Cancer stem cells (CSCs) are responsible for cancer metastasis by stimulating tumor angiogenesis via various mechanisms. To elucidate the potential of the stemness-high human colorectal cancer (CRC) ...cells (i.e., CRCSCs) in activating angiogenesis, effects of the GATA6-overexpressing HCT-116 and HT-29 human CRC clones established previously by us in promoting the angiogenesis of human umbilical vein endothelial cells (HUVECs) were examined.
Angiogenesis-promoting effects (i.e., migration, invasion, DNA synthesis, and tube formation) in HUVECs of the conditioned media (CM) from various human CRC clones were analyzed. MMP activities were assessed using a zymography assay. Western blotting and selective inhibitors were used to dissect the signaling pathway involved. IHC was used to examine the vascular density in tumor xenografts.
We found that the conditioned media (CM) collected from the GATA6-overexpressing clones enhanced angiogenesis of HUVECs more effectively which might be attributed partly to a higher MMP-9 production by HUVECs. Subsequently, elevated levels of IL-8 and VEGF-A were detected in the CM whose tube formation-enhancing activities were abolished by the co-treatment with either a VEGFR2 inhibitor or an IL-8 neutralizing antibody. Interestingly, increased production of these cytokines in the GATA6-overexpressing clones was due to an EGFR/AKT-mediated activation of NF-κB. Furthermore, not only were the levels of CD31 and endomucin but also the blood vessel density was much higher in the xenograft tumors grown from these clones.
Our findings demonstrate that human CRCSCs promote a stronger angiogenesis by producing higher amounts of angiogenic factors through activation of the EGFR/AKT/NF-κB pathway.
A good understanding of the mechanism of gene regulation that is involved in bone mineralization is critical for the design of anabolic treatments for bone deficiency diseases. Alkaline phosphatase ...(ALP) expressed by osteoblasts plays an important role in promoting bone mineralization by hydrolyzing pyrophosphate. However, the mechanism by which the expression of ALP is regulated during osteoblast differentiation has not been thoroughly investigated.
Chromatin immunoprecipitation, EMSA and mutagenesis were used to identify the Runx2 binding sites on ALP gene and to analyze the role of nuclear matrix-localization of Runx2 on the recognition and activation of ALP gene.
Using chromatin immunoprecipitation, we determined that both ectopic and endogenous Runx2 bound to ALP intron 1 in a region containing a cluster of five putative core-sites. The third one (I1C3) among those fives was bound most strongly in vitro by Runx2 and acted as a Runx2-dependent transcriptional enhancer. Furthermore, a Runx2 mutant lacking the nuclear matrix-targeting sequence (Runx2ΔNMTS) bound to the ALP gene less efficiently than the wild-type protein and a Runx2 mutant that is deficient in its ability to bind to DNA (Runx2K120A) accumulated largely in the nuclear matrix.
Nuclear matrix-localization of Runx2 influences its ALP gene recognition.
Our results showed for the first time that ALP is a direct target gene of Runx2 and illustrated that the recognition/binding and activation of the ALP by this transcription factor are dependent on its nuclear matrix-targeting.
► Alkaline phosphatase (ALP) is a direct target gene of Runx2. ► Nuclear matrix-targeting of Runx2 is essential for its recognition of ALP. ► Nuclear matrix-targeting of Runx2 is essential for its activation of ALP. ► Runx2K120A accumulates largely in the nuclear matrix fraction. ► Runx2K120A hampers the activation of ALP by wild-type Runx2.
Mirror-image pain (MIP), which occurs along with complex regional pain syndrome, rheumatoid arthritis and chronic migraine, is characterized by increased pain sensitivity of healthy body regions ...other than the actual injured or inflamed sites. A high level of peripheral inflammation may activate central or peripheral glia, triggering mirror-image pain. However, which receptors mediate inflammatory signals to contribute glial activation remains unclear. Intraplantarly injecting mice with 5-hydroxytryptamine (5-HT) or acidic buffer (proton) caused only unilateral hyperalgesia, but co-injection of 5-HT/acid induced bilateral hyperalgesia (MIP). Blocking 5-HT3 or acid-sensing ion channel 3 (ASIC3) abolished satellite glial activation, inhibiting MIP. Interestingly, intraplantar administration of a 5-HT3 agonist induced MIP, and 5-HT3–mediated MIP can be reversed by a 5-HT3 antagonist or an ASIC3 blocker. Similar results were found using a ASIC3 agonist. Furthermore, 5-HT3 was observed to co-localize with ASIC3 in DRG neurons; 5-HT3 activation-induced an increase in intracellular calcium that was inhibited by an ASIC3 blocker and vice versa. A cross-talk between 5-HT3 and ASIC3 mediates satellite glial activation, thereby triggering mirror-image pain.
