Cell-targeting conjugates of Saporin 6, a ribosome inactivating protein (RIP), were prepared using the Saporin Ala 157 Cys mutant, a small molecule inhibitor (SMI) of integrins αvβ3/αvβ5, and a potent cytotoxin, auristatin F (AF). The conjugates selectively and potently inhibited proliferation of tumor cells expressing the target integrins. We anticipate that the small molecule–RIP bioconjugate approach can be broadly applied using other small molecule drugs.

Cancer cells that enter the metastatic cascade require traits that allow them to survive within the circulation and colonize distant organ sites. As disseminating cancer cells adapt to their changing microenvironments, they also modify their metabolism and metabolite production.A mouse xenograft model of spontaneous tumor metastasis was used to determine the metabolic rewiring that occurs between primary cancers and their metastases. An “autonomous” mass spectrometry-based untargeted metabolomic workflow with integrative metabolic pathway analysis revealed a number of differentially regulated metabolites in primary mammary fat pad (MFP) tumors compared to microdissected paired lung metastases. The study was further extended to analyze metabolites in paired normal tissues which determined the potential influence of metabolites from the microenvironment.Metabolomic analysis revealed that multiple metabolites were increased in metastases, including cholesterol sulfate and phospholipids (phosphatidylglycerols and phosphatidylethanolamine). Metabolite analysis of normal lung tissue in the mouse model also revealed increased levels of these metabolites compared to tissues from normal MFP and primary MFP tumors, indicating potential extracellular uptake by cancer cells in lung metastases. These results indicate a potential functional importance of cholesterol sulfate and phospholipids in propagating metastasis. In addition, metabolites involved in DNA/RNA synthesis and the TCA cycle were decreased in lung metastases compared to primary MFP tumors.Using an integrated metabolomic workflow, this study identified a link between cholesterol sulfate and phospholipids, metabolic characteristics of the metastatic niche, and the capacity of tumor cells to colonize distant sites.

Antibody therapeutics are a promising drug class due to their high specificity and favorable pharmacokinetics. While there are many methods for the development of antibodies specific to disease associated antigens, selecting antibodies against functional epitopes with high specificity and affinity can be difficult for certain epitopes. We describe a generalizable method for synthesizing antibody mimetics by site specifically conjugating small molecules (with high affinity and specificity to disease associated antigens) to an Fc fragment to develop drugs with the benefits of an antibody. As a proof of concept, an E269pAcPhe Fc antibody Fc fragment was produced and subsequently site-specifically labeled with a linker-modified folic acid compound to generate an Fc-folic acid antibody-mimetic. This was chosen as the model system because the high-affinity folate receptor FR-α is highly expressed in a number of cancer types including breast and ovarian cancer. The specificity of the Fc-folic acid conjugate was assessed via flowcytometry with the folate-receptor positive breast cancer cell line MDA-MB-231 by measuring Fc-folic acid binding in both the absence and presence of an excess of folic acid. Fc-small molecule conjugates could be developed into a unique class of antibody-like therapeutics.

Extensive experimental evidence shows that platelets support tumour metastasis. The activation of platelets and the coagulation system have a crucial role in the progression of cancer. Within the circulatory system, platelets guard tumour cells from immune elimination and promote their arrest at the endothelium, supporting the establishment of secondary lesions. These contributions of platelets to tumour cell survival and spread suggest platelets as a new avenue for therapy.

Brain metastases occur in 20 to 40% of patients with metastatic breast cancer. The process is complex and depends on successful cancer cell evasion from the primary tumor, distribution and survival within the blood stream and cerebral microvasculature, penetration of the blood brain barrier and proliferation within the brain microenvironment. The initial steps of brain colonization are difficult to study in vivo. Therefore, in vitro assays have been developed to mimic this process. Most commonly, in vitro studies of brain colonization focus on tumor cell adhesion to brain endothelial cells and transendothelial migration. We previously investigated breast cancer brain colonization from the blood stream in vivo and defined the time and process of brain entry for five different cancer cell lines in a mouse model. We now investigated if in vitro approaches can reliably emulate the initial steps that determine successful brain colonization in vivo. To this end, we optimized an in vitro model of the vascular blood brain barrier and compared the brain invasion properties of the in vivo characterized cell models with their ability to interact with and penetrate the blood brain barrier model in vitro. Our results show that the in vitro findings correlate only poorly with the vivo results. The limitations of the in vitro approaches are discussed in light of the in vivo processes. We conclude that investigation of mechanisms supporting the earliest steps of breast cancer brain metastasis from the blood stream will depend on in vivo analyses.

