Pannexin 1 Modulates Angiogenic Activities of Human Endothelial Colony-Forming Cells Through IGF-1 Mechanism and Is a Marker of Senescence.

BACKGROUND: We examined the role of Panxs (pannexins) in human endothelial progenitor cell (EPC) senescence. METHODS: Young and replication-induced senescent endothelial colony-forming cells (ECFCs) derived from human circulating EPCs were used to examine cellular activities and senescence-associated indicators after transfection of short interference RNA specific to Panx1 or lentivirus-mediated Panx1 overexpression. Hind limb ischemia mice were used as in vivo angiogenesis model. Protein and phospho-kinase arrays were used to determine underlying mechanisms. RESULTS: Panx1 was the predominant Panx isoform in human ECFCs and upregulated in both replication-induced senescent ECFCs and circulating EPCs from aged mice and humans. Cellular activities of the young ECFCs were enhanced by Panx1 downregulation but attenuated by its upregulation. In addition, reduction of Panx1 in the senescent ECFCs could rejuvenate cellular activities with reduced senescence-associated indicators, including senescence-associated ß-galactosidase activity, p16INK4a (cyclin-dependent kinase inhibitor 2A), p21 (cyclin-dependent kinase inhibitor 1), acetyl-p53 (tumor protein P53), and phospho-histone H2A.X (histone family member X). In mouse ischemic hind limbs injected senescent ECFCs, blood perfusion ratio, salvaged limb outcome, and capillary density were all improved by Panx1 knockdown. IGF-1 (insulin-like growth factor 1) was significantly increased in the supernatant from senescent ECFCs after Panx1 knockdown. The enhanced activities and paracrine effects of Panx1 knockdown senescent ECFCs were completely inhibited by anti-IGF-1 antibodies. FAK (focal adhesion kinase), ERK (extracellular signal-regulated kinase), and STAT3 (signal transducer and activator of transcription 3) were activated in senescent ECFCs with Panx1 knockdown, in which the intracellular calcium level was reduced, and the activation was inhibited by supplemented calcium. The increased IGF-1 in Panx1-knockdown ECFCs was abrogated, respectively, by inhibitors of FAK (PF562271), ERK (U0126), and STAT3 (NSC74859) and supplemented calcium. CONCLUSIONS: Panx1 expression is upregulated in human ECFCs/EPCs with replication-induced senescence and during aging. Angiogenic potential of senescent ECFCs is improved by Panx1 reduction through increased IGF-1 production via activation of the FAK-ERK axis following calcium influx reduction. Our findings provide new strategies to evaluate EPC activities and rejuvenate senescent EPCs for therapeutic angiogenesis.

Repurposing thioridazine for inducing immunogenic cell death in colorectal cancer via eIF2α/ATF4/CHOP and secretory autophagy pathways.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

A G-quadruplex stabilizer, CX-5461 combined with two immune checkpoint inhibitors enhances in vivo therapeutic efficacy by increasing PD-L1 expression in colorectal cancer.

PURPOSE: 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. RESULTS: 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-ß 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. CONCLUSIONS: Therefore, clinically, CX-5461 combined with ICIs for CRC therapy warrants consideration because CX-5461 can turn cold tumors into hot ones.

Dinaciclib inhibits the stemness of two subtypes of human breast cancer cells by targeting the FoxM1 and Hedgehog signaling pathway.

Cyclin­dependent kinase (CDK)4/6 inhibitors in combination with endocrine therapy are the current standard of care used in the first­line treatment of hormone receptor­positive/HER2­negative metastatic breast cancer (BC). Although CDK4/6 inhibitors mainly target the cell cycle, emerging evidence has indicated further potential roles of CDKs other than regulating cell cycle progression. The G1 and G2/M transition regulators, including cyclins D and E, as well as their catalytic partners, CDK2, CDK4 and CDK6, have been reported to play crucial roles in pluripotency maintenance and cell fate decisions of human pluripotent stem cells by controlling transcription factors, signaling pathways and epigenetic regulators. Dinaciclib, a CDK1/2/5/9 inhibitor, is currently being evaluated in clinical trials against various cancer types, including BC. However, the underlying molecular mechanisms of CDK1/2/5/9 inhibitors in regulating BC stemness remain poorly understood. The present study aimed to examine the stemness­inhibitory effects of dinaciclib in MCF­7 (luminal) and HCC­1806 (triple­negative) BC cells. We found that this drug not only effectively reduced the self­renewal abilities and other malignant properties, but also dose­dependently decreased the protein expression levels of three BC stem cell markers, CD44, aldehyde dehydrogenase 1 family member A1 (ALDH1A1) and BMI1 proto­oncogene, polycomb ring finger (Bmi1), as well as three embryonic stem cell markers, Oct4, Nanog and Sox2. Moreover, the dinaciclib­induced decrease of Oct4 and Nanog protein expression was able to be restored by co­treatment with MG­132, a proteasome inhibitor. Forkhead box M1 (FoxM1), both a stemness­stimulating transcription factor and a cell cycle regulator, along with the Hedgehog signaling pathway, were identified as the therapeutic targets of dinaciclib. Collectively, the present results demonstrated a novel role of dinaciclib in suppressing BC stemness and indicated its potential use for future cancer treatments.

