Pathological and biological significance of the specific glycan, TRA-1-60, on aggressive gastric adenocarcinoma.

The glycans form a unique complex on the surface of cancer cells and play a pivotal role in tumor progression, impacting proliferation, invasion, and metastasis. TRA-1-60 is a glycan that was identified as a critical marker for the establishment of fully reprogrammed inducible pluripotent stem (iPS) cells. Its expression has been detected in multiple cancer tissues, including embryonal carcinoma, prostate cancer, and pancreatic cancer, but the biological and pathological characterization of TRA-1-60-expressing tumor cells still remains unclear within various types of malignancies. Here, we report the biological characteristics of TRA-1-60-expressing gastric cancer cells, especially those with its cell surface expression, and the therapeutic significance of targeting TRA-1-60. The cells with cell membrane expression of TRA-1-60 were mainly observed in the invasive area of patient gastric cancer tissues and correlated with advanced stages of the disease based on histopathological and clinicopathological analyses. In vitro analysis using a scirrhous gastric adenocarcinoma line, HSC-58, which highly expresses TRA-1-60 on its plasma membrane, revealed increased stress-resistant mechanisms, supported by the upregulation of glutathione synthetase (GSS) and NCF-1 (p47phox) via lipid-ROS regulatory pathways, as detected by RNA-seq analysis followed by oxidative stress gene-profiling. Our in vivo therapeutic study using the TRA-1-60-targeting antibody-drug conjugate (ADC), namely Bstrongomab conjugated Monomethyl auristatin E (MMAE), showed robust efficacy in a mouse model of peritoneal carcinomatosis induced by intraperitoneal xenograft of HSC-58, by markedly reducing massive tumor ascites. Thus, targeting the specific cell surface glycan, TRA-1-60, shows significant therapeutic impact in advanced-stage gastric cancers. (243 words).

Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface.

Background: Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-ß1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain. Methods: Cell invasion was assessed using a transwell-based assay, protein-protein interactions by an immunoprecipitation-Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography. Results: We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion. Conclusion: We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion.

The prolyl hydroxylase OGFOD1 promotes cancer cell proliferation by regulating the expression of cell cycle regulators.

OGFOD1, a prolyl-hydroxylase, has been reported to translocate from the nucleus to the cytoplasm in response to cellular stress. Here, we demonstrate that OGFOD1 regulates the transcription and post-transcriptional stabilization of cell cycle-related genes. OGFOD1 knockdown in lung cancer cells induced cell cycle arrest through the specific depletion of cyclin-dependent kinase (CDK) 1, CDK2 and cyclin B1 (CCNB1) mRNAs and the nuclear accumulation of p21Cip1 . Analysis of the mRNA dynamics in these cells revealed that CDK1 decreased in a time-dependent manner, reflecting post-transcriptional regulation by OGFOD1 and the RNA-binding protein HuR. In contrast, the depletion of CDK2 and CCNB1 resulted from decreased transcription mediated by OGFOD1. These results indicate that OGFOD1 is required to maintain the function of specific cell cycle regulators during cancer cell proliferation.

Toll-like receptor 4 promotes bladder cancer progression upon S100A8/A9 binding, which requires TIRAP-mediated TPL2 activation.

Bladder cancer is an often widely disseminated and deadly cancer. To block the malignant outgrowth of bladder cancer, we must elucidate the molecular-level characteristics of not only bladder cancer cells but also their surrounding milieu. As part of this effort, we have long been studying extracellular S100A8/A9, which is elevated by the inflammation associated with certain cancers. Extracellularly enriched S100A8/A9 can hasten a shift to metastatic transition in multiple types of cancer cells. Intriguingly, high-level S100A8/A9 has been detected in the urine of bladder-cancer patients, and the level increases with the stage of malignancy. Nonetheless, S100A8/A9 has been investigated mainly as a potential biomarker of bladder cancers, and there have been no investigations of its role in bladder-cancer growth and metastasis. We herein report that extracellular S100A8/A9 induces upregulation of growth, migration and invasion in bladder cancer cells through its binding with cell-surface Toll-like receptor 4 (TLR4). Our molecular analysis revealed the TLR4 downstream signal that accelerates such cancer cell events. Tumor progression locus 2 (TPL2) was a key factor facilitating the aggressiveness of cancer cells. Upon binding of S100A8/A9 with TLR4, TPL2 activation was enhanced by an action with a TLR4 adaptor molecule, TIR domain-containing adaptor protein (TIRAP), which in turn led to activation of the mitogen-activated protein kinase (MAPK) cascade of TPL2. Finally, we showed that sustained inhibition of TLR4 in cancer cells effectively dampened cancer survival in vivo. Collectively, our results indicate that the S100A8/A9-TLR4-TPL2 axis influences the growth, survival, and invasive motility of bladder cancer cells.

Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells.

The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis.

Intercellular adhesion molecule-1-targeted near-infrared photoimmunotherapy of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and conventional chemotherapy and molecular-targeted therapies show limited efficacy. Near-infrared photoimmunotherapy (NIR-PIT) is a new anticancer treatment that selectively damages the cell membrane of cancer cells based on NIR light-induced photochemical reactions of the antibody (Ab)-photoabsorber (IRDye700Dx) conjugate and the cell membrane. TNBC is known to express several adhesion molecules on the cell surface providing a potential new target for therapy. Here, we investigated the therapeutic efficacy of intercellular adhesion molecule-1 (ICAM-1)-targeted NIR-PIT using xenograft mouse models subcutaneously inoculated with two human ICAM-1-expressing TNBC cell lines, MDAMB468-luc and MDAMB231 cells. In vitro ICAM-1-targeted NIR-PIT damaged both cell types in a NIR light dose-dependent manner. In vivo ICAM-1-targeted NIR-PIT in both models showed early histological signs of cancer cell damage, such as cytoplasmic vacuolation. Even among the cancer cells that appeared to be morphologically intact within 2 h post treatment, abnormal distribution of the actin cytoskeleton and a significant decrease in Ki-67 positivity were observed, indicating widespread cellular injury reflected in cytoplasmic degeneration. Such damage to cancer cells by NIR-PIT significantly inhibited subsequent tumor growth and improved survival. This study suggests that ICAM-1-targeted NIR-PIT could have potential clinical application in the treatment of TNBC.

Crumbs3 is expressed in oral squamous cell carcinomas and promotes cell migration and proliferation by affecting RhoA activity.

Despite the recent progression of treatments, the 5-year survival rate of patients with oral squamous cell carcinoma (OSCC) is still poor. One of the most critical factors affecting prognosis is tumor metastasis. Developing novel molecular targeted therapies by analyzing the molecular pathway of OSCC metastasis is an urgent issue. The present study aimed to characterize the expression and function of crumbs3 (Crb3) in OSCC cell migration. Immunohistochemistry and immunoblotting revealed that Crb3 was expressed in tissues from patients with OSCC and OSCC cell lines. The motility of OSCC cell lines was decreased by knockdown of Crb3 without affecting proliferation. However, Crb3-knockout (KO) clones exhibited decreases in both cell migration and proliferation. The expression of epithelial-mesenchymal transition markers was not altered in Crb3-KO clones compared with parent cells. A xenograft mouse model of lung metastasis revealed that the metastatic potential of Crb3-KO clones was reduced. As seen with Crb3-KO clones, the motility of OSCC cells was decreased by treatment with inhibitors of RhoA activation. Serum-induced activation of RhoA in OSCC cells was evaluated by comparing the amount of GTP-bound RhoA using affinity matrices, revealing that RhoA activation was decreased in Crb3-KO clones. To the best of our knowledge, the present study was the first to demonstrate that Crb3 was expressed in squamous cell carcinoma tissues and promoted cell migration and proliferation, which was associated with RhoA activation in OSCC cells.

PLOD2-driven IL-6/STAT3 signaling promotes the invasion and metastasis of oral squamous cell carcinoma via activation of integrin ß1.

We previously reported that high expression of procollagen­lysine 2­oxoglutarate 5­dioxygenase 2 (PLOD2) leads to stabilization and plasma membrane translocation of integrin ß1 to promote the invasion and metastasis of oral squamous cell carcinoma (SCC). The present study aimed to further understand the relationship between PLOD2­integrin ß1 signaling and the tumor microenvironment. This study provided further advanced insights indicating that elevated interleukin (IL)­6 in the tumor microenvironment acts as a key molecule that triggers PLOD2­integrin ß1 axis­derived acceleration of tumor invasion and metastasis. It was found using the dual­luciferase reporter assay system that signal transducer and activator of transcription 3 (STAT3) activation by IL­6 was essential for increasing the expression levels of PLOD2 through direct activation of the PLOD2 promoter in oral SCC, whereas IL­6 stimulation did not contribute to integrin ß1 expression or the subsequent maturation process towards a functional form on the plasma membrane. Furthermore, the expression of IL­6 in oral SCC tissues was mainly observed in the tumor stroma. Finally, with double immunofluorescence staining, it was found that IL­6 expression occurred in CD163­positive M2 macrophages distributed around the tumor nest. These results combined with our previous results indicate that as IL­6 significantly increases STAT3­mediated PLOD2 promoter activity, IL­6 released by M2­type tumor­associated macrophages is a crucial factor that promotes PLOD2­integrin ß1 axis­enhanced invasion and metastasis of oral SCC cells.

