Expression Of Drug Targets Research Articles

Malaria Heat Shock Proteins: Drug Targets that Chaperone other Drug Targets

Ongoing research into the chaperone systems of malaria parasites, and particularly of Plasmodium falciparum, suggests that heat shock proteins (Hsps) could potentially be an excellent class of drug targets. The P. falciparum genome encodes a vast range and large number of chaperones, including 43 Hsp40, six Hsp70, and three Hsp90 proteins (PfHsp40s, PfHsp70s and PfHsp90s), which are involved in a number of fundamental cellular processes including protein folding and assembly, protein translocation, signal transduction and the cellular stress response. Despite the fact that Hsps are relatively conserved across different species, PfHsps do exhibit a considerable number of unique structural and functional features. One PfHsp90 is thought to be sufficiently different to human Hsp90 to allow for selective targeting. PfHsp70s could potentially be used as drug targets in two ways: either by the specific inhibition of Hsp70s by small molecule modulators, as well as disruption of the interactions between Hsp70s and co-chaperones such as the Hsp70/Hsp90 organising protein (Hop) and Hsp40s. Of the many PfHsp40s present on the parasite, there are certain unique or essential members which are considered to have good potential as drug targets. This review critically evaluates the potential of Hsps as malaria drug targets, as well as the use of chaperones as aids in the heterologous expression of other potential malarial drug targets.

Tyrosine Kinase Inhibitors Gefitinib, Lapatinib and Sorafenib Induce Rapid Functional Alterations in Breast Cancer Cells

Alterations in tyrosine kinase expression or functionality have been linked to tumor growth, and detailed analysis of tyrosine kinase pathways has led to the development of novel anticancer drugs based on their inhibition. The aim of the present work was to examine the cytotoxicity and cellular alterations correlated with multidrug resistance mechanisms induced by three tyrosine kinase inhibitors, lapatinib, sorafenib and gefitinib. The study was performed on three breast cancer cell lines (BRC-230, MCF-7 and SkBr3). Drug-induced growth inhibition was detected by Sulforhodamine B analysis. Apoptosis, cytosolic calcium alteration, extrusion pump activity and mitochondrial membrane depolarization were assessed by flow cytometry. Drug efflux-related gene expression was analyzed by RT-PCR and drug target protein expression was evaluated by Western Blot. Lapatinib and gefitinib induced a cytotoxic effect and mitochondrial membrane depolarization in BRC-230 and SkBr3 cells, while sorafenib induced apoptosis and a high and rapid dissipation of mitochondrial potential in all cell lines. Moreover, all three drugs produced a rapid cytosolic calcium mobilization from endoplasmic reticulum stores in the investigated cell lines and a strong decrease in multidrug transporter activity in BRC-230 and MCF-7 cells. Mitochondrial membrane depolarization and inhibition of multidrug transporter activity induced by tyrosine kinase inhibitors were independent of cytosolic calcium mobilization. These data suggest that the investigated drugs possess mechanisms of action that are independent of drug target expression, opening up further possibilities for the development of new therapeutic strategies.

Association of circulating tumor cells are associated with lymph node metastasis in squamous cell carcinoma of the head and neck region (SCCHN).