Advanced metastatic disease is difficult to manage and specific therapeutic targets are rare. We showed earlier that metastatic breast cancer cells use the activated conformer of adhesion receptor integrin αvβ3 for dissemination. We now investigated if targeting this form of the receptor can impact advanced metastatic disease, and we analyzed the mechanisms involved. Treatment of advanced multi-organ metastasis in SCID mice with patient-derived scFv antibodies specific for activated integrin αvβ3 caused stagnation and regression of metastatic growth. The antibodies specifically localized to tumor lesions in vivo and inhibited αvβ3 ligand binding at nanomolar levels in vitro. At the cellular level, the scFs associated rapidly with high affinity αvβ3 and dissociated extremely slowly. Thus, the scFvs occupy the receptor on metastatic tumor cells for prolonged periods of time, allowing for inhibition of established cell interaction with natural αvβ3 ligands. Potential apoptosis inducing effects of the antibodies through interaction with caspase-3 were studied as potential additional mechanism of treatment response. However, in contrast to a previous concept, neither the RGD-containing ligand mimetic scFvs nor RGD peptides bound or activated caspase-3 at the cellular or molecular level. This indicates that the treatment effects seen in the animal model are primarily due to antibody interference with αvβ3 ligation. Inhibition of advanced metastatic disease by treatment with cancer patient derived single chain antibodies against the activated conformer of integrin αvβ3 identifies this form of the receptor as a suitable target for therapy.

The incidence of brain metastasis is rising and poses a severe clinical problem, as we lack effective therapies and knowledge of mechanisms that control metastatic growth in the brain. Here we demonstrate a crucial role for high-affinity tumor cell integrin αvβ3 in brain metastatic growth and recruitment of blood vessels. Although αvβ3 is frequently up-regulated in primary brain tumors and metastatic lesions of brain homing cancers, we show that it is the αvβ3 activation state that is critical for brain lesion growth. Activated, but not non-activated, tumor cell αvβ3 supports efficient brain metastatic growth through continuous up-regulation of vascular endothelial growth factor (VEGF) protein under normoxic conditions. In metastatic brain lesions carrying activated αvβ3, VEGF expression is controlled at the post-transcriptional level and involves phosphorylation and inhibition of translational respressor 4E-binding protein (4E-BP1). In contrast, tumor cells with non-activated αvβ3 depend on hypoxia for VEGF induction, resulting in reduced angiogenesis, tumor cell apoptosis, and inefficient intracranial growth. Importantly, the microenvironment critically influences the effects that activated tumor cell αvβ3 exerts on tumor cell growth. Although it strongly promoted intracranial growth, the activation state of the receptor did not influence tumor growth in the mammary fat pad as a primary site. Thus, we identified a mechanism by which metastatic cells thrive in the brain microenvironment and use the high-affinity form of an adhesion receptor to grow and secure host support for proliferation. Targeting this molecular mechanism could prove valuable for the inhibition of brain metastasis.

Combinatorial antibody libraries have the potential to display the entire immunological record of an individual, allowing one to detect and recover any antibody ever made, irrespective of whether it is currently being produced. We have termed this the “fossil record” of an individual's antibody response. To determine whether cancer patients have ever made antibodies with disease-fighting potential, we screened combinatorial antibody libraries from cancer patients for immunoglobulins that can identify metastatic tumor cells. This strategy yielded human antibodies specific for the activated conformation of the adhesion receptor integrin αvβ3 that is associated with a metastatic phenotype. In a remarkable example of convergent evolution, two of these antibodies were shown to contain the Arg-Gly-Asp integrin recognition motif of the natural ligand within the third complementarity-determining region of the heavy chain. These antibodies interfered with lung colonization by human breast cancer cells in a mouse model and inhibited existing metastatic disease. Our data imply that, at least at some time, these antibodies were part of a patient's surveillance system against metastatic cells, targeting the activated conformer of integrin αvβ3 and disrupting its functions. The ligand-mimetic nature of these antibodies, combined with specificity for a single receptor, is unique in the integrin–ligand repertoire. The convergent evolution of critical sequences in antibodies and other ligands that bind to the same target means that the immune response has sufficient power to find a best chemical solution for the optimization of binding energy, even though antibodies evolve in real time, as compared with billions of years for the natural ligand.

