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Background: One of the main reasons for the failure of cancer treatments is that the biological function of therapeutic target molecules is only studied in cancer cells. Therefore, the dynamic and complex interactions between cancer and stromal cells in the tumor microenvironment (TME) should be investigated. TME-specific stromal cells include various cells, with tumor-associated macrophages (TAMs) being one of the major players. TAM are differentiated from monocytes, as immature monocytes recognize molecules derived from infected cells or foreign substances, become activated, and may differentiate into anti-tumoral M1 macrophages or pro-tumoral M2 macrophages (called TAMs). TAMs induce immune evasion of cancer cells in the TME, leading to active proliferation and metastasis of cancer cells. The presence of TAMs in the TME is associated with poor prognosis in various cancers. PAUF is a protein that is secreted by cancer cells, and it exerts tumor-promoting effects through autocrine and paracrine mechanisms. Compared to the autocrine actions, the paracrine effects of PAUF on immune cells in the TME are less understood. Objective: This study aimed to evaluate 1) the role of PAUF as a TME modulator, and specifically, a TAM inducer and 2) the potentiality of PAUF antibody as a blocker of TAM transformation. Methods and Results: In a monocyte-PAUF co-culture system, PAUF (tested at two concentrations, 0.1 and 0.5 μg/mL) demonstrated strong chemoattractant effects on monocytes (~400% of that caused by PBS), which were blocked by TLR2/4 inhibitors. PAUF also induced differentiation of monocytes into macrophages, which were identified to be TAM, using flow cytometric analysis and ELISA method. Moreover, these PAUF-induced TAM demonstrated T cell suppressing capacities. Finally, in a CFPAC-1 human cancer orthotopic mouse model, a humanized monoclonal PAUF-antibody diminished 60-70% of infiltrated TAM (p Conclusion: In conclusion, this study provides direct evidence that PAUF can differentiate monocytes into TAM of immune-suppressing functions, and the PAUF neutralization-induced TAM infiltration blocking suggests potential combination use of PAUF antibody with immunotherapeutics for cancer treatments. Citation Format: Fen Jiang, Yeon Jeong Kim, Yun Yong Park, Tae Heung Kang, Sang Seok Koh. PAUF stimulates monocytes and induces their differentiation into tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1347.

The soluble protein pancreatic adenocarcinoma upregulated factor (PAUF) is overexpressed in pancreatic cancer. We have recently reported that PAUF plays a crucial role in the progression and metastasis of pancreatic cancer cells. However its role in angiogenesis, which is crucial feature of tumor development, has not yet been understood. We here show that PAUF promotes angiogenesis and induces vascular permeability via endothelial cell activation. PAUF increased angiogenic traits of HUVEC cells such as proliferation, migration, and vascular network formation in vitro, which were abrogated by treatment with antibodies against PAUF. Consistently, PAUF stimulated the sprouting of microvessels from aortic rings ex vivo and the development of blood vessels into matrigel plug loaded with PAUF in vivo. We also found that PAUF increased permeability of endothelial cells as a result of the phosphorylation of VE-cadherin and promoted vascular leakiness of mouse skin. Intracellular signaling molecules ERK, AKT, and eNOS were activated in endothelial cell by PAUF, and chemical inhibitors for these signaling molecules inhibited PAUF-induced angiogenesis and vascular permeability. Expression analysis of several angiogenic factors indicated that these observed angiogenic effects of PAUF were independent of VEGF. We will discuss potential mechanism of angiogenesis mediated by PAUF. Collectively, our studies demonstrate PAUF as a novel angiogenic factor promoting angiogenesis and vascular permeability and suggest that targeting for PAUF can be an effective therapeutic treatment strategy to reduce tumor angiogenesis correlated with tumor progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5137. doi:10.1158/1538-7445.AM2011-5137

The tight junction protein claudin-4 is aberrantly up-regulated in gastric cancer, but its clinical significance and the molecular mechanism underlying claudin-4 overexpression in gastric cancer remain unclear. Here, we investigated its roles and epigenetic mechanism regulating CLDN4 expression in gastric cancer. We show that increased membranous expression of claudin-4 in gastric carcinoma is associated with better patient prognosis, whereas cytoplasmic claudin-4 expression didn't show a significant association with prognosis. Consistent with the correlation of increased membranous claudin-4 with favorable clinicopathological factors, claudin-4 overexpression inhibited the migration and invasion of gastric cancer cells; in contrast, it didn't affect cell growth. Claudin-4 expression also increased the barrier function of tight junctions. Claudin-4 up-regulation was strongly correlated with DNA hypomethylation in both gastric tissues and gastric cancer cells. Moreover, loss of repressive histone methylations and gain of active histone modifications were associated with CLDN4 overexpression in gastric cancer cells. Interestingly, CLDN4 repression could be markedly derepressed by combined treatments that simultaneously target both histone modifications and DNA demethylation in CLDN4-hypermethylated cells, whereas concomitant changes in histone methylations and acetylations are required for CLDN4 induction in CLDN4-repressed cells with low DNA methylation. Taken together, this study reveals that membranous claudin-4 expression is associated with gastric cancer progression as well as it is an independent positive prognosis marker in gastric carcinoma. Furthermore, our findings suggest that epigenetic derepression may be a possible mechanism underlying CLDN4 overexpression in gastric cancer and that claudin-4 may have potential as a promising target for the treatment of gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 101. doi:10.1158/1538-7445.AM2011-101

Pancreatic cancer is one of the deadliest human tumors and, due to lack of effective therapeutic agents and diagnostic tools, has a poor prognosis with approximately a five-year survival rate of less than 5%. This disease is characterized by early metastatic spread but the process of tumor cell dissemination is largely unknown. Recently, we have shown that a novel soluble protein named PAUF (Pancreatic Adenocarcinoma Up-regulated Factor) plays an important role in the metastasis and progression of the disease. The expression of CXCR4 is induced by PAUF, implicating a role of PAUF in the organ specific metastasis of pancreatic cancer. The tumorigenic and metastatic activity of PAUF suggests that PAUF could be a target for pancreatic cancer immunotherapy. To examine the therapeutic potential of PAUF, a human monoclonal antibody against PAUF (8F3) has been generated. The 8F3 antibody exerted inhibition of migration, invasion, adhesion and proliferation of pancreatic cancer cells in vitro. In combination with gemcitabine, 8F3 showed additive effect on reduction of cell growth. In orthotopically xenografted mouse model, these anti-tumor activities of 8F3 converted to growth retardation and repression of metastasis. The level of PAUF in tumor and serum from 8F3 treated mouse was lower than that of control mouse. The immunohistochemical analysis revealed that the treatment of 8F3 inhibited the proliferation and CXCR4 expression in both primary and metastasized tumors. The 8F3 antibody also alters intracellular signaling which was induced by PAUF. We will also discuss about the effect of 8F3 in tumor microenvironment, especially recruitment of immune cells. Collectively, these findings indicate that PAUF has a therapeutic potential for treatment of human pancreatic cancer and the 8F3 antibody can be developed for therapeutic agent for this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4406.