Selectively Targeting Tumor Hypoxia With the Hypoxia-Activated Prodrug CP-506.

Hypoxia-activated prodrugs (HAP) are a promising class of antineoplastic agents that can selectively eliminate hypoxic tumor cells. This study evaluates the hypoxia-selectivity and antitumor activity of CP-506, a DNA alkylating HAP with favorable pharmacologic properties. Stoichiometry of reduction, one-electron affinity, and back-oxidation rate of CP-506 were characterized by fast-reaction radiolytic methods with observed parameters fulfilling requirements for oxygen-sensitive bioactivation. Net reduction, metabolism, and cytotoxicity of CP-506 were maximally inhibited at oxygen concentrations above 1 µmol/L (0.1% O2). CP-506 demonstrated cytotoxicity selectively in hypoxic 2D and 3D cell cultures with normoxic/anoxic IC50 ratios up to 203. Complete resistance to aerobic (two-electron) metabolism by aldo-keto reductase 1C3 was confirmed through gain-of-function studies while retention of hypoxic (one-electron) bioactivation by various diflavin oxidoreductases was also demonstrated. In vivo, the antitumor effects of CP-506 were selective for hypoxic tumor cells and causally related to tumor oxygenation. CP-506 effectively decreased the hypoxic fraction and inhibited growth of a wide range of hypoxic xenografts. A multivariate regression analysis revealed baseline tumor hypoxia and in vitro sensitivity to CP-506 were significantly correlated with treatment response. Our results demonstrate that CP-506 selectively targets hypoxic tumor cells and has broad antitumor activity. Our data indicate that tumor hypoxia and cellular sensitivity to CP-506 are strong determinants of the antitumor effects of CP-506.

Cell-penetrating Alphabody protein scaffolds for intracellular drug targeting.

The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets. Here, we demonstrate that the Alphabody scaffold can be engineered into a cell-penetrating protein antagonist against induced myeloid leukemia cell differentiation protein MCL-1, an intracellular target in cancer, by grafting the critical B-cell lymphoma 2 homology 3 helix of MCL-1 onto the Alphabody and tagging the scaffold's termini with designed cell-penetration polypeptides. Introduction of an albumin-binding moiety extended the serum half-life of the engineered Alphabody to therapeutically relevant levels, and administration thereof in mouse tumor xenografts based on myeloma cell lines reduced tumor burden. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. Collectively, we provide proof of concept for the use of Alphabodies against intracellular disease mediators, which, to date, have remained in the realm of small-molecule therapeutics.

An osteoblast-derived proteinase controls tumor cell survival via TGF-beta activation in the bone microenvironment.

BACKGROUND: Breast to bone metastases frequently induce a "vicious cycle" in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment. METHODOLOGY/PRINCIPAL FINDINGS: To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry). Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (µCT, histomorphometry). Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1) the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay); and 2) that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFß, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays). CONCLUSION/SIGNIFICANCE: Collectively, these studies identify a novel "mini-vicious cycle" between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFß would be beneficial for the treatment of bone metastases.

Differential effects between the loss of MMP-2 and MMP-9 on structural and tissue-level properties of bone.

Matrix metalloproteinases (MMPs) are capable of processing certain components of bone tissue, including type 1 collagen, a determinant of the biomechanical properties of bone tissue, and they are expressed by osteoclasts and osteoblasts. Therefore, we posit that MMP activity can affect the ability of bone to resist fracture. To explore this possibility, we determined the architectural, compositional, and biomechanical properties of bones from wild-type (WT), Mmp2(-/-) , and Mmp9(-/-) female mice at 16 weeks of age. MMP-2 and MMP-9 have similar substrates but are expressed primarily by osteoblasts and osteoclasts, respectively. Analysis of the trabecular compartment of the tibia metaphysis by micro-computed tomography (µCT) revealed that these MMPs influence trabecular architecture, not volume. Interestingly, the loss of MMP-9 improved the connectivity density of the trabeculae, whereas the loss of MMP-2 reduced this parameter. Similar differential effects in architecture were observed in the L(5) vertebra, but bone volume fraction was lower for both Mmp2(-/-) and Mmp9(-/-) mice than for WT mice. The mineralization density and mineral-to-collagen ratio, as determined by µCT and Raman microspectroscopy, were lower in the Mmp2(-/-) bones than in WT control bones. Whole-bone strength, as determined by three-point bending or compression testing, and tissue-level modulus and hardness, as determined by nanoindentation, were less for Mmp2(-/-) than for WT bones. In contrast, the Mmp9(-/-) femurs were less tough with lower postyield deflection (more brittle) than the WT femurs. Taken together, this information reveals that MMPs play a complex role in maintaining bone integrity, with the cell type that expresses the MMP likely being a contributing factor to how the enzyme affects bone quality.

