Search

Antibody drug conjugates (ADCs), consisting of a cancer-specific antibody and cytotoxic payload, are shown to be a potent class of anticancer therapeutics, with enhanced therapeutic efficacy and reduced &ldquo, off-target&rdquo, side effects. However, the therapeutic window of ADCs is narrowed by problems such as difficulty in site-specific conjugation of payload, changes in antibody stability due to payload conjugation, and difficulty in tissue penetration. In this respect, aptamers have advantages in drug-delivery, as they can be easily and stably conjugated with cytotoxic drugs. We previously reported that oligobody, an aptamer-antibody complex, is a novel delivery method for aptamer-based therapeutics. In the current study, we describe DOligobody, a drug-conjugated oligobody comprising an aptamer-drug conjugate and an antibody. A cotinine-conjugated anti-HER2 aptamer (cot-HER2apt) was specifically bound to HER2-positive NCI-N87 cells, and underwent receptor-mediated endocytosis. Further, HER2-DOligobody, a cot-HER2apt-conjugated monomethyl auristatin E (cot-HER2apt-MMAE) oligobody, inhibited the growth of HER2-positive NCI-N87 cells. Finally, systemic administration of HER2-DOligobody significantly reduced tumor growth in a xenograft mouse model. Taken together, these results suggest that our DOligobody strategy may be a powerful platform for rapid, low-cost and effective cancer therapy.

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide. Cetuximab, a human/mouse chimeric monoclonal antibody, is effective in a limited number of CRC patients because of cetuximab resistance. This study aimed to identify novel therapeutic targets in cetuximab-resistant CRC in order to improve clinical outcomes. Through phage display technology, we isolated a fully human antibody strongly binding to the cetuximab-resistant HCT116 cell surface and identified the target antigen as glucose-regulated protein 94 (GRP94) using proteomic analysis. Short interfering RNA-mediated GRP94 knockdown showed that GRP94 plays a key role in HCT116 cell growth. In vitro functional studies revealed that the GRP94-blocking antibody we developed strongly inhibits the growth of various cetuximab-resistant CRC cell lines. We also demonstrated that GRP94 immunoglobulin G monotherapy significantly reduces HCT116 cell growth more potently compared to cetuximab, without severe toxicity in vivo. Therefore, cell surface GRP94 might be a potential novel therapeutic target in cetuximab-resistant CRC, and antibody-based targeting of GRP94 might be an effective strategy to suppress GRP94-expressing cetuximab-resistant CRC.

The tubby protein (Tub), a putative transcription factor, plays important roles in the maintenance and function of neuronal cells. A splicing defect-causing mutation in the 3'-end of the tubby gene, which is predicted to disrupt the carboxy-terminal region of the Tub protein, causes maturity-onset obesity, blindness, and deafness in mice. Although this pathological Tub mutation leads to a loss of function, the precise mechanism has not yet been investigated. Here, we found that the mutant Tub proteins were mostly localized to puncta found in the perinuclear region and that the C-terminus was important for its solubility. Immunocytochemical analysis revealed that puncta of mutant Tub co-localized with the aggresome. Moreover, whereas wild-type Tub was translocated to the nucleus by extracellular signaling, the mutant forms failed to undergo such translocation. Taken together, our results suggest that the malfunctions of the Tub mutant are caused by its misfolding and subsequent localization to aggresomes. [BMB Reports 2017; 50(1): 37-42].

Tetraspanin 8 (TSPAN8) is a member of the tetraspanin superfamily that forms TSPAN8-mediated protein complexes by interacting with themselves and other various cellular signaling molecules. These protein complexes help build tetraspanin-enriched microdomains (TEMs) that efficiently mediate intracellular signal transduction. In physiological conditions, TSPAN8 plays a vital role in the regulation of biological functions, including leukocyte trafficking, angiogenesis and wound repair. Recently, reports have increasingly shown the functional role and clinical relevance of TSPAN8 overexpression in the progression and metastasis of several cancers. In this review, we will highlight the physiological and pathophysiological roles of TSPAN8 in normal and cancer cells. Additionally, we will cover the current status of monoclonal antibodies specifically targeting TSPAN8 and the importance of TSPAN8 as an emerging therapeutic target in cancers for monoclonal antibody therapy.