Ruxolitinib Combined with Gemcitabine against Cholangiocarcinoma Growth via the JAK2/STAT1/3/ALDH1A3 Pathway.

Cholangiocarcinoma is the most common primary malignant tumor of the bile duct. The current standard first-line treatment for advanced or metastatic cholangiocarcinoma is gemcitabine and cisplatin. However, few effective treatment choices exist for refractory cholangiocarcinoma, and additional therapeutic drugs are urgently required. Our previous work demonstrated that the ALDH isoform 1A3 plays a vital role in the malignant behavior of cholangiocarcinoma and may serve as a new therapeutic target. In this study, we found a positive correlation between ALDH1A3 protein expression levels and the cell migration abilities of three cholangiocarcinoma cell lines, which was verified using ALDH1A3-overexpressing and ALDH1A3-knockdown clones. We also used ALDH1A3-high and ALDH1A3-low populations of cholangiocarcinoma cell lines from the library of integrated network-based cellular signatures (LINCS) program and assessed the effects of ruxolitinib, a commercially available JAK2 inhibitor. Ruxolitinib had a higher cytotoxic effect when combined with gemcitabine. Furthermore, the nuclear translocation STAT1 and STAT3 heterodimers were markedly diminished by ruxolitinib treatment, possibly resulting in decreased ALDH1A3 activation. Notably, ruxolitinib alone or combined with gemcitabine led to significantly reduced tumor size and weight. Collectively, our studies suggest that ruxolitinib might suppress the ALDH1A3 activation through the JAK2/STAT1/3 pathway in cholangiocarcinoma, and trials should be undertaken to evaluate its efficacy in clinical therapy.

The Stemness-High Human Colorectal Cancer Cells Promote Angiogenesis by Producing Higher Amounts of Angiogenic Cytokines via Activation of the Egfr/Akt/Nf-κB Pathway.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

GATA6 enhances the stemness of human colon cancer cells by creating a metabolic symbiosis through upregulating LRH-1 expression.

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.

MicroRNA-203 diminishes the stemness of human colon cancer cells by suppressing GATA6 expression.

The interaction between hyaluronan and CD44, an important cancer stem-cell marker, stimulates various tumor cell-specific functions such as the stemness of tumor cells. microRNA-203 (miR-203) can be downregulated by this interaction in human colorectal cancer (CRC) cells, which increases their stemness; however, the underlying mechanism is not yet defined. Here, we show that overexpression and sequestration of miR-203 in HCT-116 and HT-29 human CRC cells reduces and enhances their stemness, respectively. We also show that GATA-binding factor 6 (GATA6) is a direct target of miR-203. Our results indicate that upregulated expression of this transcription factor not only restores the self-renewal abilities of miR-203-overexpressing HCT-116 and HT-29 cells but also promotes the stemness properties of their parental counterparts. More important, we show that silencing the expression of either LRH-1 or Hes-1 is sufficient to diminish the stemness-promoting effects of GATA6 in human CRC cells. Together, our findings delineate the stemness-inhibitory mechanism of miR-203 in human CRC cells and suggest that this miR is a potential therapeutic agent for colorectal cancer.

Elucidation of the mechanism underlying CD44v6-induced transformation of IEC-6 normal intestinal epithelial cells.

