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Summary Background The extensive alveolar capillary network of the lungs is an attractive route for the administration of several agents. One key functional attribute is the rapid onset of systemic action due to the absence of first-pass metabolism. Methods Here, we applied a combinatorial approach for ligand-directed pulmonary delivery as a unique route for systemic targeting in vaccination. Findings We screened a phage display random peptide library in vivo to select, identify, and validate a ligand (CAKSMGDIVC) that specifically targets and is internalized through its receptor, α3β1 integrin, on the surface of cells lining the lung airways and alveoli and mediates CAKSMGDIVC-displaying phage binding and systemic delivery without compromising lung homeostasis. As a proof-of-concept, we show that the pulmonary delivery of targeted CAKSMGDIVC-displaying phage particles in mice and non-human primates elicits a systemic and specific humoral response. Conclusions This broad methodology blueprint represents a robust and versatile platform tool enabling new ligand-receptor discovery with many potential translational applications. Funding Cancer Center Support Grants to the University of Texas M.D. Anderson Cancer Center ( CA016672 ), University of New Mexico Comprehensive Cancer Center ( CA118100 ), Rutgers Cancer Institute of New Jersey ( CA072720 ), research awards from the Gillson-Longenbaugh Foundation , and National Institutes of Health (NIH) grant no. 1R01CA226537 .

Triple-negative breast cancer (TNBC) is an aggressive tumor with limited treatment options and poor prognosis. We applied the in vivo phage display technology to isolate peptides homing to the immunosuppressive cellular microenvironment of TNBC as a strategy for non-malignant target discovery. We identified a cyclic peptide (CSSTRESAC) that specifically binds to a vitamin D receptor, protein disulfide-isomerase A3 (PDIA3) expressed on the cell surface of tumor-associated macrophages (TAM), and targets breast cancer in syngeneic TNBC, non-TNBC xenograft, and transgenic mouse models. Systemic administration of CSSTRESAC to TNBC-bearing mice shifted the cytokine profile toward an antitumor immune response and delayed tumor growth. Moreover, CSSTRESAC enabled ligand-directed theranostic delivery to tumors and a mathematical model confirmed our experimental findings. Finally, in silico analysis showed PDIA3-expressing TAM in TNBC patients. This work uncovers a functional interplay between a cell surface vitamin D receptor in TAM and antitumor immune response that could be therapeutically exploited.

The Polb gene encodes DNA polymerase beta (Pol β), a DNA polymerase that functions in base excision repair (BER) and microhomology-mediated end-joining. The Pol β-Y265C protein exhibits low catalytic activity and fidelity, and is also deficient in microhomology-mediated end-joining. We have previously shown that the Polb(Y265C/+) and Polb(Y265C/C) mice develop lupus. These mice exhibit high levels of antinuclear antibodies and severe glomerulonephritis. We also demonstrated that the low catalytic activity of the Pol β-Y265C protein resulted in accumulation of BER intermediates that lead to cell death. Debris released from dying cells in our mice could drive development of lupus. We hypothesized that deletion of the Neil1 and Ogg1 DNA glycosylases that act upstream of Pol β during BER would result in accumulation of fewer BER intermediates, resulting in less severe lupus. We found that high levels of antinuclear antibodies are present in the sera of Polb(Y265C/+) mice deleted of Ogg1 and Neil1 DNA glycosylases. However, these mice develop significantly less severe renal disease, most likely due to high levels of IgM in their sera.

The androgen receptor (AR) is the major driver of prostate cancer growth and survival. However, almost all patients relapse with castration-resistant disease (CRPC) when treated with anti-androgen therapy. In CRPC, AR is often aberrantly activated independent of androgen. Targeting survival pathways downstream of AR could be a viable strategy to overcome CRPC. Surprisingly, little is known about how AR drives prostate cancer survival. Furthermore, CRPC tumors in which Pten is lost are also resistant to eradication by PI3K inhibitors. We sought to identify the mechanism by which AR drives tumor survival in CRPC to identify ways to overcome resistance to PI3K inhibition. We found that integrins α6β1 and Bnip3 are selectively elevated in CRPC downstream of AR. While integrin α6 promotes survival and is a direct transcriptional target of AR, the ability of AR to induce Bnip3 is dependent on adhesion to laminin and integrin α6β1-dependent nuclear translocation of HIF1α. Integrins α6β1 and Bnip3 were found to promote survival of CRPC cells selectively on laminin through the induction of autophagy and mitophagy. Furthermore, blocking Bnip3 or integrin α6β1 restored sensitivity to PI3K inhibitors in Pten-negative CRPC. We identified an AR driven pathway that cooperates with laminin and hypoxia to drive resistance to PI3K inhibitors. These findings can help explain in part why PI3K inhibitors have failed in clinical trials to overcome AR-dependent CRPC.