Thinking outside the box: using metastasis suppressors as molecular tools.

Metastasis, the process in which tumor cells move from a primary tumor through the circulation, lodge, and grow in distant locations, is a significant contributor to cancer patient morbidity and mortality, yet remains poorly understood. The molecular processes regulating tumorigenicity and metastasis are distinguishable, suggesting that it is possible to design therapeutic interventions to specifically control metastasis formation. Metastasis suppressors, which specifically regulate metastasis, are being used in "reverse genetics" approaches to discover the phenotypic alterations caused by modulating their levels and/or activity. This strategy is allowing the identification of tumor-host interactions that are crucial for efficient colonization and their disruption can be targeted to suppress metastases formation. In this review we discuss studies addressing invasion and migration, key functions for both early and late in the metastatic process. Metastasis suppressor functions, which modulate lodging and subsequent colonization of the secondary site, are also described. In sum this review focuses on metastasis suppressors that have yielded insight into mechanisms controlling metastasis formation. These serve as platform for out of the box thinking which will enable the discovery of new paradigms in metastasis research.

In vitro metastatic colonization of human ovarian cancer cells to the omentum.

Despite the potentially crucial contributions of the omentum in the regulation of ovarian cancer metastatic growth, it remains a poorly understood organ. Due to its anatomic location and structural fragility, the omentum presents inherent challenges to mechanism-based in vivo studies. Thus, the availability of an ex vivo omental model would, in part, address some of these difficulties posed. Here we describe a technique for identifying, isolating and maintaining ex vivo cultures of omenta from immune-compromised and -competent mice. Ex vivo culture conditions were developed that maintain tissue viability, architecture, and function for up to 10 days. Further experiments demonstrate that the ex vivo culture conditions allow for the proliferation of ovarian cancer cells in vitro and support a similar pattern of microscopic lesions after either intraperitoneal injection of ovarian cancer cells or co-culture of ovarian cancer cells with the omentum. In agreement with previous studies from our laboratory, histologic evaluation of these specimens found that ovarian cancer cells, as well as other peritoneal cancer cells, preferentially accumulate in, and colonize, omental areas rich in immune cells. We now recognize that these are specific, functional structures referred to as milky spots. In sum, these are foundational studies of a readily accessible model, which is easily manipulated and can be immediately used to study the dynamic process of omental colonization. It is hoped that investigators will use the data herein as a starting point for refinements and modifications which will enable them to tailor the model to the specific needs of the experimental question(s) they wish to pursue.

Osteoclast-derived matrix metalloproteinase-7, but not matrix metalloproteinase-9, contributes to tumor-induced osteolysis.