The transformation abilities of CD44s and CD44v6 in normal intestinal epithelial cells have not yet been reported. Herein, we established both CD44s and CD44v6 overexpressing stable clones from rat IEC-6 cells and demonstrated that the CD44v6 clones had higher saturation density and anchorage independence. Additionally, CD44v6 clones were more resistant to oxaliplatin and irinotecan which might be attributed to a significantly increased B-cell lymphoma 2 level and a reduced DNA damage response in these cells. Moreover, c-Met and vascular endothelial growth factor receptor 2 signalings were involved in modulating the saturation density in CD44v6 clones. Interestingly, higher activation of both AKT and extracellular-signal-regulated kinase (ERK) were detected in CD44v6 clones which might account in part for the cell density-independent nuclear localization of Yes-associated protein (YAP). To no surprise, increases of both saturation density and anchorage independence in CD44v6 clones were markedly diminished by PI3K, AKT, MEK, and ERK inhibitors as well as YAP knockdown. By contrast, overexpression of a constitutively active YAP robustly increased the aforementioned phenotypes in IEC-6 cells. Collectively, our results suggest that upregulation of CD44v6, but not CD44s, induces the transformation of normal intestinal epithelial cells possibly via activating the c-Met/AKT/YAP pathway which might also explain the important role of CD44v6 in the initiation of various carcinomas.

Two novel SHP-1 agonists, SC-43 and SC-78, are more potent than regorafenib in suppressing the in vitro stemness of human colorectal cancer cells.

Signal transducer and activator of transcription 3 (STAT3) has been shown to play a critical role in the maintenance of cancer stem cells (CSCs). Hence, the inhibition of STAT3 signaling has been suggested to be a viable therapeutic approach for cancers. Moreover, the efficacy of combinations of chemotherapeutic drugs and napabucasin, a small-molecule STAT3 inhibitor, have been assessed in various clinical trials, including those involving patients with metastatic colorectal cancer (CRC). Two recently developed small-molecule STAT3 inhibitors, SC-43 and SC-78, which can stimulate SHP-1 to inactivate STAT3, were found to have anti-tumor activity. In this study, the inhibitory effects of SC-43, SC-78, and regorafenib (a reference drug) on cell viability, STAT3 phosphorylation, and various stemness properties [e.g., sphere-forming and soft agar colony-forming abilities, CD133+/CD44+ (stem cell-like) subpopulations, and the expression of several CSC markers] were examined for both HCT-116 and HT-29 human CRC cells. We found that SC-43 and SC-78 but not regorafenib inhibited constitutive and IL-6-induced STAT3 phosphorylation in HCT-116 and HT-29 cells, respectively. Moreover, SC-43 and SC-78 were more potent than regorafenib in suppressing the stemness properties (except stem cell-like subpopulations) of these cells. As expected, SHP-1 knockdown almost completely abolished the suppressive effects of SC-43 and SC-78 on the sphere formation in both cell lines. Furthermore, SC-43 and SC-78 showed synergistic inhibitory effects with oxaliplatin and/or irinotecan on sphere formation. Overall, our results suggest that SC-43 and SC-78 are potent STAT3 inhibitors that may potentially be used in combination therapy for CRC.

G2A as a Threshold Regulator of Inflammatory Hyperalgesia Modulates Chronic Hyperalgesia.

Tissue injury, pathogen infection, and diseases are often accompanied by inflammation to release mediators that sensitize nociceptors and further recruit immune cells, which can lead to chronic hyperalgesia and inflammation. Tissue acidosis, occurring at the inflammatory site, is a major factor contributing to pain and hyperalgesia. The receptor G2 accumulation (G2A), expressed in neurons and immune cells, responds to protons or oxidized free fatty acids such as 9-hydroxyoctadecadienoic acid produced by injured cells or oxidative stresses. We previously found increased G2A expression in mouse dorsal root ganglia (DRG) at 90 min after complete Freund's adjuvant (CFA)-induced inflammatory pain, but whether G2A is involved in the inflammation or hyperalgesia remained unclear. In this study, we overexpressed or knocked-down G2A gene expression in DRG to explore the roles of G2A. G2A overexpression reduced the infiltration of acute immune cells (granulocytes) and attenuated hyperalgesia at 90 to 240 min after CFA injection. G2A knockdown increased the number of immune cells before CFA injection and prolonged the inflammatory hyperalgesia after CFA injection. G2A may serve as a threshold regulator in neurons to attenuate the initial nociceptive and inflammatory signals, modulating the chronic state of hyperalgesia.

Peripheral 5-HT3 mediates mirror-image pain by a cross-talk with acid-sensing ion channel 3.

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.

Identification of MALT1 as both a prognostic factor and a potential therapeutic target of regorafenib in cholangiocarcinoma patients.

