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Cancer-associated fibroblasts (CAFs) play an essential role in supporting cancer progression. However, the details and consequent effects in response to the communication between CAFs and angiogenesis remain largely uninvestigated, especially in anticancer drug treatments. We found that cisplatin and 5-fluorouracil could induce fibroblast differentiation toward myofibroblasts via CCAAT/enhancer-binding protein delta (CEBPD) and consequently promote proliferation, migration, and in vitro tube formation of vascular endothelial cells and angiogenesis in vivo. Stromal-cell-derived factor 4 (SDF4) is responsive to anticancer drugs via CEBPD activation in CAFs and contributes to create a permissive environment for tumor cell angiogenesis and promotion of distant metastasis. Importantly, we demonstrated that SDF4 interacts with CXCR4 to trigger VEGFD expression through the activation of the ERK1/2 and p38 pathways in endothelial cells. Taken together, our novel findings support that SDF4 can be a therapeutic target in inhibition of angiogenesis for chemotherapy drug-administrated cancer patients.

Pancreatic cancer is refractory and is characterized by extensively surrounding and intratumor fibrotic reactions that are contributed by activated pancreatic stellate cells (PSCs). Herein, we show that CCAAT/enhancer-binding protein δ (CEBPD) responds to transforming growth factor-β1 (TGF-β1) through reciprocal loop regulation and that activated hypoxia inducible factor-1α (HIF-1α) further contributes to the upregulation of the hepatoma-derived growth factor (HDGF) gene. Secreted HDGF contributes to the antiapoptosis of PSCs and consequently leads to the synthesis and deposition of extracellular matrix proteins for stabilizing PSC/pancreatic cancer cell (PCC) tumor foci. This result agrees with the observation that severe stromal growth positively correlated with stromal HDGF and CEBPD expression in pancreatic cancer specimens. Collectively, the identification of the TGF-β1-activated CEBPD/HIF-1α/HDGF axis provides new insights into novel discoveries of HDGF in the antiapoptosis and profibrosis of PSCs and the outgrowth of PCCs.

Pancreatic cancer is refractory and characterized by extensively surrounding- and intra-tumor fibrotic reactions that are contributed by activated pancreatic stellate cells (PSCs). Activation of PSCs plays a pivotal role for developing fibrotic reactions to affect themselves or pancreatic cancer cells (PCCs). In the current study, we demonstrated that hepatoma-derived growth factor (HDGF) was secreted from transforming growth factor-β1 (TGF-β1)-treated PSCs. We found that HDGF contributed to anti-apoptosis of PSCs and led to synthesis and depositions of extracellular matrix proteins for stabilizing PSCs/PCCs tumor foci. CCAAT/enhancer binding protein δ (CEBPD) responds to TGF-β1 through a reciprocal loop regulation and further activated hypoxia inducible factor-1α (HIF-1α) contributed to up-regulation ofHDGFgene. It agrees with the observation that severe stromal growth positively correlated with stromal HDGF and CEBPD in pancreatic cancer specimens. Collectively, the identification of TGF-β1-activated CEBPD/HIF-1α/HDGF axis provides new insights for the novel discoveries of HDGF in anti-apoptosis and pro-fibrosis of PSCs and outgrowth of pancreatic cancer cells.

The tumor microenvironment has been suggested to participate in tumorigenesis, but the nature of the communication between cancer cells and the microenvironment, especially in response to anticancer drugs, remains obscure. We determined that activation of the CCAAT/enhancer binding protein delta (CEBPD) response to Cisplatin and 5-Fluorouracil in cancer-associated macrophages and fibroblasts contributed to the metastasis, invasion, acquired chemoresistance and stemness of cancer cells by in vitro and in vivo assays. Specifically, reporter and in vivo DNA binding assays were used to determine that Pentraxin 3 (PTX3) is a CEBPD responsive gene and serves a protumor role upon anticancer drug treatment. Finally, a PTX3 peptide inhibitor RI37 was developed and assessed the antitumor effects by in vivo assays. RI37 could function as a promising inhibitor for preventing cancer progression and the metastasis, invasion and progression of drug-resistant cancers. The identification of PTX3 provided a new insight in the interaction between host and tumor and the RI37 peptide showed a great opportunity to largely reduce the risk of invasion and metastasis of cancer and drug-resistant cancers.

