Inhibition of Nogo-B reduces the progression of pancreatic cancer by regulation NF-κB/GLUT1 and SREBP1 pathways.

Pancreatic cancer (PC) is a lethal disease and associated with metabolism dysregulation. Nogo-B is related to multiple metabolic related diseases and types of cancers. However, the role of Nogo-B in PC remains unknown. In vitro, we showed that cell viability and migration was largely reduced in Nogo-B knockout or knockdown cells, while enhanced by Nogo-B overexpression. Consistently, orthotopic tumor and metastasis was reduced in global Nogo knockout mice. Furthermore, we indicated that glucose enhanced cell proliferation was associated to the elevation expression of Nogo-B and nuclear factor κB (NF-κB). While, NF-κB, glucose transporter type 1 (GLUT1) and sterol regulatory element-binding protein 1 (SREBP1) expression was reduced in Nogo-B deficiency cells. In addition, we showed that GLUT1 and SREBP1 was downstream target of NF-κB. Therefore, we demonstrated that Nogo deficiency inhibited PC progression is regulated by the NF-κB/GLUT1 and SREBP1 pathways, and suggested that Nogo-B may be a target for PC therapy.

Interruption of KLF5 acetylation promotes PTEN-deficient prostate cancer progression by reprogramming cancer-associated fibroblasts.

PTEN inactivation is prevalent in human prostate cancer and causes high-grade adenocarcinoma with a long latency. Cancer associated fibroblasts (CAFs) play a pivotal role in tumor progression, but it remains elusive whether and how PTEN-deficient prostate cancers reprogram CAFs to overcome the barriers for tumor progression. Herein, we report that PTEN deficiency induces KLF5 acetylation; and interruption of KLF5 acetylation orchestrates intricate interactions between cancer cells and CAFs that enhance FGFR1 signaling and promote tumor growth. Deacetylated KLF5 promotes tumor cells to secrete TNF-α, which stimulates inflammatory CAFs to release FGF9. CX3CR1 inhibition blocks FGFR1 activation triggered by FGF9 and sensitizes PTEN-deficient prostate cancer to AKT inhibitor capivasertib. This study reveals the role of KLF5 acetylation in reprogramming CAFs and provides a rational for combined therapies using inhibitors of AKT and CX3CR1.

CD36 inhibition reduces non-small-cell lung cancer development through AKT-mTOR pathway.

Lung cancer is the most common cause of cancer-related deaths worldwide and is caused by multiple factors, including high-fat diet (HFD). CD36, a fatty acid receptor, is closely associated with metabolism-related diseases, including cardiovascular disease and cancer. However, the role of CD36 in HFD-accelerated non-small-cell lung cancer (NSCLC) is unclear. In vivo, we fed C57BL/6J wild-type (WT) and CD36 knockout (CD36-/-) mice normal chow or HFD in the presence or absence of pitavastatin 2 weeks before subcutaneous injection of LLC1 cells. In vitro, A549 and NCI-H520 cells were treated with free fatty acids (FFAs) to mimic HFD situation for exploration the underlying mechanisms. We found that HFD promoted LLC1 tumor growth in vivo and that FFAs increased cell proliferation and migration in A549 and NCI-H520 cells. The enhanced cell or tumor growth was inhibited by the lipid-lowering agent pitavastatin, which reduced lipid accumulation. More importantly, we found that plasma soluble CD36 (sCD36) levels were higher in NSCLC patients than those in healthy ones. Compared to that in WT mice, the proliferation of LLC1 cells in CD36-/- mice was largely suppressed, which was further repressed by pitavastatin in HFD group. At the molecular level, we found that CD36 inhibition, either with pitavastatin or plasmid, reduced proliferation- and migration-related protein expression through the AKT/mTOR pathway. Taken together, we demonstrate that inhibition of CD36 expression by pitavastatin or other inhibitors may be a viable strategy for NSCLC treatment.

The pterostilbene-dihydropyrazole derivative Ptd-1 ameliorates vascular calcification by regulating inflammation.

