this disappointing result. Like all proteins, cells make VEGF by “transcribing” its DNA blueprint into an mRNA copy (vegf mRNA), the coding region of which is “translated” into the VEGF protein. Other, “noncoding” regions of vegf mRNA control when and where VEGF is made. Scientists have recently discovered that the noncoding regions of some mRNAs suppress tumor development. In this study, therefore, the researchers investigate whether vegf mRNA has an unrecognized function in tumor cells that could explain the disappointing clinical results of anti-VEGF therapeutics.
What Did the Researchers Do and Find?
The researchers first used a technique called small interfering (si) RNA knockdown to stop VEGF expression in human colon cancer cells growing in dishes. siRNAs are short RNAs that bind to and destroy specific mRNAs in cells, thereby preventing the translation of those mRNAs into proteins. The treatment of human colon cancer cells with vegf-targeting siRNAs made the cells more sensitive to chemotherapy-induced apoptosis (a type of cell death). This sensitivity was only partly reversed by adding VEGF to the cells. By contrast, cancer cells engineered to make more vegf mRNA had increased resistance to chemotherapy-induced apoptosis. Treatment of these cells with an antibody that inhibited VEGF function did not completely block this resistance. Together, these results suggest that both vegf mRNA and VEGF protein have anti-apoptotic effects. The researchers show that the anti-apoptotic activity of vegf mRNA requires a noncoding part of the mRNA called the 5′ UTR, and that whereas human colon cancer cells expressing this 5′ UTR form tumors in mice, cells expressing a mutated 5′ UTR do not. Finally, they report that the expression of several pro-apoptotic genes and of an anti-tumor pathway known as the interferon/STAT1 tumor suppression pathway is down-regulated in tumors that express the vegf 5′ UTR.
What Do These Findings Mean?
These findings suggest that some cancer cells have a survival system that is regulated by vegf mRNA and are the first to show that a 5′UTR of mRNA can promote tumor growth. They indicate that VEGF and its mRNA work together to promote their development and to increase their resistance to chemotherapy drugs. They suggest that combining therapies that prevent the production of vegf mRNA (for example, siRNA-based gene silencing) with therapies that block the function of VEGF might improve survival times for patients whose tumors overexpress VEGF.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050094.
This study is discussed further in a PLoS Medicine Perspective by Hughes and Jones
The US National Cancer Institute provides information about all aspects of cancer, including information on angiogenesis, and on bevacizumab, an anti-VEGF therapeutic (in English and Spanish)
CancerQuest, from Emory University, provides information on all aspects of cancer, including angiogenesis (in several languages)
Cancer Research UK also provides basic information about what causes cancers and how they develop, grow, and spread, including information about angiogenesis
Wikipedia has pages on VEGF and on siRNA (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050094
PMCID: PMC2386836  PMID: 18494554
7.  Comparative kinome analysis to identify putative colon tumor biomarkers 
Kinase domains are the type of protein domain most commonly found in genes associated with tumorigenesis. Because of this, the human kinome (the protein kinase component of the genome) represents a promising source of cancer biomarkers and potential targets for novel anti-cancer therapies. Alterations in the human colon kinome during the progression from normal colon (NC) through adenoma (AD) to adenocarcinoma (AC) were investigated using integrated transcriptomic and proteomic datasets. Two hundred thirty kinase genes and 42 kinase proteins showed differential expression patterns (fold change ≥ 1.5) in at least one tissue pair-wise comparison (AD vs. NC, AC vs. NC, and/or AC vs. AD). Kinases that exhibited similar trends in expression at both the mRNA and protein levels were further analyzed in individual samples of NC (n = 20), AD (n = 39), and AC (n = 24) by quantitative reverse transcriptase PCR. Individual samples of NC and tumor tissue were distinguishable based on the mRNA levels of a set of 20 kinases. Altered expression of several of these kinases, including chaperone activity of bc1 complex-like (CABC1) kinase, bromodomain adjacent to zinc finger domain protein 1B (BAZ1B) kinase, calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D), serine/threonine-protein kinase 24 (STK24), vaccinia-related kinase 3 (VRK3), and TAO kinase 3 (TAOK3), has not been previously reported in tumor tissue. These findings may have diagnostic potential and may lead to the development of novel targeted therapeutic interventions for colorectal cancer.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-011-0831-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s00109-011-0831-6
PMCID: PMC3307991  PMID: 22095101
Protein kinase; Colorectal cancer; Transcriptomics; Proteomics; TAOK3
8.  Kinase inhibitors can produce off-target effects and activate linked pathways by retroactivity 
BMC Systems Biology  2011;5:156.
Background
It has been shown in experimental and theoretical work that covalently modified signaling cascades naturally exhibit bidirectional signal propagation via a phenomenon known as retroactivity. An important consequence of retroactivity, which arises due to enzyme sequestration in covalently modified signaling cascades, is that a downstream perturbation can produce a response in a component upstream of the perturbation without the need for explicit feedback connections. Retroactivity may, therefore, play an important role in the cellular response to a targeted therapy. Kinase inhibitors are a class of targeted therapies designed to interfere with a specific kinase molecule in a dysregulated signaling pathway. While extremely promising as anti-cancer agents, kinase inhibitors may produce undesirable off-target effects by non-specific interactions or pathway cross-talk. We hypothesize that targeted therapies such as kinase inhibitors can produce off-target effects as a consequence of retroactivity alone.
Results
We used a computational model and a series of simple signaling motifs to test the hypothesis. Our results indicate that within physiologically and therapeutically relevant ranges for all parameters, a targeted inhibitor can naturally induce an off-target effect via retroactivity. The kinetics governing covalent modification cycles in a signaling network were more important for propagating an upstream off-target effect in our models than the kinetics governing the targeted therapy itself. Our results also reveal the surprising and crucial result that kinase inhibitors have the capacity to turn "on" an otherwise "off" parallel cascade when two cascades share an upstream activator.
Conclusions
A proper and detailed characterization of a pathway's structure is important for identifying the optimal protein to target as well as what concentration of the targeted therapy is required to modulate the pathway in a safe and effective manner. We believe our results support the position that such characterizations should consider retroactivity as a robust potential source of off-target effects induced by kinase inhibitors and other targeted therapies.
doi:10.1186/1752-0509-5-156
PMCID: PMC3257213  PMID: 21970676
9.  Targeting of Interferon-Beta to Produce a Specific, Multi-Mechanistic Oncolytic Vaccinia Virus 
PLoS Medicine  2007;4(12):e353.
Background
Oncolytic viruses hold much promise for clinical treatment of many cancers, but a lack of systemic delivery and insufficient tumor cell killing have limited their usefulness. We have previously demonstrated that vaccinia virus strains are capable of systemic delivery to tumors in mouse models, but infection of normal tissues remains an issue. We hypothesized that interferon-beta (IFN-β) expression from an oncolytic vaccinia strain incapable of responding to this cytokine would have dual benefits as a cancer therapeutic: increased anticancer effects and enhanced virus inactivation in normal tissues. We report the construction and preclinical testing of this virus.