The Korean traditional medicine, HangAmDan (HAD), was developed in 1996 for use as an antitumor agent, and has since been modified to HAD‑B (an altered form of HAD), in order to potentiate its therapeutic effects. In the present study, the effect of HAD‑B on the proliferation and invasion of NIH:OVCAR‑3 and SKOV‑3 human ovarian cancer cell lines was investigated. In addition, the expression of major signal transduction molecules and changes in the proteome in these cells were measured. HAD‑B treatment effectively induced a reduction in the levels of cell proliferation in serum‑free conditioned media. However, unaltered levels of PARP and caspase‑3 indicated that HAD‑B does not reduce proliferation by inducing apoptotic cell death. Fluorescence‑activated cell sorting analysis revealed no significant change in apoptosis following HAD-B treatment. Invasion assay results indicated a reduced rate of invasion following HAD‑B treatment. HAD‑B also influenced the expression of major signal transduction molecules; the phosphorylation of mTOR and AKT was reduced, while that of ERK was increased. Alterations in the proteomes of the two cell lines were investigated following HAD‑B treatment. Among the 9 proteins with differential expression, heat‑shock protein β‑1 (HSP27) was downregulated in NIH:OVCAR‑3 cells treated with HAD‑B. The reduced expression of HSP27 was associated with human epidermal growth factor receptor 2 (Her2) downregulation in these cells. In conclusion, the results of the current proteome assessment suggest that HAD‑B has the potential to suppress the proliferation and invasion of human ovarian cancer cells. HAD‑B treatment of NIH:OVCAR‑3 cells suppressed HSP27 expression and was also associated with Her2 downregulation.

Aptamers, composed of single-stranded DNA or RNA oligonucleotides that interact with target molecules through a specific three-dimensional structure, are selected from pools of combinatorial oligonucleotide libraries. With their high specificity and affinity for target proteins, ease of synthesis and modification, and low immunogenicity and toxicity, aptamers are considered to be attractive molecules for development as anticancer therapeutics. Two aptamers - one targeting nucleolin and a second targeting CXCL12 - are currently undergoing clinical trials for treating cancer patients, and many more are under study. In this mini-review, we present the current clinical status of aptamers and aptamer-based cancer therapeutics. We also discuss advantages, limitations, and prospects for aptamers as cancer therapeutics. [BMB Reports 2015; 48(4): 234-237]

The soluble protein pancreatic adenocarcinoma up-regulated factor (PAUF) plays an important role in pancreatic tumor progression and has begun to attract attention as a therapeutic target for pancreatic cancer. We herein present PAUF RNA-targeting gene therapy strategies with both targeting and therapeutic function using trans-splicing ribozyme (TSR) in pancreatic cancer. We developed adenoviral PAUF-targeting TSR (Rz) containing a PAUF-specific internal guide sequence (IGS) determined by library screening. This Rz harbors suicide gene, herpes simplex virus thymidine kinase (HSV-tk) or firefly luciferase (Luc) as a transgene for 3' exon replacement of PAUF RNAs. Ad-Rz-TK, Rz harboring the HSV-tk, showed significant inhibition of tumor growth in vivo as well as PAUF-dependent cell death in vitro via a successful trans-splicing reaction. Selective induction of Rz-controlled transgene in PAUF-expressing pancreatic cancer was confirmed through noninvasive in vivo imaging; a luminescence signal from Rz harboring Luc (Ad-Rz-Luc) was detectable only in pancreatic tumor sites, not in normal mice. In addition, a [(125)I] FIAU signal reflecting thymidine kinase expression through SPECT and ex vivo biodistribution was co-localized with the tumor sites when we treated with Ad-Rz-TK in orthotopic xenograft model. Taken together, these results imply that PAUF-targeting TSR can contribute to successful targeted gene therapy for pancreatic cancer.

