Enhanced Efficacy of Combination of Gemcitabine and Phosphatidylserine-Targeted Nanovesicles against Pancreatic Cancer.

Phosphatidylserine (PS) is often externalized in viable pancreatic cancer cells and is therapeutically targetable using PS-selective drugs. One of the first-line treatments for advanced pancreatic cancer disease, gemcitabine (GEM), provides only marginal benefit to patients. We therefore investigated the therapeutic benefits of combining GEM and the PS-targeting drug, saposin C-dioleoylphosphatidylserine (SapC-DOPS), for treating pancreatic ductal adenocarcinoma (PDAC). Using cell-cycle analyses and a cell surface PS-based sorting method in vitro, we observed an increase in surface PS as cells progress through the cell cycle from G1 to G2/M. We also observed that GEM treatment preferentially targets G1 phase cells that have low surface PS, resulting in an increased median surface PS level of PDAC cells. Inversely, SapC-DOPS preferentially targets high surface PS cells that are predominantly in the G2/M phase. Finally, combination therapy in subcutaneous and orthotopic PDAC tumors in vivo with SapC-DOPS and GEM or Abraxane (Abr)/GEM (one of the current standards of care) significantly inhibits tumor growth and increases survival compared with individual treatments. Our studies confirm a surface PS and cell cycle-based enhancement of cancer cytotoxicity following SapC-DOPS treatment in combination with GEM or Abr/GEM. Thus, PDAC patients treated with Abr/GEM may benefit from concurrent administration of SapC-DOPS.

SapC-DOPS nanovesicles induce Smac- and Bax-dependent apoptosis through mitochondrial activation in neuroblastomas.

BACKGROUND: High toxicity, morbidity and secondary malignancy render chemotherapy of neuroblastoma inefficient, prompting the search for novel compounds. Nanovesicles offer great promise in imaging and treatment of cancer. SapC-DOPS, a stable nanovesicle formed from the lysosomal protein saposin C and dioleoylphosphatidylserine possess strong affinity for abundantly exposed surface phosphatidylserine on cancer cells. Here, we show that SapC-DOPS effectively targets and suppresses neuroblastoma growth and elucidate the molecular mechanism of SapC-DOPS action in neuroblastoma in vitro. METHODS: In vivo targeting of neuroblastoma was assessed in xenograft mice injected intravenously with fluorescently-labeled SapC-DOPS. Xenografted tumors were also used to demonstrate its therapeutic efficacy. Apoptosis induction in vivo was evaluated in tumor sections using the TUNEL assay. The mechanisms underlying the induction of apoptosis by SapC-DOPS were addressed through measurements of cell viability, mitochondrial membrane potential (ΔΨM), flow cytometric DNA fragmentation assays and by immunoblot analysis of second mitochondria-derived activator of caspases (Smac), Bax, Cytochrome c (Cyto c) and Caspase-3 in the cytosol or in mitochondrial fractions of cultured neuroblastoma cells. RESULTS: SapC-DOPS showed specific targeting and prevented the growth of human neuroblastoma xenografts in mice. In neuroblastoma cells in vitro, apoptosis occurred via a series of steps that included: (1) loss of ΔΨM and increased mitochondrial superoxide formation; (2) cytosolic release of Smac, Cyto c, AIF; and (3) mitochondrial translocation and polymerization of Bax. ShRNA-mediated Smac knockdown and V5 peptide-mediated Bax inhibition decreased cytosolic Smac and Cyto c release along with caspase activation and abrogated apoptosis, indicating that Smac and Bax are critical mediators of SapC-DOPS action. Similarly, pretreatment with the mitochondria-stabilizing agent bongkrekic acid decreased apoptosis indicating that loss of ΔΨM is critical for SapC-DOPS activity. Apoptosis induction was not critically dependent on reactive oxygen species (ROS) production and Cyclophilin D, since pretreatment with N-acetyl cysteine and cyclosporine A, respectively, did not prevent Smac or Cyto c release. CONCLUSIONS: Taken together, our results indicate that SapC-DOPS acts through a mitochondria-mediated pathway accompanied by an early release of Smac and Bax. Specific tumor-targeting capacity and anticancer efficacy of SapC-DOPS supports its potential as a dual imaging and therapeutic agent in neuroblastoma therapy.