Methods and Findings
In vitro screening of viral strains by cytotoxicity and replication assay was coupled to cellular characterization by phospho-flow cytometry in order to select a novel oncolytic vaccinia virus. This virus was then examined in vivo in mouse models by non-invasive imaging techniques. A vaccinia B18R deletion mutant was selected as the backbone for IFN-β expression, because the B18R gene product neutralizes secreted type-I IFNs. The oncolytic B18R deletion mutant demonstrated IFN-dependent cancer selectivity and efficacy in vitro, and tumor targeting and efficacy in mouse models in vivo. Both tumor cells and tumor-associated vascular endothelial cells were targeted. Complete tumor responses in preclinical models were accompanied by immune-mediated protection against tumor rechallenge. Cancer selectivity was also demonstrated in primary human tumor explant tissues and adjacent normal tissues. The IFN-β gene was then cloned into the thymidine kinase (TK) region of this virus to create JX-795 (TK−/B18R−/IFN-β+). JX-795 had superior tumor selectivity and systemic intravenous efficacy when compared with the TK−/B18R− control or wild-type vaccinia in preclinical models.
Conclusions
By combining IFN-dependent cancer selectivity with IFN-β expression to optimize both anticancer effects and normal tissue antiviral effects, we were able to achieve, to our knowledge for the first time, tumor-specific replication, IFN-β gene expression, and efficacy following systemic delivery in preclinical models.
Stephen Thorne and colleagues describe, in a mouse model, an oncolytic vaccinia virus with interferon-dependent cancer selectivity that allows tumor-specific replication; it also expresses the IFN-β gene and hence has efficacy against tumors.
Editors' Summary
Background.
Normally, throughout life, cell division (which produces new cells) and cell death are carefully balanced to keep the body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—disorganized masses of cells. Cancers can develop anywhere in the body and, as they develop, their cells acquire other genetic changes that enable them to move and start new tumors (metastases) elsewhere. Chemotherapy drugs kill rapidly dividing cancer cells but, because some normal cells are also sensitive to these drugs, it is hard to destroy the cancer without causing serious side effects. Consequently, researchers are trying to develop “targeted” therapies that attack the changes in cancer cells that allow them to divide uncontrollably but leave normal cells unscathed. One promising class of targeted therapies is oncolytic viruses. These viruses make numerous copies of themselves inside cancer cells (but not inside normal cells). Eventually the cancer cell bursts open (lyses), releases more of the therapeutic agent, and dies.
Why Was This Study Done?
Existing oncolytic viruses have two major disadvantages: they have to be injected directly into tumors, and therefore they can't destroy distant metastases; and they don't kill cancer cells particularly efficiently. In this study, the researchers have tried to adapt vaccinia virus (a virus that infects humans and which has recently been shown to kill tumor cells when injected into the bloodstream) in two ways: to both infect cancer cells selectively and then to kill them effectively.
They hypothesized that putting a gene that causes expression of a protein called interferon-beta (IFN-β) in a particular virus strain that is itself incapable of responding to IFN-β might achieve these aims. Human cells infected with viruses usually release IFNs, which induce an antiviral state in nearby cells. But vaccinia virus makes anti-IFN proteins that prevent IFN release. If the viral genes that encode these proteins are removed from the virus, the virus cannot spread through normal cells. However, many cancer cells have defective IFN signaling pathways so the virus can spread through them. IFN-β expression by the virus, however, should improve its innate anticancer effects because IFN-β stops cancer cells dividing, induces an antitumor immune response, and stops tumors developing good blood supplies.
What Did the Researchers Do and Find?
The researchers selected a vaccinia virus strain called WR-delB18R in which the B18R gene, which encodes an anti-IFN protein, had been removed from the virus. (WR is a wild-type virus.) In laboratory experiments, IFN treatment blocked the spread of WR-delB18R in normal human cells but not in human tumor cells. After being injected into the veins of tumor-bearing mice, WR-delB18R was rapidly cleared from normal tissues but persisted in the tumors. A single injection of WR-delB18R directly into the tumor killed most of the tumor cells. A similar dose injected into a vein was less effective but nevertheless increased the survival time of some of the mice by directly killing the tumor cells, by targeting the blood supply of the tumors, and by inducing antitumor immunity. Finally, when the researchers inserted the IFN-β gene into this WR-delB18R, the new virus—JX-795—was much better at killing tumors after intravenous injection than either WR or WR-delB18R.
What Do These Findings Mean?
These findings indicate that the vaccinia virus can be adapted so that it replicates only in tumor cells and kills these cells effectively after intravenous injection. In particular, they show that the strategy adopted by the researchers both optimizes the anticancer effects of the virus and minimizes viral replication in normal tissues. JX-795 is a promising oncolytic virus, therefore, particularly since vaccinia virus has been safely used for many years to vaccinate people against smallpox. Nevertheless, it will be some years before JX-795 can be used clinically. Vaccinia virus constructs like this need to be tested extensively in the laboratory and in animals before any attempt is made to test them in people and, even if they passes all these preclinical tests with flying colors, only clinical trials will reveal whether they can treat human cancer. Several related strains of vaccinia virus are currently undergoing clinical testing.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/:10.1371/journal.pmed.0040353.
The US National Cancer Institute provides information on all aspects of cancer (in English and Spanish)
CancerQuest, from Emory University, provides information on all aspects of cancer (in several languages)
The UK charity Cancerbackup also provides information on all aspects of cancer
Wikipedia has a page on oncolytic viruses (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
A short interview about oncolytic viruses with researcher Dr. John Bell is available on the Insidermedicine Web site
The Oncolytic virus Web page provides lists of oncolytic viruses classified by type
doi:10.1371/journal.pmed.0040353
PMCID: PMC2222946  PMID: 18162040
10.  Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain 
PLoS Medicine  2006;3(12):e485.
Background
Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy.
Methods and Findings
Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors.
Conclusions
Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.
Ingo Mellinghoff and colleagues sequenced theEGFR gene in glioblastoma samples and cell lines and identified missense mutations in the extracellular domain that suggest a new mechanism for EGFR activation.
Editors' Summary
Background.
Normally, cell division (which produces new cells) and cell death are finely balanced to keep the tissues and organs of the human body in working order. But sometimes, cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—life-threatening, disorganized masses of cells. Cancer treatments often involve drugs that kill rapidly dividing cells but, although these hit cancer cells hardest, they also damage some normal tissues. Now, though, some of the specific changes that allow cancer cells to divide uncontrollably have been identified and drugs that attack only these abnormal cells are being developed. One of these—erlotinib—inhibits the activity of epidermal growth factor receptor (EGFR), a “receptor tyrosine kinase” that sits in the cell membrane. The interaction of epidermal growth factor (EGF)—a messenger protein—with the extracellular portion (or domain) of EGFR activates its intracellular part (a kinase enzyme). This adds phosphate groups to tyrosine (an amino acid) in proteins that form part of a signaling cascade that tells cells to divide. Cancer cells often have alterations in EGFR signaling. Some have extra copies of the EGFR gene (EGFR amplification); others make a short version of EGFR that is always active because it lacks the extracellular domain that binds EGF; yet others contain EGFR that is permanently active because of mutations in its kinase domain.
Why Was This Study Done?
Erlotinib can help only patients whose tumor growth is dependent on EGFR signaling. To identify these patients it is necessary to have a detailed catalog of the mutations that occur in EGFR in tumors and to know which mutations drive uncontrolled cell growth. In this study, the researchers have catalogued and characterized the mutations in EGFR that occur in glioblastoma, a deadly type of brain tumor. The researchers chose this tumor type for their study because EGFR amplification and loss of the extracellular domain of EGFR are both common in glioblastomas and because about one in five patients with glioblastoma responds well to EGFR kinase inhibitors.
