Current Cancer Drug Targets

ISSN: 1568-0096

Current Cancer Drug Targets
Volume 5, Number 7, November 2005

Contents

Novel Non-Cytotoxic Approaches For Cancer Therapy: Potential Targets and Pathways Amenable to Single Agent Multimodal Therapeutic Approaches
Guest Editor: Andrew Mazar


Editorial Pp.469-470


Hypomethylation of Urokinase (uPA) Promoter in Breast and Prostate Cancer: Prognostic and Therapeutic Implications Pp. 471-488
Pouya Pakneshan, Moshe Szyf and Shafaat A. Rabbani
[Abstract]


Targeting Invasion Induction as a Therapeutic Strategy for the Treatment of Cancer Pp. 489-503
Donna L. Livant
[Abstract]


Cancer Therapy Through Control of Cell Migration Pp. 505-518
Eugene G. Levin
[Abstract]


Inhibition of Angiogenesis by Cleaved High Molecular Weight Kininogen (HKa) and HKa Domain 5 Pp. 519-528
Keith R. McCrae, Fernando Doñate, Sergei Merkulov, Danyu Sun, Xiaoping Qi and David E. Shaw
[Abstract]


Anti-Angiogenic and Anti-Tumor Properties of Proteasome Inhibitors Pp. 529-541
Kenyon G. Daniel, Deborah J. Kuhn, Aslamuzzaman Kazi and Q. Ping Dou
[Abstract]


Control of Copper Status for Cancer Therapy Pp. 543-549
Vicki L. Goodman, George J. Brewer and Sofia D. Merajver
[Abstract]


Cellular and Molecular Surrogate Markers to Monitor Targeted and Non-Targeted Antiangiogenic Drug Activity and Determine Optimal Biologic Dose Pp. 551-559
Yuval Shaked, Guido Bocci, Raquel Munoz, Shan Man, John M.L. Ebos, Danie J. Hicklin, Francesco Bertolini, Robert D’Amato and Robert S. Kerbel
[Abstract]




Absctracts


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Editorial
Andrew P. Mazar

Historically, the development of novel cancer treatments has been approached from a perspective of mutual exclusion. For many years, cancer treatment has focused on killing as many cancer cells as possible without regard for the operative biology in a particular tumor type. Despite the diversity of oncology drugs with substantially different mechanisms of action that have been developed over the years, a single endpoint of activity, objective response at the maximum tolerated dose (MTD) has been used as the major determinant of active drug dose (one might argue that the routine use of the MTD to establish dose in cancer drug development may have even stymied the understanding of how to really use certain oncology drugs). It was only when non-cytotoxic agents began to enter clinical trials several years ago that oncology drug developers were forced to explore more representative endpoints of the biological activity of a drug as an alternative to the MTD. In addition, the tumor microenvironment has also recently been validated as a legitimate target for controlling tumor growth and thus, the tumor cell itself is no longer the only target for cancer therapy. Initial forays into the development of non-cytotoxic agents have presented a number of challenges, such as the identification of biomarkers that are representative of the biological activity of a particular drug, and initially focused on developing drugs that were highly specific for a particular target e.g. first generation MMP inhibitors, early kinase inhibitors such as SU5416. However, it has become apparent that human tumors are much too clever to be affected by highly specific agents and that a number of redundant or compensatory pathways can overcome specifically targeted therapies, revealing yet another mechanism of drug resistance. As is often the case in drug development, the interjection of some serendipity with elegant science was required to demonstrate the clinical activity of one of the first non-hormonal targeted agents (Gleevec). As it turns out, despite the initial insistence that this was a highly targeted agent specific for the Abl tyrosine kinase, Gleevec likely exhibits anti-tumor activity because it is not entirely specific for one target and inhibits a number of kinases that are important to both tumor growth as well as angiogenesis.