Vascular calcification is an independent risk factor for cardiovascular disease. However, there is still a lack of adequate treatment. This study aimed to examine the potential of (E)-1-(5-(2-(4-fluorobenzyloxy)Styryl)-4,6-dimethoxyphenyl)-3-methyl-4,5-dihydro-1H-pyrazole-1-yl) ethyl ketone (Ptd-1) to alleviate vascular calcification. ApoE-deficient mice were fed a high-fat diet for 12/16 weeks to induce intimal calcification, and wild-type mice were induced with a combination of nicotine and vitamin D3 to induce medial calcification. Human aortic smooth muscle cells (HASMCs) and aortic osteogenic differentiation were induced in vitro with phosphate. In the mouse model of atherosclerosis, Ptd-1 significantly ameliorated the progression of atherosclerosis and intimal calcification, and there were significant reductions in lipid deposition and calcium salt deposition in the aorta and aortic root. In addition, Ptd-1 significantly improved medial calcification in vivo and osteogenic differentiation in vitro. Mechanistically, Ptd-1 reduced the levels of the inflammatory factors IL-1ß, TNFα and IL-6 in vivo and in vitro. Furthermore, we demonstrated that Ptd-1 could attenuate the expression of p-ERK1/2 and ß-catenin, and that the levels of inflammatory factors were elevated in the presence of ERK1/2 and ß-catenin agonists. Interestingly, we determined that activation of the ERK1/2 pathway promoted ß-catenin expression, which further regulated the IL-6/STAT3 signaling pathway. Ptd-1 blocked ERK1/2 signaling, leading to decreased expression of inflammatory factors, which in turn improved vascular calcification. Taken together, our study reveals that Ptd-1 ameliorates vascular calcification by regulating the production of inflammatory factors, providing new ideas for the treatment of vascular calcification.

Hydroxypropyl-ß-cyclodextrin inhibits the development of triple negative breast cancer by enhancing antitumor immunity.

Triple negative breast cancer (TNBC) is regarded as one of the most aggressive forms of breast cancer. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) has been used as a therapeutic agent for Niemann-Pick disease Type C (NPC). However, the exact actions and mechanisms of HP-ß-CD on TNBC are not fully understood. To examine the influence of HP-ß-CD on the proliferation and migration of TNBC cell lines, particularly 4T1 and MDA-MB-231 cells, a range of assays, including MTT, scratch, cell cycle, and clonal formation assays, were performed. Furthermore, the effectiveness of HP-ß-CD in the treatment of TNBC was assessed in vivo using a 4T1 tumor-bearing BALB/c mouse model. We demonstrated the anti-proliferation and anti-migration effect of HP-ß-CD on TNBC both in vitro and in vivo. High cholesterol diet can attenuate HP-ß-CD-inhibited TNBC growth. Mechanistically, HP-ß-CD reduced tumor cholesterol levels by increasing ABCA1 and ABCG1-mediated cholesterol reverse transport. HP-ß-CD promoted the infiltration of T cells into the tumor microenvironment (TME) and improved exhaustion of CD8+ T cells via reducing immunological checkpoint molecules expression. Additionally, HP-ß-CD inhibited the recruitment of tumor associated macrophages to the TME via reducing CCL2-p38MAPK-NF-κB axis. HP-ß-CD also inhibited the epithelial mesenchymal transition (EMT) of TNBC cells mediated by the TGF-ß signaling pathway. In summary, our study suggests that HP-ß-CD effectively inhibited the proliferation and metastasis of TNBC, highlighting HP-ß-CD may hold promise as a potential antitumor drug.

sFgl2 gene-modified MSCs regulate the differentiation of CD4+ T cells in the treatment of autoimmune hepatitis.