What Did the Researchers Do and Find?
The researchers sequenced the whole coding sequence of the EGFR gene in more than 100 glioblastomas. Nearly 15% of the tumors contained missense mutations—changes that alter the amino acid sequence of EGFR. Only one tumor had a mutation in the EGFR kinase domain; the rest had mutations in its extracellular domain. To test whether these newly identified mutations might contribute to cancer development (oncogenesis), the researchers introduced mutated or normal EGFR genes into nontumorigenic mouse cells. Only the cells that contained the mutated EGFR genes formed tumors when injected into mice, indicating that the nontumorigenic cells had been “transformed” into cancer cells by the mutated EGFR genes. Finally, the researchers showed that EGFR containing the extracellular missense mutations had kinase activity in the absence of EGF when expressed in human and mouse cells, and that the growth of cells transformed by expression of the mutated genes was sensitive to erlotinib.
What Do These Findings Mean?
These findings identify missense mutations in the extracellular domain of EGFR as a new way to oncogenically activate this protein. Until now researchers have concentrated on the kinase domain of this and other receptor tyrosine kinases in their search for oncogenic mutations, but the results of this study suggest that future searches should be much broader. The distribution of EGFR missense mutations in glioblastoma contrasts with that in lung cancer, in which alterations in EGFR signaling are also implicated in cancer development but all the oncogenic mutations are in the kinase domain. Fortunately, EGFR kinase inhibitors like erlotinib have broad activity: They inhibit the growth of cells transformed by the expression of EGFR containing extracellular domain mutations or kinase mutations, or by the expression of the short EGFR variant. This bodes well for the use of these drugs in patients with glioblastoma. However, before these inhibitors become a standard part of cancer treatments, sensitive techniques need to be developed to analyze tumors for these mutations so that the patients who will benefit from these targeted therapies can be identified.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030485.
MedlinePlus encyclopedia entries on cancer and on brain tumors
US National Cancer Institute information for patients and professionals on brain tumors
Wikipedia pages on protein kinases, epidermal growth factor receptor, and erlotinib (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030485
PMCID: PMC1702556  PMID: 17177598
11.  Polymorphisms, Mutations, and Amplification of the EGFR Gene in Non-Small Cell Lung Cancers 
PLoS Medicine  2007;4(4):e125.
Background
The epidermal growth factor receptor (EGFR) gene is the prototype member of the type I receptor tyrosine kinase (TK) family and plays a pivotal role in cell proliferation and differentiation. There are three well described polymorphisms that are associated with increased protein production in experimental systems: a polymorphic dinucleotide repeat (CA simple sequence repeat 1 [CA-SSR1]) in intron one (lower number of repeats) and two single nucleotide polymorphisms (SNPs) in the promoter region, −216 (G/T or T/T) and −191 (C/A or A/A). The objective of this study was to examine distributions of these three polymorphisms and their relationships to each other and to EGFR gene mutations and allelic imbalance (AI) in non-small cell lung cancers.
Methods and Findings
We examined the frequencies of the three polymorphisms of EGFR in 556 resected lung cancers and corresponding non-malignant lung tissues from 336 East Asians, 213 individuals of Northern European descent, and seven of other ethnicities. We also studied the EGFR gene in 93 corresponding non-malignant lung tissue samples from European-descent patients from Italy and in peripheral blood mononuclear cells from 250 normal healthy US individuals enrolled in epidemiological studies including individuals of European descent, African–Americans, and Mexican–Americans. We sequenced the four exons (18–21) of the TK domain known to harbor activating mutations in tumors and examined the status of the CA-SSR1 alleles (presence of heterozygosity, repeat number of the alleles, and relative amplification of one allele) and allele-specific amplification of mutant tumors as determined by a standardized semiautomated method of microsatellite analysis. Variant forms of SNP −216 (G/T or T/T) and SNP −191 (C/A or A/A) (associated with higher protein production in experimental systems) were less frequent in East Asians than in individuals of other ethnicities (p < 0.001). Both alleles of CA-SSR1 were significantly longer in East Asians than in individuals of other ethnicities (p < 0.001). Expression studies using bronchial epithelial cultures demonstrated a trend towards increased mRNA expression in cultures having the variant SNP −216 G/T or T/T genotypes. Monoallelic amplification of the CA-SSR1 locus was present in 30.6% of the informative cases and occurred more often in individuals of East Asian ethnicity. AI was present in 44.4% (95% confidence interval: 34.1%–54.7%) of mutant tumors compared with 25.9% (20.6%–31.2%) of wild-type tumors (p = 0.002). The shorter allele in tumors with AI in East Asian individuals was selectively amplified (shorter allele dominant) more often in mutant tumors (75.0%, 61.6%–88.4%) than in wild-type tumors (43.5%, 31.8%–55.2%, p = 0.003). In addition, there was a strong positive association between AI ratios of CA-SSR1 alleles and AI of mutant alleles.
Conclusions
The three polymorphisms associated with increased EGFR protein production (shorter CA-SSR1 length and variant forms of SNPs −216 and −191) were found to be rare in East Asians as compared to other ethnicities, suggesting that the cells of East Asians may make relatively less intrinsic EGFR protein. Interestingly, especially in tumors from patients of East Asian ethnicity, EGFR mutations were found to favor the shorter allele of CA-SSR1, and selective amplification of the shorter allele of CA-SSR1 occurred frequently in tumors harboring a mutation. These distinct molecular events targeting the same allele would both be predicted to result in greater EGFR protein production and/or activity. Our findings may help explain to some of the ethnic differences observed in mutational frequencies and responses to TK inhibitors.
Masaharu Nomura and colleagues examine the distribution ofEGFR polymorphisms in different populations and find differences that might explain different responses to tyrosine kinase inhibitors in lung cancer patients.
Editors' Summary
Background.
Most cases of lung cancer—the leading cause of cancer deaths worldwide—are “non-small cell lung cancer” (NSCLC), which has a very low cure rate. Recently, however, “targeted” therapies have brought new hope to patients with NSCLC. Like all cancers, NSCLC occurs when cells begin to divide uncontrollably because of changes (mutations) in their genetic material. Chemotherapy drugs treat cancer by killing these rapidly dividing cells, but, because some normal tissues are sensitive to these agents, it is hard to kill the cancer completely without causing serious side effects. Targeted therapies specifically attack the changes in cancer cells that allow them to divide uncontrollably, so it might be possible to kill the cancer cells selectively without damaging normal tissues. Epidermal growth factor receptor (EGRF) was one of the first molecules for which a targeted therapy was developed. In normal cells, messenger proteins bind to EGFR and activate its “tyrosine kinase,” an enzyme that sticks phosphate groups on tyrosine (an amino acid) in other proteins. These proteins then tell the cell to divide. Alterations to this signaling system drive the uncontrolled growth of some cancers, including NSCLC.
Why Was This Study Done?