Tumor-homing peptide and its utility for advanced cancer medicine.

Cell-penetrating peptides, such as antibodies, have gained great attention as tools for the development of specific delivery systems for payloads, which might be applied as non-invasive carriers in vivo. Among these, tumor-homing peptides recently have been studied for use in tumor medicine. Tumor-homing peptides are oligopeptides, usually consisting of 30 or fewer amino acids that are efficiently and specifically incorporated into tumor cells, suggesting their potential use in establishing novel non-invasive tumor imaging systems for diagnostic and therapeutic applications. Here, we briefly introduce the biological characteristics of our tumor-homing peptides, focusing especially on those developed using a random peptide library constructed using mRNA display technology. The advantage of the tumor-homing peptides is their biological safety, given that these molecules do not show significant cytotoxicity against non-neoplastic cells; lack serious antigenicity, which alternatively might evoke unfavorable immune responses and inflammation in vivo; and are rapidly incorporated into target cells/tissues, with rates exceeding those seen for antibodies. Given their small size, tumor-homing peptides also are easy to modify and redesign. Based on these merits, tumor-homing peptides are expected to find wide application in various aspects of tumor medicine, including imaging diagnostics (eg, with dye-conjugated probes for direct visualization of invasive/metastatic tumor lesions in vivo) and therapeutics (eg, using peptide-drug conjugates [PDCs] for tumor targeting). Although further evidence will be required to demonstrate their practical utility, tumor-homing peptides are expected to show great potential as a next-generation bio-tool contributing to precision medicine for cancer patients.

PLOD2 Is Essential to Functional Activation of Integrin ß1 for Invasion/Metastasis in Head and Neck Squamous Cell Carcinomas.

Identifying the specific functional regulator of integrin family molecules in cancer cells is critical because they are directly involved in tumor invasion and metastasis. Here we report high expression of PLOD2 in oropharyngeal squamous cell carcinomas (SCCs) and its critical role as a stabilizer of integrin ß1, enabling integrin ß1 to initiate tumor invasion/metastasis. Integrin ß1 stabilized by PLOD2-mediated hydroxylation was recruited to the plasma membrane, its functional site, and accelerated tumor cell motility, leading to tumor metastasis in vivo, whereas loss of PLOD2 expression abrogated it. In accordance with molecular analysis, examination of oropharyngeal SCC tissues from patients corroborated PLOD2 expression associated with integrin ß1 at the invasive front of tumor nests. PLOD2 is thus implicated as the key regulator of integrin ß1 that prominently regulates tumor invasion and metastasis, and it provides important clues engendering novel therapeutics for these intractable cancers.

PODXL1 promotes metastasis of the pancreatic ductal adenocarcinoma by activating the C5aR/C5a axis from the tumor microenvironment.

Pancreatic invasive ductal adenocarcinoma (PDAC) is a representative intractable malignancy under the current cancer therapies, and is considered a scirrhous carcinoma because it develops dense stroma. Both PODXL1, a member of CD34 family molecules, and C5aR, a critical cell motility inducer, have gained recent attention, as their expression was reported to correlate with poor prognosis for patients with diverse origins including PDAC; however, previous studies reported independently on their respective biological significance. Here we demonstrate that PODXL1 is essential for metastasis of PDAC cells through its specific interaction with C5aR. In vitro assay demonstrated that PODXL1 bound to C5aR, which stabilized C5aR protein and recruited it to cancer cell plasma membranes to receive C5a, an inflammatory chemoattractant factor. PODXL1 knockout in PDAC cells abrogated their metastatic property in vivo, emulating the liver metastatic mouse model treated with anti-C5a neutralizing antibody. In molecular studies, PODXL1 triggered EMT on PDAC cells in response to stimulation by C5a, corroborating PODXL1 involvement in PDAC cellular invasive properties via specific interaction with the C5aR/C5a axis. Confirming the molecular assays, histological examination showed coexpression of PODXL1 and C5aR at the invasive front of primary cancer nests as well as in liver metastatic foci of PDAC both in the mouse metastasis model and patient tissues. Hence, the novel direct interaction between PODXL1 and the C5aR/C5a axis may provide a better integrated understanding of PDAC biological characteristics including its tumor microenvironment factors.