NHERF1/EBP50 (Na+/H+ exchanger regulating factor 1; Ezrin-binding phosphoprotein of 50 kDa) organizes stable protein complexes beneath the apical membrane of polar epithelial cells. By contrast, in cancer cells without any fixed polarity, NHERF1 often localizes in the cytoplasm. The regulation of cytoplasmic NHERF1 and its role in cancer progression remain unclear. In this study, we found that, upon lysophosphatidic acid (LPA) stimulation, cytoplasmic NHERF1 rapidly translocated to the plasma membrane, and subsequently to cortical protrusion structures, of ovarian cancer cells. This movement depended on direct binding of NHERF1 to C-terminally phosphorylated ERM proteins (cpERMs). Moreover, NHERF1 depletion downregulated cpERMs and further impaired cpERM-dependent remodeling of the cell cortex, suggesting reciprocal regulation between these proteins. The LPA-induced protein complex was highly enriched in migratory pseudopodia, whose formation was impaired by overexpression of NHERF1 truncation mutants. Consistent with this, NHERF1 depletion in various types of cancer cells abolished chemotactic cell migration toward a LPA gradient. Taken together, our findings suggest that the high dynamics of cytosolic NHERF1 provide cancer cells with a means of controlling chemotactic migration. This capacity is likely to be essential for ovarian cancer progression in tumor microenvironments containing LPA.

Angiogenesis is essential for tumor growth and vascular endothelial cell growth factor (VEGF) plays a key role in this process. Conversely, sphingosine 1-phosphate (S1P) is a biologically active sphingolipid known to play a key role in cancer progression by regulating endothelial cell proliferation and migration. In this study, the authors found that S1P increases the level of VEGF mRNA in human umbilical vein endothelial cells (HUVECs) and immortalized HUVECs (iHUVECs). Additionally, S1P was found to increase VEGF promoter activity in MS-1 mouse pancreatic islet endothelial cells. Furthermore, a pharmacological inhibitory study revealed that G(alpha i/o)-mediated phospholipase C, Akt, Erk, and p38 MAPK signaling are involved in this S1P-induced expression of VEGF. A component of AP1 transcription factor is important for S1P-induced VEGF expression. Taken together, these findings suggest that S1P enhances endothelial cell proliferation and migration by upregulating the expression of VEGF mRNA.

O-Linked β-N-acetylglucosamine (O-GlcNAc) modification, a reversible post-translational modification, has been implicated in the regulation of protein stability, subcellular localization of proteins and protein–protein interaction. Here, we demonstrate that O-GlcNAc modification regulates the expression of osteocalcin, an osteoblast-specific marker, via Runx2 transcriptional activity in osteoblastic differentiation. Protein-associated O-GlcNAc was increased during osteoblastic differentiation in MC3T3-E1 preosteoblasts. In addition, PUGNAc, an inhibitor of O-GlcNAcase, potentiated the expression of osteocalcin caused by ascorbic acid, parathyroid hormone (PTH) and forskolin. By conducting activity assays of the osteocalcin promoter and transcription factor, we found that the OSE2 site in the osteocalcin promoter and Runx2 were important for increased osteocalcin promoter activity by PUGNAc. Furthermore, PUGNAc led to increased O-GlcNAc modification of Runx2, which regulated the transcription of its target gene osteocalcin. Thus, these data provide evidence that O-GlcNAc modification may be a new mode of osteoblastic differentiation regulation.

Lysophosphatidic acid (LPA) activates a family of cognate G protein-coupled receptors and is involved in various pathophysiological processes. However, it is not clearly understood how these LPA receptors are specifically coupled to their downstream signaling molecules. This study found that LPA(2), but not the other LPA receptor isoforms, specifically interacts with Na(+)/H(+) exchanger regulatory factor2 (NHERF2). In addition, the interaction between them requires the C-terminal PDZ domain-binding motif of LPA(2) and the second PDZ domain of NHERF2. Moreover, the stable expression of NHERF2 potentiated LPA-induced phospholipase C-beta (PLC-beta) activation, which was markedly attenuated by either a mutation in the PDZ-binding motif of LPA(2) or by the gene silencing of NHERF2. Using its second PDZ domain, NHERF2 was found to indirectly link LPA(2) to PLC-beta3 to form a complex, and the other PLC-beta isozymes were not included in the protein complex. Consistently, LPA(2)-mediated PLC-beta activation was specifically inhibited by the gene silencing of PLC-beta3. In addition, NHERF2 increases LPA-induced ERK activation, which is followed by cyclooxygenase-2 induction via a PLC-dependent pathway. Overall, the results suggest that a ternary complex composed of LPA(2), NHERF2, and PLC-beta3 may play a key role in the LPA(2)-mediated PLC-beta signaling pathway.