BACKGROUND: Autoimmune hepatitis (AIH) is a T-cell-mediated autoimmune liver disease that can lead to liver injury and has a poor long-term prognosis. Mesenchymal stromal cells (MSCs) have immunosuppressive effects and can treat AIH. CD4+ T cells express the unique inhibitory Fcγ receptor (FcγRIIB), which is the only receptor for the immunosuppressive factor soluble fibrinogen-like protein 2 (sFgl2). This study aimed to examine the therapeutic effect of sFgl2 gene-modified MSCs (sFgl2-MSCs) on AIH. METHODS: MSCs were obtained from the inguinal fat of mice and cocultured with CD4+ T cells sorted from mouse spleens. FcγRIIB expression on CD4+ T cells was determined by flow cytometry. sFgl2 expression in MSCs transfected with lentiviral vectors carrying the Fgl2 gene and a green fluorescent protein-encoding sequence was determined by enzyme-linked immunosorbent assay. The percentages of Th1 cells Th17 cells and regulatory T cells (Tregs) were determined by flow cytometry And the levels of p-SHP2 and p-SMAD2/3 were detected by Western blotting after the cells were cocultured with MSCs for 72 h. After locating MSCs by in vivo imaging Con A-induced experimental AIH mice were randomly divided into 4 groups and administered different treatments. After 24 h histopathological scores liver function and cytokine levels were examined and the proportions of CD4+ T cells CD8+ T cells Tregs Th17 cells and Th1 cells in the spleen and liver were determined by flow cytometry. In addition immunohistochemical staining was used to detect the liver infiltration of T-bet-, Foxp3- and RORγ-positive cells. RESULTS: FcγRIIB expression on CD4+ T cells was upregulated after coculture with MSCs. After coculture with sFgl2-MSCs, the proportion of Tregs among CD4+ T cells increased, the proportion of Th17 and Th1 cells decreased, and the levels of p-SHP2 and p-SMAD2/3 increased. In vivo, sFgl2-MSCs significantly improved liver function, decreased liver necrosis area, decreased tumor necrosis factor-α, interleukin (IL)-1ß and IL-6 expression, increased IL-10 expression, reduced liver infiltration of CD4+ T and CD8+ T cells, increased the proportion of Tregs and reduced the proportions of Th17 and Th1 cells in mice. CONCLUSION: By promoting Tregs differentiation and inhibiting Th17 and Th1 cell differentiation, sFgl2 gene-modified MSCs have a more powerful therapeutic effect on Con A-induced experimental AIH and may represent a strategy for the clinical treatment of AIH.

Nitazoxanide inhibits acetylated KLF5-induced bone metastasis by modulating KLF5 function in prostate cancer.

BACKGROUND: Castration-resistant prostate cancer often metastasizes to the bone, and such bone metastases eventually become resistant to available therapies, leading to the death of patients. Enriched in the bone, TGF-ß plays a pivotal role in bone metastasis development. However, directly targeting TGF-ß or its receptors has been challenging for the treatment of bone metastasis. We previously found that TGF-ß induces and then depends on the acetylation of transcription factor KLF5 at K369 to regulate multiple biological processes, including the induction of EMT, cellular invasiveness, and bone metastasis. Acetylated KLF5 (Ac-KLF5) and its downstream effectors are thus potential therapeutic targets for treating TGF-ß-induced bone metastasis in prostate cancer. METHODS: A spheroid invasion assay was applied to prostate cancer cells expressing KLF5K369Q, which mimics Ac-KLF5, to screen 1987 FDA-approved drugs for invasion suppression. Luciferase- and KLF5K369Q-expressing cells were injected into nude mice via the tail artery to model bone metastasis. Bioluminescence imaging, micro-CT), and histological analyses were applied to monitor and evaluate bone metastases. RNA-sequencing, bioinformatic, and biochemical analyses were used to understand nitazoxanide (NTZ)-regulated genes, signaling pathways, and the underlying mechanisms. The binding of NTZ to KLF5 proteins was evaluated using fluorescence titration, high-performance liquid chromatography (HPLC), and circular dichroism (CD) analysis. RESULTS: NTZ, an anthelmintic agent, was identified as a potent invasion inhibitor in the screening and validation assays. In KLF5K369Q-induced bone metastasis, NTZ exerted a potent inhibitory effect in preventive and therapeutic modes. NTZ also inhibited osteoclast differentiation, a cellular process responsible for bone metastasis induced by KLF5K369Q. NTZ attenuated the function of KLF5K369Q in 127 genes' upregulation and 114 genes' downregulation. Some genes' expression changes were significantly associated with worse overall survival in patients with prostate cancer. One such change was the upregulation of MYBL2, which functionally promotes bone metastasis in prostate cancer. Additional analyses demonstrated that NTZ bound to the KLF5 protein, KLF5K369Q bound to the promoter of MYBL2 to activate its transcription, and NTZ attenuated the binding of KLF5K369Q to the MYBL2 promoter. CONCLUSIONS: NTZ is a potential therapeutic agent for bone metastasis induced by the TGF-ß/Ac-KLF5 signaling axis in prostate cancer and likely other cancers.

