Fatima W. Khwaja

Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis

Glioblastomas, like other solid tumors, have extensive areas of hypoxia and necrosis. The importance of hypoxia in driving tumor growth is receiving increased attention. Hypoxia-inducible factor 1 (HIF-1) is one of the master regulators that orchestrate the cellular responses to hypoxia. It is a heterodimeric transcription factor composed of alpha and beta subunits. The alpha subunit is stable in hypoxic conditions but is rapidly degraded in normoxia. The function of HIF-1 is also modulated by several molecular mechanisms that regulate its synthesis, degradation, and transcriptional activity. Upon stabilization or activation, HIF-1 translocates to the nucleus and induces transcription of its downstream target genes. Most important to gliomagenesis, HIF-1 is a potent activator of angiogenesis and invasion through its upregulation of target genes critical for these functions. Activation of the HIF-1 pathway is a common feature of gliomas and may explain the intense vascular hyperplasia often seen in glioblastoma multiforme. Activation of HIF results in the activation of vascular endothelial growth factors, vascular endothelial growth factor receptors, matrix metalloproteinases, plasminogen activator inhibitor, transforming growth factors alpha and beta, angiopoietin and Tie receptors, endothelin-1, inducible nitric oxide synthase, adrenomedullin, and erythropoietin, which all affect glioma angiogenesis. In conclusion, HIF is a critical regulatory factor in the tumor microenvironment because of its central role in promoting proangiogenic and invasive properties. While HIF activation strongly promotes angiogenesis, the emerging vasculature is often abnormal, leading to a vicious cycle that causes further hypoxia and HIF upregulation.

Ibuprofen Inhibits Survival of Bladder Cancer Cells by Induced Expression of the p75NTR Tumor Suppressor Protein

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used to reduce inflammation and as analgesics by inhibition of cyclooxygenase-2. At higher concentrations, some NSAIDs inhibit proliferation and induce apoptosis of cancer cells. Although several molecular mechanisms have been postulated to explain the anticancer effects of NSAIDs, they do not involve merely the inhibition of cyclooxygenase-2, and a more proximate initiator molecule may be regulated by NSAIDs to inhibit growth. The p75 neurotrophin receptor (p75NTR) is a proximate cell membrane receptor glycoprotein that has been identified as a tumor and metastasis suppressor. We observed that NSAID treatment of cell lines from bladder and other organs induced expression of the p75NTR protein. Of the different types of NSAIDs examined, ibuprofen was more efficacious than aspirin and acetaminophen and comparable with (R)-flurbiprofen and indomethacin in induction of p75NTR protein expression. This rank order NSAID induction of the p75NTR protein correlated with the ability of these NSAIDs to reduce cancer cell survival. To examine a mechanistic relationship between ibuprofen induction of p75NTR protein and inhibition of survival, bladder cancer cells were transfected with ponasterone A-inducible vectors that expressed a death domain-deleted (ΔDD) or intracellular domain-deleted (ΔICD) p75NTR product that acts as a dominant negative antagonist of the intact p75NTR protein. Expression of ΔDD and ΔICD rescued cells from ibuprofen inhibition of growth. These observations suggest that p75NTR is an important upstream modulator of the anticancer effects of NSAIDs and that ibuprofen induction of the p75NTR protein establishes an alternate mechanism by which ibuprofen may exert an anticancer effect.

Proteomic identification of the wt-p53-regulated tumor cell secretome.

Tumor–stroma interactions play a major role in tumor development, maintenance and progression. Yet little is known on how the genetic alterations that underlie cell transformation elicit cell extrinsic changes modulating heterotypic cell interactions. We hypothesized that these events involve a modification in the complement of secreted proteins by the cell, acting as mediators of intercellular communication. To test this hypothesis, we examined the role of wt-p53, a major tumor suppressor, on the tumor microenvironment through its regulation of secreted factors. Using a combination of 2-DE and cICAT proteomic techniques, we found a total of 111 secreted proteins, 39 of which showed enhanced and 21 inhibited secretion in response to wt-p53 expression. The majority of these were not direct targets of p53 transcription factor activity, suggesting a novel role for wt-p53 in the control of intracellular protein trafficking and/or secreted protein stability. Evidence for p53-controlled post-translational modifications on nine secreted proteins was also found. These findings will enhance our understanding of wt-p53 modulated interactions of the tumor with its environment.

The aryl propionic acid R-flurbiprofen selectively induces p75NTR-dependent decreased survival of prostate tumor cells.

