Marina Cardó-Vila

PRUNE2 is a human prostate cancer suppressor regulated by the intronic long noncoding RNA PCA3.

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. Prostate cancer antigen 3 (PCA3) is the most specific prostate cancer biomarker but its function remains unknown. Here we identify PRUNE2, a target protein-coding gene variant, which harbors the PCA3 locus, thereby classifying PCA3 as an antisense intronic long noncoding (lnc)RNA. We show that PCA3 controls PRUNE2 levels via a unique regulatory mechanism involving formation of a PRUNE2/PCA3 double-stranded RNA that undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing. PRUNE2 expression or silencing in prostate cancer cells decreased and increased cell proliferation, respectively. Moreover, PRUNE2 and PCA3 elicited opposite effects on tumor growth in immunodeficient tumor-bearing mice. Coregulation and RNA editing of PRUNE2 and PCA3 were confirmed in human prostate cancer specimens, supporting the medical relevance of our findings. These results establish PCA3 as a dominant-negative oncogene and PRUNE2 as an unrecognized tumor suppressor gene in human prostate cancer, and their regulatory axis represents a unique molecular target for diagnostic and therapeutic intervention.

Discovery and horizontal follow-up of an autoantibody signature in human prostate cancer

Significance For the management of prostate cancer it has remained a significant clinical challenge to identify biomarkers that can be used as prognostic indicators to facilitate early treatment decisions and indicate patients at risk for castrate-resistant bone-metastatic prostate cancer in need of more aggressive treatment. In this report, serum antibodies to alpha-2–Heremans–Schmidt glycoprotein (fetuin-A) were demonstrated to display increased reactivity with concomitant development of metastatic castrate-resistant disease in a large cohort of prostate cancer patients. Furthermore, metastatic prostate cancer cell lines and bone metastasis samples displayed robust fetuin-A expression. To our knowledge, this is the first report to indicate that serum autoantibodies reactive to fetuin-A show utility as a prognostic indicator for prostate cancer patients at risk for progressing to metastatic disease. In response to an urgent need for improved diagnostic and predictive serum biomarkers for management of metastatic prostate cancer, we used phage display fingerprinting to analyze sequentially acquired serum samples from a patient with advancing prostate cancer. We identified a peptide ligand, CTFAGSSC, demonstrating an increased recovery frequency over time. Serum antibody reactivity to this peptide epitope increased in the index patient, in parallel with development of deteriorating symptoms. The antigen mimicking the peptide epitope was identified as alpha-2–Heremans–Schmid glycoprotein, also known as fetuin-A. Metastatic prostate cancer cell lines and bone metastasis samples displayed robust fetuin-A expression, and we demonstrated serum immune reactivity to fetuin-A with concomitant development of metastatic castrate-resistant disease in a large cohort of prostate cancer patients. Whereas fetuin-A is an established tumor antigen in several types of cancer, including breast cancer, glioblastoma, and pancreas cancer, this report is to our knowledge the first study implicating fetuin-A in prostate cancer and indicating that autoantibodies specific for fetuin-A show utility as a prognostic indicator for prostate cancer patients prone to progress to metastatic disease.

Selection and identification of ligand peptides targeting a model of castrate-resistant osteogenic prostate cancer and their receptors

Significance This study shows how phage display technology can be applied successfully to in vivo models and can advance molecular oncology through the identification of tumor-homing peptides and their target receptors. Treatment options are still limited for prostate cancer patients who have progressed to develop castrate-resistant osteoblastic bone metastases. The peptides identified in this study may lead to breakthroughs in fighting metastatic androgen-independent prostate cancer by enabling drug targeting and nanotechnology-based therapeutic strategies and may lead to significant advances in the management and therapy of this frequently lethal disease. 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.

Integrated nanotechnology platform for tumor-targeted multimodal imaging and therapeutic cargo release

Significance The main goal in the emerging field of cancer nanomedicine is to generate, standardize, and produce multifunctional carriers designed to improve the response of drugs against tumors. Here we report the design, development, and preclinical validation of a ligand-directed bioinorganic platform that integrates tumor targeting, receptor-mediated cell internalization, photon-to-heat conversion, and drug delivery. This enabling hydrogel-based technology can accommodate a broad variety of ligands, nanoparticles, and payloads. We show experimental proof-of-concept in mouse models of breast and prostate cancer with molecular imaging and marked reduction of tumor growth. However, with future proof that this technology is translatable, medical applications beyond cancer may also be leveraged. 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.