Liver X receptor agonists exert antitumor effects against hepatocellular carcinoma via inducing REPS2 expression.

Recent studies show that liver X receptor (LXR) agonists exert significant antitumor effects in a variety of tumor cell lines including hepatocellular carcinoma (HCC). But the molecular mechanisms underlying LXR antitumor activity are not fully understood. In this study we investigated the effect of LXR agonist T0901317 (T317) on HCC development and its relationship with RalA binding protein 1 (RALBP1)-associated EPS domain containing 2 (REPS2)/epidermal growth factor receptor (EGFR) signaling axis. We showed that T317 (0.1-0.5 µM) dose-dependently increased REPS2 expression in normal hepatocytes (BNLCL.2 and LO2) and HCC cells (HepG2 and Huh-7). Using promoter activity assay and chromatin immunoprecipitation (CHIP) assay we demonstrated that T317 enhanced REPS2 expression at the transcriptional level via promoting the binding of LXR protein to the LXR-response element (LXRE) in the REPS2 promoter region. We showed that the inhibitory effect of T317 on the proliferation and migration of HCC cells was closely related to REPS2. Moreover, we revealed that T317 (400 nM) increased expression of REPS2 in HepG2 cells, thus inhibiting epidermal growth factor (EGF)-mediated endocytosis of EGFR as well as the downstream activation of AKT/NF-κB, p38MAPK, and ERK1/2 signaling pathways. Clinical data analysis revealed that REPS2 expression levels were inversely correlated with the development of HCC and reduced REPS2 expression associated with poor prognosis, suggesting that REPS2 might be involved in the development of HCC. In conclusion, this study provides new insights into the potential mechanisms of LXR agonist-inhibited HCC.

Targeting aldehyde dehydrogenase for prostate cancer therapies.

Prostate cancer (PCa) is the most common cancer in men in the United States. About 10 - 20% of PCa progress to castration-resistant PCa (CRPC), which is accompanied by metastasis and therapeutic resistance. Aldehyde dehydrogenase (ALDH) is famous as a marker of cancer stem-like cells in different cancer types, including PCa. Generally, ALDHs catalyze aldehyde oxidation into less toxic carboxylic acids and give cancers a survival advantage by reducing oxidative stress caused by aldehyde accumulation. In PCa, the expression of ALDHs is associated with a higher tumor stage and more lymph node metastasis. Functionally, increased ALDH activity makes PCa cells gain more capabilities in self-renewal and metastasis and reduces the sensitivity to castration and radiotherapy. Therefore, it is promising to target ALDH or ALDHhigh cells to eradicate PCa. However, challenges remain in moving the ALDH inhibitors to PCa therapy, potentially due to the toxicity of pan-ALDH inhibitors, the redundancy of ALDH isoforms, and the lack of explicit understanding of the metabolic signaling transduction details. For targeting PCa stem-like cells (PCSCs), different regulators have been revealed in ALDHhigh cells to control cell proliferation and tumorigenicity. ALDH rewires essential signaling transduction in PCa cells. It has been shown that ALDHs produce retinoic acid (RA), bind with androgen, and modulate diverse signaling. This review summarizes and discusses the pathways directly modulated by ALDHs, the crucial regulators that control the activities of ALDHhigh PCSCs, and the recent progress of ALDH targeted therapies in PCa. These efforts will provide insight into improving ALDH-targeted treatment.

Measurement of Bone Metastatic Tumor Growth by a Tibial Tumorigenesis Assay.

Bone metastasis is a frequent and lethal complication of many cancer types (i.e., prostate cancer, breast cancer, and multiple myeloma), and a cure for bone metastasis remains elusive. To recapitulate the process of bone metastasis and understand how cancer cells metastasize to bone, intracardiac injection and intracaudal arterial animal models were developed. The intratibial injection animal model was established to investigate the communication between cancer cells and the bone microenvironment and to mimic the setting of prostate cancer patients with bone metastasis. Given that detailed protocols of intratibial injection and its quantitative analysis are still insufficient, in this protocol, we provide hands-on procedures for how to prepare cells, perform the tibial injection, monitor tibial tumor growth, and quantitatively evaluate the tibial tumors in pathological samples. This manuscript provides a ready-to-use experimental protocol for investigating cancer cell behaviors in bone and developing novel therapeutic strategies for bone metastatic cancer patients.

