Antao Chang

ATF4 is directly recruited by TLR4 signaling and positively regulates TLR4-trigged cytokine production in human monocytes.

Toll-like receptors (TLRs) are sentinels of the host defense system, which recognize a large number of microbial pathogens. The host defense system may be inefficient or inflammatory diseases may develop if microbial recognition by TLRs and subsequent TLR-triggered cytokine production are deregulated. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in several pathophysiological processes. In this report, we found that ATF4 is also involved in the TLR-mediated innate immune response, which participates in TLR4 signal transduction and mediates the secretion of a variety of cytokines. We observed that ATF4 is activated and translocates to the nucleus following lipopolysaccharide (LPS) stimulation via the TLR4-MyD88-dependent pathway. Additionally, a cytokine array assay showed that some key inflammatory cytokines, such as IL-6, IL-8 and RANTES, are positively regulated by ATF4. We also demonstrate that c-Jun directly binds to ATF4, thereby promoting the secretion of inflammatory cytokines. Taken together, these results indicate that ATF4 acts as a positive regulator in TLR4-triggered cytokine production.

Yes-associated protein (YAP) increases chemosensitivity of hepatocellular carcinoma cells by modulation of p53

The yes-associated protein (YAP) transcription co-activator has been reported either as an oncogene candidate or a tumor suppressor. Liver tissue chips revealed that about 51.4% human hepatocellular carcinoma (HCC) samples express YAP and 32.9% HCC samples express phosphorylated YAP. In this study, we found that chemotherapy increased YAP protein expression and nuclear translocation in HepG2 cells, as well as p53 protein expression and nuclear translocation. However, little is known about YAP functions during chemotherapy. Our results show that overexpression of YAP increases chemosensitivity of HepG2 cells during chemotherapy. Dominant negative transfection of Flag-S94A (TEAD binding domain mutant) or Flag-W1W2 (WW domain mutant) to HepG2 cells decreases p53 expression/ nuclear translocation and chemosensitivity when compared with control HepG2 cells. Furthermore, rescue transfection of Flag-5SA-S94A or Flag-5SA-W1W2, respectively to HepG2 cells regains p53 expression/nuclear translocation and chemosensitivity. These results indicate that YAP promotes chemosensitivity by modulating p53 during chemotherapy and both TEAD and WW binding domains are required for YAP-mediated p53 function. ChIP assay results also indicated that YAP binds directly to the p53 promoter to improve its expression. In addition, p53 could positively feedback YAP expression through binding to the YAP promoter. Taken together, our current data indicate that YAP functions as a tumor suppressor that enhances apoptosis by modulating p53 during chemotherapy.

TIFA suppresses hepatocellular carcinoma progression via MALT1-dependent and -independent signaling pathways

TIFA, also called T2BP, was first identified using yeast two-hybrid screening. Our previous work showed that TIFA suppresses hepatocellular carcinoma (HCC) progression via apoptosis and cell cycle arrest. However, the mechanism by which this TIFA suppression occurs remains unclear. Here we demonstrated that TIFA-induced apoptosis demonstrates two distinct time patterns (i.e., at 48 h and >7 days) when TIFA reconstitution occurs. Moreover, we found that MALT1 (a competitor of TIFA) plays a crucial role in short-duration TIFA reconstitution. In this regard, MALT1 silencing with shRNA markedly enhances TIFA-induced apoptosis in vitro and in vivo. In addition, TIFA overexpression triggers JNK and p38 activation in long-duration TIFA reconstitution through TRAF6 binding. In particular, JNK activation leads to TIFA-induced apoptosis while p38 activation governs TIFA-induced cell cycle arrest by p53-p21 signaling in vitro and in vivo. Our data suggest a novel mechanism by which TIFA suppresses HCC progression via both MALT1-dependent and MALT1-independent signaling pathways. This may provide insights into a novel targets where HCC progression may be vulnerable to clinical treatment.

Tumour-derived Interleukin 35 promotes pancreatic ductal adenocarcinoma cell extravasation and metastasis by inducing ICAM1 expression

Interleukin 35 (IL-35) is a novel member of the IL-12 family, consisting of an EBV-induced gene 3 (EBI3) subunit and a P35 subunit. IL-35 is an immune-suppressive cytokine mainly produced by regulatory T cells. However, the role of IL-35 in cancer metastasis and progression is not well understood. Here we demonstrate that IL-35 is overexpressed in human pancreatic ductal adenocarcinoma (PDAC) tissues, and that IL-35 overexpression is associated with poor prognosis in PDAC patients. IL-35 has critical roles in PDAC cell extravasation and metastasis by facilitating the adhesion to endothelial cells and transendothelial extravasation. Mechanistically, IL-35 promotes ICAM1 overexpression through a GP130-STAT1 signalling pathway, which facilitates endothelial adhesion and transendothelial migration via an ICAM1–fibrinogen–ICAM1 bridge. In an orthotopic xenograft model, IL-35 promotes spontaneous pancreatic cancer metastasis in an ICAM1-dependent manner. Together, our results indicate additional functions of IL-35 in promoting PDAC metastasis through mediating ICAM1 expression.

