Mahaveer S. Bhojani

CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-associated vasculature

Chemokines and chemokine receptors have been posited to have important roles in several common malignancies, including breast and lung cancer. Here, we demonstrate that CXCR7 (RDC1, CCX-CKR2), recently deorphanized as a chemokine receptor that binds chemokines CXCL11 and CXCL12, can regulate these two common malignancies. Using a combination of overexpression and RNA interference, we establish that CXCR7 promotes growth of tumors formed from breast and lung cancer cells and enhances experimental lung metastases in immunodeficient as well as immunocompetent mouse models of cancer. These effects did not depend on expression of the related receptor CXCR4. Furthermore, immunohistochemistry of primary human tumor tissue demonstrates extensive CXCR7 expression in human breast and lung cancers, where it is highly expressed on a majority of tumor-associated blood vessels and malignant cells but not expressed on normal vasculature. In addition, a critical role for CXCR7 in vascular formation and angiogenesis during development is demonstrated by using morpholino-mediated knockdown of CXCR7 in zebrafish. Taken together, these data suggest that CXCR7 has key functions in promoting tumor development and progression.

Vascular Targeted Nanoparticles for Imaging and Treatment of Brain Tumors

Purpose: Development of new therapeutic drug delivery systems is an area of significant research interest. The ability to directly target a therapeutic agent to a tumor site would minimize systemic drug exposure, thus providing the potential for increasing the therapeutic index. Experimental Design: Photodynamic therapy (PDT) involves the uptake of a sensitizer by the cancer cells followed by photoirradiation to activate the sensitizer. PDT using Photofrin has certain disadvantages that include prolonged cutaneous photosensitization. Delivery of nanoparticles encapsulated with photodynamic agent specifically to a tumor site could potentially overcome the drawbacks of systemic therapy. In this study, we have developed a multifunctional polymeric nanoparticle consisting of a surface-localized tumor vasculature targeting F3 peptide and encapsulated PDT and imaging agents. Results: The nanoparticles specifically bound to the surface of MDA-435 cells in vitro and were internalized conferring photosensitivity to the cells. Significant magnetic resonance imaging contrast enhancement was achieved in i.c. rat 9L gliomas following i.v. nanoparticle administration. Serial magnetic resonance imaging was used for determination of pharmacokinetics and distribution of nanoparticles within the tumor. Treatment of glioma-bearing rats with targeted nanoparticles followed by PDT showed a significant improvement in survival rate when compared with animals who received PDT after administration of nontargeted nanoparticles or systemic Photofrin. Conclusions: This study reveals the versatility and efficacy of the multifunctional nanoparticle for the targeted detection and treatment of cancer.

Noninvasive real-time imaging of apoptosis

Strict coordination of proliferation and programmed cell death (apoptosis) is essential for normal physiology. An imbalance in these two opposing processes results in various diseases including AIDS, neurodegenerative disorders, myelodysplastic syndromes, ischemia/reperfusion injury, cancer, autoimmune disease, among others. Objective and quantitative noninvasive imaging of apoptosis would be a significant advance for rapid and dynamic screening as well as validation of experimental therapeutic agents. Here, we report the development of a recombinant luciferase reporter molecule that when expressed in mammalian cells has attenuated levels of reporter activity. In cells undergoing apoptosis, a caspase-3-specific cleavage of the recombinant product occurs, resulting in the restoration of luciferase activity that can be detected in living animals with bioluminescence imaging. The ability to image apoptosis noninvasively and dynamically over time provides an opportunity for high-throughput screening of proapoptotic and antiapoptotic compounds and for target validation in vivo in both cell lines and transgenic animals.

Molecular imaging of Akt kinase activity.

The serine/threonine kinase Akt mediates mitogenic and anti-apoptotic responses that result from activation of multiple signaling cascades. It is considered a key determinant of tumor aggressiveness and is a major target for anticancer drug development. Here, we describe a new reporter molecule whose bioluminescence activity within live cells and in mice can be used to measure Akt activity. Akt activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to activation or inhibition of receptor tyrosine kinase, inhibition of phosphoinositide 3-kinase, or direct inhibition of Akt. The results provide unique insights into the pharmacokinetics and pharmacodynamics of agents that modulate Akt activity, revealing the usefulness of this reporter for rapid dose and schedule optimization in the drug development process.