Osteosarcoma occurs predominantly in children and young adults. High-grade tumors require multidisciplinary treatment consisting of chemotherapy in the neoadjuvant and adjuvant settings, along with surgical intervention. Despite this approach, death from respiratory failure secondary to the development and progression of pulmonary metastases remains a significant problem. Here, we identify the IL-11 receptor α subunit (IL-11Rα) as a cell surface marker of tumor progression that correlates with poor prognosis in patients with osteosarcoma. We also show that both IL-11Rα and its ligand, IL-11, are specifically up-regulated in human metastatic osteosarcoma cell lines; engagement of this autocrine loop leads to tumor cell proliferation, invasion, and anchorage-independent growth in vitro. Consistently, IL-11Rα promotes lung colonization by human metastatic osteosarcoma cells in vivo in an orthotopic mouse model. Finally, we evaluate the IL-11Rα-targeted proapoptotic agent bone metastasis-targeting peptidomimetic (BMTP-11) in preclinical models of primary intratibial osteosarcomas, observing marked inhibition of both tumor growth and lung metastases. This effect was enhanced when BMTP-11 was combined with the chemotherapeutic drug gemcitabine. Our combined data support the development of approaches targeting IL-11Rα, and establish BMTP-11 as a leading drug candidate for clinical translation in patients with high-risk osteosarcoma.

The Pim and AKT serine/threonine protein kinases are implicated as drivers of cancer. Their regulation of tumor growth is closely tied to the ability of these enzymes to mainly stimulate protein synthesis by activating mTORC1 (mammalian target of rapamycin complex 1) signaling, although the exact mechanism is not completely understood. mTORC1 activity is normally suppressed by amino acid starvation through a cascade of multiple regulatory protein complexes, e.g., GATOR1, GATOR2, and KICSTOR, that reduce the activity of Rag GTPases. Bioinformatic analysis revealed that DEPDC5 (DEP domain containing protein 5), a component of GATOR1 complex, contains Pim and AKT protein kinase phosphorylation consensus sequences. DEPDC5 phosphorylation by Pim and AKT kinases was confirmed in cancer cells through the use of phospho-specific antibodies and transfection of phospho-inactive DEPDC5 mutants. Consistent with these findings, during amino acid starvation the elevated expression of Pim1 overcame the amino acid inhibitory protein cascade and activated mTORC1. In contrast, the knockout of DEPDC5 partially blocked the ability of small molecule inhibitors against Pim and AKT kinases both singly and in combination to suppress tumor growth and mTORC1 activity in vitro and in vivo. In animal experiments knocking in a glutamic acid (S1530E) in DEPDC5, a phospho mimic, in tumor cells induced a significant level of resistance to Pim and the combination of Pim and AKT inhibitors. Our results indicate a phosphorylation-dependent regulatory mechanism targeting DEPDC5 through which Pim1 and AKT act as upstream effectors of mTORC1 to facilitate proliferation and survival of cancer cells.

Significance Prostate cancer has an unpredictable natural history: While most tumors are clinically indolent, some patients display lethal phenotypes. Serum prostate-specific antigen is the most often used test in prostate cancer but screening is controversial. Treatment options are limited for metastatic disease, hence the need for early diagnosis. Prostate cancer antigen 3 ( PCA3 ), a long noncoding RNA, is the most specific biomarker identified and approved as a diagnostic test. However, its inherent biological function (if any) has remained elusive. We uncovered a negative transdominant oncogenic role for PCA3 that down-regulates an unrecognized tumor suppressor gene, PRUNE2 (a human homolog of the Drosophila prune gene) thereby promoting malignant cell growth. This work defines a unique biological function for PCA3 in prostate cancer.

Six members of the microRNA-17 (miR-17) family were mapped to three different chromosomes, although they share the same seed sequence and are predicted to target common genes, among which are those encoding hypoxia-inducible factor-1α (HIF1A) and VEGFA. Here, we evaluated the in vivo expression profile of the miR-17 family in the murine retinopathy of prematurity (ROP) model, whereby Vegfa expression is highly enhanced at the early stage of retinal neovascularization, and we found simultaneous reduction of all miR-17 family members at this stage. Using gene reporter assays, we observed binding of these miRs to specific sites in the 3' UTRs of Hif1a and Vegfa. Furthermore, overexpression of these miRs decreased HIF1A and VEGFA expression in vitro. Our data indicate that this miR-17 family elicits a regulatory synergistic down-regulation of Hif1a and Vegfa expression in this biological model. We propose the existence of a coordinated regulatory network, in which diverse miRs are synchronously regulated to target the Hif1a transcription factor, which in turn, potentiates and reinforces the regulatory effects of the miRs on Vegfa to trigger and sustain a significant physiological response.