Molecules that inhibit the tyrosine kinase activity of EGFR (for example, gefitinib) dramatically shrink some NSCLCs, particularly those in East Asian patients. Tumors shrunk by tyrosine kinase inhibitors (TKIs) often (but not always) have mutations in EGFR's tyrosine kinase. However, not all tumors with these mutations respond to TKIs, and other genetic changes—for example, amplification (multiple copies) of the EGFR gene—also affect tumor responses to TKIs. It would be useful to know which genetic changes predict these responses when planning treatments for NSCLC and to understand why the frequency of these changes varies between ethnic groups. In this study, the researchers have examined three polymorphisms—differences in DNA sequences that occur between individuals—in the EGFR gene in people with and without NSCLC. In addition, they have looked for associations between these polymorphisms, which are present in every cell of the body, and the EGFR gene mutations and allelic imbalances (genes occur in pairs but amplification or loss of one copy, or allele, often causes allelic imbalance in tumors) that occur in NSCLCs.
What Did the Researchers Do and Find?
The researchers measured how often three EGFR polymorphisms (the length of a repeat sequence called CA-SSR1, and two single nucleotide variations [SNPs])—all of which probably affect how much protein is made from the EGFR gene—occurred in normal tissue and NSCLC tissue from East Asians and individuals of European descent. They also looked for mutations in the EGFR tyrosine kinase and allelic imbalance in the tumors, and then determined which genetic variations and alterations tended to occur together in people with the same ethnicity. Among many associations, the researchers found that shorter alleles of CA-SSR1 and the minor forms of the two SNPs occurred less often in East Asians than in individuals of European descent. They also confirmed that EGFR kinase mutations were more common in NSCLCs in East Asians than in European-descent individuals. Furthermore, mutations occurred more often in tumors with allelic imbalance, and in tumors where there was allelic imbalance and an EGFR mutation, the mutant allele was amplified more often than the wild-type allele.
What Do These Findings Mean?
The researchers use these associations between gene variants and tumor-associated alterations to propose a model to explain the ethnic differences in mutational frequencies and responses to TKIs seen in NSCLC. They suggest that because of the polymorphisms in the EGFR gene commonly seen in East Asians, people from this ethnic group make less EGFR protein than people from other ethnic groups. This would explain why, if a threshold level of EGFR is needed to drive cells towards malignancy, East Asians have a high frequency of amplified EGFR tyrosine kinase mutations in their tumors—mutation followed by amplification would be needed to activate EGFR signaling. This model, though speculative, helps to explain some clinical findings, such as the frequency of EGFR mutations and of TKI sensitivity in NSCLCs in East Asians. Further studies of this type in different ethnic groups and in different tumors, as well as with other genes for which targeted therapies are available, should help oncologists provide personalized cancer therapies for their patients.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040125.
US National Cancer Institute information on lung cancer and on cancer treatment for patients and professionals
MedlinePlus encyclopedia entries on NSCLC
Cancer Research UK information for patients about all aspects of lung cancer, including treatment with TKIs
Wikipedia pages on lung cancer, EGFR, and gefitinib (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0040125
PMCID: PMC1876407  PMID: 17455987
12.  Kinase/phosphatase overexpression reveals pathways regulating hippocampal neuron morphology 
Kinases and phosphatases that regulate neurite number versus branching versus extension are weakly correlated.The kinase family that most strongly enhances neurite growth is a family of non-protein kinases; sugar kinases related to NADK.Pathway analysis revealed that genes in several cancer pathways were highly active in enhancing neurite growth.
In neural development, neuronal precursors differentiate, migrate, extend long axons and dendrites, and finally establish connections with their targets. Clinical conditions such as spinal cord injury, traumatic brain injury, stroke, multiple sclerosis, Parkinson's disease, Huntington's disease, and Alzheimer's disease are often associated with a loss of axon and/or dendrite connectivity and treatment strategies would be enhanced by new therapies targeting cell intrinsic mechanisms of axon elongation and regeneration.
Phosphorylation controls most cellular processes, including the cell cycle, proliferation, metabolism, and apoptosis. Neuronal differentiation, including axon formation and elongation, is also regulated by a wide range of kinases and phosphatases. For example, the non-receptor tyrosine kinase Src is required for cell adhesion molecule-dependent neurite outgrowth. In addition to individual kinases and phosphatases, signaling pathways like the MAPK, growth factor signaling, PIP3, cytoskeletal, and calcium-dependent pathways have been shown to impinge on or control neuronal process development. Recent results have implicated GSK3 and PTEN as therapeutically relevant targets in axonal regeneration after injury. However, these and other experiments have studied only a small fraction of the total kinases and phosphatases in the genome. Because of recent advances in genomic knowledge, large-scale cDNA production, and high-throughput phenotypic analysis, it is now possible to take a more comprehensive approach to understanding the functions of kinases and phosphatases in neurons.
We performed a large, unbiased set of experiments to answer the question ‘what effect does the overexpression of genes encoding kinases, phosphatases, and related proteins have on neuronal morphology?' We used ‘high-content analysis' to obtain detailed results about the specific phenotypes of neurons. We studied embryonic rat hippocampal neurons because of their stereotypical development in vitro (Dotti et al, 1988) and their widespread use in studies of neuronal differentiation and signaling. We transfected over 700 clones encoding kinases and phosphatases into hippocampal neurons and analyzed the resulting changes in neuronal morphology.
Many known genes, including PP1a, ERK1, ErbB2, atypical PKC, Calcineurin, CaMK2, IGF1R, FGFR, GSK3, and PIK3 were observed to have significant effects on neurite outgrowth in our system, consistent with earlier findings in the literature.
We obtained quantitative data for many cellular and neuronal morphological parameters from each neuron imaged. These included nuclear morphology (nuclear area and Hoechst dye intensity), soma morphology (tubulin intensity, area, and shape), and numerous parameters of neurite morphology (e.g. tubulin intensity along the neurites, number of primary neurites, neurite length, number of branches, distance from the cell body to the branches, number of crossing points, width and area of the neurites, and longest neurite; Supplementary Figure 1). Other parameters were reported on a ‘per well' basis, including the percentage of transfected neurons in a condition, as well as the percentage of neurons initiating neurite growth. Data for each treatment were normalized to a control (pSport CAT) within the same experiment, then aggregated across replicate experiments.
Correlations among the 19 normalized parameters were analyzed for neurons transfected with all kinase and phosphatase clones (Figure 2). On the basis of this analysis, the primary variables that define the neurite morphology are primary neurite count, neurite average length, and average branches. Interestingly, primary neurite count was not well correlated with neurite length or branching. The Pearson correlation coefficient (r2) between the number of primary neurites and the average length of the neurites was 0.3, and between the number of primary neurites and average branching was 0.2. In contrast, the correlation coefficient of average branching with neurite average length was 0.7. The most likely explanation is that signaling mechanisms underlying the neurite number determination are different than those controlling length/branching of the neurites.
Related proteins are often involved in similar neuronal functions. For example, families of receptor protein tyrosine phosphatases are involved in motor axon extension and guidance in both Drosophila and in vertebrates, and a large family of Eph receptor tyrosine kinases regulates guidance of retinotectal projections, motor axons, and axons in the corpus callosum. We therefore asked whether families of related genes produced similar phenotypes when overexpressed in hippocampal neurons. Our set of genes covered 40% of the known protein kinases, and many of the non-protein kinases and phosphatases.
Gene families commonly exhibit redundant function. Redundant gene function has often been identified when two or more knockouts are required to produce a phenotype. Our technique allowed us to measure whether different members of gene families had similar (potentially redundant) or distinct effects on neuronal phenotype.
