Xiaoren Zhang

NF-κB Signaling Pathway, Inflammation and Colorectal Cancer

There is growing evidence for a connection between inflammation and tumor development, and the nuclear factor kappa B (NF-κB), a proinflammatory transcription factor, is hypothesized to promote tumorigenesis. Although the genetic evidence for the hypothesis has been lacking, recent papers have lent credence to this hypothesis. It has been reported that constitutive NF-κB activation in inflammatory bowel diseases (IBDs) increases risk of colorectal cancer (CRC) in the patients with the number of years of active disease. NF-κB activation might induce cellular transformation, mediate cellular proliferation, prevent the elimination of pre-neoplastic and fully malignant cells by up-regulating the anti-apoptosis proteins. Furthermore, NF-κB may contribute to the progression of CRC by regulating the expression of diverse target genes that are involved in cell proliferation (Cyclin D1), angiogenesis (VEGF, IL-8, COX2), and metastasis (MMP9). These findings implicate NF-κB inhibition as an important therapeutic target in CRC. However, due to lack of knowledge about the specific roles of different NF-κB subunits in different stage of carcinogenesis, and compounds to block specific subunits of NF-κB family, it will be a long time before the coming of targeting NF-κB in CRC therapy.

Reciprocal expression of TRAIL and CD95L in Th1 and Th2 cells: role of apoptosis in T helper subset differentiation.

AbstractUpon activation, naïve T helper cells can differentiate into two major distinct subsets, T helper 1 (Th1) and T helper 2 (Th2), as defined by their effector functions and cytokine secretion patterns. Cytokine milieu and costimulatory molecules have been shown to play an essential role in determining T helper differentiation. However, it is still unclear how the effects of signals of costimulatory molecules and cytokines are exerted during T helper differentiation. We show evidence suggesting that while cytokine signals initiate the differentiation program, the selective action of death effectors determines the end point balance of differentiating T helper subsets. We examined the expression of TNF-related apoptosis-inducing ligand (TRAIL) and CD95L in cloned and in vitro differentiated Th1 and Th2 cells. We found that activation-induced expression of TRAIL is exclusively observed in Th2 clones and primary T helper cells differentiated under the Th2 condition, while the expression of CD95L is mainly in Th1 cells. Furthermore, these two subsets exhibit distinct susceptibilities to TRAIL- and CD95L-mediated apoptosis. Th2 cells are more resistant to either TRAIL- or CD95L-induced apoptosis than Th1 cells. More importantly, both Th1 and Th2 cells could induce apoptosis in labeled Th1 but not Th2 cells. Blocking TRAIL and CD95L significantly enhance IFN-γ production in vitro. Likewise, young MRL/MpJ-lpr/lpr mice also showed more Th1 response to ovalbumin immunization as compared to MRL/MpJ+/+. Therefore, apoptosis mediated by CD95L and TRAIL is critical in determining the fate of differentiating T helper cells.

An Osteopontin‐Integrin Interaction Plays a Critical Role in Directing Adipogenesis and Osteogenesis by Mesenchymal Stem Cells

An imbalance between normal adipogenesis and osteogenesis by mesenchymal stem cells (MSCs) has been shown to be related to various human metabolic diseases, such as obesity and osteoporosis; however, the underlying mechanisms remain elusive. We found that the interaction between osteopontin (OPN), an arginine‐glycine‐aspartate‐containing glycoprotein, and integrin αv/β1 plays a critical role in the lineage determination of MSCs. Although OPN is a well‐established marker during osteogenesis, its role in MSC differentiation is still unknown. Our study reveals that blockade of OPN function promoted robust adipogenic differentiation, while inhibiting osteogenic differentiation. Re‐expression of OPN restored a normal balance between adipogenesis and osteogenesis in OPN−/− MSCs. Retarded bone formation by OPN−/− MSCs was also verified by in vivo implantation with hydroxyapatite‐tricalcium phosphate, a bone‐forming matrix. The role of extracellular OPN in MSC differentiation was further demonstrated by supplementation and neutralization of OPN. Blocking well‐known OPN receptors integrin αv/β1 but not CD44 also affected MSC differentiation. Further studies revealed that OPN inhibits the C/EBPs signaling pathway through integrin αv/β1. Consistent with these in vitro results, OPN−/− mice had a higher fat to total body weight ratio than did wild‐type mice. Therefore, our study demonstrates a novel role for OPN‐integrin αv/β1 in regulating MSC differentiation. Stem Cells 2014;32:327–337

The role of activation-induced cell death in the differentiation of T-helper-cell subsets