In Alzheimer's disease (AD), large populations of endothelial cells undergo angiogenesis due to brain hypoxia and inflammation. Substantial evidence from epidemiologic, pathologic, and clinical reports suggests that vascular factors are critical for the pathogenesis of AD. However, the precise mechanistic correlation between inflammation and angiogenesis in AD has not been well elucidated. Prostaglandin E2 (PGE2), a key factor of the inflammatory response, has been known to promote angiogenesis. In this study, we demonstrated that PGE2 acts through EP4 receptor and protein kinase A to modulate CCAAT/enhancer-binding protein delta (CEBPD) abundance in astrocytes. Attenuated vessel formation was observed in the brains of AppTg/Cebpd(-/-) mice. We showed that miR135a was responsive to the induction of CEBPD and further negatively regulated thrombospondin 1 (THBS1) transcription by directly targeting its 3'-untranslated region (3'UTR) in astrocytes. Furthermore, conditioned media from astrocytes expressing miR135a promoted Human umbilical vein endothelial cells (HUVECs) tube-like formation, which correlated with the effects of PGE2 on angiogenesis. Our results indicated that CEBPD contributes to the repression of THBS1 transcription by activating the expression of miR135a in astrocytes following PGE2 treatment. We provided new evidence that astrocytic CEBPD increases angiogenesis during AD pathogenesis. This discovery supports the negative influence of CEBPD activation in astrocytes with respect to AD pathogenesis and implies that the CEBPD/miR135a/THBS1 axis could be a therapeutic target of AD.

Aristolochic acid (AA) is a common cause of Chinese herb nephropathy. The mechanisms involved in the pathogenesis of AA nephropathy (AAN) are intricate. One well-documented effect of AA in the kidney is its pro-fibrotic activity. Nitric oxide (NO), a messenger gas generated from l-arginine, is the product of nitric oxide synthase (NOS). NO is involved in renal hemodynamics and exerts cytoprotective effects against renal injury. In the present study, the role of NO in AAN was investigated in MES-13 cells, a glomerular mesangial cell line. NO endogenously generated by the induction of inducible nitric oxide synthase (iNOS) with lipopolysaccharide (LPS)/interferon-γ (IFN-γ) significantly downregulated connective tissue growth factor (CTGF) protein expression in MES-13 cells. AA significantly suppressed LPS/IFN-γ-induced NO production and reversed CTGF expression that was downregulated by LPS/IFN-γ. AA decreased iNOS gene and protein expressions in a concentration-dependent manner. AA caused declines in LPS/IFN-γ-induced signal transducer and activator of transcription-1α (STAT-1α) phosphorylation and interferon response factor-1 (IRF-1) mRNA expression. Furthermore, AA attenuated IκB phosphorylation and reduced NF-κB translocation to the nuclear fraction. Taken together, our data indicate that AA reversed the CTGF expression inhibited by LPS/IFN-γ treatment via suppression of NO and iNOS expressions in MES-13 cells through inhibition of the JAK/STAT-1α and NF-κB signaling pathways. NO potentially exerts antifibrotic activity by down regulation of CTGF in MES-13 cells and inhibition of the iNOS gene by AA might partially account for the fibrotic effects of AA in nephropathy.