Epidemiologic studies show that patients chronically consuming nonsteroidal anti-inflammatory drugs (NSAID) for arthritis exhibit a reduced incidence of prostate cancer. In addition, some NSAIDs show anticancer activity in vitro. NSAIDs exert their anti-inflammatory effects by inhibiting cyclooxygenase (COX) activity; however, evidence suggests that COX-independent mechanisms mediate decreased prostate cancer cell survival. Hence, we examined the effect of selected aryl propionic acid NSAIDs and structurally related compounds on the decreased survival of prostate cancer cell lines PC-3, DU-145, and LNCaP by induction of the p75(NTR) protein. p75(NTR) has been shown to function as a tumor suppressor in the prostate by virtue of its intracellular death domain that can initiate apoptosis and inhibit growth. The most efficacious compounds for induction of p75(NTR) and decreased survival, in rank-order, were R-flurbiprofen, ibuprofen, oxaprozin, fenoprofen, naproxen, and ketoprofen. Because R-flurbiprofen and ibuprofen exhibited the greatest efficacy, we examined their dose-dependent specificity of induction for p75(NTR) relative to other members of the death receptor family. Whereas treatment with R-flurbiprofen or ibuprofen resulted in a massive induction of p75(NTR) protein levels, the expression of Fas, p55(TNFR), DR3, DR4, DR5, and DR6 remained largely unchanged. Moreover, transfection of either cell line before R-flurbiprofen or ibuprofen treatment with a dominant negative form of p75(NTR) to antagonize p75(NTR) activity or p75(NTR) small interfering RNA to prevent p75(NTR) protein expression rescued both cell lines from decreased survival. Hence, R-flurbiprofen and ibuprofen selectively induce p75(NTR)-dependent decreased survival of prostate cancer cells independently of COX inhibition.

Attractin Is Elevated in the Cerebrospinal Fluid of Patients with Malignant Astrocytoma and Mediates Glioma Cell Migration

Purpose: There are a limited number of noninvasive methods available for the monitoring of neoplastic disease in the central nervous system. The goal of our study was to find reliable markers that could be used for disease monitoring as well as to identify new targets for the therapeutic intervention for malignant astrocytoma (WHO grades 3 and 4). Experimental Design: We employed proteomic techniques to identify secreted proteins in the cerebrospinal fluid that were specific to patients with malignant astrocytoma. Results: Among 60 cerebrospinal fluid samples of patients with various central nervous system diseases, attractin was consistently found to be elevated in the samples of patients with malignant astrocytoma. To independently validate these results, we examined attractin expression in a new set of 108 normal and tumoral brain tissue specimens and found elevated expression in 97% of malignant astrocytomas, with the highest levels in grade 4 tumors. Using immunohistochemistry, we further showed that attractin is produced and secreted by the tumor cells. Finally, we showed that cerebrospinal fluid from brain tumor patients induces glioma cell migration and that attractin is largely responsible for this promigratory activity. Conclusions: Our results find attractin to be a reliable secreted marker for high-grade gliomas. Additionally, our migration studies suggest that it may be an important mediator of tumor invasiveness, and thus, a potential target in future therapies.

Regulation of Bim by TCR Signals in CD4/CD8 Double-Positive Thymocytes

Bim, a BH3-only Bcl-2 family member, is required for apoptosis of thymocytes in response to negative selection signals. Regulation of the apoptotic activity of Bim during negative selection is not understood. In this study we demonstrate that in murine thymocytes undergoing apoptosis in response to anti-CD3ε injection, levels of Bim protein expression do not change. In immature thymocytes, Bim is associated with mitochondria before stimulation and is not regulated by a change in subcellular localization during apoptosis. We also show that BimEL is rapidly phosphorylated in thymocytes in response to CD3ε cross-linking both in vivo and in vitro, and that phosphorylation is sustained for at least 24 h. Analysis of MHC-deficient mice shows that phosphorylation of Bim occurs in CD4/CD8 double-positive thymocytes and does not depend on activation of mature T cells. We also find that TCR cross-linking on thymocytes induces an increase in the proportion of Bcl-xL bound to Bim at late time points. Our results favor a model in which strong TCR signals regulate the apoptotic activity of Bim by phosphorylation and subsequent changes in binding to Bcl-xL in immature thymocytes.