Activation-induced cell death (AICD) has been demonstrated in T-cell hybridomas, immature thymocytes, and activated mature T cells. However, the molecular mechanisms of AICD and its physiological role in T-helper-cell differentiation remain uncertain. Recently, we have shown that Th1 and Th2 cells have distinct mechanisms of AICD. Our findings suggest that signaling from cytokines initiates the differentiation program, but that the selective action of death effectors determines the fate of differentiating T-helper cells, and thus, the ultimate balance between T-helper subpopulations. Among T cells, activation-induced expression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is observed exclusively in Th2 clones and primary T-helper cells differentiated under Th2 conditions, while the expression of CD95L (Fas ligand) occurs mainly in Th1 cells. Furthermore, Th1 cells are more susceptible than Th2 cells to apoptosis induced through either TRAIL or CD95L, and radiolabeled Th1 cells can be induced into apoptosis via fratricide by both Th1 and Th2 cells, while Th2 cells are spared. The pan-caspase inhibitor, z-VAD, prevents AICD in Th1 cells, but not Th2 cells, indicating different mechanisms of AICD in each T-helper subtype. Antibody blockade of TRAIL and CD95L significantly boosts interferon-γ (IFN-γ) production in vitro. Also, young mice with mutant CD95 (MRL/MpJ-lpr/lpr) have a stronger Th1 response to ovalbumin immunization than do controls. We conclude that apoptosis mediated by CD95L and TRAIL is critical in the selective removal of differentiating T helper cells.

The histone H3 lysine-27 demethylase Jmjd3 plays a critical role in specific regulation of Th17 cell differentiation

Interleukin (IL) 17-producing T helper (Th17) cells play critical roles in the clearance of extracellular bacteria and fungi as well as the pathogenesis of various autoimmune diseases, such as multiple sclerosis, psoriasis, and ulcerative colitis. Although a global transcriptional regulatory network of Th17 cell differentiation has been mapped recently, the participation of epigenetic modifications in the differentiation process has yet to be elucidated. We demonstrated here that histone H3 lysine-27 (H3K27) demethylation, predominantly mediated by the H3K27 demethylase Jmjd3, crucially regulated Th17 cell differentiation. Activation of naïve CD4(+) T cells immediately induced high expression of Jmjd3. Genetic depletion of Jmjd3 in CD4(+) T cells specifically impaired Th17 cell differentiation both in vitro and in vivo. Ectopic expression of Jmjd3 largely rescued the impaired differentiation of Th17 cells in vitro in Jmjd3-deficient CD4(+) T cells. Importantly, Jmjd3-deficient mice were resistant to the induction of experimental autoimmune encephalomyelitis (EAE). Furthermore, inhibition of the H3K27 demethylase activity with the specific inhibitor GSK-J4 dramatically suppressed Th17 cell differentiation in vitro. At the molecular level, Jmjd3 directly bound to and reduced the level of H3K27 trimethylation (me3) at the genomic sites of Rorc, which encodes the master Th17 transcription factor Rorγt, and Th17 cytokine genes such as Il17, Il17f, and Il22. Therefore, our studies established a critical role of Jmjd3-mediated H3K27 demethylation in Th17 cell differentiation and suggest that Jmjd3 can be a novel therapeutic target for suppressing autoimmune responses.