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•Peripheral 5-HT3 and ASIC3 are essential to mediate mirror-image pain through activation of satellite glial cells.•5-HT3 is cross-talked with ASIC3 to regulate downstream signaling that affecting activation of satellite glial cells.•5-HT2B-mediated an inhibitory signal to prevent 5-HT3-mediated mirror-image pain.
The purpose of this study was to isolate cancer stem cells (CSCs, also called tumor-initiating cells, TICs) from established human colorectal carcinoma (CRC) cell lines, characterize them extensively ...and dissect the mechanism for their stemness. Freshly isolated CD44+ and CD44− cells from the HCT-15 human colon cancer cell line were subjected to various analyses. Interestingly, CD44+ cells exhibited higher soft agar colony-forming ability and in vivo tumorigenicity than CD44− cells. In addition, the significant upregulation of the protein Snail and the downregulation of miR-203, a stemness inhibitor, in CD44+ cells suggested that this population possessed higher invasion/metastasis and differentiation potential than CD44− cells. By manipulating the expression of CD44 in HCT-15 and HCT-116 cells, we found that the levels of several EMT activators and miR-203 were positively and negatively correlated with those of CD44, respectively. Further analyses revealed that miR-203 levels were repressed by Snail, which was shown to bind to specific E-box(es) present in the miR-203 promoter. In agreement, silencing miR-203 expression in wild-type HCT-116 human colon cancer cells also resulted in an increase of their stemness. Finally, we discovered that c-Src kinase activity was required for the downregulation of miR-203 in HCT-15 cells, which was stimulated by the interaction between hyaluronan (HA) and CD44.
Taken together, CD44 is a critical molecule for modulating stemness in CSCs. More importantly, we show for the first time that the downregulation of miR-203 by HA/CD44 signaling is the main reason for stemness-maintenance in colon cancer cells.
•CD44 is required to maintain stem-like properties of HCT-15 and HCT-116 cells.•MiR-203 functions as a stemness-inhibitor in HCT-15, HT-29 and HCT-116 cells.•CD44 triggers a Snail-mediated miR-203 suppression to maintain the stemness of CSC.
•CX-5461 may enhance immune checkpoint inhibitor (ICI) therapeutic efficacy.•CX-5461 triggers cGAS-STING-IFN pathway, eliciting STAT1-mediated PD-L1 expression.•CX-5461+ICI leads to improved ...immunotherapeutic effects in CRC.•They increase cytotoxic T-cell and reduce myeloid-derived suppressor cell numbers.•CX-5461 can turn cold CRC tumors into hot ones.
Immune checkpoint inhibitors (ICIs) alone or in combination with chemotherapy can improve the limited efficacy of colorectal cancer (CRC) immunotherapy. CX-5461 causes substantial DNA damage and genomic instability and can increase ICIs’ therapeutic efficacies through tumor microenvironment alteration.
We analyzed whether CX-5461 enhances ICIs’ effects in CRC and discovered that CX-5461 causes severe DNA damage, including cytosolic dsDNA appearance, in various human and mouse CRC cells. Our bioinformatics analysis predicted CX-5461-based interferon (IFN) signaling pathway activation in these cells, which was verified by the finding that CX-5461 induces IFN-α and IFN-β secretion in these cells. Next, cGAMP, phospho-IRF3, CCL5, and CXCL10 levels exhibited significant posttreatment increases in CRC cells, indicating that CX-5461 activates the cGAS-STING-IFN pathway. CX-5461 also enhanced PD-L1 expression through STAT1 activation. CX-5461 alone inhibited tumor growth and prolonged survival in mice. CX-5461+anti-PD-1 or anti-PD-L1 alone exhibited synergistic growth-suppressive effects against CRC and breast cancer. CX-5461 alone or CX-5461+anti-PD-1 increased cytotoxic T-cell numbers and reduced myeloid-derived suppressor cell numbers in mouse spleens.