To determine whether groups of related genes affect neuronal morphology in similar ways, we used sequence alignment information to construct gene clusters (Figure 6). Genes were clustered at nine different thresholds of similarity (called ‘tiers'). The functional effect for a particular parameter was then averaged within each cluster of a given tier, and statistics were performed to determine the significance of the effect. We analyzed the results for three key neurite parameters (average neurite length, primary neurite count, and average branching). Genes that perturbed each of these phenotypes are grouped in Figure 6. Eight families, most with only a few genes, produced significant changes for one or two parameters. A diverse family of non-protein kinases had a positive effect on neurite outgrowth in three of the four parameters analyzed. This family of kinases consisted of a variety of enzymes, mostly sugar and lipid kinases. A similar analysis was performed using pathway cluster analysis with pathways from the KEGG database, rather than sequence homology. Interestingly, pathways involved in cancer cell proliferation potentiated neurite extension and branching.
Our studies have identified a large number of kinases and phosphatases, as well as structurally and functionally defined families of these proteins, that affect neuronal process formation in specific ways. We have provided an analytical methodology and new tools to analyze functional data, and have implicated genes with novel functions in neuronal development. Our studies are an important step towards the goal of a molecular description of the intrinsic control of axodendritic growth.
Development and regeneration of the nervous system requires the precise formation of axons and dendrites. Kinases and phosphatases are pervasive regulators of cellular function and have been implicated in controlling axodendritic development and regeneration. We undertook a gain-of-function analysis to determine the functions of kinases and phosphatases in the regulation of neuron morphology. Over 300 kinases and 124 esterases and phosphatases were studied by high-content analysis of rat hippocampal neurons. Proteins previously implicated in neurite growth, such as ERK1, GSK3, EphA8, FGFR, PI3K, PKC, p38, and PP1a, were confirmed to have effects in our functional assays. We also identified novel positive and negative neurite growth regulators. These include neuronal-developmentally regulated kinases such as the activin receptor, interferon regulatory factor 6 (IRF6) and neural leucine-rich repeat 1 (LRRN1). The protein kinase N2 (PKN2) and choline kinase α (CHKA) kinases, and the phosphatases PPEF2 and SMPD1, have little or no established functions in neuronal function, but were sufficient to promote neurite growth. In addition, pathway analysis revealed that members of signaling pathways involved in cancer progression and axis formation enhanced neurite outgrowth, whereas cytokine-related pathways significantly inhibited neurite formation.
doi:10.1038/msb.2010.52
PMCID: PMC2925531  PMID: 20664637
bioinformatics; development; functional genomics; metabolic and regulatory networks; neuroscience
13.  A Novel Requirement for Janus Kinases as Mediators of Drug Resistance Induced by Fibroblast Growth Factor-2 in Human Cancer Cells 
PLoS ONE  2011;6(5):e19861.
The development of resistance to chemotherapy is a major cause of cancer-related death. Elucidating the mechanisms of drug resistance should thus lead to novel therapeutic strategies. Fibroblast growth factor (FGF)-2 signaling induces the assembly of a multi-protein complex that provides tumor cells with the molecular machinery necessary for drug resistance. This complex, which involves protein kinase C (PKC) ε, v-raf murine sarcoma viral oncogene homolog B1 (B-RAF) and p70 S6 kinase β (S6K2), enhances the selective translation of anti-apoptotic proteins such as B-cell leukaemia/lymphoma-2 (BCL-2) and inhibitors of apoptosis protein (IAP) family members and these are able to protect multiple cancer cell types from chemotherapy-induced cell death. The Janus kinases (JAKs) are most noted for their critical roles in mediating cytokine signaling and immune responses. Here, we show that JAKs have novel functions that support their consideration as new targets in therapies aimed at reducing drug resistance. As an example, we show that the Janus kinase TYK2 is phosphorylated downstream of FGF-2 signaling and required for the full phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Moreover, TYK2 is necessary for the induction of key anti-apoptotic proteins, such as BCL-2 and myeloid cell leukemia sequence (MCL) 1, and for the promotion of cell survival upon FGF-2. Silencing JAK1, JAK2 or TYK2 using RNA interference (RNAi) inhibits FGF2-mediated proliferation and results in the sensitization of tumor cells to chemotherapy-induced killing. These effects are independent of activation of signal transducer and activator of transcription (STAT) 1, STAT3 and STAT5A/B, the normal targets of JAK signaling. Instead, TYK2 associates with the other kinases previously implicated in FGF-2-mediated drug resistance. In light of these findings we hypothesize that TYK2 and other JAKs are important modulators of FGF-2-driven cell survival and that inhibitors of these kinases will likely improve the effectiveness of other cancer therapies.
doi:10.1371/journal.pone.0019861
PMCID: PMC3098828  PMID: 21625473
14.  Use of Proteins as Biomarkers and Their Role in Carcinogenesis 
Journal of Cancer  2015;6(1):9-18.
Summary: Improved diagnostic methods and medical therapies are necessary for early detection and treatment and an improved prognosis. It is thus vital to both examine and evaluate the role of the various existing proteins as biomarkers in carcinogenesis and to assess the contribution of these proteins in anti-cancer activity, for consideration in therapeutic strategies. It is essential to both examine and evaluate the role of the various existing proteins as biomarkers in carcinogenesis and to assess the contribution of these proteins in anti-cancer activity, for consideration in therapeutic strategies. The purpose of this review is twofold. Firstly, it is to evaluate recent data about which proteins can be utilized as biomarkers in carcinogenesis. The proteins reviewed include: CPTP, IL-6, CCN, and S100. Secondly, it is to evaluate the contribution of dietary proteins in cancer activity. Specifically, how whey protein, soy proteins and lectin, a phytochemical could be useful in cancer prevention and treatment.
Recent Findings: Whey protein, present in dairy products, is an excellent source of the sulphur amino acid cysteine, the rate limiting substrate in glutathione synthesis. Notably, this protein survives digestion and has been shown to have anti-carcinogenic properties in animal studies. Lectins are phytochemicals present in plant foods, and have active components which alters cancer initiation, promotion and progression. Lectins have been characterized as a useful tool in biochemistry, cell biology, immunology and in diagnostic and therapeutic purposes in cancer research. Soy proteins contain various compounds, including isoflavones, protease inhibitors and protein kinase inhibitors, which have been proven effective in tumor growth inhibition. They have therefore, been greatly emphasized in cancer prevention and treatment. It has been proved that soy food consumption was associated with decreased risk of death and recurrence of breast cancer. CPTP is a recently discovered protein whose main role is to transport C1P, a pro-inflammatory molecule. The discovery of CPTP may shine a light on the mechanism of inflammatory diseases, and hopefully offer a potential target for therapeutic purposes in cancer research. Interleukin-6 is a multifunctional cytokine that affects the activity of cancer cells. It is involved in tumor growth, and elevated levels is associated with an increased risk of cancer. S100B is a well-established biomarker for malignant melanoma, and useful in assessing tumor load, stage and prognosis for patients with this disease. Other members of this family of proteins include S100A4, which has been associated with several malignancies and S100A2, which has been found to be decreased in some cancers. CCN are a group of regulatory proteins, located in the extracellular matrix (maricellular). They are involved in cellular adhesion, mitogenesis, chemotaxis, cell survival, and wound healing. CCN proteins are also able to modulate the signals of several proteins, which may also influence skeletal development and angiogenesis. Many of the functions of these proteins are thus also related to tumor growth. Furthermore, CCN interacts with estrogen in the development of cancer, and is implicated in some breast and ovarian cancers.
doi:10.7150/jca.10560
PMCID: PMC4278910  PMID: 25553084
biomarkers; proteins; lung cancer.