Crumbs3 regulates the expression of glycosphingolipids on the plasma membrane to promote colon cancer cell migration.

The cell polarity regulator Crumbs3 (Crb3) promotes colon cancer cell migration and metastasis. However, the underlying mechanism of cancer cell migration regulated by Crb3 has not been fully elucidated. Here, we demonstrated that Crb3 is associated with cell migration by regulating glycosphingolipid (GSL) expression in human colon cancer cells. Crb3-knockout (KO) cells showed a remarkable increase in ganglioside GM3 (GM3) on the cell surface. Reduced migration by Crb3-KO cells was restored by forced expression of both Crb3 and Neuraminidase3 (Neu3). Immunofluorescent staining revealed that most Crb3 is colocalized with the recycling endosome marker Rab11. These findings show that Crb3 may promote colon cancer cell migration by regulating the expression of GSLs on the cell surface.

Upregulation of Mobility in Pancreatic Cancer Cells by Secreted S100A11 Through Activation of Surrounding Fibroblasts.

S100A11, a member of the S100 family of proteins, is actively secreted from pancreatic ductal adenocarcinoma (PDAC) cells. However, the role of the extracellular S100A11 in PDAC progression remains unclear. In the present study, we investigated the extracellular role of S100A11 in crosstalking between PDAC cells and surrounding fibroblasts in PDAC progression. An abundant S100A11 secreted from pancreatic cancer cells stimulated neighboring fibroblasts through receptor for advanced glycation end products (RAGE) upon S100A11 binding and was followed by not only an enhanced cancer cell motility in vitro but also an increased number of the PDAC-derived circulating tumor cells (CTCs) in vivo. Mechanistic investigation of RAGE downstream in fibroblasts revealed a novel contribution of a mitogen-activated protein kinase kinase kinase (MAPKKK), tumor progression locus 2 (TPL2), which is required for positive regulation of PDAC cell motility through induction of cyclooxygenase 2 (COX2) and its catalyzed production of prostaglandin E2 (PGE2), a strong chemoattractive fatty acid. The extracellularly released PGE2 from fibroblasts was required for the rise in cellular migration as well as infiltration of their adjacent PDAC cells in a coculture setting. Taken together, our data reveal a novel role of the secretory S100A11 in PDAC disseminative progression through activation of surrounding fibroblasts triggered by the S100A11-RAGE-TPL2-COX2 pathway. The findings of this study will contribute to the establishment of a novel therapeutic antidote to PDACs that are difficult to treat by regulating cancer-associated fibroblasts (CAFs) through targeting the identified pathway.

Critical role of the MCAM-ETV4 axis triggered by extracellular S100A8/A9 in breast cancer aggressiveness.

Metastatic breast cancer is the leading cause of cancer-associated death in women. The progression of this fatal disease is associated with inflammatory responses that promote cancer cell growth and dissemination, eventually leading to a reduction of overall survival. However, the mechanism(s) of the inflammation-boosted cancer progression remains unclear. In this study, we found for the first time that an extracellular cytokine, S100A8/A9, accelerates breast cancer growth and metastasis upon binding to a cell surface receptor, melanoma cell adhesion molecule (MCAM). Our molecular analyses revealed an important role of ETS translocation variant 4 (ETV4), which is significantly activated in the region downstream of MCAM upon S100A8/A9 stimulation, in breast cancer progression in vitro as well as in vivo. The MCAM-mediated activation of ETV4 induced a mobile phenotype called epithelial-mesenchymal transition (EMT) in cells, since we found that ETV4 transcriptionally upregulates ZEB1, a strong EMT inducer, at a very high level. In contrast, downregulation of either MCAM or ETV4 repressed EMT, resulting in greatly weakened tumor growth and lung metastasis. Overall, our results revealed that ETV4 is a novel transcription factor regulated by the S100A8/A9-MCAM axis, which leads to EMT through ZEB1 and thereby to metastasis in breast cancer cells. Thus, therapeutic strategies based on our findings might improve patient outcomes.

Crumbs3 is a critical factor that regulates invasion and metastasis of colon adenocarcinoma via the specific interaction with FGFR1.