The p38 MAPK Pathway Mediates Aryl Propionic Acid–Induced Messenger RNA Stability of p75NTR in Prostate Cancer Cells

The p75(NTR) acts as a tumor suppressor in the prostate, but its expression is lost as prostate cancer progresses and is minimal in established prostate cancer cell lines such as PC-3, DU-145, and LNCaP. Previously, we showed that treatment with R-flurbiprofen or ibuprofen induced p75(NTR) expression in PC-3 and DU-145 cells leading to p75(NTR)-mediated decreased survival. Here, we investigate the mechanism by which these drugs induce p75(NTR) expression. We show that the observed increase in p75(NTR) protein due to R-flurbiprofen and ibuprofen treatment was accompanied by an increase in p75(NTR) mRNA, and this increase in mRNA was the result of increased mRNA stability and not by an up-regulation of transcription. In addition, we show that treatment with R-flurbiprofen or ibuprofen led to sustained activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Furthermore, inhibition of the p38 MAPK pathway with the p38 MAPK-specific inhibitor SB202190 or by small interfering RNA (siRNA) knockdown of p38 MAPK protein prevented induction of p75(NTR) by R-flurbiprofen and ibuprofen. We also observed that siRNA knockdown of MAPK-activated protein kinase (MK)-2 and MK3, the kinases downstream of p38 MAPK that are responsible for the mRNA stabilizing effects of the p38 MAPK pathway, also prevented an induction of p75(NTR) by R-flurbiprofen and ibuprofen. Finally, we identify the RNA stabilizing protein HuR and the posttranscriptional regulator eukaryotic translation initiation factor 4E as two possible mechanisms by which the p38 MAPK pathway may increase p75(NTR) expression. Collectively, the data suggest that R-flurbiprofen and ibuprofen induce p75(NTR) expression by increased mRNA stability that is mediated through the p38 MAPK pathway.

Proteomic Discovery of Biomarkers in the Cerebrospinal Fluid of Brain Tumor Patients

Central nervous system (CNS) diseases often induce changes in the protein composition of the cerebrospinal fluid (CSF) as this liquid bathes the brain and collects its secreted products. The detection and monitoring of such pathology-related changes can be exploited for their relation to tumor growth in the brain. The potential of using differential proteomic profiling in CNS malignancies to identify diagnostic, prognostic, and therapeutic response markers as well as therapeutic targets is currently being actively investigated. Furthermore, elucidation of the CSF oncoproteome may yield important biological insights into the pathogenesis of CNS neoplasms. New proteomic technology forecasts rapid screening, low sample consumption, and accurate protein identification in the CSF. These technological improvements will permit the gathering of reliable data on CSF protein composition in a clinically relevant microchip format in the foreseeable future. Here we review the current status of proteomic technology and its application to CNS cancers, with a focus on translational studies.

Abstract 4181: Retrospective tissue microarray analysis finds potential predictors of nodal metastasis in early tongue cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Pakistan has one of the highest incidences of oral cancer in the world. In Shaukat Khanum Memorial Cancer Hospital and Research Center (SKMCH&RC), Squamous Cell Carcinoma of Tongue (SCCOT) is the second most common head and neck malignancy. To date only neck dissection, a highly invasive and costly procedure, can predict regional and distant recurrence of disease. However, only 20-30% of patients with early tongue carcinoma will actually have metastatic disease. Therefore, there is a pressing need to find low cost, and sensitive methods to identify patients requiring this invasive procedure. In this study we aim to examine a subset of proteins through tissue microarray (TMA) that could potentially serve as prognostic markers of nodal metastasis in early SCCOT in order to minimize unnecessary neck dissection in patients with early disease. Method: Retrospective analysis was performed in a double-blind manner on 2 tissue microarrays (5 cores/patient) created from paraffin-embedded specimens from 67 patients with well documented clinical history of the disease. The training set included 30 of the patients and was used to identify potential markers. The top two markers were then validated using the remaining 37 patients as the test set. Twenty five different proteins were tested as potential biomarkers of metastasis based on published literature on SCCOT and analyzed through IHC. Results: The partial protein profiles from TMA analyses recovered differential protein expression pattern in patients with recurrence versus those who remain disease free upto 36 months post treatment. Among these a glycoprotein, podoplanin (p<0.0001) and two cytokines, β-2 microglobulin (p<0.0001) and interleukin-8 (p = 0.004) were found up regulated and E-cadherin (p = 0.007) was down regulated in samples that metastasized following surgery. The expression of these three proteins combined in a potential biomarker panel could reliably predict outcome in 100% of the cases using k-nearest neighbor algorithm. These results were further confirmed using a verification set of 46 patients. In this new set, the potential biomarker panel was sufficient in predicting metastasis in 97.2% of SCCOT patients coming in with early disease. Conclusion: This study provides proof of principle data to show that individual proteins can be found through statistical analyses to compile a biomarker panel that can distinguish between patients with node positive versus node negative disease. These markers can be used either individually or in the form of a panel to optimize staging and treatment of SCCOT patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4181. doi:10.1158/1538-7445.AM2011-4181