The IκB family member Bcl-3 stabilizes c-Myc in colorectal cancer

Dear Editor, The proto-oncogene c-myc has been thought to play a critical role during the tumor-initiating process in multiple human cancers. Among others, colorectal cancer (CRC) is particularly associated with deregulated expression of c-Myc (Meyer and Penn, 2008; Wilkins and Sansom, 2008). Physiologically, Myc mRNA and protein levels are tightly regulated, and the Myc protein is highly unstable. The high levels of Myc protein in human CRC could be attributed to the altered Myc turnover and aberrant transcriptional activation of the myc genes (Ikegaki et al., 1986; Welcker and Clurman, 2008). The atypical member of the IkB family Bcl-3 can bind to p50 and p52 homodimers on DNA, thereby positively or negatively regulating the expression of NF-kB target genes, depending on the context (Fujita et al., 1993; Wang et al., 2012). Recently, high levels of Bcl-3 have been noted in a variety of solid cancers including CRC (Puvvada et al., 2010; Maldonado and Melendez-Zajgla, 2011). However, the function of Bcl-3 in colorectal tumorigenesis remains to be elucidated. We found that human CRC tissues exhibited increased levels of Bcl-3 compared with colorectal normal tissues (Figure 1A and B). In order to explore the role of Bcl-3 in human colorectal tumorigenesis, we transduced the human CRC cell line HCT116 with a teton lentiviral vector containing the shRNA against bcl-3 gene to establish the doxycycline (DOX)-inducible Bcl-3 knockdown cell line (HCT116/shBcl-3) (Supplementary Figure S1A). The in vitro cell growth was significantly slower upon Bcl-3 knockdown in HCT116 cells (Figure 1C and Supplementary Figure S2A). Bcl-3 knockdown also significantly suppressed the ability of HCT116 cells to form colonies in plate and soft agar (Supplementary Figure S2B and C). The inhibition was associated with a block in the G1/S transition of cell cycle (Supplementary Figure S2D). The inhibitory effects of Bcl-3 knockdown on in vitro CRC cell growth were also observed in mouse CRC cell line CT26 WT (Supplementary Figures S1B, S3A–C), excluding that the growth inhibition was due to the off-target effects of Bcl-3 knockdown. These results indicate that Bcl-3 knockdown suppresses colorectal tumor cell growth in vitro. To elucidate the mechanism by which Bcl-3 regulated the cell cycle, we compared the expressions of cell cycle-related genes between wild-type and Bcl-3 knockdown cells. We found that the level of c-Myc protein, but not the mRNA, was markedly decreased upon Bcl-3 knockdown. Accordingly, p21, which is negatively regulated by c-Myc, was increased. Bcl-3 knockdown, however, did not significantly affect the expression of other genes such as cyclin D1, cyclin E1, Skp2, p27, and IL-6 (Figure 1D and Supplementary Figure S4A). This suggests that reduced c-Myc protein level is associated with cell growth inhibition by Bcl-3 knockdown. We over-expressed c-Myc in Bcl-3 knockdown HCT116 cells, and found that the inhibited cell growth by Bcl-3 knockdown could be partially rescued by the over-expression of c-Myc (Figure 1E), indicating that reduced c-Myc protein contributes to reduced cell proliferation upon Bcl-3 knockdown. To verify that Bcl-3 regulates c-Myc protein level, we determined the half-life of c-Myc protein after applying the protein synthesis inhibitor cyclohemimide (CHX). c-Myc levels decreased faster in Bcl-3 knockdown cells than in control cells, while cyclin D1 levels decreased similarly in both cells (Figure 1F). Intriguingly, the inhibition of proteasomal function by MG-132 restored the decreased c-Myc protein in Bcl-3 knockdown cells to levels seen in control cells (Figure 1G). Moreover, Bcl-3 over-expression in a Bcl-3-deficient cell line significantly extended the half-life of c-Myc protein and reduced the levels of ubiquitinated c-Myc (Supplementary Figure S4B and C). The results above suggest that Bcl-3 regulates ubiquitination-mediated degradation of c-Myc. c-Myc protein stability can be differentially regulated by phosphorylation at threonine 58 (Thr58) and at serine 62 (Ser62). The phosphorylation at Thr58 by GSK-3 leads to degradation of c-Myc, while the phosphorylation at Ser62 likely mediated by ERK1/2 is required for RAS-induced stabilization of the c-Myc protein (Sears et al., 1999, 2000). In this study, we did not observe any notable difference on AKT phosphorylation or the expression of its targets such as p27 and cyclin E1 upon the absence of Bcl-3 in HCT116 cells. By contrast, we consistently found reduced levels of c-Myc, p-c-Myc at Ser62 and p-ERK1/2 upon Bcl-3 knockdown (Figure 1H and Supplementary Figure S5). Collectively, our data indicate that Bcl-3 may stabilize c-Myc protein by enhancing the ERK1/2-mediated phosphorylation of c-Myc at Ser62. Cell growth inhibition in vitro by Bcl-3 knockdown prompted us to investigate the effect of Bcl-3 on tumor cell growth in vivo. Bcl-3 knockdown induced by Dox significantly inhibited tumor cell growth in vivo when compared with tumor growth in control mice without Dox treatment (Figure 1I–K). There were significantly fewer Ki67 positive cells in xenograft tumors of Bcl-3 knockdown cells compared with controls (Supplementary Figure S6). We also noted decreased levels of c-Myc protein and ERK phosphorylation, but not c-myc mRNA, in xenograft tumor tissue grown under Bcl-3 knockdown conditions (Figure 1L and Supplementary Figure S7). Similar inhibitory effects of Bcl-3 280 | Journal of Molecular Cell Biology (2013), 5, 280–282 doi:10.1093/jmcb/mjt020 Published online June 20, 2013