15.  A Cell-Cell Communication Marker for Identifying Targeted Tumor Therapies 
Current bioactive compounds  2013;9(3):255-262.
Cell-cell communication through gap junctions is aberrant or absent in a majority of human cancer cells, compared to cells in corresponding normal tissues. This and other evidence has led to the hypothesis that gap junction channels, comprised of connexin proteins, are important in growth control and cancer progression. The major goal of this ongoing study was to identify bioactive compounds that specifically upregulate gap junction channel-mediated cell-cell communication as potential anti-tumor therapies. Control of cell-cell communication is linked to growth regulatory intracellular signaling pathways; we therefore further aimed to identify signaling pathways modulated by these compounds in order to assess their potential as targeted anti-tumor therapies. Compounds were screened for their ability to upregulate gap junction-mediated cell-cell communication by using a fluorescent dye transfer assay to measure cell-cell communication between tumor promoter-treated astroglial cells or ras-transformed epithelial cells. Western blotting using connexin-specific and phosphorylation site-specific antibodies was used to monitor phosphorylation changes in signaling pathway proteins. Our results identified three compounds that upregulate gap junction-mediated cell-cell communication in our screening assays, chaetoglobosin K(ChK), 4-phenyl-3-butenoic acid (PBA) and the methyl ester of PBA (PBA-Me). Further analyses demonstrated that in tumorigenic cells, ChK downregulates phosphorylation of Akt kinase, an enzyme in the PI3-kinase signaling pathway that is found to be upregulated in a number of human cancers, on a key activation site. However, ChK did not inhibit PI-3 kinase in vitro as did the classic PI-3 kinase inhibitor, Wortmannin. PBA and PBA-Me were found to upregulate phosphorylation of p38 MAPK on a key activation site in tumorigenic cells, which is downregulated in several human cancer cell types. ChK and PBA also decreased activation of SAPK/JNK, another kinase found to be upregulated in a number of human cancers. These studies highlight the potential of monitoring gap junction intercellular communication for identifying experimental anti-tumor compounds.
doi:10.2174/157340720903140119155322
PMCID: PMC4232852  PMID: 25404879
Gap junction; p38 MAPK; JNK; Akt kinase
16.  Continuum of care with anti-angiogenic therapies in metastatic colorectal cancer 
Inhibition of tumor angiogenesis has emerged as an important therapeutic component in the management of metastatic colorectal cancer. Three anti-angiogenic agents are currently approved in this clinical setting: bevacizumab, ziv-aflibercept, and regorafenib. Bevacizumab, a monoclonal antibody that targets the angiogenesis-driving ligand vascular endothelial growth factor A (VEGF-A), is the only anti-angiogenic agent approved in first-line therapy for metastatic colorectal cancer, where it can be used in combination with intravenous 5-fluorouracil-containing chemotherapy regimens. In conjunction with second-line chemotherapies, bevacizumab also has anti-cancer activity, both for the management of metastatic colorectal cancer in patients who received it as a part of their first line therapy and for those who are naïve to it. Ziv-aflibercept also has demonstrated clinical activity in conjunction with the chemotherapeutic regimen FOLFIRI in the second line management of patients with metastatic colorectal cancer; it functions by binding VEGF-A to the vascular endothelial growth factor proteins VEGF-B and PIGF (placental growth factor). Regorafenib, which inhibits multiple tyrosine kinases, including the VEGF receptors, has proven clinical benefit in the management of patients with metastatic colorectal cancer refractory to all other therapies. For patients’ whose cancers are refractory to all other therapies, there is also evidence for the use of bevacizumab with fluoropyrimidine monotherapy, but only in the bevacizumab-naïve patient subset. Presently, it is not clear if any one agent as more activity in a particular line of therapy than another, has greater efficacy when paired with a particular chemotherapy backbone, or if a particular patient subset is more likely to benefit from these agents. Given the present benefit and tolerance data, an anti-angiogenic agent should be considered in all lines of therapy in the management of metastatic colorectal cancer, with the evidence for the use of these agents in each specific line of therapy and in specific chemotherapeutic combinations driving agent selection.
doi:10.3978/j.issn.2078-6891.2013.030
PMCID: PMC3712300  PMID: 23997941
Colorectal cancer; metastatic; angiogenesis; bevacizumab; ziv-aflibercept; regorafenib
17.  PI3K inhibition results in enhanced HER signaling and acquired ERK dependency in HER2-overexpressing breast cancer 
Oncogene  2011;30(22):2547-2557.
There is a strong rationale to therapeutically target the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway in breast cancer since it is highly deregulated in this disease and it also mediates resistance to anti-HER2 therapies. However, initial studies with rapalogs, allosteric inhibitors of mTORC1, have resulted in limited clinical efficacy probably due to the release of a negative regulatory feedback loop that triggers AKT and ERK signaling. Since activation of AKT occurs via PI3K, we decided to explore whether PI3K inhibitors prevent the activation of these compensatory pathways. Using HER2-overexpressing breast cancer cells as a model, we observed that PI3K inhibitors abolished AKT activation. However, PI3K inhibition resulted in a compensatory activation of the ERK signaling pathway. This enhanced ERK signaling occurred as a result of activation of HER family receptors as evidenced by induction of HER receptors dimerization and phosphorylation, increased expression of HER3 and binding of adaptor molecules to HER2 and HER3. The activation of ERK was prevented with either MEK inhibitors or anti-HER2 monoclonal antibodies and tyrosine kinase inhibitors. Combined administration of PI3K inhibitors with either HER2 or MEK inhibitors resulted in decreased proliferation, enhanced cell death and superior anti-tumor activity compared with single agent PI3K inhibitors. Our findings indicate that PI3K inhibition in HER2-overexpressing breast cancer activates a new compensatory pathway that results in ERK dependency. Combined anti-MEK or anti-HER2 therapy with PI3K inhibitors may be required in order to achieve optimal efficacy in HER2-overexpressing breast cancer. This approach warrants clinical evaluation.
doi:10.1038/onc.2010.626
PMCID: PMC3107390  PMID: 21278786
PI3K/mTOR; HER2; feedback; ERK; BEZ235
18.  Emergence of Protein Kinase CK2 as a Key Target in Cancer Therapy 
BioFactors (Oxford, England)  2010;36(3):187-195.
Protein kinase CK2, a protein serine/threonine kinase, plays a global role in activities related to cell growth, cell death and cell survival. CK2 has a large number of potential substrates localized in diverse locations in the cell including, e.g., NF-κB as an important downstream target of the kinase. In addition to its involvement in cell growth and proliferation it is also a potent suppressor of apoptosis, raising its key importance in cancer cell phenotype. CK2 interacts with diverse pathways which illustrates the breadth of its impact on the cellular machinery of both cell growth and cell death giving it the status of a “master regulator” in the cell. With respect to cancer, CK2 has been found to be dysregulated in all cancers examined demonstrating increased protein expression levels and nuclear localization in cancer cells compared with their normal counterparts. We originally proposed CK2 as a potentially important target for cancer therapy. Given the ubiquitous and essential for cell survival nature of the kinase, an important consideration would be to target it specifically in cancer cells while sparing normal cells. Towards that end, our design of a tenascin based sub-50 nm (i.e., less than 50 nm size) nanocapsule in which an anti-CK2 therapeutic agent can be packaged is highly promising because this formulation can specifically deliver the cargo intracellularly to the cancer cells in vivo. Thus, appropriate strategies to target CK2 especially by molecular approaches may lead to a highly feasible and effective approach to eradication of a given cancer.
doi:10.1002/biof.96
PMCID: PMC2916697  PMID: 20533398
Protein kinase CK2; casein kinase 2; signaling; cancer; NF-κB; nanocapsule; nanoparticle; tenfibgen; tenascin; antisense; siRNA
19.  BPR1K653, a Novel Aurora Kinase Inhibitor, Exhibits Potent Anti-Proliferative Activity in MDR1 (P-gp170)-Mediated Multidrug-Resistant Cancer Cells 
PLoS ONE  2011;6(8):e23485.