Targeting of elevated cell surface phosphatidylserine with saposin C-dioleoylphosphatidylserine nanodrug as individual or combination therapy for pancreatic cancer.

Pancreatic cancer is one of the deadliest of cancers with a five-year survival of roughly 8%. Current therapies are: surgery, radiation and chemotherapy. Surgery is curative only if the cancer is caught very early, which is rare, and the latter two modalities are only marginally effective and have significant side effects. We have developed a nanosome comprised of the lysosomal protein, saposin C (SapC) and the acidic phospholipid, dioleoylphosphatidylserine (DOPS). In the acidic tumor microenvironment, this molecule, SapC-DOPS, targets the phosphatidylserine cancer-biomarker which is predominantly elevated on the surface of cancer cells. Importantly, SapC-DOPS can selectively target pancreatic tumors and metastases. Furthermore, SapC-DOPS has exhibited an impressive safety profile with only a few minor side effects in both preclinical experiments and in phase I clinical trials. With the dismal outcomes for pancreatic cancer there is an urgent need for better treatments and SapC-DOPS is a good candidate for addition to the oncologist's toolbox.

SapC-DOPS as a Novel Therapeutic and Diagnostic Agent for Glioblastoma Therapy and Detection: Alternative to Old Drugs and Agents.

Glioblastoma multiforme (GBM), the most common type of brain cancer, is extremely aggressive and has a dreadful prognosis. GBM comprises 60% of adult brain tumors and the 5 year survival rate of GBM patients is only 4.3%. Standard-of-care treatment includes maximal surgical removal of the tumor in combination with radiation and temozolomide (TMZ) chemotherapy. TMZ is the "gold-standard" chemotherapy for patients suffering from GBM. However, the median survival is only about 12 to 18 months with this protocol. Consequently, there is a critical need to develop new therapeutic options for treatment of GBM. Nanomaterials have unique properties as multifunctional platforms for brain tumor therapy and diagnosis. As one of the nanomaterials, lipid-based nanocarriers are capable of delivering chemotherapeutics and imaging agents to tumor sites by enhancing the permeability of the compound through the blood-brain barrier, which makes them ideal for GBM therapy and imaging. Nanocarriers also can be used for delivery of radiosensitizers to the tumor to enhance the efficacy of the radiation therapy. Previously, high-atomic-number element-containing particles such as gold nanoparticles and liposomes have been used as radiosensitizers. SapC-DOPS, a protein-based liposomal drug comprising the lipid, dioleoylphosphatidylserine (DOPS), and the protein, saposin C (SapC), has been shown to be effective for treatment of a variety of cancers in small animals, including GBM. SapC-DOPS also has the unique ability to be used as a carrier for delivery of radiotheranostic agents for nuclear imaging and radiotherapeutic purposes. These unique properties make tumor-targeting proteo-liposome nanocarriers novel therapeutic and diagnostic alternatives to traditional chemotherapeutics and imaging agents. This article reviews various treatment modalities including nanolipid-based delivery and therapeutic systems used in preclinical and clinical trial settings for GBM treatment and detection.

Lack of NFATc1 SUMOylation prevents autoimmunity and alloreactivity.

Posttranslational modification with SUMO is known to regulate the activity of transcription factors, but how SUMOylation of individual proteins might influence immunity is largely unexplored. The NFAT transcription factors play an essential role in antigen receptor-mediated gene regulation. SUMOylation of NFATc1 represses IL-2 in vitro, but its role in T cell-mediated immune responses in vivo is unclear. To this end, we generated a novel transgenic mouse in which SUMO modification of NFATc1 is prevented. Avoidance of NFATc1 SUMOylation ameliorated experimental autoimmune encephalomyelitis as well as graft-versus-host disease. Elevated IL-2 production in T cells promoted T reg expansion and suppressed autoreactive or alloreactive immune responses. Mechanistically, increased IL-2 secretion counteracted IL-17 and IFN-γ expression through STAT5 and Blimp-1 induction. Then, Blimp-1 repressed IL-2 itself, as well as the induced, proliferation-associated survival factor Bcl2A1. Collectively, these data demonstrate that prevention of NFATc1 SUMOylation fine-tunes T cell responses toward lasting tolerance. Thus, targeting NFATc1 SUMOylation presents a novel and promising strategy to treat T cell-mediated inflammatory diseases.