miR-449a inhibits colorectal cancer progression by targeting SATB2

miR-449a has been reported to act as a tumor suppressor in several cancers, however, it is controversial whether it inhibits tumor growth in colorectal cancer. The mechanisms underlying its expression and functions in colorectal cancers are still largely unknown. SATB2 is a sensitive and specific marker for CRC diagnosis. However, the mechanisms by which the expression and functions of SATB2 are regulated still remain to be clarified. We investigated the expression and functional significance of miR-449a and SATB2 and the mechanisms of their dysregulation in human CRC cells. miR-449a overexpression or SATB2 depletion inhibited tumor growth and promoted apoptosis in colorectal tumor cells in vitro and in xenograft mouse model, partially by downregulating SATB2. Expression of miR-449a was increased epigenetically via knocking down their targets, particularly SATB2. miR-449a was downregulated and STAB2 expression was upregulated in human CRCs. Their expressions were significantly associated with overall survival of CRC patients. Our findings demonstrate the existence of a miR-449a-SATB2 negative feedback loop that maintains low levels of miR-449a as well as high level of SATB2, thereby promoting CRC development.

p57Kip2 is an unrecognized DNA damage response effector molecule that functions in tumor suppression and chemoresistance

The DNA damage response (DDR) helps to maintain genome integrity, suppress tumorigenesis and mediate the radiotherapeutic and chemotherapeutic effects on cancer. Here we report that p57Kip2, a cyclin-dependent kinase (CDK) inhibitor implicated in the development of tumor-prone Beckwith–Wiedemann syndrome, is an effector molecule of the DNA-damage response. Genotoxic stress induces p57Kip2 expression via the bone morphogenetic protein-Smad1 and Atm-p38MAPK-Atf2 pathways in p53-proficient or -deficient cells and requires the Smad1-Atf2 complex that facilitates their recruitment to the p57Kip2 promoter. Elevated p57Kip2 induces G1/S phase cell cycle arrest but inhibits cell death in response to DNA damage and acts in parallel with p53 to suppress cell transformation and tumor formation. p57Kip2 is also upregulated in stage I and II clinical rectal tumor samples, likely due to genome instability of precancerous and/or early cancer cells. Targeting p57Kip2 in primary rectal cancer cells and tumor models resulted in increased sensitivity to doxorubicin, suggesting that p57Kip2 has a role in chemoresistance, which is consistent with its pro-survival function. These findings place p57Kip2 in DDR and uncover molecular mechanisms by which p57Kip2 suppresses tumorigenesis and causes chemoresistance.

Established Thymic Epithelial Progenitor/Stem Cell-Like Cell Lines Differentiate into Mature Thymic Epithelial Cells and Support T Cell Development

Common thymic epithelial progenitor/stem cells (TEPCs) differentiate into cortical and medullary thymic epithelial cells (TECs), which are required for the development and selection of thymocytes. Mature TEC lines have been widely established. However, the establishment of TEPC lines is rarely reported. Here we describe the establishment of thymic epithelial stomal cell lines, named TSCs, from fetal thymus. TSCs express some of the markers present on tissue progenitor/stem cells such as Sca-1. Gene expression profiling verifies the thymic identity of TSCs. RANK stimulation of these cells induces expression of autoimmune regulator (Aire) and Aire-dependent tissue-restricted antigens (TRAs) in TSCs in vitro. TSCs could be differentiated into medullary thymic epithelial cell-like cells with exogenously expressed NF-κB subunits RelB and p52. Importantly, upon transplantation under the kidney capsules of nude mice, TSCs are able to differentiate into mature TEC-like cells that can support some limited development of T cells in vivo. These findings suggest that the TSC lines we established bear some characteristics of TEPC cells and are able to differentiate into functional TEC-like cells in vitro and in vivo. The cloned TEPC-like cell lines may provide useful tools to study the differentiation of mature TEC cells from precursors.

Bcl-3 regulates TGFβ signaling by stabilizing Smad3 during breast cancer pulmonary metastasis.

Transforming growth factor beta (TGFβ) signaling in breast cancer is selectively associated with pulmonary metastasis. However, the underlying mechanisms remain unclear. Here we show that Bcl-3, a member of the IκB family, serves as a critical regulator in TGFβ signaling to modulate breast cancer pulmonary metastasis. Bcl-3 expression was significantly associated with metastasis-free survival in breast cancer patients. Bcl-3 deletion inhibited the migration and invasion of breast cancer cells in vitro, as well as breast cancer lung metastasis in vivo. Bcl-3 was required for the expression of downstream TGFβ signaling genes that are involved in breast cancer lung metastasis. Bcl-3 knockdown enhanced the degradation of Smad3 but not Smad2 following TGFβ treatment. Bcl-3 could bind to Smad3 and prevent the ubiquitination and degradation of Smad3 protein. These results indicate that Bcl-3 serves as a promising target to prevent breast tumor lung metastasis.