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.

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CD44 is a single-pass cell surface glycoprotein that is distinguished as the first molecule used to identify cancer stem cells in solid tumors based on its expression. In this regard, the CD44high cell population demonstrates not only the ability to regenerate a heterogeneous tumor, but also the ability to self-regenerate when transplanted into immune-deficient mice. However, the exact role of CD44 in cancer stem cells remains unclear in part because CD44 exists in various isoforms due to alternative splicing.Methods: Gain- and loss-of-function methods in different models were used to investigate the effects of CD44 on breast cancer stemness. Cancer stemness was analyzed by detecting SOX2, OCT4 and NANOG expression, ALDH activity, side population (SP) and sphere formation. Glucose consumption, lactate secretion and reactive oxygen species (ROS) levels were detected to assess glycolysis. Western blot, immunohistochemical staining, ELISA and TCGA dataset analysis were performed to determine the association of CD44ICD and PFKFB4 with clinical cases. A PFKFB4 inhibitor, 5MPN, was used in a xenograft model to inhibit breast cancer development.Results: In this report, we found that the shortest CD44 isoform (CD44s) inhibits breast cancer stemness, whereas the cleaved product of CD44 (CD44ICD) promotes breast cancer stemness. Furthermore, CD44ICD interacts with CREB and binds to the promoter region of PFKFB4, thereby regulating PFKFB4 transcription and expression. The resultant PFKFB4 expression facilitates the glycolysis pathway (vis-à-vis oxidative phosphorylation) and promotes stemness of breast cancer. In addition, we found that CD44ICD and PFKFB4 expressions are generally up-regulated in the tumor portion of breast cancer patient samples. Most importantly, we found that 5MPN (a selective inhibitor of PFKFB4) suppresses CD44ICD-induced tumor development.Conclusion: CD44ICD promotes breast cancer stemness via PFKFB4-mediated glycolysis, and therapies that target PFKFB4 (e.g., 5MPN therapy) may lead to improved outcomes for cancer patients.

Bacteria/virus-induced chronic inflammation is involved in both tumor initiation and tumor progression. Toll-like receptor 4 (TLR4) has been implicated in the development of several types of cancer. In this study, we explored the impact of TLR4 activation by lipopolysaccharide (LPS) on breast cancer metastasis and associated signaling molecules. We first examined TLR4 expression levels in breast tissue using a human breast tissue microarray and breast cell lines. We then studied the role of TLR4 activation by LPS stimulation in breast cancer metastasis using both in vitro and in vivo models. Finally, we investigated signaling molecules involved in the process using Western blotting and fluorescent immunohistochemistry staining. The results showed that TLR4 expression levels increased in breast cancer tissue compared to normal breast tissue. In addition, our results also showed that TLR4 pathway activation by LPS stimulation in MCF7 and MDA-MB-231 breast cancer cells caused the following actions: (1) promotes migration of breast cancer cells, (2) triggers the β-catenin signaling pathway via PI3K/Akt/GSK3β, and (3) promotes transcription of downstream β-catenin target genes leading to breast cancer metastasis. This study substantiates and further extends the relationship between TLR4 activation by LPS and breast cancer using both in vitro and in vivo models. The results suggest that the Akt/GSK3β/β-catenin signal transduction pathway may serve as a viable clinical treatment target in breast cancer. Anat Rec, 300:1219-1229, 2017. © 2017 Wiley Periodicals, Inc.

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.

As an important component of tumor microenvrionment, CD4+CD25+ Tregs reduce antitumor immunity, promote angiogenesis and metastasis in breast cancer. However, their function in regulating the “sternness” of tumor cells and the communication between Tregs and cancer stem cells (CSCs) remain elusive. Here, we disclose that the primarily cultured Tregs isolated from breast-tumor-bearing Foxp3-EGFP mouse upregulate the stemness property of breast cancer cells. Tregs increased the side-population and the Aldehyde dehydrogenase-bright population of mouse breast cancer cells, promoted their sphere formation in a paracrine manner, and enhanced the expression of stemness genes, such as Sox2 and so forth. In addition, Tregs increased tumorigenesis, metastasis and chemoresistance of breast cancer cells. Furthermore, Sox2-overexpression tumor cells acitivated NF-κB-CCL1 signaling to recruit Tregs through reducing the binding of H3K27Me3 on promoter regions of p65 and Ccl1. These findings reveal the functional interaction between Tregs and CSCs and indicate that targeting on the communication between them is a promising strategy in breast cancer therapy.

Ferropotsis is among the most important mechanisms of cancer suppression, which could be harnessed for cancer therapy. However, no natural small-molecule compounds with cancer inhibitory activity have been identified to date. In the present study, we reported the discovery of a novel ferroptosis inducer, talaroconvolutin A (TalaA), and the underlying molecular mechanism. We discovered that TalaA killed colorectal cancer cells in dose-dependent and time-dependent manners. Interestingly, TalaA did not induce apoptosis, but strongly triggered ferroptosis. Notably, TalaA was significantly more effective than erastin (a well-known ferroptosis inducer) in suppressing colorectal cancer cells via ferroptosis. We revealed a dual mechanism of TalaA’ action against cancer. On the one hand, TalaA considerably increased reactive oxygen species levels to a certain threshold, the exceeding of which induced ferroptosis. On the other hand, this compound downregulated the expression of the channel protein solute carrier family 7 member 11 (SLC7A11) but upregulated arachidonate lipoxygenase 3 (ALOXE3), promoting ferroptosis. Furthermore, in vivo experiments in mice evidenced that TalaA effectively suppressed the growth of xenografted colorectal cancer cells without obvious liver and kidney toxicities. The findings of this study indicated that TalaA could be a new potential powerful drug candidate for colorectal cancer therapy due to its outstanding ability to kill colorectal cancer cells via ferroptosis induction.

Since reports on the clinical significance of legumain in cancer have shown inconsistent results, we systematically evaluated clinical indicators of legumain in cancer. We searched the Cochrane Library, PubMed, Embase, and EBSCO databases and the Wangfang and CNKI databases in China by using “legumain” and (“neoplasms” OR “cancer”) as search terms. We included case-controlled studies of legumain and cancer. The quality of the studies was evaluated by using Lichtenstein’s guidelines, and valid data was extracted for analysis. In total, 10 articles were included in this study. Meta-analysis showed that legumain was overexpressed in cancer compared with in normal tissue and was higher in stage III–IV disease than in I–II disease. Moreover, legumain overexpression was correlated with poor prognosis and clinical stage. Furthermore, Cancer Genome Atlas data showed that among patients with rectal cancer, those with tumors overexpressing legumain had shorter overall survival than those in the low expression group (P < 0.05). Legumain appears to be involved in tumor development and deterioration; thus, it can potentially be developed into both a marker for monitoring and diagnosing tumors and a therapeutic target.