Targeting Podoplanin for the Treatment of Osteosarcoma.

PURPOSE: Osteosarcoma, the most common bone malignancy in children, has a poor prognosis, especially when the tumor metastasizes to the lungs. Therefore, novel therapeutic strategies targeting both proliferation and metastasis of osteosarcoma are required. Podoplanin (PDPN) is expressed by various tumors and is associated with tumor-induced platelet activation via its interaction with C-type lectin-like receptor 2 (CLEC-2) on platelets. We previously found that PDPN contributed to osteosarcoma growth and metastasis through platelet activation; thus, in this study, we developed an anti-PDPN humanized antibody and evaluated its effect on osteosarcoma growth and metastasis. EXPERIMENTAL DESIGN: Nine osteosarcoma cell lines and two osteosarcoma patient-derived cells were collected, and we evaluated the efficacy of the anti-DPN-neutralizing antibody PG4D2 and the humanized anti-PDPN antibody AP201, which had IgG4 framework region. The antitumor and antimetastasis effect of PG4D2 and AP201 were examined in vitro and in vivo. In addition, growth signaling by the interaction between PDPN and CLEC-2 was analyzed using phospho-RTK (receptor tyrosine kinase) array, growth assay, or immunoblot analysis under the supression of RTKs by knockout and inhibitor treatment. RESULTS: We observed that PG4D2 treatment significantly suppressed tumor growth and pulmonary metastasis in osteosarcoma xenograft models highly expressing PDPN. The contribution of PDGFR activation by activated platelet releasates to osteosarcoma cell proliferation was confirmed, and the humanized antibody, AP201, suppressed in vivo osteosarcoma growth and metastasis without significant adverse events. CONCLUSIONS: Targeting PDPN with a neutralizing antibody against PDPN-CLEC-2 without antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity is a novel therapeutic strategy for PDPN-positive osteosarcoma.

Mitochondrial complex I inhibitors suppress tumor growth through concomitant acidification of the intra- and extracellular environment.

The disruption of the tumor microenvironment (TME) is a promising anti-cancer strategy, but its effective targeting for solid tumors remains unknown. Here, we investigated the anti-cancer activity of the mitochondrial complex I inhibitor intervenolin (ITV), which modulates the TME independent of energy depletion. By modulating lactate metabolism, ITV induced the concomitant acidification of the intra- and extracellular environment, which synergistically suppressed S6K1 activity in cancer cells through protein phosphatase-2A-mediated dephosphorylation via G-protein-coupled receptor(s). Other complex I inhibitors including metformin and rotenone were also found to exert the same effect through an energy depletion-independent manner as ITV. In mouse and patient-derived xenograft models, ITV was found to suppress tumor growth and its mode of action was further confirmed. The TME is usually acidic owing to glycolytic cancer cell metabolism, and this condition is more susceptible to complex I inhibitors. Thus, we have demonstrated a potential treatment strategy for solid tumors.

Platelet-derived lysophosphatidic acid mediated LPAR1 activation as a therapeutic target for osteosarcoma metastasis.

Osteosarcoma is the most common primary malignant bone cancer, with high rates of pulmonary metastasis. Osteosarcoma patients with pulmonary metastasis have worse prognosis than those with localized disease, leading to dramatically reduced survival rates. Therefore, understanding the biological characteristics of metastatic osteosarcoma and the molecular mechanisms of invasion and metastasis of osteosarcoma cells will lead to the development of innovative therapeutic intervention for advanced osteosarcoma. Here, we identified that osteosarcoma cells commonly exhibit high platelet activation-inducing characteristics, and molecules released from activated platelets promote the invasiveness of osteosarcoma cells. Given that heat-denatured platelet releasate maintained the ability to promote osteosarcoma invasion, we focused on heat-tolerant molecules, such as lipid mediators in the platelet releasate. Osteosarcoma-induced platelet activation leads to abundant lysophosphatidic acid (LPA) release. Exposure to LPA or platelet releasate induced morphological changes and increased invasiveness of osteosarcoma cells. By analyzing publicly available transcriptome datasets and our in-house osteosarcoma patient-derived xenograft tumors, we found that LPA receptor 1 (LPAR1) is notably upregulated in osteosarcoma. LPAR1 gene KO in osteosarcoma cells abolished the platelet-mediated osteosarcoma invasion in vitro and the formation of early pulmonary metastatic foci in experimental pulmonary metastasis models. Of note, the pharmacological inhibition of LPAR1 by the orally available LPAR1 antagonist, ONO-7300243, prevented pulmonary metastasis of osteosarcoma in the mouse models. These results indicate that the LPA-LPAR1 axis is essential for the osteosarcoma invasion and metastasis, and targeting LPAR1 would be a promising therapeutic intervention for advanced osteosarcoma.