Gleevec is an example of an evolving paradigm in oncology drug discovery and development which will be referred to for the purposes of this introduction as single agent multimodal cancer treatment. Multimodal cancer treatment is usually defined as combining several agents or approaches to treat a particular tumor type (for example, the combination of an anti-angiogenic agent and a chemotherapeutic agent or the combination of a chemotherapeutic agent and radiation therapy). However, through the choice of appropriate pathways to target in a tumor, it is conceivable that a single agent could exhibit multimodal activity by interfering with several signaling pathways important to tumor progression in multiple tumor-associated cell types simultaneously, leading to a more robust anti-tumor response than would be expected with an agent that targeted a single pathway or cell type within a tumor. Such agents would be expected to demonstrate single agent activity but would also be suitable to be combined with chemotherapy, radiation therapy or other single multimodal agents that affected complimentary pathways implicated in tumor growth. There are a number of examples of such agents that have recently been approved or are currently in development. For example, several agents targeting the EGF pathway e.g. erlotinib, cetuximab have recently been approved for the treatment of lung cancer and colon cancer, respectively. The EGF pathway is central to a number of signaling pathways implicated in tumor progression and although it is amenable to inhibition by single agents, it mediates a number of signaling events in parallel. For example, the inhibition of the EGF pathway will also inhibit crosstalk with pathways mediated by Ras, B-raf and PTEN in tumor cells and may have direct and indirect effects on angiogenesis as well. In addition, compounds such as SU11248 (sunitinib, which inhibits VEGFR, c-Kit, PDGFR and Flt-3) and BAY 43-9006 (sorafenib, which inhibits RAF/MEK/ERK, VEGFR, PDGFR and c-Kit), both of which are in late stage clinical development, have demonstrated clinical activity in a number of tumor types. Similar to EGF inhibitors, SU11248 and BAY 43-9006 both target multiple pathways important to tumor progression in multiple tumor compartments, consistent with a single agent multimodal effect. Finally, there are a number of compounds in discovery and early clinical development with similar attributes.

This Hot Topics issue, entitled “Novel Non-Cytotoxic Approaches for Cancer Therapy: Potential Targets and Pathways Amenable to Single Agent Multimodal Therapeutic Approaches,” features seven reviews describing new advances in the identification and validation of targets and pathways that are thought to have promiscuous effects on tumor progression. Thus, therapeutic targeting of these targets and pathways could affect multiple downstream processes important to tumor growth. In addition, the therapeutic targeting of these pathways would also be expected to affect multiple cell types within a tumor including angiogenic endothelial cells, tumor cells and tumor-associated macrophages (which are also thought to promote tumor growth). In keeping with this multimodal theme, the article by Drs. Pakneshan, Szyf and Rabbani introduces the urokinase plasminogen activator (uPA) system and describes the epigenetic control of expression of uPA by methylation, the loss of which is associated with the transition to more aggressive forms of cancer. The up-regulation of the uPA system is thought to be central to a number of processes associated with tumor growth, invasion, metastasis, and angiogenesis including the activation of matrix metalloproteases (MMPs), the activation of extracellular matrix (ECM)-sequestered growth factors, ECM remodeling, and the induction of intracellular signaling leading to proliferation and migration. Dr. Donna Livant further elaborates on the role of the uPA system in cell migration and invasion and its interactions with integrins and ECM in cell migration and invasion. Both of these reviews highlight the promiscuous role that the uPA system plays in tumor progression, highlighting the number of pathways and tumor-associated cell types that could be inhibited simultaneously by therapeutically targeting this system.

The idea of cell motility (migration, invasion) as a cancer therapeutic target is further examined in the review by Dr. Gene Levin. Targeting migration, which is a complex process involving a number of pathways that are operational simultaneously, with a single molecule implies that a number of pathways must be inhibited concurrently. Dr. Levin describes a novel, differentially translated isoform of FGF-2 that, unlike the more familiar 18 kDa isoform, which promotes endothelial cell proliferation and migration, is able to very potently inhibit cell migration in vitro and suppress tumor growth in vivo. The effects on migration are observed with both endothelial cells and tumor cells, indicating the potential to simultaneously affect multiple compartments within a tumor. The article by McCrae et al. describes a novel anti-angiogenic agent derived from an ubiquitous plasma protein, kininogen (HK). The role of HK in tumor progression is complex since when HK is cleaved, it releases two fragments, bradykinin, which may promote tumor growth, and HKa (activated HK), which was originally described by Dr. McCrae and colleagues as having anti-angiogenic activity through the inhibition of endothelial cell proliferation. Recent studies have also demonstrated that peptide regions derived from HKa inhibit tumor cell invasion in vitro and cancer metastasis in vivo indicating that this protein, similar to the FGF-2 isoform described by Dr. Levin, may also affect multiple processes and multiple tumor-associated cell types influencing tumor progression [1, 2].