Intrahepatic cholangiocarcinoma (CCA) is an aggressive cancer that lacks an effective targeted therapy. Here, we assessed the therapeutic efficacy of regorafenib in CCA, as well as elucidated its underlying mechanism. We first demonstrated that regorafenib not only inhibited growth but also induced apoptosis in human CCA cells. Subsequently, we used in silico approaches to identify MALT1 (Mucosa-associated lymphoid tissue protein 1), which plays an important role in activating NF-κB, as a potential target of regorafenib. Overexpression of Elk-1, but not Ets-1, in HuCCT1 cells markedly reduced their sensitivity to regorafenib, which might be attributed to a significant increase in MALT1 levels. Our results further demonstrated that this drug drastically inhibited MALT1 expression by suppressing the Raf/Erk/Elk-1 pathway. The efficacy of regorafenib in decreasing in vivo CCA growth was confirmed in animal models. Regorafenib efficacy was observed in two MALT1-positive CCA patients who failed to respond to several other lines of therapy. Finally, MALT1 was also identified as an independent poor prognostic factor for patients with intrahepatic CCA. In conclusion, our study identified MALT1 to be a downstream mediator of the Raf/Erk/Elk-1 pathway and suggested that MALT1 may be a new therapeutic target for successful treatment of CCA by regorafenib.

Heteromerization of G2A and OGR1 enhances proton sensitivity and proton-induced calcium signals.

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.

ALDH1A3, the Major Aldehyde Dehydrogenase Isoform in Human Cholangiocarcinoma Cells, Affects Prognosis and Gemcitabine Resistance in Cholangiocarcinoma Patients.

PURPOSE: Intrahepatic cholangiocarcinoma is a fatal primary liver cancer resulting from diagnosis at an advanced stage. Understanding the mechanisms of drug resistance and metastasis of cholangiocarcinoma may improve the disease prognosis. Enhanced aldehyde dehydrogenase (ALDH) activity is suggested to be associated with increased drug resistance and the metastasis. This study aims to investigate the roles of the ALDH isoforms in cholangiocarcinoma. EXPERIMENTAL DESIGN: Aldefluor assays, RT-PCR, and Western blot analysis were used to identify the major ALDH isoforms contributing to Aldefluor activity in human cholangiocarcinoma cell lines. We manipulated isoform expression in HuCCT1 cells to elucidate the role of ALDH1A3 in the malignant progression of these cells. Finally, we used immunohistochemical staining to evaluate the clinical significance of ALDH1A3 in 77 hepatectomized cholangiocarcinoma patients and an additional 31 patients with advanced cholangiocarcinoma who received gemcitabine-based therapy. RESULTS: ALDH(high) cholangiocarcinoma cells not only migrated faster but were more resistant to gemcitabine. Among the 19 ALDH isoforms studied, ALDH1A3 was found to be the main contributor to Aldefluor activity. In addition, we also found that knockdown of ALDH1A3 expression in HuCCT1 cells markedly reduced not only their sensitivity to gemcitabine, which might be attributed to a decreased expression of ribonucleotide reductase M1, but also their migration. Most importantly, this enzyme was also identified as an independent poor prognostic factor for patients with intrahepatic cholangiocarcinoma, as well as a prognostic biomarker of gemcitabine-treated patients. CONCLUSIONS: ALDH1A3 plays an important role in enhancing malignant behavior of cholangiocarcinoma and serves as a new therapeutic target. Clin Cancer Res; 22(16); 4225-35. ©2016 AACR.

Serotonin Receptor 2B Mediates Mechanical Hyperalgesia by Regulating Transient Receptor Potential Vanilloid 1.

Serotonin [5-hydroxytryptamine (5-HT)], an inflammatory mediator, contributes to inflammatory pain. The presence of multiple 5-HT subtype receptors on peripheral and central nociceptors complicates the role of 5-HT in pain. Previously, we found that 5-HT2B/2C antagonist could block 5-HT-induced mechanical hyperalgesia. However, the types of neurons or circuits underlying this effect remained unsolved. Here, we demonstrate that the Gq/11-phospholipase Cß-protein kinase Cε (PKCε) pathway mediated by 5-HT2B is involved in 5-HT-induced mechanical hyperalgesia in mice. Administration of a transient receptor potential vanilloid 1 (TRPV1) antagonist inhibited the 5-HT-induced mechanical hyperalgesia. 5-HT injection enhanced 5-HT- and capsaicin-evoked calcium signals specifically in isolectin B4 (IB4)-negative neurons; signals were inhibited by a 5-HT2B/2C antagonist and PKCε blocker. Thus, 5-HT2B mediates 5-HT-induced mechanical hyperalgesia by regulating TRPV1 function.