Expression of adhesion receptor integrin αvβ3 in an activated functional form strongly promotes metastasis in human breast cancer cells. Here, we report that αvβ3 cooperates with matrix metalloproteinase type 9 (MMP-9) in breast cancer cell migration. This cooperation is regulated by the activation state of the integrin. Expression of activated αvβ3 in metastatic variants of MDA-MB 435 human breast cancer cells and primary metastatic cells from breast cancer patients strongly enhanced migration toward vitronectin and fibrinogen. This enhancement was mediated by a soluble factor produced by breast cancer cells expressing activated αvβ3. When transferred, this factor also up-regulated αvβ3-dependent migration of breast cancer cells that express the nonactivated integrin. The factor was identified as metalloproteinase MMP-9. Whereas all tested breast cancer cell variants produced latent MMP-9, only those with activated αvβ3 produced the mature form of this metalloproteinase. Recombinant mature MMP-9, but not latent MMP-9 or either form of MMP-2, enhanced αvβ3-dependent breast cancer cell migration. The migratory response was inhibited by tissue inhibitors of metalloproteinase or when MMP-9 was depleted from the inducing supernatants. The results indicate a causal relationship between the expression of activated integrin αvβ3 and production of enzymatically active MMP-9 in metastatic breast cancer cells. These molecules cooperate to enhance breast cancer cell migration toward specific matrix proteins, and this may contribute to the strongly enhanced metastatic capacity of breast cancer cells that express activated αvβ3.

Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin αvβ3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin αvβ3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated αvβ3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant αvβ3D723R, but not αvβ3WT, in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin αvβ3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer.

•Reduction in NAMPT expression enhances metastatic aggressiveness in breast cancer.•Low NAMPT is associated with upregulation of αvβ3 and β1 integrins in tumor cells.•Reduction in NAMPT expression enhances adhesion to extracellular matrix proteins.•Data suggest that non-lethal inhibition of NAMPT may increase tumor aggressiveness.NAD+ metabolism is an essential regulator of cellular redox reactions, energy pathways, and a substrate provider for NAD+ consuming enzymes. We recently demonstrated that enhancement of NAD+/NADH levels in breast cancer cells with impaired mitochondrial NADH dehydrogenase activity, through augmentation of complex I or by supplementing tumor cell nutrients with NAD+ precursors, inhibits tumorigenicity and metastasis. To more fully understand how aberrantly low NAD+ levels promote tumor cell dissemination, we here asked whether inhibition of NAD+ salvage pathway activity by reduction in nicotinamide phosphoribosyltransferase (NAMPT) expression can impact metastasis and tumor cell adhesive functions. We show that knockdown of NAMPT, the enzyme catalyzing the rate-limiting step of the NAD+ salvage pathway, enhances metastatic aggressiveness in human breast cancer cells and involves modulation of integrin expression and function. Reduction in NAMPT expression is associated with upregulation of select adhesion receptors, particularly αvβ3 and β1 integrins, and results in increased breast cancer cell attachment to extracellular matrix proteins, a key function in tumor cell dissemination. Interestingly, NAMPT downregulation prompts expression of integrin αvβ3 in a high affinity conformation, known to promote tumor cell adhesive interactions during hematogenous metastasis. NAMPT has been selected as a therapeutic target for cancer therapy based on the essential functions of this enzyme in NAD+ metabolism, cellular redox, DNA repair and energy pathways. Notably, our results indicate that incomplete inhibition of NAMPT, which impedes NAD+ metabolism but does not kill a tumor cell can alter its phenotype to be more aggressive and metastatic. This phenomenon could promote cancer recurrence, even if NAMPT inhibition initially reduces tumor growth.

Cell-targeting conjugates of Saporin 6, a ribosome inactivating protein (RIP), were prepared using the Saporin Ala 157 Cys mutant, a small molecule inhibitor (SMI) of integrins αvβ3/αvβ5, and a potent cytotoxin, auristatin F (AF). The conjugates selectively and potently inhibited proliferation of tumor cells expressing the target integrins. We anticipate that the small molecule–RIP bioconjugate approach can be broadly applied using other small molecule drugs.