5523 Background: Lymph node metastasis frequently occurs in SCCHN and is generally associated with poor prognosis. The molecular mechanisms regulating tumor cell dissemination and thus the potential targets for novel treatment strategies are still largely unresolved. In this study we tested whether circulating tumor cells (CTCs) are associated with lymph node metastasis in SCCHN. We further established a method to characterize CTCs for the expression of potential drug targets and started with their characterization for EGFR expression. Methods: After informed patient consent, 7.5 ml blood was collected into heparinized tubes. After lysis of erythrocytes samples were enriched for CTCs by depletion of CD45+ blood leukocytes. Samples were stained with fluorescence-labeled antibodies to EpCAM, pan-cytokeratin, EGFR and CD45 or the relevant isotype control antibodies. Absolute numbers of EpCAM+Cytokeratin+CD45- CTCs and their EGFR expression profile were determined by flow cytometry. Results: Currently, a total of 40 SCCHN patients mainly with advanced disease (76% T3-4 stage; 70% N1 or beyond), but all without clinically detectable distant metastases, have been enrolled in this study with the most frequent tumor localization being the oropharynx (65%). Two or more CTCs per 3.75 ml of blood were detected in 40 percent of SCCHN patients (mean ± SD: 3.8 ± 2.6; range: 2-10) whereas none were found in peripheral blood samples from normal control subjects (n=10). The frequency of CTCs depended on the extent of lymph node metastasis with significantly lower CTC numbers in cases with none or only one affected lymph node (p=.01) but did not correlate with T stage (p=.69). In 47% of the cases with detectable CTCs these cells expressed EGFR. Conclusions: The presence of CTCs correlated with lymph node metastasis in SCCHN. The frequency of patients with detectable CTCs was within the range of the reported frequency of patients who will also develop distant metastases. Further studies will clarify whether the presence of circulating tumor cells may be a predictive factor for subsequent hematogenous metastases. In addition, EGFR was identified as one of the potential targets in this rare cell population. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Merck Serono Merck Serono Merck Serono

Abstract 809: Selecting ovarian cancer patients for clinical trials using a broad panel of molecular markers

Abstract Our understanding of the molecular biology of ovarian cancer is rapidly expanding, in part through efforts such as The Cancer Genome Atlas (TCGA) project, which is comprehensively cataloguing the genomic aberrations of 500 advanced serous ovarian carcinomas. These and other studies have shown that ovarian cancers are molecularly complex and highly heterogeneous. Importantly, the prevalence of over-expression or alteration of any one drug target is very low in ovarian cancer (with the exception of p53 mutations, which occur in 70-90% of advanced stage serous carcinomas). Therefore, the identification of patients for clinical trials of targeted drug therapies by measuring expression of a single target or pathway has limited usefulness. To improve the efficiency of trial enrollment and also the likelihood of successful therapeutic outcomes, we propose to evaluate every patient using a broad tumor marker panel. In feasibility studies, we have analyzed ovarian tumors for a panel of markers that includes molecular targets such as EGFR, HER2, VEGF, PDGFR, IGF1R, c-met, c-kit, ER, and AR, as well as signaling pathway proteins (e.g., HIF1alpha, COX2, PTEN). Because targeted agents are frequently combined with chemotherapy in ovarian cancer clinical trials, we also included proteins associated with response to representative agents (e.g., platinums, taxanes, anthracyclines), such as drug transporters (BCRP, MRP1, PGP) and proteins involved in DNA repair/modification (ERCC1, MGMT) or synthesis (TS, RRM1, TOPO1/2A). Formalin-fixed, paraffin-embedded tumor blocks were obtained following patient consent and protein expression was measured by immunohistochemical analyses at CLIA-certified laboratories. Expression data were recorded as histoscores (% tumor cells stained x intensity). To date, 44 tumor specimens (histology: 28 serous, 6 clear cell, 1 endometrioid, 1 MMMT, and 2 granulosa cell; 3 stage I, 3 stage II, 31 stage III, 2 stage IV, 5 unknown) have been profiled. Approximately half of the specimens were derived from the ovary; the others were recurrent lesions or metastases (peritoneum, pelvis, omentum, liver, and colon) discovered at diagnosis. Two were pleural effusion or lung nodule biopsies. The markedly heterogeneous nature of ovarian tumors is apparent from the results of this analysis. One exception is the expression of the angiogenic factor VEGF, which is highly expressed in >80% of these tumors. PDGFR alpha and beta were highly co-expressed in multiple tumors. IGF1R was highly expressed in ∼25%, ER alpha in ∼40%, and AR in ∼20% of ovarian tumors. No tumor over-expressed HER2 and EGFR was highly detected in only 10% of the cases. Importantly, hierarchical clustering analyses based upon drug target expression revealed the existence of several sub-groups of ovarian cancer patients and provide clues to select from treatment options. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 809.