Patients with inoperable or unresectable pancreatic neuroendocrine tumors (NETs) have limited treatment options. These rare human tumors often express somatostatin receptors (SSTRs) and thus are clinically responsive to certain relatively stable somatostatin analogs, such as octreotide. Unfortunately, however, this tumor response is generally short-lived. Here we designed a hybrid adeno-associated virus and phage (AAVP) vector displaying biologically active octreotide on the viral surface for ligand-directed delivery, cell internalization, and transduction of an apoptosis-promoting tumor necrosis factor (TNF) transgene specifically to NETs. These functional attributes of AAVP-TNF particles displaying the octreotide peptide motif (termed Oct-AAVP-TNF) were confirmed in vitro, in SSTR type 2-expressing NET cells, and in vivo using cohorts of pancreatic NET-bearing Men1 tumor-suppressor gene KO mice, a transgenic model of functioning (i.e., insulin-secreting) tumors that genetically and clinically recapitulates the human disease. Finally, preclinical imaging and therapeutic experiments with pancreatic NET-bearing mice demonstrated that Oct-AAVP-TNF lowered tumor metabolism and insulin secretion, reduced tumor size, and improved mouse survival. Taken together, these proof-of-concept results establish Oct-AAVP-TNF as a strong therapeutic candidate for patients with NETs of the pancreas. More broadly, the demonstration that a known, short, biologically active motif can direct tumor targeting and receptor-mediated internalization of AAVP particles may streamline the potential utility of myriad other short peptide motifs and provide a blueprint for therapeutic applications in a variety of cancers and perhaps many nonmalignant diseases as well.

A major challenge of targeted molecular imaging and drug delivery in cancer is establishing a functional combination of ligand-directed cargo with a triggered release system. Here we develop a hydrogel-based nanotechnology platform that integrates tumor targeting, photon-to-heat conversion, and triggered drug delivery within a single nanostructure to enable multimodal imaging and controlled release of therapeutic cargo. In proof-of-concept experiments, we show a broad range of ligand peptide-based applications with phage particles, heat-sensitive liposomes, or mesoporous silica nanoparticles that self-assemble into a hydrogel for tumor-targeted drug delivery. Because nanoparticles pack densely within the nanocarrier, their surface plasmon resonance shifts to near-infrared, thereby enabling a laser-mediated photothermal mechanism of cargo release. We demonstrate both noninvasive imaging and targeted drug delivery in preclinical mouse models of breast and prostate cancer. Finally, we applied mathematical modeling to predict and confirm tumor targeting and drug delivery. These results are meaningful steps toward the design and initial translation of an enabling nanotechnology platform with potential for broad clinical applications.

Circulating cancer cells can putatively colonize distant organs to form metastases or to reinfiltrate primary tumors themselves through a process termed "tumor self-seeding." Here we exploit this biological attribute to deliver tumor necrosis factor alpha (TNF), a potent antitumor cytokine, directly to primary and metastatic tumors in a mechanism that we have defined as "tumor self-targeting." For this purpose, we genetically engineered mouse mammary adenocarcinoma (TSA), melanoma (B16-F10), and Lewis lung carcinoma cells to produce and release murine TNF. In a series of intervention trials, systemic administration of TNF-expressing tumor cells was associated with reduced growth of both primary tumors and metastatic colonies in immunocompetent mice. We show that these malignant cells home to tumors, locally release TNF, damage neovascular endothelium, and induce massive cancer cell apoptosis. We also demonstrate that such tumor-cell-mediated delivery avoids or minimizes common side effects often associated with TNF-based therapy, such as acute inflammation and weight loss. Our study provides proof of concept that genetically modified circulating tumor cells may serve as targeted vectors to deliver anticancer agents. In a clinical context, this unique paradigm represents a personalized approach to be translated into applications potentially using patient-derived circulating tumor cells as self-targeted vectors for drug delivery.