Self-targeting of TNF-releasing cancer cells in preclinical models of primary and metastatic tumors

Significance A recent paradigm shift has established “tumor cell seeding” as an intriguing biological phenomenon in cancer biology. However, the clinical implications of a bidirectional flow of cancer cells remains largely unexplored. We show that systemic administration of TNF-expressing cancer cells reduces growth of both primary tumors and metastatic colonies defined here as “tumor self-targeting.” Our findings support a provocative concept in which circulating tumor cells genetically manipulated ex vivo and readministered into the circulating bloodstream may indeed serve as tumor-targeted cellular vectors in preclinical settings and could potentially open a field of translational investigation. 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.

AAVP displaying octreotide for ligand-directed therapeutic transgene delivery in neuroendocrine tumors of the pancreas

Significance There are literally thousands of biologically active, clinically relevant peptide motifs in mammalian species. Surprisingly, however, despite this abundance of potential peptide reagents for ligand-directed delivery, applications for targeted gene therapy are generally lacking. Here we used a hybrid AAV/phage (AAVP) vector for octreotide ligand-directed therapeutic gene delivery to pancreatic neuroendocrine tumors in a transgenic mouse model that faithfully recapitulates the cognate human disease. This platform is readily available for a translational clinical trial. In a broader context, this proof-of-concept work establishes a unique targeting paradigm in which existing ligand/receptors may be exploited in nature while minimizing or eliminating several rate-limiting steps of conventional phage display library selection that require cumbersome experimental discovery work. 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.

The peptidomimetic Vasotide targets two retinal VEGF receptors and reduces pathological angiogenesis in murine and nonhuman primate models of retinal disease

A stable small peptide that binds to two retinal VEGF receptors blocks angiogenesis in murine and nonhuman primate models of human blinding retinal diseases. A new peptide for treating retinal disease In a new study, Sidman et al. report the inhibitory effect of a peptide drug called Vasotide on blood vessel overgrowth in the retinas of three animal models of human blinding retinal diseases (two rodent and one nonhuman primate). Delivery of Vasotide in eye drops prevented a blood vessel growth-promoting molecule, VEGF, from binding to two different receptors, VEGF receptor-1 and neuropilin-1, expressed by the retinal endothelial cells that line the inner surface of blood vessels. Blood vessel growth from preexisting vessels (angiogenesis) underlies many severe diseases including major blinding retinal diseases such as retinopathy of prematurity (ROP) and aged macular degeneration (AMD). This observation has driven development of antibody inhibitors that block a central factor in AMD, vascular endothelial growth factor (VEGF), from binding to its receptors VEGFR-1 and mainly VEGFR-2. However, some patients are insensitive to current anti-VEGF drugs or develop resistance, and the required repeated intravitreal injection of these large molecules is costly and clinically problematic. We have evaluated a small cyclic retro-inverted peptidomimetic, D(Cys-Leu-Pro-Arg-Cys) [D(CLPRC)], and hereafter named Vasotide, that inhibits retinal angiogenesis by binding selectively to the VEGF receptors VEGFR-1 and neuropilin-1 (NRP-1). Delivery of Vasotide via either eye drops or intraperitoneal injection in a laser-induced monkey model of human wet AMD, a mouse genetic knockout model of the AMD subtype called retinal angiomatous proliferation (RAP), and a mouse oxygen-induced model of ROP decreased retinal angiogenesis in all three animal models. This prototype drug candidate is a promising new dual receptor inhibitor of the VEGF ligand with potential for translation into safer, less-invasive applications to combat pathological angiogenesis in retinal disorders.

Targeted molecular-genetic imaging and ligand-directed therapy in aggressive variant prostate cancer

Significance Aggressive variant prostate cancer (AVPC) is a clinically defined tumor with neuroendocrine or small-cell differentiation, visceral metastases, low prostate-specific antigen, androgen receptor insensitivity, and poor/brief responses to androgen-deprivation or platinum-based chemotherapy. AVPC incidence has markedly increased, yielding an unmet diagnostic/therapeutic need. Here we adapted a patient-derived xenograft model and tumor samples to demonstrate ligand-directed theranostics of AVPC in vivo. We engineered human Herpes simplex virus thymidine kinase type-1 as a noninvasive imaging reporter/suicide transgene into adeno-associated virus/phage (AAVP) particles displaying motif ligands to cell surface-associated glucose-regulated protein 78kD (GRP78), toward a clinic-ready system. Although individual components of the AAVP system have been extensively investigated, this study is evidence of successful application in relevant preclinical models of untreatable and hard to diagnose aggressive tumor variants. Aggressive variant prostate cancers (AVPC) are a clinically defined group of tumors of heterogeneous morphologies, characterized by poor patient survival and for which limited diagnostic and treatment options are currently available. We show that the cell surface 78-kDa glucose-regulated protein (GRP78), a receptor that binds to phage-display-selected ligands, such as the SNTRVAP motif, is a candidate target in AVPC. We report the presence and accessibility of this receptor in clinical specimens from index patients. We also demonstrate that human AVPC cells displaying GRP78 on their surface could be effectively targeted both in vitro and in vivo by SNTRVAP, which also enabled specific delivery of siRNA species to tumor xenografts in mice. Finally, we evaluated ligand-directed strategies based on SNTRVAP-displaying adeno-associated virus/phage (AAVP) particles in mice bearing MDA-PCa-118b, a patient-derived xenograft (PDX) of castration-resistant prostate cancer bone metastasis that we exploited as a model of AVPC. For theranostic (a merging of the terms therapeutic and diagnostic) studies, GRP78-targeting AAVP particles served to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) gene, which has a dual function as a molecular-genetic sensor/reporter and a cell suicide-inducing transgene. We observed specific and simultaneous PET imaging and treatment of tumors in this preclinical model of AVPC. Our findings demonstrate the feasibility of GPR78-targeting, ligand-directed theranostics for translational applications in AVPC.