Abstract 3089: Heterogenous high level HER-2 amplification in a small subset of colorectal cancers

Abstract HER-2 is the molecular target for antibody-based treatment of breast cancer (trastuzumab). The potential benefit of anti-HER-2 therapy is currently investigated in several other HER-2 amplified cancers. For example, trastuzumab was recently shown to be effective in HER-2 positive gastric cancer. To address the potential applicability of anti-HER-2 therapy in CRC, tissue microarray (TMA) sections and colorectal resection specimens of 1851 CRC were analyzed for HER-2 overexpression and amplification using FDA approved reagents for immunohistochemistry and fluorescence in situ hybridization. HER-2 amplification was seen in 2.5% and HER-2 overexpression in 2.7 % of 1439 interpretable CRC. Amplification was often high level with HER-2 copies ranging from 4 to 60 per tumor cell and was strongly related to protein overexpression. HER-2 amplification and overexpression were unrelated to histological tumor type, tumor localization, grading, pT, pN, pM or survival. As heterogeneity of drug target expression could represent a major drawback for targeted cancer therapy we next studied HER-2 heterogeneity in selected cases. Extensive evaluation of all available large sections from patients with HER-2 positive CRC revealed heterogenous findings in 3 of 4 cases. In summary, high level HER-2 amplification occurs in a small fraction of CRCs. Heterogeneity of amplification may limit the utility of anti HER-2 therapy in some of these tumors and therefore, adequate clinical trials are needed to further evaluate this approach. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3089.

Population-based differences in treatment outcome following anticancer drug therapies

Population-based differences in toxicity and clinical outcome following treatment with anticancer drugs have an important effect on oncology practice and drug development. These differences arise from complex interactions between biological and environmental factors, which include genetic diversity affecting drug metabolism and the expression of drug targets, variations in tumour biology and host physiology, socioeconomic disparities, and regional preferences in treatment standards. Some well-known examples include the high prevalence of activating epidermal growth factor receptor (EGFR) mutations in pulmonary adenocarcinoma among northeast (China, Japan, Korea) and parts of southeast Asia (excluding India) non-smokers, which predict sensitivity to EGFR kinase inhibitors, and the sharp contrast between Japan and the west in the management and survival outcome of gastric cancer. This review is a critical overview of population-based differences in the four most prevalent cancers in the world: lung, breast, colorectal, and stomach cancer. Particular attention is given to the clinical relevance of such knowledge in terms of the individualisation of drug therapy and in the design of clinical trials.

Abstract B262: Tyrosine kinase inhibitors induce rapid cellular alterations in breast cancer cells in vitro

Abstract Purpose: Alterations in tyrosine kinase (TK) expression or functionality have been linked to tumor growth, and the unraveling of TK pathways has led to the discovery of novel anticancer drugs. The aims of this study were to evaluate cytotoxic and multidrug resistance mechanisms, such as Ca2+ homeostasis and efflux pump activity, of three tyrosine kinase inhibitors (TKIs), lapatinib, sorafenib and gefitinib, in two breast cancer cell lines. Methods: Two human breast cancer cell lines, Estrogen Receptor (ER) positive MCF-7 and ER negative BRC-230 were exposed to the investigated drugs for 20, 40, 60, 120 and 240 min. Cytotoxicity, cytosolic Ca2+ levels, extrusion pump activity and mitochondrial membrane depolarization were assessed by flow cytometry. Expression of drug efflux genes was determined by RT-PCR, and protein expression of drug targets by Western blot. Results: MCF-7 cells did not express EGFR, p-EGFR, Her-2 or p-Her-2, while BRC-230 were positive for EGFR and p-EGFR only. ERK1/2 was equally expressed in both cell lines, but only BRC-230 were positive for p-ERK1/2. MEK and p-MEK were expressed in both cell lines, albeit more intensively in BRC-230. As expected, based on their drug target expression profiles, gefitinib and lapatinib were found to induce cytocidal effects and mitochondrial membrane depolarization in BRC-230 only, while sorafenib caused apoptosis and loss of mitochondrial potential in both cell lines. All three drugs triggered a rapid and biphasic increase in cytosolic [Ca2+] (after a 20 min exposure), and a decline in xenobiotic extrusion pump activity that was independent of efflux gene downregulation. The thapsigargin-induced depletion of endoplasmic reticulum Ca2+ blocked the increase in cytosolic Ca2+, but had no effect on multidrug transporter activity or on mitochondrial membrane depolarization. Conclusions: Gefitinib, lapatinib and sorafenib induced a rapid increase in cytosolic [Ca2+] and mitochondrial membrane depolarization, in addition to blocking drug transporter activity, independently of the expression of specific drug targets and of drug-induced cytotoxicity. These data suggest that TKIs may elicit different molecular mechanisms to those previously reported in the literature. Further research of these pathways is warranted in order to identify new, more effective strategies for cancer treatment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B262.