The mycotoxin, citrinin (CTN), is a secondary metabolite of the fermented products of Monascus. The mycotoxin can either suppress or stimulate immune responses. In the present study, the immunomodulatory role of CTN in nitric oxide (NO) production, a proinflammatory mediator in the process of inflammation, was investigated. NO is well known as a mediator of immune responses. Overproduction of NO catalyzed by inducible nitric oxide synthase (iNOS) protects host cells against microbial invasion, while aberrant iNOS induction is associated with the pathophysiology of inflammatory events. Herein, we report that CTN significantly suppressed lipopolysaccharide (LPS)/interferon (IFN)-γ-induced NO production in MES-13 cells, a glomerular mesangial cell line. The percentage of NO reduction caused by CTN was far greater than that of the decline in cell viability. CTN decreased iNOS gene and protein expressions in concentration-dependent manners. CTN caused declines in LPS/IFN-γ-induced signal transducer and activator of transcription-1α (STAT-1α) phosphorylation. Furthermore, LPS/IFN-γ's induction of interferon response factor-1 (IRF-1) mRNA expression was inhibited by CTN. Moreover, CTN attenuated IκB-α phosphorylation and reduced NF-κB's translocation to the nuclear fraction. Taken together, our data indicated that CTN significantly suppressed NO and iNOS expressions in MES-13 cells via inhibition of the JAK/STAT-1α and NF-κB signaling pathways.

Reactive astrogliosis is a cellular manifestation of neuroinflammation and occurs in response to all forms and severities of the central nervous system (CNS)’s injury and disease. Both astroglial proliferation and antiapoptotic processes are aspects of astrogliosis. However, the underlying mechanism of this response remains poorly understood. In addition, little is known about why activated astrocytes are more resistant to stress and inflammation. CCAAT/enhancer binding protein delta (CEBPD) is a transcription factor found in activated astrocytes that surround β-amyloid plaques. In this study, we found that astrocytes activation was attenuated in the cortex and hippocampus of APPswe/PS1 E9 (AppTg)/Cebpd−/−mice. Furthermore, an increase in apoptotic astrocytes was observed in AppTg/Cebpd−/−mice, suggesting that CEBPD plays a functional role in enhancing the antiapoptotic ability of astrocytes. We found that Zinc Finger Protein 179 (ZNF179) was a CEBPD-regulated gene that played an antiapoptotic, but not proliferative, role in astrocytes. The transcriptions of the proapoptotic genes, insulin-like growth factor binding protein 3 (IGFBP3) and BCL2-interacting killer (BIK), were suppressed by ZNF179 via its interaction with the promyelocytic leukemia zinc finger (PLZF) protein in astrocytes. This study provides the first evidence that ZNF179, PLZF, IGFBP3, and BIK contributed to the novel CEBPD-induced antiapoptotic feature of astrocytes. Electronic supplementary material The online version of this article (doi:10.1007/s12035-014-8714-9) contains supplementary material, which is available to authorized users.

The DNA mismatch-repair (MMR) system corrects replicative errors and minimizes mutations that occur at a high rate in microsatellites. Patients with chronic inflammation or inflammation-associated cancer display microsatellite instability (MSI), indicating a possible MMR inactivation. In fact, H2O2-generated oxidative stress inactivates the MMR function and increases mutation accumulation in a reporter microsatellite. However, it remains unclear whether MSI induced by oxidative stress is preventable because of the lack of a sufficiently sensitive detection assay. Here, we developed and characterized a dual-fluorescent system, utilizing DsRed harboring the (CA)13 microsatellite as a reporter and GFP for normalization, in near-isogenic human colorectal cancer cell lines. Via flow cytometry, this reporter sensitively detected H2O2-generated oxidative microsatellite mutations in a dose-dependent manner. The reporter further revealed that glutathione or N-acetylcysteine was better than aspirin and ascorbic acid for suppressing oxidative microsatellite mutations. These two thiol compounds also partially suppressed oxidative frameshift mutations in the coding microsatellites of the hMSH6 and CHK1 genes based on a fluoresceinated PCR-based assay. MSI suppression by N-acetylcysteine appears to be mediated through reduction of oxidative frameshift mutations in the coding microsatellite of hMSH6 and protection of hMSH6 and other MMR protein levels from being decreased by H2O2. Our findings suggest a linkage between oxidative damage, MMR deficiency, and MSI. The two thiol compounds are potentially valuable for preventing inflammation-associated MSI. The dual-fluorescent reporter with improved features will facilitate identification of additional compounds that modulate MSI, which is relevant to cancer initiation and progression.