In the fifth article of this Hot Topics issue, Daniel et al. explore the proteosome as a therapeutic target for the inhibition of angiogenesis. Targeting the proteosome has already been validated clinically as an anti-tumor strategy through the recent approval of bortezomib (Velcade) and the ability of proteosome inhibitors to also inhibit angiogenesis suggests the potential for broader and more robust activity than initially observed clinically. In their contribution, Daniel et al. review a number of other proteosome inhibitors in addition to bortezomib and describe how the binding of copper by novel organic compounds leads to the inhibition of proteosome activity. Copper has been implicated in a number of steps in tumor progression including angiogenesis and tumor cell proliferation and the formation of organocopper complexes in situ may provide one explanation for how depleting copper at the molecular level leads to the inhibition of angiogenesis and tumor growth. Drs. Goodman, Brewer and Merajver explore the role of copper in tumor growth and angiogenesis further. Copper has pleiotropic effects on tumor growth and angiogenesis and may regulate a number of pathways important to tumor growth. The depletion of copper in cancer patients clinically has provided preliminary evidence of anti-tumor effects, and Drs. Goodman et al. also discuss the data.

The issue is concluded with the contribution of Shaked et al., which describes cellular and molecular surrogates that may be useful for helping to establish the biologically active dose of a wide variety of cancer drugs, regardless of mechanism of action and without necessitating dosing to MTD. A number of chemotherapeutic and targeted drugs that affect both the endothelial and the tumor cell compartments have demonstrated similar abilities to affect VEGFR-2+ circulating cells, some of which may be circulating endothelial cell progenitors. These cells may represent a universal biomarker for evaluating a number of multimodal approaches (single and multiple agent) in the clinic without regard for the specific pathways being affected by these compounds. Although validation of this approach and the correlation of anti-tumor effects with depletion of VEGFR-2+ circulating cells remains to be established in the clinic, the correlation of anti-tumor effects with the depletion of these cells in a uniform manner by a number of drugs with widely differing mechanisms of action in preclinical studies nevertheless raises the possibility that this approach could have generic utility as a pharmacodynamic biomarker in cancer clinical trials, and could move oncology drug development, at least in some cases, away from its reliance on dose escalating until a MTD is established.

Many of the concepts put forward in this introduction and in the reviews that comprise this Hot Topics issue are controversial and in fact, are meant to be provocative. Recent advances in our understanding of tumor biology have presented a formidable challenge of how to translate these findings into the clinic and I hope that this Hot Topics issue will serve as an introduction to the next generation of therapeutic approaches for the treatment of cancer. I would like to thank all of the authors that contributed to this issue and hope that anyone reading this issue will learn as much as I did in assembling this group of articles.

Andrew P. Mazar
Attenuon, LLC,
11535 Sorrento Valley Rd., Suite 401,
San Diego, CA 92121, USA
E-mail: mazar@attenuon.com

ACKNOWLEDGEMENT
I would like to thank my wife, Nicole, for editorial assistance with the manuscripts.

REFERENCES
[1] Kamiyama, F.; Maeda, T.; Yamane, T.; Li, Y. H.; Ogukubo, O.; Otsuka, T.; Ueyama, H.; Takahashi, S.; Ohkubo, I.; Matsui, N. Inhibition of vitronectin-mediated haptotaxis and haptoinvasion of MG-63 cells by domain 5 (D5(H)) of human high-molecular-weight kininogen and identification of a minimal amino acid sequence. Biochem. Biophys. Res. Commun. 2001, 288, 975-980.
[2] Kawasaki, M.; Maeda, T.; Hanasawa, K.; Ohkubo, I.; Tani, T. Effect of His-Gly-Lys motif derived from domain 5 of high molecular weight kininogen on suppression of cancer metastasis both in vitro and in vivo. J. Biol. Chem. 2003, 278, 49301-49307.