Cancer-Specific Targeting of Taurine-Upregulated Gene 1 Enhances the Effects of Chemotherapy in Pancreatic Cancer.

Overcoming drug resistance is one of the biggest challenges in cancer chemotherapy. In this study, we examine whether targeting the long noncoding RNA taurine upregulated gene 1 (TUG1) could be an effective therapeutic approach to overcome drug resistance in pancreatic ductal adenocarcinoma (PDAC). TUG1 was expressed at significantly higher levels across 197 PDAC tissues compared with normal pancreatic tissues. Overall survival of patients with PDAC who had undergone 5-FU-based chemotherapy was shorter in high TUG1 group than in low TUG1 group. Mechanistically, TUG1 antagonized miR-376b-3p and upregulated dihydropyrimidine dehydrogenase (DPD). TUG1 depletion induced susceptibility to 5-FU in BxPC-3 and PK-9 pancreatic cell lines. Consistently, the cellular concentration of 5-FU was significantly higher under TUG1-depleted conditions. In PDAC xenograft models, intravenous treatment with a cancer-specific drug delivery system (TUG1-DDS) and 5-FU significantly suppressed PDAC tumor growth compared with 5-FU treatment alone. This novel approach using TUG1-DDS in combination with 5-FU may serve as an effective therapeutic option to attenuate DPD activity and meet appropriate 5-FU dosage requirements in targeted PDAC cells, which can reduce the systemic adverse effects of chemotherapy. SIGNIFICANCE: Targeting TUG1 coupled with a cancer-specific drug delivery system effectively modulates 5-FU catabolism in TUG1-overexpressing PDAC cells, thus contributing to a new combinatorial strategy for cancer treatment. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/7/1654/F1.large.jpg.

γ-Glutamyltranspeptidase (GGT)-Activatable Fluorescence Probe for Durable Tumor Imaging.

γ-Glutamyltranspeptidase (GGT) is overexpressed in several types of cancer. Existing GGT-targeting fluorescence probes can image these cancers, but the fluorescent hydrolysis product leaks from the target cancer cells during prolonged incubation or fixation. Here, we present a functionalized fluorescence probe for GGT, 4-CH2 F-HMDiEtR-gGlu, which is designed to generate an azaquinone methide intermediate during activation by GGT; this intermediate reacts with intracellular nucleophiles to generate a fluorescent adduct that is trapped inside the cells, without loss of the target enzyme activity. Application of the probe to patient-derived xenograft (PDX) mice enabled in vivo cancer imaging for a prolonged period and was also compatible with fixation and immunostaining of the cancer tissue.

Fascin protein stabilization by miR-146a implicated in the process of a chronic inflammation-related colon carcinogenesis model.

OBJECTIVE: In sporadic colon tumors, multistep process of well-known genetic alterations accelerates carcinogenesis; however, this does not appear to be the case in inflammation-related ones. We previously established a model of inflammation-related colon carcinogenesis using human colonic adenoma cells, and identified fascin as a driver gene of this process. We analyzed the microRNAs involved in the stable fascin expression in colon adenocarcinoma cells. MATERIALS AND METHODS: miRNA microarray analysis was performed using FPCK-1-1 adenoma cells and its-derived FPCKpP1-4 adenocarcinoma cells through chronic inflammation. To assess the involvement of miRNA in the inflammation-related carcinogenesis, sphere-forming ability, expression of colon cancer stemness markers, and stability of fascin protein via the proteasome using tough decoy RNA technique. RESULTS: We found that 17 miRNAs including miR-146a were upregulated and 16 miRNAs were downregulated in FPCKpP1-4 adenocarcinoma cells. We revealed that miR-146a in the adenocarcinoma cells brought about acquisition of sphere formation, cancer stemness, and inhibition of proteasomal degradation of the fascin protein. CONCLUSIONS: We found that stable fascin expression is brought about via the inhibition of proteasome degradation by miR-146a in the process of a chronic inflammation-related colon carcinogenesis.

Fascin regulates chronic inflammation-related human colon carcinogenesis by inhibiting cell anoikis.