Regulatory T cells induce transplant immune tolerance.

Organ transplantation is the preferred treatment option for end-stage organ failure. Although immunosuppressants are effective for preventing the occurrence of acute rejection, they also cause a series of side effects in transplant recipients. To improve the quality of patient survival, a new therapeutic strategy that has fewer side effects than current immunosuppressive regimens and can induce allograft immune tolerance and effectively prevent transplant rejection is needed. In this context, regulatory T cells (Tregs) are considered to be promising research targets. With the increasing understanding of the immunomodulatory role of Tregs, the use of Treg-based cellular therapies has shifted from prevention/treatment of autoimmune diseases to clinical trials for organ transplantation. This review describes the phenotype and in vitro expansion of Tregs and the mechanisms by which they exert immunomodulatory effects in transplantation immunity, highlights recent clinical trial data on Treg-based cellular therapies in transplantation, and describes future directions and limitations.

Novel Gene Signatures Predictive of Patient Recurrence-Free Survival and Castration Resistance in Prostate Cancer.

Molecular signatures predictive of recurrence-free survival (RFS) and castration resistance are critical for treatment decision-making in prostate cancer (PCa), but the robustness of current signatures is limited. Here, we applied the Robust Rank Aggregation (RRA) method to PCa transcriptome profiles and identified 287 genes differentially expressed between localized castration-resistant PCa (CRPC) and hormone-sensitive PCa (HSPC). Least absolute shrinkage and selection operator (LASSO) and stepwise Cox regression analyses of the 287 genes developed a 6-gene signature predictive of RFS in PCa. This signature included NPEPL1, VWF, LMO7, ALDH2, NUAK1, and TPT1, and was named CRPC-derived prognosis signature (CRPCPS). Interestingly, three of these 6 genes constituted another signature capable of distinguishing CRPC from HSPC. The CRPCPS predicted RFS in 5/9 cohorts in the multivariate analysis and remained valid in patients stratified by tumor stage, Gleason score, and lymph node status. The signature also predicted overall survival and metastasis-free survival. The signature's robustness was demonstrated by the C-index (0.55-0.74) and the calibration plot in all nine cohorts and the 3-, 5-, and 8-year area under the receiver operating characteristic curve (0.67-0.77) in three cohorts. The nomogram analyses demonstrated CRPCPS' clinical applicability. The CRPCPS thus appears useful for RFS prediction in PCa.

Acetylation of KLF5 maintains EMT and tumorigenicity to cause chemoresistant bone metastasis in prostate cancer.

Advanced prostate cancer (PCa) often develops bone metastasis, for which therapies are very limited and the underlying mechanisms are poorly understood. We report that bone-borne TGF-ß induces the acetylation of transcription factor KLF5 in PCa bone metastases, and acetylated KLF5 (Ac-KLF5) causes osteoclastogenesis and bone metastatic lesions by activating CXCR4, which leads to IL-11 secretion, and stimulating SHH/IL-6 paracrine signaling. While essential for maintaining the mesenchymal phenotype and tumorigenicity, Ac-KLF5 also causes resistance to docetaxel in tumors and bone metastases, which is overcome by targeting CXCR4 with FDA-approved plerixafor. Establishing a mechanism for bone metastasis and chemoresistance in PCa, these findings provide a rationale for treating chemoresistant bone metastasis of PCa with inhibitors of Ac-KLF5/CXCR4 signaling.

ZFHX3 Promotes the Proliferation and Tumor Growth of ER-Positive Breast Cancer Cells Likely by Enhancing Stem-Like Features and MYC and TBX3 Transcription.