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Hypomethylation of Urokinase (uPA) Promoter in Breast and Prostate Cancer: Prognostic and Therapeutic Implications
Pouya Pakneshan, Moshe Szyf and Shafaat A. Rabbani

Methylation of CpG islands of tumor suppressor genes, growth factors, and hormone receptors among other genes causes epigenetic changes in chromatin structure without altering DNA sequence to regulate transcription of these genes. This epigenetic regulation of gene expression plays an important role in the process of tumor invasion, growth and metastasis in malignancies. In hormone dependent malignancies such as breast and prostate cancer, sex steroids play an important role in the process of tumor initiation and progression. These malignancies are often initiated as a less aggressive hormone-responsive type that gradually progresses to become highly invasive and hormone-insensitive. At the early stages, cells lose a functional hormone receptor due to mutations, blockage of signaling pathway or hormone receptor gene silencing. This transition of cancer cells causes them to become refractory to the standard hormone therapies. In later stages, important factors like growth factors, cytokines and proteases promote tumor growth, invasion and metastases. The most commonly implicated protease in these processes is urokinase type plasminogen activator (uPA), which is known to be expressed in a number of malignancies including breast and prostate cancer and is directly associated with the higher invasive and metastatic potential of malignancies. In this chapter, we will review DNA methylation as the underlying molecular mechanism regulating uPA gene expression and its potential diagnostic, prognostic and therapeutic implication.


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Targeting Invasion Induction as a Therapeutic Strategy for the Treatment of Cancer
Donna L. Livant

The spread of cancer cells from the primary tumor to a distant site involves many of the invasive processes normally required for wound healing, including migration through the local connective tissue, invasion of the vasculature, extravasation, invasion of the connective tissue at a distant site, and angiogenesis. Thus, the abilities of tumor cells to invade the host, and to induce endothelial cell invasion and neovascularization, are central to malignant progression. The plasminogen activator system, which plays a direct role in stimulating α5β1 integrin fibronectin receptor-mediated invasion during wound healing, is also very important in tumor cell invasion and metastasis, as well as in angiogenesis. Therefore, the α5β1 receptor and the plasminogen activator system may be promising targets for directed anticancer therapies.


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Cancer Therapy Through Control of Cell Migration
Eugene G. Levin

Cell migration plays a pivotal role in a many biological process that are essential for development, repair, and pathogenesis. Thus, inhibition of migration has the potential of limiting or suppressing the development of various diseases. Much of the focus on the therapeutic treatment of cancer has involved compounds that target cell proliferation and subsequent cell death. However, targeting migration is another approach that has not been pursued but holds promise for alternative means of therapy. One such potential therapeutic is a small protein that inhibits the migration of a number of cell types. This protein is derived from the amino terminal end of the 24 kDa form of fibroblast growth factor, and suppresses migration in the presence of a variety of growth factors. Analysis of the protein in mouse models shows that it inhibits in vivo angiogenesis and tumor growth at low concentrations. Thus, inhibition of migration is a viable alternative to more traditional methods of therapeutically treating tumors. Further study of the mechanism of inhibition can lead to the development of novel drugs targeting a distinctive cell process.


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Inhibition of Angiogenesis by Cleaved High Molecular Weight Kininogen (HKa) and HKa Domain 5
Keith R. McCrae, Fernando Doñate, Sergei Merkulov, Danyu Sun, Xiaoping Qi and David E. Shaw

High molecular weight kininogen (HK) is an abundant, multi-domain plasma protein that circulates in plasma primarily in its single chain form. Proteolytic cleavage of HK by plasma kallikrein releases the vasoactive nanopeptide bradykinin (BK), and converts HK into two-chain HK (HKa). BK appears to have pro-angiogenic activity, most likely mediated through binding to B1 and B2 receptors on endothelial cells. Conversely, HKa and its domain 5, but not (single chain) HK, have potent anti-angiogenic activity comparable to other endogenous angiogenesis inhibitors. The mechanism by which HKa exerts its anti-angiogenic activity remains controversial, but appears to involve binding to cell surface tropomyosin and induction of apoptosis of proliferating endothelial cells. A role for tropomyosin in mediating the anti-angiogenic signals of other anti-angiogenic proteins such as endostatin and histidine-proline-rich glycoprotein (HPRG) has also been reported. Here we review the physiological importance of high molecular weight kininogen in angiogenesis, with emphasis on the mechanism(s) by which this activity is mediated.