By a proteomics-based approach, we identified an overexpression of fascin in colon adenocarcinoma cells (FPCKpP-3) that developed from nontumorigenic human colonic adenoma cells (FPCK-1-1) and were converted to tumorigenic by foreign-body-induced chronic inflammation in nude mice. Fascin overexpression was also observed in the tumors arising from rat intestinal epithelial cells (IEC 6) converted to tumorigenic in chronic inflammation which was induced in the same manner. Upregulation of fascin expression in FPCK-1-1 cells by transfection with sense fascin cDNA converted the cells tumorigenic, whereas antisense fascin-cDNA-transfected FPCKpP-3 cells reduced fascin expression and lost their tumor-forming ability in vivo. The tumorigenic potential by fascin expression was consistent with their ability to survive and grow in the three-dimensional multicellular spheroids. We found that resistance to anoikis (apoptotic cell death as a consequence of insufficient cell-to-substrate interactions), which is represented by the three-dimensional growth of solid tumors in vivo, was regulated by fascin expression through caspase-dependent apoptotic signals. From these, we demonstrate that fascin is a potent suppressor to caspase-associated anoikis and accelerator of the conversion of colonic adenoma cells into adenocarcinoma cells by chronic inflammation.

Autonomous cure of damaged human intestinal epithelial cells by TLR2 and TLR4-dependent production of IL-22 in response to Spirulina polysaccharides.

In order to analyze the damage of human epithelial cells, we used human quasi-normal FPCK-1-1 cells derived from a colonic polyp in a patient with familial adenomatous polyposis as a monolayer, which is co-cultured with peptidoglycan (PGN)-stimulated THP-1 cells. Co-cultured FPCK-1-1 cells showed a decreased transepithelial electrical resistance (TER) and the lower level of claudin-2. When Spirulina complex polysaccharides were added one day before the start of the co-culture, there was no decrease of TER and claudin-2 (early phase damage). In contrast, when Spirulina complex polysaccharides were added to FPCK-1-1 cells after the level of TER had decreased, there was no recovery at the level of claudin-2, though the TER level recovered (late phase damage). The mucosa reconstitution is suggested to be involved in the recovery from the damaged status. Interestingly, autonomous recovery of FPCK-1-1 cells from both the early and late phase damage requires the production of IL-22, because anti-IL-22 antibodies inhibited recovery in these cases. Antibodies against either TLR2 or TLR4 inhibited the production of IL-22 from FPCK-1-1 colon epithelial cells, suggesting that signals through TLR2 and TLR4 are necessary for autonomous recovery of FPCK-1-1 colon epithelial cells by producing IL-22. In conclusion, we have established a useful model for the study of intestinal damage and recovery using human colon epithelial cells and our data suggest that damage to human colon epithelial cells can, at least in part, be recovered by the autonomous production of IL-22 in response to Spirulina complex polysaccharides.

Chronic inflammation-derived nitric oxide causes conversion of human colonic adenoma cells into adenocarcinoma cells.

It has been suggested that nitric oxide (NO) derived from chronically inflamed tissues is a cause of carcinogenesis. We herein demonstrated that administration of an inducible NO synthase inhibitor, aminoguanidine, significantly suppressed the tumorigenic conversion of human colonic adenoma (FPCK-1-1) cells into adenocarcinoma (FPCK/Inflam) cells accelerated by foreign body-induced chronic inflammation in nude mice. To determine whether NO directly promotes carcinogenesis, we exposed FPCK-1-1 cells continuously to chemically generated NO (FPCK/NO), and periodically examined their tumorigenicity. FPCK/NO cells formed tumors, whereas vehicle-treated cells (FPCK/NaOH) did not. We selected a tumorigenic population from FPCK/NO cells kept it in three-dimensional (3D) culture where in vivo-like multicellular spheroidal growth was expected. FPCK/Inflam cells developed large spheroids whereas FPCK/NO cells formed tiny but growing compact aggregates in 3D culture. Meanwhile, FPCK-1-1 and FPCK/NaOH cells underwent anoikis (apoptotic cell death consequential on insufficient cell-to-substrate interactions) through activation of caspase 3. The survived cells in the 3D culture (FPCK/NO/3D), which were derived from FPCK/NO cells, showed a similar tumor incidence to that of FPCK/Inflam cells. These results showed that NO was one of the causative factors for the acceleration of colon carcinogenesis, especially in the conversion from adenoma to adenocarcinoma in the chronic inflammatory environment.

Whole-exome sequencing of human pancreatic cancers and characterization of genomic instability caused by MLH1 haploinsufficiency and complete deficiency.