Breast cancer is a common malignancy, but the understanding of its cellular and molecular mechanisms is limited. ZFHX3, a transcription factor with many homeodomains and zinc fingers, suppresses prostatic carcinogenesis but promotes tumor growth of liver cancer cells. ZFHX3 regulates mammary epithelial cells' proliferation and differentiation by interacting with estrogen and progesterone receptors, potent breast cancer regulators. However, whether ZFHX3 plays a role in breast carcinogenesis is unknown. Here, we found that ZFHX3 promoted the proliferation and tumor growth of breast cancer cells in culture and nude mice; and higher expression of ZFHX3 in human breast cancer specimens was associated with poorer prognosis. The knockdown of ZFHX3 in ZFHX3-high MCF-7 cells decreased, and ZFHX3 overexpression in ZFHX3-low T-47D cells increased the proportion of breast cancer stem cells (BCSCs) defined by mammosphere formation and the expression of CD44, CD24, and/or aldehyde dehydrogenase 1. Among several transcription factors that have been implicated in BCSCs, MYC and TBX3 were transcriptionally activated by ZFHX3 via promoter binding, as demonstrated by luciferase-reporter and ChIP assays. These findings suggest that ZFHX3 promotes breast cancer cells' proliferation and tumor growth likely by enhancing BCSC features and upregulating MYC, TBX3, and others.

TGF-ß causes Docetaxel resistance in Prostate Cancer via the induction of Bcl-2 by acetylated KLF5 and Protein Stabilization.

Prostate cancer is the second leading cause of cancer-related death in the United States. As a first line treatment for hormone-refractory prostate cancer, docetaxel (DTX) treatment leads to suboptimal effect since almost all patients eventually develop DTX resistance. In this study, we investigated whether and how TGF-ß affects DTX resistance of prostate cancer. Methods: Cytotoxicity of DTX in DU 145 and PC-3 cells was measured by CCK-8 and Matrigel colony formation assays. Resistance to DTX in DU 145 cells was examined in a xenograft tumorigenesis model. A luciferase reporter system was used to determine transcriptional activities. Gene expression was analyzed by RT-qPCR and Western blotting. Results: We found that KLF5 is indispensable in TGF-ß-induced DTX resistance. Moreover, KLF5 acetylation at lysine 369 mediates DTX resistance in vitro and in vivo. We showed that the TGF-ß/acetylated KLF5 signaling axis activates Bcl-2 expression transcriptionally. Furthermore, DTX-induced Bcl-2 degradation depends on a proteasome pathway, and TGF-ß inhibits DTX-induced Bcl-2 ubiquitination. Conclusion: Our study demonstrated that the TGF-ß-acetylated KLF5-Bcl-2 signaling axis mediates DTX resistance in prostate cancer and blockade of this pathway could provide clinical insights into chemoresistance of prostate cancer.

MiR-669b-3p regulates CD4+ T cell function by down-regulating indoleamine-2, 3-dioxygenase.

OBJECTIVE: Acute rejection is a major cause of morbidity and mortality after solid organ transplantation. Therefore, optimizing treatment strategies and improving curative effect is urgent and necessary. Reliable biomarkers for acute rejection and the underlying molecular mechanisms remain to be determined. METHODS: In this study, we established a mouse-to-mouse cardiac transplantation model and identified miR-669b-3p as a potential biomarker of acute rejection using a microRNA polymerase chain reaction (PCR)-based chip assay. RESULTS: Further analyses showed that miR-669b-3p negatively regulated indoleamine-2,3-dioxygenase (IDO), a rate-limiting enzyme of tryptophan catabolism inhibiting T cell function. Using mixed lymphocyte reaction assay, we showed that miR-669b-3p increased proliferation stimulation index and inhibited apoptosis in CD4+ T cells. Moreover, miR-669b-3p regulated the expression of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and Interleukin 10 (IL-10) and contributed to cytokine shift towards a Th2-dominant response. CONCLUSION: Our results advance the current understanding of the immune regulatory function of miRNA and shed light on the role of miR-669b-3p in CD4+ T cells, suggesting that miR-669b-3p is a potential target for acute allograft rejection.

Mesenchymal stem cells transfected with sFgl2 inhibit the acute rejection of heart transplantation in mice by regulating macrophage activation.