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Anti-Angiogenic and Anti-Tumor Properties of Proteasome Inhibitors
Kenyon G. Daniel, Deborah J. Kuhn, Aslamuzzaman Kazi and Q. Ping Dou

Tumor growth and metastasis depend on the formation of blood vessels, angiogenesis, to supply the developing mass with nutrients, oxygen, and waste removal. The proteasome, a massive multisubunit catabolic body, exerts a regulatory influence on angiogenesis. Inhibition of the proteasome activity has been found to inhibit angiogenesis and induce apoptosis in human cancer cells with limited toxicity to normal cells. Therefore, the dual action of angiogenesis inhibition and cell death induction makes proteasome inhibition an attractive modality for chemotherapy. A variety of proteasome inhibitors have been studied including: antibiotics such as lactacystin, the green tea polyphenols, and the boronic acid Velcade (MLN-341). Most recently, certain classes of copper compounds have been found to act as potent proteasome inhibitors. The potential of particular organic compounds, such as 8-hydroxyquinoline, to spontaneously bind with tumor cellular copper and form proteasome inhibitors provides a new modality of anti-proteasome and anti-angiogenesis chemotherapy. This review examines angiogenesis, the proteasome, representative proteasome inhibitors, and the emerging role of copper.

The formation of new blood vessels, or angiogenesis, is an important and necessary function in both embryonic development and wound repair [1, 2]. Therefore, the ability to regenerate or form new vessels for blood flow is essential. The control of angiogenic pathways is tightly regulated in normal differentiated adult cells, which generally do not stimulate blood vessel growth unless injury occurs. However, cancerous tissues stimulate angiogenesis that in turn leads to increased tumor formation and possible metastases [3]. Many of the factors involved in angiogenesis are regulated by the proteasome, which recently has become a focus in anti-cancer therapies due to its involvement in cell cycle and apoptosis control [4, 5]. Here we discuss angiogenesis and its relation to the proteasome. Additionally, current modalities of anti-angiogenic treatment, mainly proteasome inhibitory strategies, are reviewed. Furthermore, proteasome inhibitors, both natural and synthetic, and their anti-angiogenic effects as well as future approaches to anti-angiogenic chemotherapies are also discussed.


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Control of Copper Status for Cancer Therapy
Vicki L. Goodman, George J. Brewer and Sofia D. Merajver

Copper is a trace element which is tightly regulated in mammals and lower animals. Disruptions of copper homeostasis in humans are rare and they cause serious disorders such as Wilson’s disease and Menke’s disease. Copper plays an important role in promoting physiological and malignant angiogenesis. Formation of new blood vessels by a tumor enables tumor growth, invasion, and metastasis are copper requiring processes. The copper chelator tetrathiomolybdate (TM), which quickly and effectively depletes copper stores, is under investigation as an anti-angiogenic agent. Promising results from in vitro experiments, in pre-clinical animal models, and in a phase I clinical trial have led to several phase II trials of TM in patients with advanced cancers.


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Cellular and Molecular Surrogate Markers to Monitor Targeted and Non-Targeted Antiangiogenic Drug Activity and Determine Optimal Biologic Dose
Yuval Shaked, Guido Bocci, Raquel Munoz, Shan Man, John M.L. Ebos, Danie J. Hicklin, Francesco Bertolini, Robert D’Amato and Robert S. Kerbel

Perhaps the most significant recent advance in oncology therapeutics has been the approval of various “molecularly targeted” anti-cancer drugs. Currently, there are a large number of similar drugs in early or late stage development, including antiangiogenic agents. Clinical development of such drugs suffers from several handicaps including determining whether a patient’s cancer expresses the target and is functionally contributing to cancer growth, monitoring biologic activity, and determining optimal biologic dose. The last problem is related to the low frequency of objective tumor responses (tumor shrinkage) caused by such drugs, or the lack of dose limiting toxicities necessary to define a maximum tolerated dose (MTD), or expression of optimal therapeutic activity at doses below the MTD, when one can be defined. These problems necessitate the development of alternative pharmacodynamic surrogate markers.

Here we summarize several such promising markers for monitoring targeted antiangiogenic activity, and establishing optimal therapeutic/biologic dosing. The first is molecular - plasma VEGF – levels of which are rapidly and significantly increased in a dose dependent manner after injection of normal or tumor bearing mice with anti-VEGFR-2 antibodies. The second is a cellular marker, and more generic in nature - circulating VEGF receptor-2 positive cells found in peripheral blood, some of which may be circulating endothelial progenitor cells. Levels of such cells are suppressed in a dose dependent manner which correlate with previously determined optimal biologic/therapeutic anti-tumor activity of various antiangiogenic drugs or treatments. Finally, another promising marker we discuss is soluble VEGFR-2.