Transcriptional Program Associated with IFN- α Response of Renal Cell Carcinoma

Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Journal of Interferon & Cytokine Research (Impact Factor: 2). 04/2006; 26(3):156-70. DOI: 10.1089/jir.2006.26.156
Source: PubMed


Metastatic renal cell carcinoma (RCC) is refractory to therapy; however, 10%-20% of patients respond favorably with interferon-alpha (IFN-alpha) treatment. To understand the molecular basis of response to IFN-alpha therapy, we performed global gene expression analysis of sensitive and resistant RCC cell lines in the absence and in the presence of IFN-alpha, using high-density oligonucleotide arrays to detect differentially expressed genes. In the absence of IFN-alpha, no significant differences in gene expression were observed between six sensitive and six resistant cell lines. Gene expression analysis following a time course of IFN-alpha2b treatment in one sensitive (SK-RC-17) and one resistant (SK-RC-12) cell line revealed that 484 and 354 transcripts, respectively, were modulated. A considerable number of these transcripts were similarly modulated between the two cell types that included several known targets of IFN signaling associated with antiviral and immunomodulatory activity. A further analysis of gene expression pattern in response to IFN revealed that several transcripts associated with proapoptotic function were upregulated in the sensitive cells. In the resistant cells, transcripts associated with cell survival and proliferation were induced, and key apoptotic molecules were suppressed. This study suggests that the IFN-alpha response of individual RCC tumors is determined by the expression pattern of genes in the apoptosis vs. survival and proliferation pathways rather than by alterations in expression of one or more individual genes.