Epithelial cell polarity regulator Crumbs3 (Crb3), a mammalian homolog within the Drosophila Crb gene family, was initially identified as an essential embryonic development factor. It is recently implicated in tumor suppression, though its specific functions are controversial. We here demonstrate that Crb3 strongly promotes tumor invasion and metastasis of human colon adenocarcinoma cells. Crb3 centrality to tumor migration was supported by strong expression at invasive front and metastatic foci of colonic adenocarcinoma of the patient tissues. Accordingly, two different Crb3-knockout (KO) lines, Crb3-KO (Crb3 -/-) DLD-1 and Crb3-KO WiDr from human colonic adenocarcinomas, were generated by the CRISPR-Cas9 system. Crb3-KO DLD-1 cells exhibited loss of cellular mobility in vitro and dramatic suppression of liver metastases in vivo in contrast to the wild type of DLD-1. Unlike DLD-1, Crb3-KO WiDr mobility and metastasis were unaffected, which were similar to wild-type WiDr. Proteome analysis of Crb3-coimmunopreciptated proteins identified different respective fibroblast growth factor receptor (FGFR) isotypes specifically bound to Crb3 isoform a through their intracellular domain. In DLD-1, Crb3 showed membranous localization of FGFR1 leading to its functional activation, whereas Crb3 bound to cytoplasmic FGFR4 in WiDr without FGFR1 expression, leading to cellular growth. Correlative expression between Crb3 and FGFR1 was consistently detected in primary and metastatic colorectal cancer patient tissues. Taking these together, Crb3 critically accelerates cell migration, namely invasion and metastasis of human colon cancers, through specific interaction to FGFR1 on colon cancer cells.

Melanoma cell adhesion molecule is the driving force behind the dissemination of melanoma upon S100A8/A9 binding in the original skin lesion.

Since metastasis accounts for the majority of cancer-associated deaths, studies on the mechanisms of metastasis are needed to establish innovative strategies for cancer treatment. We previously reported that melanoma cell adhesion molecule (MCAM) functions as a critical receptor for S100A8/A9, and binding of S100A8/A9 to MCAM results in the migration of melanoma cells to lung tissue. However, the critical role of MCAM in the original melanoma skin lesion is still not clear. In this study, we aimed to determine the importance of the S100A8/A9-MCAM axis in melanoma dissemination in a skin lesion as a critical early step for metastasis. Mechanistic studies revealed the downstream signaling of MCAM that signaled the induction of metastasis. S100A8/A9-MCAM binding activates mitogen-activated protein kinase kinase kinase 8 (MAP3K8), also termed TPL2, leading to strong activation of the transcription factor ETV4 and subsequent induction of matrix metalloproteinase-25 (MMP25), and finally to induction of melanoma lung tropic metastasis. Collectively, our results demonstrate a crucial role of the S100A8/A9-MCAM signaling axis in metastatic onset of melanoma cells and indicate that strategies targeting the identified pathway may be useful for the establishment of innovative anti-cancer therapies.

Extracellular S100A11 Plays a Critical Role in Spread of the Fibroblast Population in Pancreatic Cancers.

The fertile stroma in pancreatic ductal adenocarcinomas (PDACs) has been suspected to greatly contribute to PDAC progression. Since the main cell constituents of the stroma are fibroblasts, there is crosstalking(s) between PDAC cells and surrounding fibroblasts in the stroma, which induces a fibroblast proliferation burst. We have reported that several malignant cancer cells including PDAC cells secrete a pronounced level of S100A11, which in turn stimulates proliferation of cancer cells via the receptor for advanced glycation end products (RAGE) in an autocrine manner. Owing to the RAGE+ expression in fibroblasts, the extracellular abundant S100A11 will affect adjacent fibroblasts. In this study, we investigated the significance of the paracrine axis of S100A11-RAGE in fibroblasts for their proliferation activity. In in vitro settings, extracellular S100A11 induced upregulation of fibroblast proliferation. Our mechanistic studies revealed that the induction is through RAGE-MyD88-mTOR-p70 S6 kinase upon S100A11 stimulation. The paracrine effect on fibroblasts is linked mainly to triggering growth but not cellular motility. Thus, the identified pathway might become a potential therapeutic target to suppress PDAC progression through preventing PDAC-associated fibroblast proliferation.

Neuroplastin-ß mediates S100A8/A9-induced lung cancer disseminative progression.