BACKGROUND: Mesenchymal stem cells (MSCs) have become a promising candidate for cell-based immune therapy for acute rejection (AR) after heart transplantation due to possessing immunomodulatory properties. In this study, we evaluated the efficacy of soluble fibronectin-like protein 2 (sFgl2) overexpressing mesenchymal stem cells (sFgl2-MSCs) in inhibiting AR of heart transplantation in mice by regulating immune tolerance through inducing M2 phenotype macrophage polarization. METHODS AND RESULTS: The sFgl2, a novel immunomodulatory factor secreted by regulatory T cells, was transfected into MSCs to enhance their immunosuppressive functions. After being co-cultured for 72 h, the sFgl2-MSCs inhibited M1 polarization whereas promoted M2 of polarization macrophages through STAT1 and NF-κB pathways in vitro. Besides, the sFgl2-MSCs significantly enhanced the migration and phagocytosis ability of macrophages stimulated with interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Further, the application potential of sFgl2-MSCs in AR treatment was demonstrated by heterotopic cardiac transplantation in mice. The tissue damage and macrophage infiltration were evaluated by H&E and immunohistochemistry staining, and the secretion of inflammatory cytokines was analyzed by ELISA. The results showed that sFgl2-MSCs injected intravenously were able to locate in the graft, promote the M2 polarization of macrophages in vivo, regulate the local and systemic immune response, significantly protect tissues from damaging, and finally prolonged the survival time of mice heart grafts. CONCLUSION: sFgl2-MSCs ameliorate AR of heart transplantation by regulating macrophages, which provides a new idea for the development of anti-AR treatment methods after heart transplantation.

The transcription factor ZFHX3 is crucial for the angiogenic function of hypoxia-inducible factor 1α in liver cancer cells.

Angiogenesis is a hallmark of tumorigenesis, and hepatocellular carcinoma (HCC) is hypervascular and therefore very dependent on angiogenesis for tumor development and progression. Findings from previous studies suggest that in HCC cells, hypoxia-induced factor 1α (HIF1A) and zinc finger homeobox 3 (ZFHX3) transcription factors functionally interact in the regulation of genes in HCC cells. Here, we report that hypoxia increases the transcription of the ZFHX3 gene and enhances the binding of HIF1A to the ZFHX3 promoter in the HCC cell lines HepG2 and Huh-7. Moreover, ZFHX3, in turn, physically associated with and was functionally indispensable for HIF1A to exert its angiogenic activity, as indicated by in vitro migration and tube formation assays of human umbilical vein endothelial cells (HUVECs) and microvessel formation in xenograft tumors of HCC cells. Mechanistically, ZFHX3 was required for HIF1A to transcriptionally activate the vascular endothelial growth factor A (VEGFA) gene by binding to its promoter. Functionally, down-regulation of ZFHX3 in HCC cells slowed their tumor growth, and addition of VEGFA to conditioned medium from ZFHX3-silenced HCC cells partially rescued the inhibitory effect of this medium on HUVEC tube formation. In human HCC, ZFHX3 expression was up-regulated, and this up-regulation correlated with both HIF1A up-regulation and worse patient survival, confirming a functional association between ZFHX3 and HIF1A in human HCC. We conclude that ZFHX3 is an angiogenic transcription factor that is integral to the HIF1A/VEGFA signaling axis in HCC cells.

KLF5 Is Crucial for Androgen-AR Signaling to Transactivate Genes and Promote Cell Proliferation in Prostate Cancer Cells.

Androgen/androgen receptor (AR) signaling drives both the normal prostate development and prostatic carcinogenesis, and patients with advanced prostate cancer often develop resistance to androgen deprivation therapy. The transcription factor Krüppel-like factor 5 (KLF5) also regulates both normal and cancerous development of the prostate. In this study, we tested whether and how KLF5 plays a role in the function of AR signaling in prostate cancer cells. We found that KLF5 is upregulated by androgen depending on AR in LNCaP and C4-2B cells. Silencing KLF5, in turn, reduced AR transcriptional activity and inhibited androgen-induced cell proliferation and tumor growth in vitro and in vivo. Mechanistically, KLF5 occupied the promoter of AR, and silencing KLF5 repressed AR transcription. In addition, KLF5 and AR physically interacted with each other to regulate the expression of multiple genes (e.g., MYC, CCND1 and PSA) to promote cell proliferation. These findings indicate that, while transcriptionally upregulated by AR signaling, KLF5 also regulates the expression and transcriptional activity of AR in androgen-sensitive prostate cancer cells. The KLF5-AR interaction could provide a therapeutic opportunity for the treatment of prostate cancer.