IKKβ Enforces a LIN28B/TCF7L2 Positive Feedback Loop That Promotes Cancer Cell Stemness and Metastasis

Considerable evidence suggests that proinflammatory pathways drive self-renewal of cancer stem-like cells (CSC), but the underlying mechanisms remain mainly undefined. Here we report that the let7 repressor LIN28B and its regulator IKBKB (IKKβ) sustain cancer cell stemness by interacting with the Wnt/TCF7L2 (TCF4) signaling pathway to promote cancer progression. We found that LIN28B expression correlated with clinical progression and stemness marker expression in breast cancer patients. Functional studies demonstrated that the stemness properties of LIN28B-expressing human breast and lung cancer cells were enhanced by IKKβ, whereas loss of LIN28B abolished stemness properties in these settings. These phenomena were driven through interactions with TCF7L2, which enhanced LIN28B expression by direct binding to intron 1 of the LIN28B gene, which in turn promoted TCF7L2 mRNA translation through a positive feedback loop. Notably, RNAi-mediated silencing of LIN28B or pharmacologic inhibition of IKKβ was sufficient to suppress primary and metastatic tumor growth in vivo. Together, our results establish the LIN28B/TCF7L2 interaction loop as a central mediator of cancer stemness driven by proinflammatory processes during progression and metastasis, possibly offering a new therapeutic target for generalized interventions in advanced cancers.

Activating transcription factor 4 increases chemotherapeutics resistance of human hepatocellular carcinoma

It has been reported that activating transcription factor 4 (ATF4) increases the processes of tumor growth, metastasis and drug resistance. However, the role played by ATF4 in chemoresistance of hepatocellular carcinoma (HCC) remains unknown. Clarification of this role of ATF4 in HCC could greatly benefit the efficacy of clinical treatment of HCC. In this study, we found that ATF4 was overexpressed in about 50.7% of HCC tissues. In fact knockdown of ATF4 significantly increased the cytotoxicity of cisplatin in both in vitro and in vivo assays, while overexpression of this molecule dramatically decreased the sensitivity of HCC cell lines to cisplatin. Additionally, we found that synthesis of glutathione was significantly reduced in HCC cell lines subjected to ATF4 knockdown. Taken together, these results demonstrate that ATF4 can increase resistance to cisplatin in HCC by increased biosynthesis of glutathione, and that this may be a potent novel target for the future development of anti-HCC drugs.

TIFA, an inflammatory signaling adaptor, is tumor suppressive for liver cancer

TIFA (TNF receptor associated factor (TRAF)-interacting protein with a Forkhead-associated (FHA) domain), also called T2BP, was first identified using a yeast two-hybrid screening. TIFA contains a FHA domain, which directly binds phosphothreonine and phosphoserine, and a consensus TRAF6-binding motif. TIFA-mediated oligomerization and poly-ubiquitinylation of TRAF6 mediates signaling downstream of the Tumor necrosis factor alpha receptor 1 (TNFaR-I) and interleukin-1/Toll-like receptor 4 (TLR4) pathways. Examining TIFA expression in hepatocellular carcinoma (HCC) tissues microarrays, we noted marked decreases TIFA reactivity in tumor versus control samples. In agreement, we found that HCC cell lines show reduced TIFA expression levels versus normal liver controls. Reconstituting TIFA expression in HCC cell lines promoted two independent apoptosis signaling pathways: the induction of p53 and cell cycle arrest, and the activation of caspase-8 and caspase-3. In contrast, the expression of a non-oligomerizing mutant of TIFA impacted cells minimally, and suppression of TIFA expression protected cells from apoptosis. Mice bearing TIFA overexpression hepatocellular xenografts develop smaller tumors versus TIFA mutant tumors; terminal deoxynucleotidyl transferase dUTP nick end labeling staining demonstrates increased cell apoptosis, and decreased proliferation, reflecting cell cycle arrest. Interestingly, p53 has a greater role in decreased proliferation than cell death, as it appeared dispensable for TIFA-induced cell killing. The findings demonstrate a novel suppressive role of TIFA in HCC progression via promotion of cell death independent of p53.