Compiling evidence indicates an unusual role of extracellular S100A8/A9 in cancer metastasis. S100A8/A9 secreted from either cancer cells or normal cells including epithelial and inflammatory cells stimulates cancer cells through S100A8/A9 sensor receptors in an autocrine or paracrine manner, leading to cancer cell metastatic progression. We previously reported a novel S100A8/A9 receptor, neuroplastin-ß (NPTNß), which plays a critical role in atopic dermatitis when it is highly activated in keratinocytes by an excess amount of extracellular S100A8/A9 in the inflammatory skin lesion. Interestingly, our expression profiling of NPTNß showed significantly high expression levels in lung cancer cell lines in a consistent manner. We hence aimed to determine the significance of NPTNß as an S100A8/A9 receptor in lung cancer. Our results showed that NPTNß has strong ability to induce cancer-related cellular events, including anchorage-independent growth, motility and invasiveness, in lung cancer cells in response to extracellular S100A8/A9, eventually leading to the expression of a cancer disseminative phenotype in lung tissue in vivo. Mechanistic investigation revealed that binding of S100A8/A9 to NPTNß mediates activation of NFIA and NFIB and following SPDEF transcription factors through orchestrated upstream signals from TRAF2 and RAS, which is linked to anchorage-independent growth, motility and invasiveness. Overall, our results indicate the importance of the S100A8/A9-NPTNß axis in lung cancer disseminative progression and reveal a pivotal role of its newly identified downstream signaling, TRAF2/RAS-NFIA/NFIB-SPDEF, in linking to the aggressive development of lung cancers.

Identification of TRA-1-60-positive cells as a potent refractory population in follicular lymphomas.

Despite receiving rituximab-combined chemotherapy, follicular lymphoma (FL) patients often suffer tumor recurrence and understand that the cause of relapse in FL would thus significantly ameliorate the tumor therapeutics. In the present study, we show that TRA-1-60-expressing cells are a unique population in FL, converge to the conventional stem cell marker Oct3/4 and ALDH1-positive population, and resist current B-lymphoma agents. TRA-1-60 expression was observed in scattered lymphoma cells in FL tissues only as well as in resting B-lymphocytes inside germinal centers. Retrospective comparison between recurrent and cognate primary tissues showed that the number of TRA-1-60-positive cells from rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (R-CHOP)-treated FL had increased relative to primary tissue, a finding corroborated by assays on different rituximab-treated FL cell lines, FL-18 and DOHH2, wherein TRA-positive cell numbers increased over 10-fold compared to the untreated sample. Concordantly, scanty TRA-1-60-positive FL-18 cells implanted s.c. into mice evinced potent tumor-initiating capacity in vivo, where tumors were 12-fold larger in volume (P = 0.0021 < 0.005) and 13-fold heavier in weight (P = 0.0015 < 0.005) compared to those xenografted from TRA-negative cells. To explain these results, gene expression profiling and qPCR analysis indicated that TRA-1-60-positive cells defined a distinct population from that of TRA-negative cells, with upregulation of multiple drug transporters and therapeutic resistance genes. Hence, TRA-1-60-expressing cells in FL are considered to be vigorously intractable against conventional therapeutic agents, which may explain its refractory recurrence.

Newly developed anti-S100A8/A9 monoclonal antibody efficiently prevents lung tropic cancer metastasis.

The metastatic dissemination of cancer cells to remote areas of the body is the most problematic aspect in cancer patients. Among cancers, melanomas are notoriously difficult to treat due to their significantly high metastatic potential even during early stages. Hence, the establishment of advanced therapeutic approaches to regulate metastasis is required to overcome the melanoma disease. An accumulating mass of evidence has indicated a critical role of extracellular S100A8/A9 in melanoma distant metastasis. Lung S100A8/A9 is induced by melanoma cells from distant organs and it attracts these cells to its enriched lung environment since melanoma cells possess several receptors that sense the S100A8/A9 ligand. We hence aimed to develop a neutralizing antibody against S100A8/A9 that would efficiently block melanoma lung metastasis. Our protocol provided us with one prominent antibody, Ab45 that efficiently suppressed not only S100A8/A9-mediated melanoma mobility but also lung tropic melanoma metastasis in a mouse model. This prompted us to make chimeric Ab45, a chimera antibody consisting of mouse Ab45-Fab and human IgG2-Fc. Chimeric Ab45 also showed significant inhibition of the lung metastasis of melanoma. From these results, we have high hopes that the newly produced antibody will become a potential biological tool to block melanoma metastasis in future clinical settings.