Heterogeneity of Drug Target Expression Among Metastatic Lesions: Lessons from a Breast Cancer Autopsy Program

The target tissue for most cancer therapeutics is metastatic disease, either the treatment of overt metastases or the prevention of colonization by micrometastases in the adjuvant setting. Despite this, few drugs are tested in the metastatic setting in preclinical experiments, and relatively little

Technology Insight: novel imaging of molecular targets is an emerging area crucial to the development of targeted drugs

Targeted drugs hold great promise for the treatment of malignant tumors; however, there are several challenges for efficient evaluation of these drugs in preclinical and clinical studies. These challenges include identifying the 'correct', biologically active concentration and dose schedule, selecting the patients likely to benefit from treatment, monitoring inhibition of the target protein or pathway, and assessing the response of the tumor to therapy. Although anatomic imaging will remain important, molecular imaging provides several new opportunities to make the process of drug development more efficient. Various techniques for molecular imaging that enable noninvasive and quantitative imaging are now available in the preclinical and clinical settings, to aid development and evaluation of new drugs for the treatment of cancer. In this Review, we discuss the integration of molecular imaging into the process of drug development and how molecular imaging can address key questions in the preclinical and clinical evaluation of new targeted drugs. Examples include imaging of the expression and inhibition of drug targets, noninvasive tissue pharmacokinetics, and early assessment of the tumor response.

Gene Expression in Gastrointestinal Stromal Tumors Is Distinguished by KIT Genotype and Anatomic Site

Gastrointestinal stromal tumors (GISTs) are specific KIT expressing and KIT-signaling driven mesenchymal tumors of the human digestive tract, many of which have KIT-activating mutations. Previous studies have found a relatively homogeneous gene expression profile in GIST, as compared with other histological types of sarcomas. Transcriptional heterogeneity within clinically or molecularly defined subsets of GISTs has not been previously reported. We tested the hypothesis that the gene expression profile in GISTs might be related to KIT genotype and possibly to other clinicopathological factors. An HG-U133A Affymetrix chip (22,000 genes) platform was used to determine the variability of gene expression in 28 KIT-expressing GIST samples from 24 patients. A control group of six intra-abdominal leiomyosarcomas was also included for comparison. Statistical analyses (t tests) were performed to identify discriminatory gene lists among various GIST subgroups. The levels of expression of various GIST subsets were also linked to a modified version of the growth factor/KIT signaling pathway to analyze differences at various steps in signal transduction. Genes involved in KIT signaling were differentially expressed among wild-type and mutant GISTs. High gene expression of potential drug targets, such as VEGF, MCSF, and BCL2 in the wild-type group, and Mesothelin in exon 9 GISTs were found. There was a striking difference in gene expression between stomach and small bowel GISTs. This finding was validated in four separate tumors, two gastric and two intestinal, from a patient with familial GIST with a germ-line KIT W557R substitution. GISTs have heterogeneous gene expression depending on KIT genotype and tumor location, which is seen at both the genomic level and the KIT signaling pathway in particular. These findings may explain their variable clinical behavior and response to therapy.