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May 2, 2013

The Tumor Microenvironment as a Drug Target: Chasing Slippery Targets

This first part of a two-part series discusses how researchers are looking at ways to attack tumors by targeting the cellular frameworks that keep them alive.

The Tumor Microenvironment as a Drug Target: Chasing Slippery Targets

Getting at stromal targets remains challenging, as scientists search for “druggable” molecules unique to tumor stromal cells and the tumor microenvironment. [© Alex - Fotolia.com]

  • Researchers are increasingly “thinking outside the cancer cell” to provide insights into how other cell types and the tumor microenvironment (TME) support tumor growth and metastasis. These include normal host cells such as endothelial cells, fibroblasts, mesenchymal cells, and immune cells, at sites distant from and local to the site at which malignant transformation occurs.

    And scientists say that the balance of these cellular interactions both determines the natural history of the cancer and influences its response to therapy. This active tumor-host dynamic has stimulated interest in the TME as a key target for cancer drugs. The microenvironment of a cancer is now perceived as an integral part of a tumor’s anatomy and physiology that functionally cannot be totally dissociated from what has traditionally been called “cancer.” This includes distinctive microenvironmental conditions, like hypoxia, that result from the disordered vasculature characteristic of solid tumors.

    Among potential noncancer cells in the TME, cancer stromal cells including fibroblastic cells, endothelial cells, and cells of hematopoietic origin have emerged as critical players in promoting tumor proliferation. These cells, scientists hypothesize, participate in neovascularization, invasion, and metastasis as well as interacting with immune cells to “tilt the equilibrium toward a tolerogenic environment favoring the tumor cells.”

  • Fibroblast Activation Protein

    But getting at stromal targets remains challenging, as scientists search for “druggable” molecules unique to tumor stromal cells and the tumor microenvironment. Making use of these targets to fight cancer will require considerable molecular ingenuity. According to Professor Ellen Puré, Ph.D., of the Wistar Institute, the key target is the stroma itself, and finding targets that break down the barrier between tumor cells and drugs intended to kill them.

    One potential stromal drug target is FAP (fibroblast activation protein), a cell surface serine protease selectively expressed on tumor-associated fibroblasts and other mesenchymal stromal cells in epithelial tumors. This protease has emerged as a mediator of tumorigenesis, angiogenesis, and metastasis.

    Dr. Puré and her colleagues investigated the role of FAP in mouse models of epithelial-derived solid tumors. They showed that genetic deletion and pharmacologic inhibition of FAP inhibited tumor growth in an endogenous mouse model of lung cancer driven by the K-rasG12D mutant. FAP inhibition also inhibited tumor growth in a mouse model of colon cancer, in which CT26 mouse colon cancer cells were transplanted into immune competent syngeneic mice.

    The results, the authors said, indicated that FAP depletion inhibits tumor cell proliferation indirectly, increases accumulation of collagen, decreases myofibroblast content, and decreases blood vessel density in tumors. These data provide, they say, proof of principle that targeting stromal cell-mediated modifications of the tumor microenvironment may be an effective approach to treating epithelial-derived solid tumors.

    Dr. Puré told GEN that she and her colleagues “are working on developing several approaches to disrupting the tumor stroma. The stroma keeps drugs out in several ways,” she explained. “It increases intrastitial fluid pressure, so that in the tumor, the blood pressure is greater than blood vessel pressure, thereby preventing drug diffusion.”

    Further, she said, “the stroma changes the structure and maturity of a blood vessel, thereby impacting how well it delivers drug to a tumor. Stromal cells and the matrix can produce form a relatively nonporous barrier that traps, among other things, immune cells entering the tumor.”

    Dr. Puré’s laboratory focuses on the use of small molecule inhibitors to inhibit FAP activity at the molecular level; she and others are also “trying to kill off the cells that express FAP with antibody-conjugated toxin and cell-based immunotherapies.”

  • Thapsigargin and FAP

    But, scientists say, while FAP cell-associated activities may be targets for diagnosis and treatment of various cancers, coming up with drugs to inhibit their activity has proven challenging.

    Antibodies that can target or interrupt FAP activity are not yet available, and development of small molecule inhibitors of FAP is stymied because the endogenous substrates of FAP have not been identified. And researchers say, similarly as for other enzymatic targets such as kinases, the highly conserved structure of the catalytic domains of serine proteases, in particular the structural relatedness of FAP to other members of the DPP family proteases, can “defy the rationale of the highly specific small molecule inhibitors required to avoid off-target effects.”

    But rather than targeting the protease directly with for example, small molecule enzyme inhibitors, investigators at the department of pharmacology and molecular sciences, Johns Hopkins University, say that FAP's pathophysiological role in carcinogenesis may be “highly contextual” depending on both the exact nature of the tumor microenvironment present and the cancer type in question to determine its tumor-promoting or tumor-suppressing phenotype.

    As an alternative strategy, W. Nathaniel Brennen, Ph.D., and colleagues are “taking advantage” of FAP’s restricted expression and unique substrate preferences by developing a FAP-activating prodrug that targets the activation of a cytotoxic compound within the tumor stroma.

    In their test case, the investigators tested Thapsigargin (TG), a highly toxic natural plant product that triggers a rise in intracellular calcium levels with subsequent apoptosis. FAP is therefore, they said, a provocative target for the activation of prodrugs consisting of a FAP-specific peptide coupled to a potent cytotoxic analog of TG.

    The investigators tested the efficacy of FAP-activated peptidyl-TG prodrug in vitro in cell proliferation assays and assessed its effects on intracellular calcium in human cancer cell lines. The effects of FAP-activated prodrugs on tumor growth and host toxicity were also tested in Balb-C nude MCF-7 and LNCaP xenograft mice. FAP-activated prodrugs killed human cancer cells at low nanomolar concentrations; amino acid-12ADT analogs from FAP-cleaved prodrugs, but not uncleaved prodrugs, produced a rapid rise in intracellular calcium within minutes of exposure.

    Immunohistochemical analysis of xenografts exposed to FAP-prodrugs documented stromal-selective cell death of fibroblasts, pericytes, and endothelial cells of sufficient magnitude to inhibit growth of MCF-7 and LNCaP xenografts with minimal systemic toxicity, whereas non-FAP cleavable prodrugs proved inactive.

    The authors comment that strategies that attempt to exploit cellular targets within the tumor stroma offer several potential advantages over traditional approaches. These include providing a more genetically stable target that is not only less heterogeneous than its malignant epithelial counterparts but also less likely to acquire resistance to a cytotoxic agent.

    Targeting CAFs or stromal cells remains a “relatively underappreciated” treatment strategy, the scientists commented, largely due to a lack of evidence suggesting that it is a practical option in the clinical setting.

    But, they say, the surge in both pharmaceutical and academic efforts to develop FAP as a therapeutic target offers a promising outlook for changing that statement in the not-so-distant future.

    To read part II of this article, click here.


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