Whole-exome sequencing (Exome-seq) has been successfully applied in several recent studies. We here sequenced the exomes of 15 pancreatic tumor cell lines and their matched normal samples. We captured 162,073 exons of 16,954 genes and sequenced the targeted regions to a mean coverage of 56-fold. This study identified a total of 1517 somatic mutations and validated 934 mutations by transcriptome sequencing. We detected recurrent mutations in 56 genes. Among them, 41 have not been described. The mutation rates varied widely among cell lines. The diversity of the mutation rates was significantly correlated with the distinct MLH1 copy-number status. Exome-seq revealed intensive genomic instability in a cell line with MLH1 homozygous deletion, indicated by a dramatically elevated rate of somatic substitutions, small insertions/deletions (indels), as well as indels in microsatellites. Notably, we found that MLH1 expression was decreased by nearly half in cell lines with an allelic loss of MLH1. While these cell lines were negative in conventional microsatellite instability assay, they showed a 10.5-fold increase in the rate of somatic indels, e.g., truncating indels in TP53 and TGFBR2, indicating MLH1 haploinsufficiency in the correction of DNA indel errors. We further analyzed the exomes of 15 renal cell carcinomas and confirmed MLH1 haploinsufficiency. We observed a much higher rate of indel mutations in the affected cases and identified recurrent truncating indels in several cancer genes such as VHL, PBRM1, and JARID1C. Together, our data suggest that MLH1 hemizygous deletion, through increasing the rate of indel mutations, could drive the development and progression of sporadic cancers.

Epidermal growth factor-dependent enhancement of invasiveness of squamous cell carcinoma of the breast.

Factors that promote the aggressiveness of squamous cell carcinoma of the breast are not well understood. To examine the involvement of cell motility and the mechanism of this behavior, a squamous cell carcinoma cell line of the breast (HBC9) was established from a metastatic lymph node of a Japanese woman. HBC9 expressed epidermal growth factor receptor (EGFR), but was negative for Her2 or Her3.The invasive ability of HBC9 was compared with that of four breast ductal carcinoma cell lines by Matrigel invasion assay. EGF stimulation induced the formation of surface protrusions and cell migration in HBC9 cells, and significantly increased the number of cells migrating through the Matrigel. The invasive ability of HBC9 was compared with other cell lines of breast carcinoma; it was much greater than that of MCF-7, BT474, or HBC5, but did not differ significantly from that of MDA-MB-231. Observation of the surface protrusions of HBC9 by confocal laser microscopy revealed co-localization of Arp2 and N-WASP with actin polymerization, detected by visualization with phalloidin, indicating that the protrusions induced by EGF were invadopodia. In HBC9 cells, cortactin also co-localized with the N-WASP/Arp2/3 complex in the protrusions. Immunohistochemistry of 12 cases of squamous cell carcinoma of the breast revealed expression of cortactin and EGFR in all of them, and this was confirmed by western blotting in two cases. These results suggest that EGF-dependent enhancement of cell motility by formation of invadopodia associated with cortactin is a cause of the clinical aggressiveness of squamous cell carcinoma of the breast.

The selective continued linkage of centromeres from mitosis to interphase in the absence of mammalian separase.

Separase is an evolutionarily conserved protease that is essential for chromosome segregation and cleaves cohesin Scc1/Rad21, which joins the sister chromatids together. Although mammalian separase also functions in chromosome segregation, our understanding of this process in mammals is still incomplete. We generated separase knockout mice, reporting an essential function for mammalian separase. Separase-deficient mouse embryonic fibroblasts exhibited severely restrained increases in cell number, polyploid chromosomes, and amplified centrosomes. Chromosome spreads demonstrated that multiple chromosomes connected to a centromeric region. Live observation demonstrated that the chromosomes of separase-deficient cells condensed, but failed to segregate, although subsequent cytokinesis and chromosome decondensation proceeded normally. These results establish that mammalian separase is essential for the separation of centromeres, but not of the arm regions of chromosomes. Other cell cycle events, such as mitotic exit, DNA replication, and centrosome duplication appear to occur normally. We also demonstrated that heterozygous separase-deficient cells exhibited severely restrained increases in cell number with apparently normal mitosis in the absence of securin, which is an inhibitory partner of separase.

Putative gene loci associated with carcinogenesis and metastasis of endocervical adenocarcinomas of uterus determined by conventional and array-based CGH.