Molecular Imaging of Gene Expression and Efficacy following Adenoviral-Mediated Brain Tumor Gene Therapy

Cancer gene therapy is an active area of research relying upon the transfer and subsequent expression of a therapeutic transgene into tumor cells in order to provide for therapeutic selectivity. Noninvasive assessment of therapeutic response and correlation of the location, magnitude, and duration of transgene expression in vivo would be particularly useful in the development of cancer gene therapy protocols by facilitating optimization of gene transfer protocols, vector development, and prodrug dosing schedules. In this study, we developed an adenoviral vector containing both the therapeutic transgene yeast cytosine deaminase (yCD) along with an optical reporter gene (luciferase). Following intratumoral injection of the vector into orthotopic 9 L gliomas, anatomical and diffusion-weighted MR images were obtained over time in order to provide for quantitative assessment of overall therapeutic efficacy and spatial heterogeneity of cell kill, respectively. In addition, bioluminescence images were acquired to assess the duration and magnitude of gene expression. MR images revealed significant reduction in tumor growth rates associated with yCD/5-fluorocytosine (5FC) gene therapy. Significant increases in mean tumor diffusion values were also observed during treatment with 5FC. Moreover, spatial heterogeneity in tumor diffusion changes were also observed revealing that diffusion magnetic resonance imaging could detect regional therapeutic effects due to the nonuniform delivery and/or expression of the therapeutic yCD transgene within the tumor mass. In addition, in vivo bioluminescence imaging detected luciferase gene expression, which was found to decrease over time during administration of the prodrug providing a noninvasive surrogate marker for monitoring gene expression. These results demonstrate the efficacy of the yCD/5FC strategy for the treatment of brain tumors and reveal the feasibility of using multimodality molecular and functional imaging for assessment of gene expression and therapeutic efficacy.

AGTR1 overexpression defines a subset of breast cancer and confers sensitivity to losartan, an AGTR1 antagonist

Breast cancer patients have benefited from the use of targeted therapies directed at specific molecular alterations. To identify additional opportunities for targeted therapy, we searched for genes with marked overexpression in subsets of tumors across a panel of breast cancer profiling studies comprising 3,200 microarray experiments. In addition to prioritizing ERBB2, we found AGTR1, the angiotensin II receptor type I, to be markedly overexpressed in 10–20% of breast cancer cases across multiple independent patient cohorts. Validation experiments confirmed that AGTR1 is highly overexpressed, in several cases more than 100-fold. AGTR1 overexpression was restricted to estrogen receptor-positive tumors and was mutually exclusive with ERBB2 overexpression across all samples. Ectopic overexpression of AGTR1 in primary mammary epithelial cells, combined with angiotensin II stimulation, led to a highly invasive phenotype that was attenuated by the AGTR1 antagonist losartan. Similarly, losartan reduced tumor growth by 30% in AGTR1-positive breast cancer xenografts. Taken together, these observations indicate that marked AGTR1 overexpression defines a subpopulation of ER-positive, ERBB2-negative breast cancer that may benefit from targeted therapy with AGTR1 antagonists, such as losartan.

Inhibition of N-linked glycosylation disrupts receptor tyrosine kinase signaling in tumor cells.

Receptor tyrosine kinases (RTK) are therapeutic targets for the treatment of malignancy. However, tumor cells develop resistance to targeted therapies through the activation of parallel signaling cascades. Recent evidence has shown that redundant or compensatory survival signals responsible for resistance are initiated by nontargeted glycoprotein RTKs coexpressed by the cell. We hypothesized that disrupting specific functions of the posttranslational machinery of the secretory pathway would be an effective strategy to target both primary and redundant RTK signaling. Using the N-linked glycosylation inhibitor, tunicamycin, we show that expression levels of several RTKS (EGFR, ErbB2, ErbB3, and IGF-IR) are exquisitely sensitive to inhibition of N-linked glycosylation. Disrupting this synthetic process reduces both cellular protein levels and receptor activity in tumor cells through retention of the receptors in the endoplasmic reticulum/Golgi compartments. Using U251 glioma and BXPC3 pancreatic adenocarcinoma cell lines, two cell lines resistant to epidermal growth factor receptor-targeted therapies, we show that inhibiting N-linked glycosylation markedly reduces RTK signaling through Akt and radiosensitizes tumor cells. In comparison, experiments in nontransformed cells showed neither a reduction in RTK-dependent signaling nor an enhancement in radiosensitivity, suggesting the potential for a therapeutic ratio between tumors and normal tissues. This study provides evidence that enzymatic steps regulating N-linked glycosylation are novel targets for developing approaches to sensitize tumor cells to cytotoxic therapies.