The matrix metalloproteinases MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 are highly expressed in the tumor-bone microenvironment, and, of these, MMP-7 and MMP-9 were found to be localized to bone-resorbing osteoclasts in human breast-to-bone metastases. In a bid to define the roles of host-derived MMP-7 and MMP-9 in the tumor-bone microenvironment, the tibias of MMP-7 and MMP-9 null mice were injected with osteolytic luciferase-tagged mammary tumor cell lines. Our data show that osteoclast-derived MMP-7 significantly contributes to tumor growth and tumor-induced osteolysis whereas osteoclast-derived MMP-9 had no effect on these processes. MMP-7 is capable of processing a number of nonmatrix molecules to soluble active forms that have profound effects on cell-cell communication, such as RANKL, a crucial mediator of osteoclast precursor recruitment and maturation. Therefore, the ability of osteoclast-derived MMP-7 to promote RANKL solubilization in the tumor-bone microenvironment was explored. Results revealed that levels of soluble RANKL were significantly lower in the MMP-7 null mice compared with wild-type (WT) controls. In keeping with this observation, MMP-7 null mice had significantly fewer osteoclast numbers at the tumor-bone interface compared with the WT controls. In summary, we propose that the solubilization of RANKL by MMP-7 is a potential mechanism through which MMP-7 mediates mammary tumor-induced osteolysis. Our studies indicate that the selective inhibition of MMP-7 in the tumor-bone microenvironment may be of benefit for the treatment of lytic breast-to-bone metastases.

Vesicular trafficking and secretion of matrix metalloproteinases-2, -9 and tissue inhibitor of metalloproteinases-1 in neuronal cells.

Matrix metalloproteinases (MMPs) are endopeptidases that cleave matrix, soluble and membrane-bound proteins and are regulated by their endogenous inhibitors the tissue inhibitors of MMPs (TIMPs). Nothing is known about MMP/TIMP trafficking and secretion in neuronal cells. We focussed our attention on the gelatinases MMP-2 and MMP-9, and their inhibitor TIMP-1. MMPs and TIMP-1 fused to GFP were expressed in N2a neuroblastoma and primary neuronal cells to study trafficking and secretion using real time video-microscopy, imaging, electron microscopy and biochemical approaches. We show that MMPs and TIMP-1 are secreted in 160-200 nm vesicles in a Golgi-dependent pathway. These vesicles distribute along microtubules and microfilaments, co-localise differentially with the molecular motors kinesin and myosin Va and undergo both anterograde and retrograde trafficking. MMP-9 retrograde transport involves the dynein/dynactin molecular motor. In hippocampal neurons, MMP-2 and MMP-9 vesicles are preferentially distributed in the somato-dendritic compartment and are found in dendritic spines. Non-transfected hippocampal neurons also demonstrate vesicular secretion of MMP-2 in both its pro- and active forms and gelatinolytic activity localised within dendritic spines. Our results show differential trafficking of MMP and TIMP-1-containing vesicles in neuronal cells and suggest that these vesicles could play a role in neuronal and synaptic plasticity.

Effect of ablation or inhibition of stromal matrix metalloproteinase-9 on lung metastasis in a breast cancer model is dependent on genetic background.

Matrix metalloproteinases (MMP) are a family of enzymes with a myriad of functions. Lately, we have come to realize that broad-spectrum inhibition of these enzymes, as was tried unsuccessfully in multiple phase III trials in cancer patients, is likely unwise given the protumorigenic and antitumorigenic functions of various family members. Here, we used the multistage mammary tumor model MMTV-PyVT to investigate roles for either MMP7 or MMP9 in tumor progression. We found no effect of genetic ablation of MMP7 or MMP9 on the multifocal tumors that developed in the mammary glands. Lack of MMP7 also had no effect on the development of lung metastases, suggesting that MMP7 is irrelevant in this model. In contrast, MMP9 deficiency was associated with an 80% decrease in lung tumor burden. The predominant cellular source of MMP9 was myeloid cells, with neutrophils being the largest contributor in tumor-bearing lungs. Experimental metastasis assays corroborated the role of host-derived MMP9 in lung metastasis and also facilitated determination of a time frame most relevant for the MMP9-mediated effect. The lung tumors from MMP9-deficient mice showed decreased angiogenesis. Surprisingly, the antimetastatic outcome of MMP9 ablation seemed to be dependent on strain. Only mice that had genetic background derived from C57BL/6 showed reduced metastasis, whereas mice fully of the FVB/N background showed no significant effect. These strain-specific responses were also observed in a study using a highly selective pharmacologic inhibitor of MMP9 and thus suggest that responses to MMP inhibition are controlled by genetic differences.