4 Reads
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The aim of my project was to identify and functionally characterise novel human proteins that influence cancer relevant cellular processes like cell proliferation, signalling, and apoptosis upon over-expression. The focus of my work was 1) The establishment of a high throughput cell based assay to screen for proteins involved in the modulation of cell signalling pathways, specifically the activation of the ERK1/2 pathway, 2) to apply this assay in a screen of previously uncharacterised proteins, and 3) to characterise one candidate protein from this assay and to validate its association with the ERK1/2 pathway. The principle of the assay is based on the detection of phosphorylated ERK1/2 in cells over-expressing N- and C-terminal YFP tagged proteins. Data acquisition was done using a flow cytometer with an integrated 96-well plate reader. A total of 200 proteins were screened, out of which eleven novel cancer relevant modulators of ERK1/2 activation were identified. One of the candidates, the Radial spoke head like -1 (Rshl1), which was identified as an inhibitor of ERK1/2 activation was followed up, and shown to be down regulated in kidney cancer. The protein was identified as an inhibitor of proliferation in another cell based assay. The corresponding gene is located on chromosome 19q13.3 at the primary ciliary dyskinesia locus, and the encoded protein contains a radial spoke domain. However, the biological role of this protein was not described. I found that Rshl1 indeed localizes to primary cilia but also to the cytoplasm and nucleus of human kidney cells. Further, I found that its localisation is cell cycle phase dependent. Rshl1 co-localised with MEK1, ERK1/2 and CDK2 and interacts with MEK1, CDK2 and ERK3. Its role as an inhibitor of proliferation was elucidated by the finding that over-expression of Rshl1 caused a G0/G1 phase arrest in human kidney cells via an up-regulation of p57KIP2 expression and stabilization of ERK3. Rshl1 thus regulates the cell cycle by inhibiting the ERK1/2 kinase. It interacts with critical signalling proteins in the cell and maintains homeostasis by arresting cells in the G0/G1 phase. In conclusion, I screened 200 novel proteins for their influence on ERK1/2 activation and identified eleven novel modulators of ERK1/2 pathway. Detailed functional analysis of Rshl1, which was an inhibitor of ERK1/2 activation, identifies this protein as a novel player in the MAPK pathway, and shed light on its role in homeostasis and tumorigenesis. Das Ziel dieses Projektes war die Identifizierung und funktionelle Charakterisierung unbekannter Proteine die, nach Überexpression, Krebs-relevante zelluläre Prozesse wie z.B. Proliferation, Signaltransduktion und Apoptose beeinflussen. Der Fokus meiner Arbeit lag in der Etablierung eines zellbasierten Hochdurchsatz-Assays zur Untersuchung von Proteinen auf die Modulation von Zell-Signalwegen, im Speziellen der Aktivierung des ERK1/2- Signalweges. Das Prinzip des Assays basiert auf der Detektion der phosphorylierten Form von ERK1/2 in Zellen, die Fusionsproteine mit N- und C-terminalem YFP überexprimieren. Die Datenaufnahme wurde mit einem Durchflußzytometer mit integriertem 96-Well-Platten Lesegerät durchgeführt. Insgesamt wurden 200 Proteine untersucht, von denen schließlich sieben als Krebs-relevante ERK1/2-Modulatoren identifiziert wurden. Einer der Kandidaten, das Radial Spoke Head Like-1 (Rshl1) Protein, welches als Inhibitor der ERK1/2 Kinase identifiziert wurde, habe ich im Rahmen meiner Arbeit funktionell charakterisiert. In vorherigen Studien wurde gezeigt, dass Rshl1 in Nierenkrebs herunter reguliert ist und es wurde als Inhibitor der Proliferation beschrieben. Das Rshl1-Gen ist auf Chromosom 19q13.3 im Primary Ciliary Dykinesia Lokus lokalisiert und das Protein enthält eine Radial-Spoke-Domäne, jedoch ist die biologische Funktion bisher nicht bekannt. In der vorliegenden Studie habe ich die Lokalisation des Rshl1 Proteins in primären Cilien, im Cytoplasma und im Kern von Nierenzellen nachgewiesen und konnte eine Zellzyklus-abhängige Lokalisation feststellen. Ich habe gezeigt, dass Rshl1 mit den Proteinen MEK1, ERK1/2 und CDK2 co-lokalisiert und habe seine direkte Interaktion mit MEK-1, CDK2 und ERK3 nachgewiesen. Seine Rolle als Inhibitor der Proliferation wurde durch die Blockade von Nierenzellen mit Rshl1-Überexpression in der G0/G1-Phase des Zellzyklus, sowie durch die verstärkte Expression des Zellzyklus-Repressors p57KIP2 und die Stabilisierung von ERK3 erläutert. Diese Studie zeigt somit zum ersten Mal, dass Rshl1 den Zellzyklus durch die Inhibierung der ERK1/2-Kinase reguliert. Es interagiert mit Schlüsselproteinen der Signaltransduktion und erhält das Gleichgewicht während der G0/G1-Phase des Zellzyklus. Zusammengefasst habe ich 200 Proteine auf ihren Einfluss auf die ERK1/2-Aktivierung untersucht und sieben neue Modulatoren des ERK1/2-Signalweges identifiziert. Die Ergebnisse aus der detaillierten funktionellen Analyse des Proteins Rshl1, für das eine Inhibierung des ERK1/2-Siganlweges nachgewiesen wurde, bestätigt die Stärke und Effizienz dieses Ansatzes und hebt die Bedeutung einer solchen Untersuchung im Rahmen der funktionellen Genomanalyse hervor.
    Preview · Article · Jan 2006
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: This review describes some aspects of uncontrolled tumor growth and development. In the past, it has been shown that colon adenocarcinomas use several tactics to avoid cell deletion and to maintain cell viability. In particular, colorectal cancer cells resist death ligands-induced apoptosis by expressing anti-apoptotic proteins. By direct interaction with FADD, the FLIP protein inhibits the signal transmission from death receptors to their cytoplasmic targets in COLO 205 cells. Colorectal cancer cells also stimulate own survival by inhibiting cytotoxic signals induced by interferons. Moreover, IFN-alpha increases immune-resistance of colon cancer cells by activation of NF-kappaB. Additionally, the cytoplasmic retention of proapoptotic protein clusterin also supports viability of cancer cells. Upon suitable stimulation normal cells are featured by clusterin translocation to the nucleus with concomitant cell death. We found that proapoptotic activity of clusterin is dependent on calcium ions, and depletion of intracellular calcium caused extensive death of COLO 205 cells. Other type of strategy to inhibit chemotherapy-dependent cell death is the activity of multidrug resistance proteins (MDR). These cell membrane efflux pumps actively expel the drugs from the cell interior to prevent their action on intracellular targets. The reversal of P-gp efflux pump in chemoresistant COLO 320 cell line was observed upon phenothiazine derivatives. The variety of antiapoptotic mechanisms in colorectal cancer cells makes anticancer therapy a great challenge but detailed knowledge of their complexicity gives promise to sensitize cancer cells to death stimuli.
    Full-text · Article · Feb 2006 · Advances in Medical Sciences
  • [Show abstract] [Hide abstract]
    ABSTRACT: Interferons (IFNs) are a class of cytokines with pleotropic actions that regulate a variety of cellular activities. Clinical trials with recombinant IFNs (IFN-alpha2a and IFN-alpha2b) have demonstrated clinical activity in patients with advanced renal cell carcinoma (RCC). Their efficacy is characterized by a low overall tumor regression rate of < 15%, progression-free survival of 4-5 months, and overall median survival of 10-18 months. This cytokine became the standard of care for patients with metastatic RCC and was then used as the comparator arm in a series of phase II and III clinical trials that have defined a new treatment paradigm for patients with advanced RCC. This paradigm uses the tyrosine kinase inhibitors (TKIs) sorafenib and sunitinib, the mammalian target of rapamycin (mTOR) inhibitor temsirolimus, and the vascular endothelial growth factor monoclonal antibody bevacizumab. These 3 categories of agents were then investigated in combination with IFN-alpha in a series of preclinical and clinical studies. The collective data from these reports suggest the combination of IFN-alpha and bevacizumab is active and has a role in RCC therapy, whereas combinations with the TKIs or mTOR inhibitors have limited efficacy and/or excessive toxicity. The clinical and preclinical studies leading to these conclusions are reviewed herein.
    No preview · Article · Dec 2008 · Clinical Genitourinary Cancer
Show more