Background
Over-expression of Aurora kinases promotes the tumorigenesis of cells. The aim of this study was to determine the preclinical profile of a novel pan-Aurora kinase inhibitor, BPR1K653, as a candidate for anti-cancer therapy. Since expression of the drug efflux pump, MDR1, reduces the effectiveness of various chemotherapeutic compounds in human cancers, this study also aimed to determine whether the potency of BPR1K653 could be affected by the expression of MDR1 in cancer cells.
Principal Findings
BPR1K653 specifically inhibited the activity of Aurora-A and Aurora-B kinase at low nano-molar concentrations in vitro. Anti-proliferative activity of BPR1K653 was evaluated in various human cancer cell lines. Results of the clonogenic assay showed that BPR1K653 was potent in targeting a variety of cancer cell lines regardless of the tissue origin, p53 status, or expression of MDR1. At the cellular level, BPR1K653 induced endo-replication and subsequent apoptosis in both MDR1-negative and MDR1-positive cancer cells. Importantly, it showed potent activity against the growth of xenograft tumors of the human cervical carcinoma KB and KB-derived MDR1-positive KB-VIN10 cells in nude mice. Finally, BPR1K653 also exhibited favorable pharmacokinetic properties in rats.
Conclusions and Significance
BPR1K653 is a novel potent anti-cancer compound, and its potency is not affected by the expression of the multiple drug resistant protein, MDR1, in cancer cells. Therefore, BPR1K653 is a promising anti-cancer compound that has potential for the management of various malignancies, particularly for patients with MDR1-related drug resistance after prolonged chemotherapeutic treatments.
doi:10.1371/journal.pone.0023485
PMCID: PMC3160846  PMID: 21887256
20.  Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells 
Introduction
Sustained HER2 signaling at the cell surface is an oncogenic mechanism in a significant proportion of breast cancers. While clinically effective therapies targeting HER2 such as mAbs and tyrosine kinase inhibitors exist, tumors overexpressing HER2 eventually progress despite treatment. Thus, abrogation of persistent HER2 expression at the plasma membrane to synergize with current approaches may represent a novel therapeutic strategy.
Methods
We generated polyclonal anti-HER2 antibodies (HER2-VIA) by vaccinating mice with an adenovirus expressing human HER2, and assessed their signaling effects in vitro and anti-tumor effects in a xenograft model. In addition, we studied the signaling effects of human HER2-specific antibodies induced by vaccinating breast cancer patients with a HER2 protein vaccine.
Results
HER2-VIA bound HER2 at the plasma membrane, initially activating the downstream kinases extracellular signal-regulated protein kinase 1/2 and Akt, but subsequently inducing receptor internalization in clathrin-coated pits in a HER2 kinase-independent manner, followed by ubiquitination and degradation of HER2 into a 130 kDa fragment phosphorylated at tyrosine residues 1,221/1,222 and 1,248. Following vaccination of breast cancer patients with the HER2 protein vaccine, HER2-specific antibodies were detectable and these antibodies bound to cell surface-expressed HER2 and inhibited HER2 signaling through blocking tyrosine 877 phosphorylation of HER2. In contrast to the murine antibodies, human anti-HER2 antibodies induced by protein vaccination did not mediate receptor internalization and degradation.
Conclusion
These data provide new insight into HER2 trafficking at the plasma membrane and the changes induced by polyclonal HER2-specific antibodies. The reduction of HER2 membrane expression and HER2 signaling by polyclonal antibodies induced by adenoviral HER2 vaccines supports human clinical trials with this strategy for those breast cancer patients with HER2 therapy-resistant disease.
doi:10.1186/bcr3204
PMCID: PMC3446352  PMID: 22676470
21.  Carcinomatous meningitis: Leptomeningeal metastases in solid tumors 
Surgical Neurology International  2013;4(Suppl 4):S265-S288.
Leptomeningeal metastasis (LM) results from metastatic spread of cancer to the leptomeninges, giving rise to central nervous system dysfunction. Breast cancer, lung cancer, and melanoma are the most frequent causes of LM among solid tumors in adults. An early diagnosis of LM, before fixed neurologic deficits are manifest, permits earlier and potentially more effective treatment, thus leading to a better quality of life in patients so affected. Apart from a clinical suspicion of LM, diagnosis is dependent upon demonstration of cancer in cerebrospinal fluid (CSF) or radiographic manifestations as revealed by neuraxis imaging. Potentially of use, though not commonly employed, today are use of biomarkers and protein profiling in the CSF. Symptomatic treatment is directed at pain including headache, nausea, and vomiting, whereas more specific LM-directed therapies include intra-CSF chemotherapy, systemic chemotherapy, and site-specific radiotherapy. A special emphasis in the review discusses novel agents including targeted therapies, that may be promising in the future management of LM. These new therapies include anti-epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors erlotinib and gefitinib in nonsmall cell lung cancer, anti-HER2 monoclonal antibody trastuzumab in breast cancer, anti-CTLA4 ipilimumab and anti-BRAF tyrosine kinase inhibitors such as vermurafenib in melanoma, and the antivascular endothelial growth factor monoclonal antibody bevacizumab are currently under investigation in patients with LM. Challenges of managing patients with LM are manifold and include determining the appropriate patients for treatment as well as the optimal route of administration of intra-CSF drug therapy.
doi:10.4103/2152-7806.111304
PMCID: PMC3656567  PMID: 23717798
Diagnostic tools; leptomeningeal metastases; monoclonal antibody; neoplastic meningitis; solid tumors; tyrosine kinase inhibitors; targeted therapy
22.  Activation of HER3 Interferes with Antitumor Effects of Axl Receptor Tyrosine Kinase Inhibitors: Suggestion of Combination Therapy1 
Neoplasia (New York, N.Y.)  2014;16(4):301-318.