Among the phospholipase C that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate, four mammalian phospholipase C-beta (PLC-beta) isotypes (isotypes 1-4) are activated through G protein-coupled receptors (GPCRs). Although the regulation of the PLC-betas by GPCRs and heterotrimeric G proteins has been extensively studied, little is known about the molecular determinants that regulate their activity. The PLC-beta isozymes carry a putative PSD-95/Dlg/ZO-1 (PDZ) binding motif (X(S/T)X(V/L)COOH) at their carboxyl terminus, which is implicated in specific interactions with anchor proteins. Using the yeast two-hybrid system, we identified Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) as a protein that interacted with a C-terminal heptapeptide of PLC-beta3. Immunoprecipitation studies revealed that NHERF2 interacts specifically with PLC-beta3, but not with other PLC-beta isotypes. Furthermore, PLC-beta3 interacted with NHERF2 rather than with other PDZ-containing proteins. This interaction required the COOH-terminal NTQL sequence of PLC-beta3 and the second PDZ domain of NHERF2. Interestingly, NHERF2 potentiated the PLC-beta activation by carbachol in COS7 and HeLa cells, while mutant NHERF2, lacking the second PDZ domain, had no such effect. Taken together, the data suggest that NHERF2 may act as a modulator underlying the process of PLC-beta3-mediated signaling.

Previously, we reported that a novel secretory protein, pancreatic adenocarcinoma up-regulated factor (PAUF), which is highly expressed in pancreatic cancer and mediates the growth and metastasis of pancreatic cancer cells. In this study, we generated and characterized a 2'-fluoropyrimidine modified RNA aptamer (P12FR2) directed against human PAUF. P12FR2 binds specifically to human PAUF with an estimated apparent K(D) of 77nM. P12FR2 aptamer inhibits PAUF-induced migration of PANC-1, human pancreatic cancer cells, in a wound healing assay. Moreover, intraperitoneal injection of P12FR2 decreased tumor growth by about 60% in an in vivo xenograft model with CFPAC-1 pancreatic cancer cells, without causing a loss of weight in the treated mice. Taken together, we propose here that PAUF-specific RNA aptamer, P12FR2, has the potential to be effective in the therapy of human pancreatic cancer.

Although the functional significance of neuronal phospholipase D (PLD) is being recognized, little is known about its regulatory role in neuronal cells. To elucidate the regulatory mechanism of neuronal PLD, we investigated PLD(2)-binding neuronal protein from rat brain cytosol. During the fractionation of rat brain cytosol by four-column chromatography, a 62-kDa PLD(2)-interacting protein was detected by PLD(2) overlay assay and identified as collapsin response mediator protein-2 (CRMP-2), which controls neuronal axon guidance and outgrowth. Using bacterially expressed glutathione S-transferase fusion proteins, we found that two regions (amino acids 65-192 (the phagocytic oxidase domain) and 724-825) of PLD(2) and a single region (amino acids 243-300) of CRMP-2 are required for the direct binding of both proteins. A co-immunoprecipitation study in COS-7 cells also showed an in vivo interaction between CRMP-2 and PLD(2). Interestingly, CRMP-2 was found to potently inhibit PLD(2) activity in a concentration-dependent manner (IC(50) = 30 nm). Overexpression studies also showed that CRMP-2 is an in vivo inhibitor of PLD(2) in PC12 cells. Moreover, increasing the concentration of semaphorin 3A, one of the repulsive axon guidance cues, showed that PLD(2) activity can be inhibited in PC12 cells. Immunocytochemistry further revealed that PLD(2) is co-localized with CRMP-2 in the distal tips of neurites, its possible action site, in differentiated PC12 cells. Taken together, our results indicate that CRMP-2 may interact directly with and inhibit neuronal PLD(2), suggesting that this inhibitory mode of regulation may play a role in neuronal pathfinding during the developmental stage.