Radiation gastrointestinal (GI) syndrome is a major lethal toxicity that may occur after a radiation/nuclear incident. Currently, there are no prophylactic countermeasures against radiation GI syndrome lethality for first responders, military personnel, or remediation workers entering a contaminated area. The pathophysiology of this syndrome requires depletion of stem cell clonogens (SCCs) within the crypts of Lieberkühn, which are a subset of cells necessary for postinjury regeneration of gut epithelium. Recent evidence indicates that SCC depletion is not exclusively a result of DNA damage but is critically coupled to ceramide-induced endothelial cell apoptosis within the mucosal microvascular network. Here we show that ceramide generated on the surface of endothelium coalesces to form ceramide-rich platforms that transmit an apoptotic signal. Moreover, we report the generation of 2A2, an anti-ceramide monoclonal antibody that binds to ceramide to prevent platform formation on the surface of irradiated endothelial cells of the murine GI tract. Consequently, we found that 2A2 protected against endothelial apoptosis in the small intestinal lamina propria and facilitated recovery of crypt SCCs, preventing the death of mice from radiation GI syndrome after high radiation doses. As such, we suggest that 2A2 represents a prototype of a new class of anti-ceramide therapeutics and an effective countermeasure against radiation GI syndrome mortality.

Inhibition of blood vessel formation is a viable therapeutic approach in angiogenesis-dependent diseases. We previously used a combinatorial screening on vascular endothelial growth factor (VEGF)-activated endothelial cells to select the sequence CPQPRPLC and showed that the motif Arg-Pro-Leu targets VEGF receptor-1 and neuropilin-1. Here, we evaluated and validated D (LPR), a derivative molecule with strong antiangiogenesis attributes. This prototype drug markedly inhibits neovascularization in three mouse models: Matrigel-based assay, functional human/murine blood vessel formation, and retinopathy of prematurity. In addition to its systemic activity, D (LPR) also inhibits retinal angiogenesis when administered in an eye-drop formulation. Finally, in preliminary studies, we have showed targeted drug activity in an experimental tumor-bearing mouse model. These results show that drugs targeting extracellular domains of VEGF receptors are active, affect signal transduction, and have potential for clinical application. On a larger context, this study illustrates the power of ligand-directed selection plus retro-inversion for rapid drug discovery and development.

The epidermal growth factor receptor (EGFR), a tyrosine kinase, is central to human tumorigenesis. Typically, three classes of drugs inhibit tyrosine kinase pathways: blocking antibodies, small kinase inhibitors, and soluble ligand receptor traps/decoys. Only the first two types of EGFR-binding inhibitory drugs are clinically available; notably, no EGFR decoy has yet been developed. Here we identify small molecules mimicking EGFR and that functionally behave as soluble decoys for EGF and TGFα, ligands that would otherwise activate downstream signaling. After combinatorial library selection on EGFR ligands, a panel of binding peptides was narrowed by structure–function analysis. The most active motif was CVRAC (EGFR 283–287), which is necessary and sufficient for specific EGFR ligand binding. Finally, a synthetic retro-inverted derivative, D (CARVC), became our preclinical prototype of choice. This study reveals an EGFR-decoy drug candidate with translational potential.

Mammalian cell membranes provide an interface between the intracellular and extracellular compartments. It is currently thought that cytoplasmic signaling adapter proteins play no functional role within the extracellular tumor environment. Here, by selecting combinatorial random peptide libraries in tumor-bearing mice, we uncovered a direct, specific, and functional interaction between CRKL, an adapter protein [with Src homology 2 (SH2)- and SH3-containing domains], and the plexin-semaphorin-integrin domain of β 1 integrin in the extracellular milieu. Through assays in vitro, in cellulo, and in vivo, we show that this unconventional and as yet unrecognized protein–protein interaction between a regulatory integrin domain (rather than a ligand-binding one) and an intracellular adapter (acting outside of the cells) triggers an alternative integrin-mediated cascade for cell growth and survival. Based on these data, here we propose that a secreted form of the SH3/SH2 adaptor protein CRKL may act as a growth-promoting factor driving tumorigenesis and may lead to the development of cancer therapeutics targeting secreted CRKL.