Biotherapy of Brain Tumors with Phosphatidylserine-Targeted Radioiodinated SapC-DOPS Nanovesicles.

Glioblastoma multiforme (GBM), a common type of brain cancer, has a very poor prognosis. In general, viable GBM cells exhibit elevated phosphatidylserine (PS) on their membrane surface compared to healthy cells. We have developed a drug, saposin C-dioleoylphosphatidylserine (SapC-DOPS), that selectively targets cancer cells by honing in on this surface PS. To examine whether SapC-DOPS, a stable, blood-brain barrier-penetrable nanovesicle, could be an effective delivery system for precise targeted therapy of radiation, we iodinated several carbocyanine-based fluorescent reporters with either stable iodine (127I) or radioactive isotopes (125I and 131I). While all of the compounds, when incorporated into the SapC-DOPS delivery system, were taken up by human GBM cell lines, we chose the two that best accumulated in the cells (DiI (22,3) and DiD (16,16)). Pharmacokinetics were conducted with 125I-labeled compounds and indicated that DiI (22,3)-SapC-DOPS had a time to peak in the blood of 0.66 h and an elimination half-life of 8.4 h. These values were 4 h and 11.5 h, respectively, for DiD (16,16)-SapC-DOPS. Adult nude mice with GBM cells implanted in their brains were treated with 131I-DID (16,16)-SapC-DOPS. Mice receiving the radionuclide survived nearly 50% longer than the control groups. These data suggest a potential novel, personalized treatment for a devastating brain disease.

Enhanced phosphatidylserine-selective cancer therapy with irradiation and SapC-DOPS nanovesicles.

Normal living cells exhibit phosphatidylserine (PS) primarily within the intracellular leaflet of the plasma membrane. In contrast, viable cancer cells have high levels of PS on the external surface, and exhibit a broad range of surface PS, even within specific types of cancer. Agents that target surface PS have recently been developed to treat tumors and are expected to be more effective with higher surface PS levels. In this context, we examined whether surface PS is increased with irradiation. In vitro irradiation of cancer cell lines selected surviving cells that had higher surface PS in a dose- and time-dependent manner. This was more pronounced if surface PS was initially in the lower range for cancer cells. Radiation also increased the surface PS of tumor cells in subcutaneous xenografts in nude mice. We found an inverse relationship between steady state surface PS level of cancer cell lines and their sensitivity to radiation-induced cell death. In addition, serial irradiation, which selected surviving cells with higher surface PS, also increased resistance to radiation and to some chemotherapeutic drugs, suggesting a PS-dependent mechanism for development of resistance to therapy. On the other hand, fractionated radiation enhanced the effect of a novel anti-cancer, PS-targeting drug, SapC-DOPS, in some cancer cell lines. Our data suggest that we can group cancer cells into cells with low surface PS, which are sensitive to radiation, and high surface PS, which are sensitive to SapC-DOPS. Combination of these interventions may provide a potential new combination therapy.

Variation in human cancer cell external phosphatidylserine is regulated by flippase activity and intracellular calcium.

Viable cancer cells expose elevated levels of phosphatidylserine (PS) on the exoplasmic face of the plasma membrane. However, the mechanisms leading to elevated PS exposure in viable cancer cells have not been defined. We previously showed that externalized PS may be used to monitor, target and kill tumor cells. In addition, PS on tumor cells is recognized by macrophages and has implications in antitumor immunity. Therefore, it is important to understand the molecular details of PS exposure on cancer cells in order to improve therapeutic targeting. Here we explored the mechanisms regulating the surface PS exposure in human cancer cells and found that differential flippase activity and intracellular calcium are the major regulators of surface PS exposure in viable human cancer cells. In general, cancer cell lines with high surface PS exhibited low flippase activity and high intracellular calcium, whereas cancer cells with low surface PS exhibited high flippase activity and low intracellular calcium. High surface PS cancer cells also had higher total cellular PS than low surface PS cells. Together, our results indicate that the amount of external PS in cancer cells is regulated by calcium dependent flippase activity and may also be influenced by total cellular PS.