Thymosin beta-4 knockdown in IEC-6 normal intestinal epithelial cells induces DNA re-replication via downregulating Emi1.

Thymosin ß4 (Tß4 ) is a multifunctional protein already used clinically to treat various diseases; however, the promoting effect of this protein on tumor malignancy should not be neglected. Here, we assessed whether Tß4 alteration influences normal intestinal epithelial cells because Tß4 is deemed a novel target for treating colorectal cancer (CRC). For this purpose, we examined the consequences of shRNA-mediated knockdown of Tß4 in IEC-6 normal rat small intestinal cells and found that inhibiting Tß4 expression significantly suppressed their growth and induced apoptosis in some cells. Flow cytometric analysis further revealed a marked decrease of G0/G1 population but a drastic increase of polyploid ones in these cells. The increase of polyploidy likely resulted from DNA re-replication because not only the de novo DNA synthesis was greatly increased but also the expression levels of Cdc6 (a replication-licensing factor), cyclin A, and phosphorylated-checkpoint kinase 1 were all dramatically elevated. Moreover, marked reductions in both RNA and protein levels of Emi1 (early mitotic inhibitor 1) were also detected in Tß4 -downregulated IEC-6 cells which might be accounted by the downregulation of E2F1, a transcription factor capable of inducing Emi1 expression, mediated by glycogen synthase-3ß (GSK-3ß). To our best knowledge, this is the first report showing that inhibiting Tß4 expression triggers DNA re-replication in normal intestinal epithelial cells, suggesting that this G-actin sequester may play a crucial role in maintaining genome stability in these cells. More importantly, clinical oncologists should take this novel activity into consideration when design CRC therapy based on targeting Tß4 .

Maintenance of the stemness in CD44(+) HCT-15 and HCT-116 human colon cancer cells requires miR-203 suppression.

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.

Promotion of osteoblast differentiation in mesenchymal cells through Cbl-mediated control of STAT5 activity.

The identification of the molecular mechanisms controlling the degradation of regulatory proteins in mesenchymal stromal cells (MSC) may provide clues to promote MSC osteogenic differentiation and bone regeneration. Ubiquitin ligase-dependent degradation of proteins is an important process governing cell fate. In this study, we investigated the role of the E3 ubiquitin ligase c-Cbl in MSC osteoblast differentiation and identified the mechanisms involved in this effect. Using distinct shRNA targeting c-Cbl, we showed that c-Cbl silencing promotes osteoblast differentiation in murine and human MSC, as demonstrated by increased alkaline phosphatase activity, expression of phenotypic osteoblast marker genes (RUNX2, ALP, type 1 collagen), and matrix mineralization in vitro. Coimmunoprecipitation analyses showed that c-Cbl interacts with the transcription factor STAT5, and that STAT5 forms a complex with RUNX2, a master transcription factor controlling osteoblastogenesis. Silencing c-Cbl decreased c-Cbl-mediated STAT5 ubiquitination, increased STAT5 protein level and phosphorylation, and enhanced STAT5 and RUNX2 transcriptional activity. The expression of insulin like growth factor-1 (IGF-1), a target gene of STAT5, was increased by c-Cbl silencing in MSC and in bone marrow stromal cells isolated from c-Cbl deficient mice, suggesting that IGF-1 contributes to osteoblast differentiation induced by c-Cbl silencing in MSC. Consistent with these findings, pharmacological inhibition of STAT5 activity, or neutralization of IGF-1 activity, abrogated the positive effect of c-Cbl knockdown on MSC osteogenic differentiation. Taken together, the data provide a novel functional mechanism by which the ubiquitin ligase c-Cbl regulates the osteoblastic differentiation program in mesenchymal cells by controlling Cbl-mediated STAT5 degradation and activity.

Nuclear matrix-targeting of the osteogenic factor Runx2 is essential for its recognition and activation of the alkaline phosphatase gene.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 (11C3) 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 (Runx2deltaNMTS) 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. CONCLUSIONS: Nuclear matrix-localization of Runx2 influences its ALP gene recognition. GENERAL SIGNIFICANCE: 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.