Liposomal Nanoparticles Carrying anti-IL6R Antibody to the Tumour Microenvironment Inhibit Metastasis in Two Molecular Subtypes of Breast Cancer Mouse Models

Tumour microenvironment (TME) contributes significantly towards potentiating the stemness and metastasis properties of cancer cells. IL6-Stat3 is one of the important cell signaling pathways in mediating the communication between tumour and immune cells. Here, we have systematically developed a novel anti-CD44 antibody-mediated liposomal nanoparticle delivery system loaded with anti-IL6R antibody, which could specifically target the TME of CD44+ breast cancer cells in different mouse models for triple negative and luminal breast cancer. This nanoparticle had an enhanced and specific tumour targeting efficacy with dramatic anti-tumour metastasis effects in syngeneic BALB/c mice bearing 4T1 cells as was in the syngeneic MMTV-PyMT mice. It inhibited IL6R-Stat3 signaling and moderated the TME, characterized by the reduced expression of genes encoding Stat3, Sox2, VEGFA, MMP-9 and CD206 in the breast tissues. Furthermore, this nanoparticle reduced the subgroups of Sox2+ and CD206+ cells in the lung metastatic foci, demonstrating its inhibitory effect on the lung metastatic niche for breast cancer stem cells. Taken together, the CD44 targeted liposomal nanoparticles encapsulating anti-IL6R antibody achieved a significant effect to inhibit the metastasis of breast cancer in different molecular subtypes of breast cancer mouse models. Our results shed light on the application of nanoparticle mediated cancer immune-therapy through targeting TME.

JMJD3 suppresses stem cell-like characteristics in breast cancer cells by downregulation of Oct4 independently of its demethylase activity

Epigenetic regulator JMJD3 plays an important role in both tumor progression and somatic cell reprogramming. Here, we explored the effect of JMJD3 on the stem cell-like characteristics of breast cancer and its underlying mechanism involving stemness-related transcription factor Oct4. Our data revealed that, in breast cancer cells lines and an orthotopic xenograph mouse model of breast cancer, ectopic overexpression of JMJD3 suppressed stem cell-like characteristics of breast cancer cells, whereas knockdown of JMJD3 promoted these characteristics. Oct4 mediated the suppressive effects of JMJD3 on the stemness of breast cancer cells. The inhibitory effect of JMJD3 on Oct4 was independent of demethylase activity, but mediated via degradation of PHF20. Furthermore, we applied an agonist of the vitamin D receptor, paricalcitol, and found that it induced JMJD3 in breast cancer cells. Our data showed that administration of paricalcitol suppressed stem cell-like characteristics and Oct4 expression. Taken together, JMJD3 inhibits the stem cell-like characteristics in breast cancer by suppression of stemness factor Oct4 in a PHF20-dependent manner. Administration of paricalcitol leads to upregulation of JMJD3 that suppresses Oct4 expression and the stem cell-like characteristics in breast cancer.

Inactivation of p38 MAPK contributes to stem cell-like properties of non-small cell lung cancer

Cancer stem cells (CSCs) are recognized as the major source for cancer initiation and recurrence. Yet, the mechanism by which the cancer stem cell properties are acquired and maintained in a cancer cell population is not well understood. In the current study, we observed that the level of active p38 MAPK is downregulated, while the level of the stemness marker SOX2 is upregulated in lung cancer tissues as compared to normal tissues. We further demonstrated that inactivation of p38 is a potential mechanism contributing to acquisition and maintenance of cancer stem cell properties in non-small cell lung cancer (NSCLC) cells. p38, in particular the p38γ and p38δ isoforms, suppresses the cancer stem cell properties and tumor initiating ability of NSCLC cells by promoting the ubiquitylation and degradation of stemness proteins such as SOX2, Oct4, Nanog, Klf4 and c-Myc, through MK2-mediated phosphorylation of Hsp27 that is an essential component of the proteasomal degradation machinery. In contrast, inactivation of p38 in lung cancer cells leads to upregulation of the stemness proteins, thus promoting the cancer stem cell properties of these cells. These findings have demonstrated a novel mechanism by which cancer stem cell properties are acquired and maintained in a cancer cell population, and have revealed a new function of the p38 pathway in suppressing cancer development. These studies have also identified a new pathway that can potentially serve as a target for cancer therapies aimed at eliminating CSCs.