Transcriptional repression by the HDAC4-RelB-p52 complex regulates multiple myeloma survival and growth.

Although transcriptional activation by NF-κB is well appreciated, physiological importance of transcriptional repression by NF-κB in cancer has remained elusive. Here we show that an HDAC4-RelB-p52 complex maintains repressive chromatin around proapoptotic genes Bim and BMF and regulates multiple myeloma (MM) survival and growth. Disruption of RelB-HDAC4 complex by a HDAC4-mimetic polypeptide blocks MM growth. RelB-p52 also represses BMF translation by regulating miR-221 expression. While the NIK-dependent activation of RelB-p52 in MM has been reported, we show that regardless of the activation status of NIK and the oncogenic events that cause plasma cell malignancy, several genetically diverse MM cells including Bortezomib-resistant MM cells are addicted to RelB-p52 for survival. Importantly, RelB is constitutively phosphorylated in MM and ERK1 is a RelB kinase. Phospho-RelB remains largely nuclear and is essential for Bim repression. Thus, ERK1-dependent regulation of nuclear RelB is critical for MM survival and explains the NIK-independent role of RelB in MM.

Phosphatidylserine-selective targeting and anticancer effects of SapC-DOPS nanovesicles on brain tumors.

Brain tumors, either primary (e.g., glioblastoma multiforme) or secondary (metastatic), remain among the most intractable and fatal of all cancers. We have shown that nanovesicles consisting of Saposin C (SapC) and dioleylphosphatidylserine (DOPS) are able to effectively target and kill cancer cells both in vitro and in vivo. These actions are a consequence of the affinity of SapC-DOPS for phosphatidylserine, an acidic phospholipid abundantly present in the outer membrane of a variety of tumor cells and tumor-associated vasculature. In this study, we first characterize SapC-DOPS bioavailability and antitumor effects on human glioblastoma xenografts, and confirm SapC-DOPS specificity towards phosphatidylserine by showing that glioblastoma targeting is abrogated after in vivo exposure to lactadherin, which binds phosphatidylserine with high affinity. Second, we demonstrate that SapC-DOPS selectively targets brain metastases-forming cancer cells both in vitro, in co-cultures with human astrocytes, and in vivo, in mouse models of brain metastases derived from human breast or lung cancer cells. Third, we demonstrate that SapC-DOPS have cytotoxic activity against metastatic breast cancer cells in vitro, and prolong the survival of mice harboring brain metastases. Taken together, these results support the potential of SapC-DOPS for the diagnosis and therapy of primary and metastatic brain tumors.

Transcriptional repression of Bim by a novel YY1-RelA complex is essential for the survival and growth of Multiple Myeloma.

Multiple Myeloma (MM) is an incurable plasma cell cancer that is caused by several chromosomal translocations and gene deletions. Although deregulation of several signaling pathways including the Nuclear Factor-Kappa B (NF-κB) pathway has been reported in MM, the molecular requirement and the crosstalk between NF-κB and its target genes in MM cell survival has been largely unclear. Here, we report that Yin Yang1 (YY1), a target gene for NF-κB, is hyperexpressed in most MM tumor cells obtained from human patients, exhibits constitutive nuclear localization, and is essential for survival of MM cells. Mechanistically, we report a novel YY1-RelA complex formation, which is essential to transcriptionally repress a proapoptotic gene Bim. In line with this, depletion of YY1 or RelA resulted in elevated levels of Bim and apoptosis. Moreover, both YY1 and RelA are recruited to the Bim promoter and are required to repress the Bim promoter. Importantly, depletion of YY1 or RelA almost completely impaired the colony forming ability of MM progenitor cells suggesting that both RelA and YY1 are essential for the survival and growth of MM progenitor cells. Moreover, depletion of either YY1 or RelA completely inhibited MM tumor growth in xenograft models for human myeloma. Thus, a novel RelA-YY1 transcriptional repression complex is an attractive drug target in MM.