Although tumors tend to be associated with immune cells and inflammation, this immune response often fails to eliminate the cancer and instead promotes cancer progression. Tumor-associated macrophages (TAMs) fail to phagocytose tumor cells, and they also produce signals that suppress the adaptive immune response. We showed that immunosuppressive prostaglandin E₂ (PGE₂) led to the production and activity of the transcription factor CCAAT/enhancer binding protein δ (C/EBPδ) by stimulating the nucleocytoplasmic shuttling of the RNA binding protein Hu antigen R (HuR), which bound to and stabilized CEBPD mRNA in macrophages. An increase in C/EBPδ abundance in macrophages in response to PGE₂ resulted in enhanced production of the immunosuppressive cytokine interleukin-10 (IL-10) and of pentraxin 3 (PTX3), which suppresses the ability of macrophages to phagocytose tumor cells. Furthermore, conditioned medium from C/EBPδ-replete, but not C/EBPδ-deficient, macrophages inhibited the phagocytosis of tumor cells by macrophages, suggesting an autocrine mode of regulation. Immunohistochemical analysis demonstrated that the amount of cytosolic HuR protein correlated with increased C/EBPδ abundance in TAMs in malignant nasopharyngeal carcinoma. Together, these data suggest that the inflammatory PGE₂-HuR-C/EBPδ axis in macrophages promotes tumor progression by preventing the phagocytosis of tumor cells and inducing immunosuppressive cytokine production.

This lack of understanding especially impacts the design of anticancer treatments. It is a common observation in the clinic that recurrent cancers, and particularly anticancer drug-resistant cancers, are more difficult to treat because they spread faster than the original tumor. CCAAT/enhancer binding protein delta (CEBPD) was activated in M2 macrophages and myofibroblasts/cancer-associated fibroblasts (CAFs) and contributed to metastasis, invasion and recurrence of cancer cells in response to PGE2 and the anticancer drugs. We further found that pentraxin 3 (PTX3) was activated in response to CEBPD induction and involved in migration and invasion of breast cancer cells, and the same phenomenon was also observed in drug-resistant cancers. Interestingly, we found that a recombinant PTX3 protein containing the C-terminal region (pPTX3) showed an opposite tumorigenic effect to the mammalian PTX3. The results suggest that PTX3 could be a promising target for preventing the metastasis/invasion of cancers and treating drug-resistant cancers. Citation Format: Jhih-Ying Chi, Ju-Ming Wang. Pentraxin 3 is responsive to the activation of CCAAT/enhancer binding delta and promotes cancer progression in the tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 926.

Many chemotherapeutic agents damage DNA and induce DNA damage response (DDR) signaling, resulting in growth arrest or apoptosis. DNA mismatch repair (MMR) system not only maintains genomic stability but also participates in DDR signaling. MMR deficiency leads to genetic hypermutability, manifested as microsatellite instability (MSI), and contributes to cancer pathogenesis and drug resistance. MSI has been linked to chemotherapy and therapy-related secondary cancer. Therefore, it is important to prevent drug-induced MSI to increase the effectiveness of chemotherapy while reducing cancer mortality. The purpose of this study is to characterize the MSI-inducing ability of individual chemotherapeutic agents and identify small compounds that suppress drug-induced MSI. In a human colorectal cancer cell model, all tested drugs individually induced the instability of reporter and endogenous microsatellites while reducing steady-state levels of certain MMR proteins. Relatively, oxaliplatin induced a higher MSI frequency than many tested drugs such as 5-FU. It is known that MMR-deficient tumors are resistant to 5-FU and not oxaliplatin. We then screened a commercially available library for small compounds that suppress oxaliplatin-induced MSI using a dual-fluorescent MSI reporter. We found that the majority of compounds were capable of reducing oxaliplatin-induced MSI. However, they also undesirably decreased the cytotoxicity of oxaliplatin. On the other hand, a small number of compounds such as I-6 and II-13 suppressed oxaliplatin-induced MSI with or without an improvement of drug cytotoxicity. Relative to oxaliplatin alone, co-treatment of 1 μM oxaliplatin with 6-10 μM compound II-13 decreased drug-induced MSI frequency by 45-70% without significantly affecting the cytotoxicity of oxaliplatin. Co-treatment of oxaliplatin with 4-10 μM compound I-6, however, decreased both drug-induced MSI and cell numbers by 50-78% in a dose-dependent manner. To our surprise, compounds such as II-13 suppressed MSI induced by oxaliplatin but not by 5-FU. In conclusion, individual drugs displayed different MSI-inducing abilities whereas MSI-suppressing compounds appear to be drug-specific. Further identification and validation of MSI-suppressing compounds specific for 5-FU or leucovorin, for example, would improve the effectiveness of combination chemotherapy such as FOLFOX. Citation Format: Christina L. Chang, Li-Yan Huang, Chang-Lin Wu, Jhih-Ying Chi. Improving the effectiveness of chemotherapy by combining compounds that suppress drug-induced microsatellite instability. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3873. doi:10.1158/1538-7445.AM2015-3873