Therefore, clinically, CX-5461 combined with ICIs for CRC therapy warrants consideration because CX-5461 can turn cold tumors into hot ones.
Colorectal cancer (CRC) is a highly prevalent cancer type with limited targeted therapies available and 5-year survival rate, particularly for late-stage patients. There have been numerous attempts ...to repurpose drugs to tackle this problem. It has been reported that autophagy inducers could augment the effect of certain chemotherapeutic agents by enhancing immunogenic cell death (ICD).
In this study, we employed bioinformatics tools to identify thioridazine (THD), an antipsychotic drug, and found that it could induce autophagy and ICD in CRC. Then in vitro and in vivo experiments were performed to further elucidate the molecular mechanism of THD in CRC.
THD was found to induce endoplasmic reticulum (ER) stress in CRC cells by activating the eIF2α/ATF4/CHOP axis and facilitating the accumulation of secretory autophagosomes, leading to ICD. In addition, THD showed a remarkable ICD-activating effect when combined with oxaliplatin (OXA) to prevent tumor progression in the mouse model.
Together, our findings suggest that the repurposed function of THD in inhibiting CRC involves the upregulation of autophagosomes and ER stress signals, promoting the release of ICD markers, and providing a potential candidate to enhance the clinical outcome for CRC treatment. Video Abstract.
Cancer stem cells play critical roles in tumor initiation, progression, and relapse. Since we previously found that GATA6 promotes the stemness in HCT‐116 and HT‐29 human colorectal cancer (CRC) ...cells, we aimed to identify the downstream mediator(s) of the stemness‐stimulating effect of GATA6 herein. LRH‐1 was found as a direct target of GATA6 and its upregulation promoted the stemness in both HCT‐116 and HT‐29 cells. Subsequently, hypoxia‐inducible factor‐1α (HIF‐1α) was identified as a direct target of LRH‐1 and its expression level and activity were significantly elevated in the LRH‐1‐overexpressing clones established from the aforementioned two CRC lines. Accordingly, the expression levels of several HIF‐1α targets were also markedly increased, resulting in a stronger glycolysis associated with dramatic elevations of the lactate levels in these cells. Strikingly, higher mitochondrial activities were also found in these clones which might be attributed to the increase of PGC‐1α stimulated by the lactate uptaken through the upregulated MCT‐1. Finally, significant increases in the self‐renewal ability, intracellular radical oxygen species levels and mitochondrial mass were detected in the CD133+/CD44+ subpopulations isolated from CRC cells regardless of their LRH‐1 expression levels. Together, our results suggest a novel metabolic symbiosis between different colorectal cancer stem cell subpopulations critical for maintaining their mutual stemness.
Our observations demonstrate a reverse Warburg effect in human colorectal cancer stem cells (CRCSCs) because the expression levels and activity of hypoxia‐inducible factor‐1α are markedly increased by LRH‐1 upregulation resulted from GATA6 overexpression in the majority of colorectal cancer (CRC) cells which undergo aerobic glycolysis to proliferate rapidly and, in the meantime, generate large amount of lactate that can be uptaken by another subset of CRC cells for mitochondrial oxidative phosphorylation. In this manner, metabolic symbiosis and energy transfer are maintained between two CRCSC subpopulations.
Proton-sensing G-protein-coupled receptors (GPCRs; OGR1, GPR4, G2A, TDAG8), with full activation at pH 6.4 ∼ 6.8, are important to pH homeostasis, immune responses and acid-induced pain. Although G2A ...mediates the G13-Rho pathway in response to acid, whether G2A activates Gs, Gi or Gq proteins remains debated. In this study, we examined the response of this fluorescence protein-tagged OGR1 family to acid stimulation in HEK293T cells. G2A did not generate detectable intracellular calcium or cAMP signals or show apparent receptor redistribution with moderate acid (pH ≥ 6.0) stimulation but reduced cAMP accumulation under strong acid stimulation (pH ≤ 5.5). Surprisingly, coexpression of OGR1- and G2A-enhanced proton sensitivity and proton-induced calcium signals. This alteration is attributed to oligomerization of OGR1 and G2A. The oligomeric potential locates receptors at a specific site, which leads to enhanced proton-induced calcium signals through channels.
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