The Axl receptor tyrosine kinase (RTK) has been established as a strong candidate for targeted therapy of cancer. However, the benefits of targeted therapies are limited due to acquired resistance and activation of alternative RTKs. Therefore, we asked if cancer cells are able to overcome targeted Axl therapies. Here, we demonstrate that inhibition of Axl by short interfering RNA or the tyrosine kinase inhibitor (TKI) BMS777607 induces the expression of human epidermal growth factor receptor 3 (HER3) and the neuregulin 1(NRG1)–dependent phosphorylation of HER3 in MDA-MB231 and Ovcar8 cells. Moreover, analysis of 20 Axl-expressing cancer cell lines of different tissue origin indicates a low basal phosphorylation of RAC-α serine/threonine-protein kinase (AKT) as a general requirement for HER3 activation on Axl inhibition. Consequently, phosphorylation of AKT arises as an independent biomarker for Axl treatment. Additionally, we introduce phosphorylation of HER3 as an independent pharmacodynamic biomarker for monitoring of anti-Axl therapy response. Inhibition of cell viability by BMS777607 could be rescued by NRG1-dependent activation of HER3, suggesting an escape mechanism by tumor microenvironment. The Axl-TKI MPCD84111 simultaneously blocked Axl and HER2/3 signaling and thereby prohibited HER3 feedback activation. Furthermore, dual inhibition of Axl and HER2/3 using BMS777607 and lapatinib led to a significant inhibition of cell viability in Axl-expressing MDA-MB231 and Ovcar8 cells. Therefore, we conclude that, in patient cohorts with expression of Axl and low basal activity of AKT, a combined inhibition of Axl and HER2/3 kinase would be beneficial to overcome acquired resistance to Axl-targeted therapies.
doi:10.1016/j.neo.2014.03.009
PMCID: PMC4094830  PMID: 24862757
23.  Inhibition of eEF-2 kinase Sensitizes Human Glioma Cells to TRAIL and Down-regulates Bcl-xL Expression 
Elongation factor-2 kinase (eEF-2 kinase, also known as calmodulin-dependent protein kinase III), is a unique calcium/calmodulin-dependent enzyme that inhibits protein synthesis by phosphorylating and inactivating elongation factor-2 (eEF-2). We previously reported that expression/activity of eEF-2 kinase was up-regulated in several types of malignancies including Gliomas, and was associated with response of tumor cells to certain therapeutic stress. In the current study, we sought to determine whether eEF-2 kinase expression affected sensitivity of glioma cells to treatment with tumor the necrosis factor-related apoptosis-inducing ligand (TRAIL), a targeted therapy able to induce apoptosis in cancer cells but causes no toxicity in most normal cells. We found that inhibition of eEF-2 kinase by RNA interference (RNAi) or by a pharmacological inhibitor (NH125) enhanced TRAIL-induced apoptosis in the human glioma cells, as evidenced by an increase in apoptosis in the tumor cells treated with eEF-2 kinase siRNA or the eEF-2 kinase inhibitor. We further demonstrated that sensitization of tumor cells to TRAIL was accompanied by a down-regulation of the anti-apoptotic protein, Bcl-xL, and that overexpression of Bcl-xL could abrogate the sensitizing effect of inhibiting eEF-2 kinase on TRAIL. The results of this study may help devise a new therapeutic strategy for enhancing the efficacy of TRAIL against malignant glioma by targeting eEF-2 kinase.
doi:10.1016/j.bbrc.2011.09.038
PMCID: PMC3210449  PMID: 21945617
eEF-2 kinase; TRAIL; Bcl-xl; apoptosis; glioblastoma
24.  Heregulin β1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells 
Introduction
Resistance to anti-epidermal growth factor receptor (anti-EGFR) therapies is an emerging clinical problem. The efficacy of anti-EGFR therapies can be influenced by the presence of heregulins (HRGs), which can bind erbB3/4 receptors and can activate alternative signalling pathways. In the present study we have examined whether HRG signalling can circumvent EGFR blockade in an EGFR-positive tamoxifen-resistant MCF-7 (Tam-R) breast cancer cell line.
Methods
Tam-R cells, incubated with the selective EGFR tyrosine kinase inhibitor gefitinib ('Iressa', ZD1839), were exposed to HRGβ1 and the effects on erbB receptor dimerization profiles and on activation of associated downstream signalling components were assessed by immunoprecipitation, western blotting and immunocytochemistry. The effects of HRGβ1 on gefitinib-treated Tam-R cell growth and invasion were also examined, and HRGβ1 expression levels were assessed in breast cancer tissue by immunohistochemistry to address the potential clinical relevance of such a resistance mechanism.
Results
In Tam-R cells, HRGβ1 promoted erbB3/erbB2 and erbB3/EGFR heterodimerization, promoted ERK1/2 and AKT pathway activation and increased cell proliferation and invasion. Gefitinib prevented HRGβ1-driven erbB3/EGFR heterodimerization, ERK1/2 activation and Tam-R cell proliferation, but HRGβ1-driven erbB3/erbB2 heterodimerization, AKT activation and Tam-R cell invasion were maintained. A combination of gefitinib and the phosphatidylinositol 3-kinase inhibitor LY294002 effectively blocked HRGβ1-mediated intracellular signalling activity, growth and invasion in Tam-R cells. Similarly, targeting erbB2 with trastuzumab in combination with gefitinib in Tam-R cells reduced HRGβ1-induced erbB2 and ERK1/2 activity; however, HRGβ1-driven AKT activity and cell growth were maintained while cell invasion was significantly enhanced with this combination. In clinical tissue all samples demonstrated cytoplasmic tumour epithelial HRGβ1 protein staining, with expression correlating with EGFR positivity and activation of both AKT and ERK1/2.
Conclusion
HRGβ1 can overcome the inhibitory effects of gefitinib on cell growth and invasion in Tam-R cells through promotion of erbB3/erbB2 heterodimerization and activation of the phosphatidylinositol 3-kinase/AKT signalling pathway. This may have implications for the effectiveness of anti-EGFR therapies in breast cancer as HRGβ1 is enriched in many EGFR-positive breast tumours.
doi:10.1186/bcr1754
PMCID: PMC2206726  PMID: 17686159
25.  Multiple Mechanisms Mediate Resistance to Sorafenib in Urothelial Cancer 
Genetic and epigenetic changes in the mitogen activated protein kinase (MAPK) signaling render urothelial cancer a potential target for tyrosine kinase inhibitor (TKI) treatment. However, clinical trials of several TKIs failed to prove efficacy. In this context, we investigated changes in MAPK signaling activity, downstream apoptotic regulators and changes in cell cycle distribution in different urothelial cancer cell lines (UCCs) upon treatment with the multikinase inhibitor sorafenib. None of the classical sorafenib targets (vascular endothelial growth factor receptor 1/-receptor 2, VEGFR1/-R2; platelet-derived growth factor receptor α/-receptor β, PDGFR-α/-β; c-KIT) was expressed at significant levels leaving RAF proteins as its likely molecular target. Low sorafenib concentrations paradoxically increased cell viability, whereas higher concentrations induced G1 arrest and eventually apoptosis. MAPK signaling remained partly active after sorafenib treatment, especially in T24 cells with an oncogenic HRAS mutation. AKT phosphorylation was increased, suggesting compensatory activation of the phosphatidylinositol-3-kinase (PI3K) pathway. Sorafenib regularly down regulated the anti-apoptotic myeloid cell leukemia 1 (Mcl-1) protein, but combinatorial treatment with ABT-737 targeting other B-cell lymphoma 2 (Bcl-2) family proteins did not result in synergistic effects. In summary, efficacy of sorafenib in urothelial cancer cell lines appears hampered by limited effects on MAPK signaling, crosstalk with further cancer pathways and an anti-apoptotic state of UCCs. These observations may account for the lack of efficacy of sorafenib in clinical trials and should be considered more broadly in the development of signaling pathway inhibitors for drug therapy in urothelial carcinoma.
doi:10.3390/ijms151120500
PMCID: PMC4264180  PMID: 25387078
tyrosine kinase inhibitor; urothelial cancer; mitogen activated protein kinase (MAPK) signaling; apoptosis

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