DNA mismatch repair (MMR) system corrects replicative errors, especially in microsatellites, and minimizes mutations caused by DNA-damaging agents. Patients with chronic and cancer-associated inflammation display microsatellite instability (MSI), indicative of an inactivated MMR system. MMR-deficient patients also display frameshift mutations in coding microsatellites of many genes, including oncogenes and tumor suppressors. We previously reported that oxidative stress, which occurs in the inflammatory settings, not only inactivates the MMR function but also increases accumulation of frameshift mutations. However, it remains unclear whether oxidative frameshift mutations are preventable due to the lack of a sufficiently sensitive detection assay. Here, we developed and characterized a dual-fluorescent system, utilizing RFP harboring the (CA)13 microsatellite as a reporter and GFP for normalization, in near-isogeneic human colorectal cancer cell lines. By flow cytometry, the new reporter sensitively detected a low level of H2O2-induced frameshift mutations in MMR-proficient cells. Without a functional MMR system, 0.5 mM H2O2 increased frameshift mutations by approximately 10 folds in the cells. In MMR-deficient cells, our reporter revealed that 5 mM glutathione or N-acetylcysteine suppressed oxidative frameshift mutations by approximately 75% or 90% respectively, without affecting cell viability. In contrast, 0.5 mM ascorbic acid augmented H2O2-induced frameshift mutations by two folds, whereas 1 mM aspirin had no effects. Glutathione and N-acetylcysteine also partially suppressed oxidative frameshift mutations in coding microsatellites of endogenous hMSH6 and CHK1 genes, determined by a fluorescinated PCR-based assay. The new utility of these thiol compounds is potentially valuable for preventing oxidative frameshift mutations in the inflammatory settings. The dual-fluorescent reporter system with improved sensitivity will facilitate identification of additional compounds that modulate frameshift mutations relevant to cancer initiation and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2543. doi:1538-7445.AM2012-2543

Manifested as microsatellite instability (MSI), DNA mismatch repair (MMR) deficiency is known to contribute to colorectal cancer pathogenesis and drug resistance. Growing evidence indicates that MMR deficiency is also widespread among secondary cancers in patients who have received chemotherapy. It is unclear what chemotherapeutic agents cause MMR deficiency. In this study, we aim at identifying the MSI-inducing anti-cancer drugs. To facilitate the identification, we developed an in-vivo dual fluorescent reporter system in a human colorectal cancer cell model using RFP that contains a microsatellite as the MSI reporter and GFP as the cell label. Based on flow cytometric analysis, we found that test anti-cancer drugs increased the MSI frequency in a dose-dependent manner. This was confirmed by fluorescent microscopy. With the NCI recommended markers, we detected a low frequency of MSI in cells after treated with a couple, but not all, of test anti-cancer drugs. Our cell-based MSI reporter assay will facilitate large scale screens of MSI-inducing and MSI-reducing drugs/compounds, which might provide treatment strategies for controlling therapy-related secondary cancers. 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 3899. doi:10.1158/1538-7445.AM2011-3899