OBJECTIVES: This study aimed to estimate the gene loci associated with carcinogenesis of endocervical adenocarcinoma of uterus (EA) and metastasis. Study design Sixteen patients with EA were studied; 6 had nodal metastasis. DNA was extracted from EAs, and subjected to both conventional comparative genomic hybridization (CGH) and array-based CGH. Copy number abnormalities were compared between cases with and without nodal metastasis. RESULTS: In all EAs, high frequencies of copy number losses were detected in genes LRP1B (on 2q21.2), DAB2 (5p13), and DCC (18q21.3), as well as regions 3p, 16q, and 22q, and copy number amplifications in genes NRAS (1p13.2), TOP2A (17q21-q22), NCOA3(AIB1) (20q12), and ARSA (22q tel). Nodal metastasis was associated with high frequencies of copy number loss in genes PGRMC2 and LAMA3 and amplification in CDK6 and NCOA3(AIB1). CONCLUSION: This is the first report of gene copy number alterations spanning the whole genome in EA. These altered genes are speculated to be associated with EAs as a tumor suppressor and/or oncogene.

Conventional and array-based comparative genomic hybridization analyses of novel cell lines harboring HPV18 from glassy cell carcinoma of the uterine cervix.

We established 2 novel human cell lines (GCCOT-1, GCCRK) from glassy cell carcinoma. Both cell lines showed dual tendencies of glandular and squamous differentiation, and thus possess the characteristics resembling reserve cells, the putative origin of most carcinomas arising from the uterine cervix. HPV type 18 DNA including E6-E7, which is commonly found in cell types other than squamous cell carcinoma of uterine cervix, was detected in both cell lines. We analyzed gene copy number alterations of the 2 cell lines using conventional comparative genomic hybridization (CGH) coupled with array-based CGH. Among the putative oncogenes demonstrating copy number gain in both cell lines, FGR(SRC2) at 1p36.2-1 and LAMC2 at 1q25-31 have not been reported to show amplification in previous analyses of conventional cervical cell lines. These oncogenes are thus speculated to be directly associated with oncogenesis of glassy cell carcinoma. On the other hand, among the putative suppressor genes demonstrating copy number loss in both cell lines, the 9q region, ATM at 11q22.3, and CYLD at 16q12-13 have not been reported to show loss in conventional cervical cancer cell lines. These sites are speculated to be important as tumor suppressors directly associated with oncogenesis of glassy cell carcinoma. This study suggests for the first time that together with the presence of HPV type 18, alterations at the above sites are closely associated with oncogenesis of glassy cell carcinoma, a special type of carcinoma in the uterine cervix.

Identification of candidate predictive markers of anticancer drug sensitivity using a panel of human cancer cell lines.

We previously investigated the correlations between the expression of 9216 genes and various chemosensitivities in a panel of 39 human cancer cell lines(1)) and found that the expression levels of AKR1B1 and CTSH were correlated with sensitivity and resistance to multiple drugs, respectively. To validate these correlations, we investigated the expression of these two genes and the chemosensitivities in 12 additional gastric cancer cell lines. The expression of AKR1B1 in the additional cell lines exhibited significant correlations with sensitivities to 8 of the 23 drugs examined, while that of CTSH displayed a significant negative correlation with only one (MS-247) of the 27 drugs examined. Their expressions were weakly correlated with sensitivity and resistance, respectively, to the remainder of the drugs. Moreover, when the 12 cell lines were divided into high-expressing and low-expressing groups, a comparison of these groups using Mann-Whitney's U test revealed that high expression levels of AKR1B1 and CTSH were related to sensitivity to 21 of the drugs and resistance to 8 of the drugs, respectively. The present results suggest that AKR1B1 and CTSH may be good markers for prediction of sensitivity to certain drugs and that our panel of 39 cell lines has the potential to identify candidate predictive marker genes.

Ectopic chromosome around centrosome in metaphase cells as a marker of high-risk human papillomavirus-associated cervical intraepithelial neoplasias.

This report describes the appearance of ectopic chromosome around centrosome (ECAC) in metaphase cell nuclei of high-risk HPV-associated cervical neoplasms. ECAC are clearly visible on HE-stained sections as a tiny (approx. 0.7 micro), round, dark structure or an aggregate of filamentous chromosome, often symmetrical at bilateral centrosomes. They appear in CINs from an early stage (CIN I), with the highest incidence in HPV16-associated CIN II-III (75%), and are less common in HPV-related invasive carcinomas (21%) and in lesions associated with high-risk HPV types other than 16. Rates for ECAC-positive nuclei in metaphase preparations (ECAC rate) for each lesion ranged 3.6-30%, the average being 14.5%. ECAC appeared very rarely in CINs associated with intermediate-risk HPVs and was never encountered in HPV6/11-induced condylomas or HPV-unrelated neoplasms in other organs. ECAC may be morphologic evidence of HPV-induced chromosomal instability working as a background mechanism in cervical carcinogenesis and also a useful marker for the histopathologic differentiation of high-risk CINs.