Towards a transcriptome-based theranostic platform for unfavorable breast cancer phenotypes

Significance Inflammatory breast cancer (IBC) is defined clinically and pathologically. Dermal lymphatic invasion is typical but is neither necessary nor sufficient for diagnosis; sentinel lymph node biopsy is contraindicated, challenging multidisciplinary management with upfront chemotherapy, surgery, and postoperative radiotherapy. Here we applied a ligand-directed “theranostic” (a combination of therapeutic and diagnostic) enabling platform to target IBC based on adeno-associated virus/phage (AAVP)-Herpes simplex virus thymidine kinase type-1 (HSVtk) particles displaying ligands to cell surface-associated 78-kD glucose-regulated protein (GRP78). In a suite of preclinical models and human tumor samples, we show simultaneous noninvasive molecular serial PET/CT imaging and targeted suicide transgene therapy. This study shows that a tumor-specific promoter, human GRP78 (hGRP78), can drive the expression of an imaging/suicide transgene in IBC and aggressive breast cancer in vivo. Inflammatory breast carcinoma (IBC) is one of the most lethal forms of human breast cancer, and effective treatment for IBC is an unmet clinical need in contemporary oncology. Tumor-targeted theranostic approaches are emerging in precision medicine, but only a few specific biomarkers are available. Here we report up-regulation of the 78-kDa glucose-regulated protein (GRP78) in two independent discovery and validation sets of specimens derived from IBC patients, suggesting translational promise for clinical applications. We show that a GRP78-binding motif displayed on either bacteriophage or adeno-associated virus/phage (AAVP) particles or loop-grafted onto a human antibody fragment specifically targets orthotopic IBC and other aggressive breast cancer models in vivo. To evaluate the theranostic value, we used GRP78-targeting AAVP particles to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) transgene, obtaining simultaneous in vivo diagnosis through PET imaging and tumor treatment by selective activation of the prodrug ganciclovir at tumor sites. Translation of this AAVP system is expected simultaneously to image, monitor, and treat the IBC phenotype and possibly other aggressive (e.g., invasive and/or metastatic) subtypes of breast cancer, based on the inducible cell-surface expression of the stress-response chaperone GRP78, and possibily other cell-surface receptors in human tumors.

Interleukin-11 Receptor Is a Candidate Target for Ligand-Directed Therapy in Lung Cancer: Analysis of Clinical Samples and BMTP-11 Preclinical Activity

We previously isolated an IL-11-mimic motif (CGRRAGGSC) that binds to IL-11 receptor (IL-11R) inxa0vitro and accumulates in IL-11R-expressing tumors inxa0vivo. This synthetic peptide ligand was used as a tumor-targeting moiety in the rational design of BMTP-11, which is a drug candidate in clinical trials. Here, we investigated the specificity and accessibility of IL-11R as a target and the efficacy of BMTP-11 as a ligand-targeted drug in lung cancer. We observed high IL-11R expression levels in a large cohort of patients (nxa0=xa0368). In matching surgical specimens (i.e., paired tumors and nonmalignant tissues), the cytoplasmic levels of IL-11R in tumor areas were significantly higher than in nonmalignant tissues (nxa0=xa036; Pxa0=xa00.003). Notably, marked overexpression of IL-11R was observed in both tumor epithelial and vascular endothelial cell membranes (nxa0=xa0301; Pxa0<xa00.0001). BMTP-11 induced inxa0vitro cell death in a representative panel of human lung cancer cell lines. BMTP-11 treatment attenuated the growth of subcutaneous xenografts and reduced the number of pulmonary tumors after tail vein injection of human lung cancer cells in mice. Our findings validate BMTP-11 as a pharmacologic candidate drug in preclinical models of lung cancer and patient-derived tumors. Moreover, the high expression level in patients with non-small cell lungxa0cancer is a promising feature for potential translational applications.