TWEAK (TNF-like weak inducer of apoptosis) is a TNF superfamily member implicated in several mechanisms. Although fibroblast growth factor inducible 14 (Fn14)/TweakR has been reported as its receptor, an as yet unrecognized surface molecule(s) might modulate TWEAK function(s). Thus, we set out to identify TWEAK-binding proteins by screening a combinatorial peptide library. Cyclic peptides containing a consensus motif (WXDDG) bound to TWEAK specifically. These peptides were similar to CD163, a scavenger receptor cysteine-rich domain family member, restricted to the monocyte/macrophage lineage and responsible for the uptake of circulating haptoglobin-hemoglobin (Hp-Hb) complexes. Sequence profile analysis suggested that TWEAK mimicked the CD163 natural ligand (Hp-Hb). Consistently, we show dose-dependent TWEAK binding to CD163 and blockade by an anti-CD163 Ab. In a competition assay, both soluble CD163 and Fn14/TweakR were able to compete off TWEAK binding to coated Fn14/TweakR or CD163, respectively. Flow-cytometry and immunofluorescence assays showed that human monocytes (Fn14/TweakR negative and CD163 positive) bind TWEAK, thus blocking the recognition of CD163 and reducing the activation mediated by a specific mAb in these cells. We demonstrate that monocytes can sequester TWEAK from supernatants, thus preventing tumor cell apoptosis; this effect was reverted by preincubation with the peptide mimicking CD163 or with a mAb anti-CD163, indicating specificity. Finally, we show that recombinant human TWEAK binding to CD163-transfected Chinese hamster ovary cells is inhibited by the presence of either unlabeled TWEAK or the Hp-Hb complex. Together, these data are consistent with the hypothesis that CD163 either acts as a TWEAK scavenger in pathological conditions or serves as an alternate receptor for TWEAK in cells lacking Fn14/TweakR.

We performed combinatorial peptide library screening in vivo on a novel human prostate cancer xenograft that is androgen-independent and induces a robust osteoblastic reaction in bonelike matrix and soft tissue. We found two peptides, PKRGFQD and SNTRVAP, which were enriched in the tumors, targeted the cell surface of androgen-independent prostate cancer cells in vitro, and homed to androgen receptor-null prostate cancer in vivo. Purification of tumor homogenates by affinity chromatography on these peptides and subsequent mass spectrometry revealed a receptor for the peptide PKRGFQD, α-2-macroglobulin, and for SNTRVAP, 78-kDa glucose-regulated protein (GRP78). These results indicate that GRP78 and α-2-macroglobulin are highly active in osteoblastic, androgen-independent prostate cancer in vivo. These previously unidentified ligand–receptor systems should be considered for targeted drug development against human metastatic androgen-independent prostate cancer.

The matricellular SPARC family member hevin (SPARC-like 1/SPARCL-1/SC1/Mast9) contributes to neural development and alters tumor progression in a range of mammalian models. The distribution of hevin in mouse tissues was reexamined with a novel monoclonal antibody that discriminates between hevin and its ortholog SPARC. We now report proteolysis of hevin in many tissues, with the most extensive processing in the brain. We demonstrate a cleavage site within the hevin sequence for the neural tissue proteinase ADAMTS4. Digestion of hevin by ADAMTS4 in vitro produced fragments similar to those present in brain lysates. Monoclonal antibodies revealed a SPARC-like fragment generated from hevin that was co-localized with ADAMTS4 in vivo. We show that proteolysis of hevin by ADAMTS4 in the mouse cerebellum is important for the normal development of this tissue. In conclusion, we have identified the fragmentation of hevin by ADAMTS4 in the mouse brain and propose that this specific proteolysis is integral to cell morphology and extracellular matrix deposition in the developing brain.

The matricellular protein SPARC (secreted protein acidic and rich in cysteine) has been implicated in development, differentiation, response to injury, and tumor biology by virtue of its regulation of extracellular matrix production/assembly and its antiadhesive and antiproliferative effects on different cell types. Despite numerous biological activities described for SPARC, cell surface receptors for this protein have not been identified. By phage display and in vitro-binding assays, we now show that SPARC interacts with stabilin-1, a scavenger receptor expressed by tissue macrophages and sinusoidal endothelial cells. The interaction is mediated by the extracellular epidermal growth factor-like region of stabilin-1 containing the sequence FHGTAC. Using FACS analysis and confocal microscopy, we demonstrate that stabilin-1 internalizes and targets SPARC to an endosomal pathway in Chinese hamster ovary cells stably transfected with this receptor. In human macrophages, stabilin-1 expression is required for receptor-mediated endocytosis of SPARC. SPARC was efficiently endocytosed by alternatively activated macrophages stimulated by IL-4 and dexamethasone, but not solely by Th1 or Th2 cytokines. A time course of ligand exposure to alternatively activated macrophages revealed that stabilin-1-mediated endocytosis of SPARC was followed by its targeting for degradation, similar to the targeting of acetylated low density lipoprotein, another stabilin-1 ligand. We propose that alternatively activated macrophages coordinate extracellular matrix remodeling, angiogenesis, and tumor progression via stabilin-1-mediated endocytosis of SPARC and thereby regulate its extracellular concentration.

SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.