Targeted Imaging and Therapy of Brain Cancer Using Theranostic Nanoparticles

The past decade has seen momentous development in brain cancer research in terms of novel imaging-assisted surgeries, molecularly targeted drug-based treatment regimens or adjuvant therapies and in our understanding of molecular footprints of initiation and progression of malignancy. However, mortality due to brain cancer has essentially remained unchanged in the last three decades. Thus, paradigm-changing diagnostic and therapeutic reagents are urgently needed. Nanotheranostic platforms are powerful tools for imaging and treatment of cancer. Multifunctionality of these nanovehicles offers a number of advantages over conventional agents. These include targeting to a diseased site thereby minimizing systemic toxicity, the ability to solubilize hydrophobic or labile drugs leading to improved pharmacokinetics and their potential to image, treat and predict therapeutic response. In this article, we will discuss the application of newer theranostic nanoparticles in targeted brain cancer imaging and treatment.

Real-time Evaluation of p53 Oscillatory Behavior In vivo Using Bioluminescent Imaging

p53 is a key mediator of cellular response to stress, and, although its function has been carefully evaluated in vitro, noninvasive evaluation of the transcriptional activity of p53 in live animals has not been reported. To this end, we developed a transgenic mouse model wherein the firefly luciferase gene expression was dependent on the p53-responsive P2 promoter from the murine double minute 2 (MDM2) gene. Bioluminescence activity following ionizing radiation was shown to be dose, time, and p53 dependent. In addition, expression of both p53 and its activated form as well as the expression of p53 target genes (MDM2 and p21) correlated with bioluminescence activity. Temporal evaluation of p53 activity following ionizing radiation showed a distinct oscillatory pattern, which confirmed the oscillations observed previously in cultured cells. In addition, the kinetics of oscillations were altered by pretreatment with radiation-modifying agents. These results show the use of this mouse model in enhancing our understanding of the transcriptional role of p53 in vivo.

Role of Epidermal Growth Factor Receptor Degradation in Cisplatin-Induced Cytotoxicity in Head and Neck Cancer

Cisplatin and its analogues are the most commonly used agents in the treatment of head and neck squamous cell carcinoma. In this study, we investigated a possible role of epidermal growth factor (EGF) receptor (EGFR) phosphorylation and degradation in cisplatin-induced cytotoxicity. Cisplatin treatment led to an increase in initial EGFR phosphorylation at Y1045, the binding site of ubiquitin ligase, Casitas B-lineage lymphoma (c-Cbl), followed by ubiquitination in the relatively cisplatin-sensitive cell lines. However, cisplatin-resistant cell lines underwent minimal EGFR phosphorylation at the Y1045 site and minimal ubiquitination. We found that EGFR degradation in response to cisplatin was highly correlated with cytotoxicity in seven head and neck cancer cell lines. Pretreatment with EGF enhanced cisplatin-induced EGFR degradation and cytotoxicity, whereas erlotinib pretreatment blocked EGFR phosphorylation, degradation, and cisplatin-induced cytotoxicity. Expression of a mutant Y1045F EGFR, which is relatively resistant to c-Cbl-mediated degradation, in Chinese hamster ovary cells and the UMSCC11B human head and neck cancer cell line protected EGFR from cisplatin-induced degradation and enhanced cell survival compared with wild-type (WT) EGFR. Transfection of WT c-Cbl enhanced EGFR degradation and cisplatin-induced cytotoxicity compared with control vector. These results show that cisplatin-induced EGFR phosphorylation and subsequent ubiquitination and degradation is an important determinant of cisplatin sensitivity. Our findings suggest that treatment with an EGFR inhibitor before cisplatin would be antagonistic, as EGFR inhibition would protect EGFR from cisplatin-mediated phosphorylation and subsequent ubiquitination and degradation, which may explain the negative results of several recent clinical trials. Furthermore, they suggest that EGFR degradation is worth exploring as an early biomarker of response and as a target to improve outcome.