Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity.

BACKGROUND: Mutant Ras plays multiple functions in tumorigenesis including tumor formation and metastasis. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a metastasis inhibitor gene, suppresses matrix metalloproteinase (MMP) activity in the metastatic cascade. Clarifying the relationship between Ras and RECK and understanding the underlying molecular mechanism may lead to the development of better treatment for Ras-related tumors. METHODS: Suppression subtractive hybridization PCR (SSH PCR) was conducted to identify Ha-ras (val12) up-regulated genes in bladder cancer cells. Stable cell lines of human breast cancer (MCF-7-ras) and mouse NIH3T3 fibroblasts (7-4) harboring the inducible Ha-ras (val12) oncogene, which could be induced by isopropylthio-ß-D-galactoside (IPTG), were used to clarify the relationship between Ras and the up-regulated genes. Chromatin immunoprecipitation (ChIP) assay, DNA affinity precipitation assay (DAPA) and RECK reporter gene assay were utilized to confirm the complex formation and binding with promoters. RESULTS: Retinoblastoma binding protein-7 (RbAp46) was identified and confirmed as a Ha-ras (val12) up-regulated gene. RbAp46 could bind with histone deacetylase (HDAC1) and Sp1, followed by binding to RECK promoter at the Sp1 site resulting in repression of RECK expression. High expression of Ras protein accompanied with high RbAp46 and low RECK expression were detected in 75% (3/4) of the clinical bladder cancer tumor tissues compared to the adjacent normal parts. Ras induced RbAp46 expression increases invasion of the bladder cancer T24 cells and MMP-9 activity was increased, which was confirmed by specific lentiviral shRNAs inhibitors against Ras and RbAp46. Similarly, knockdown of RbAp46 expression in the stable NIH3T3 cells "7-4" by shRNA decreased Ras-related lung metastasis using a xenograft nude mice model. CONCLUSIONS: We confirmed that RbAp46 is a Ha-ras (val12) up-regulated gene and binds with HDAC1 and Sp1. Furthermore, RbAp46 binds to the RECK promoter at the Sp1 site via recruitment by Sp1. RECK is subsequently activated, leading to increased MMP9 activity, which may lead to increased metastasis in vivo. Our findings of Ras upregulation of RbAp46 may lead to revealing a novel mechanism of Ras-related tumor cell metastasis.

Metformin enhances cisplatin cytotoxicity by suppressing signal transducer and activator of transcription-3 activity independently of the liver kinase B1-AMP-activated protein kinase pathway.

Metformin has been used as first-line treatment in patients with type 2 diabetes, and is reported to reduce cancer risk and progression by activating the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Cisplatin remains the main drug for treating advanced non-small-cell lung cancer. However, drug resistance often develops through several mechanisms during the treatment course, including one mechanism mediated by the activation of the IL-6/signal transducer and activator of transcription (STAT)-3 pathway, related to the generation of reactive oxygen species (ROS). This study demonstrated a correlation between STAT3 phosphorylation and cisplatin cytotoxicity, using AS2 (PC14PE6/AS2)-derived cell lines (AS2/S3C) that contained constitutively active STAT3 plasmids as a model. A STAT3 inhibitor (JSI-124) enhanced the cisplatin sensitivity in AS2 cells, whereas metformin inhibited STAT3 phosphorylation and enhanced cisplatin cytotoxicity. By contrast, another AMPK activator (5-aminoimidazole-4-carboxamide-riboside) failed to produce these effects. LKB1-AMPK silencing by small, interfering RNA or mammalian target of rapamycin (mTOR) inhibition by rapamycin or pp242 did not alter the effect of metformin on STAT3 activity suppression, suggesting that metformin can modulate the STAT3 pathway through an LKB1-AMPK-independent and probably mTOR-independent mechanism. Metformin also inhibited cisplatin-induced ROS production and autocrine IL-6 secretion in AS2 cells. Both mechanisms contributed to the ability of metformin to suppress STAT3 activation in cancer cells, which resulted in the decreased secretion of vascular endothelial growth factor by cancer cells. The growth of subcutaneous tumor xenografts was significantly delayed by a combination of cisplatin and metformin. This is the first study to demonstrate that metformin suppresses STAT3 activation via LKB1-AMPK-mTOR-independent but ROS-related and autocrine IL-6 production-related pathways. Thus, metformin helps to overcome tumor drug resistance by targeting STAT3.

Mitochondrial uncoupling protein 2 regulates the effects of paclitaxel on Stat3 activation and cellular survival in lung cancer cells.

Growing evidence suggests that Stat3 contributes to chemoresistance. However, the impact of chemotherapy on Stat3 activity is unclear. We found that paclitaxel activated Stat3 in the human lung cancer cell lines PC14PE6AS2 (AS2) and H157, whereas it reduced Stat3 activation in A549 and H460 cells. Pretreatment of AS2 and H157 cells with rotenone, an inhibitor of mitochondrially produced reactive oxygen species (ROS), or carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP), a mitochondrial uncoupler, suppressed the paclitaxel-induced activation of Stat3. Uncoupling protein 2 (UCP-2), located in the inner membrane of the mitochondria, can reduce ROS production in conditions of oxidative stress. UCP-2 protein expression in the four cancer cell lines was higher than that in normal lung epithelial cells (NL-20), but its expression was lower in AS2 and H157 cells relative to A549 and H460 cells. Silencing high UCP-2 expression with small interfering RNA (siRNA) in A549 and H460 cells restored paclitaxel-induced Stat3 activation. In addition, paclitaxel-induced Stat3 activation led to the upregulation of survivin and Mcl-1, which in turn facilitated cell survival. Moreover, the CL1-5 subline had lower UCP-2 expression relative to the parental CL1-0 cells. Treatment with paclitaxel activated Stat3 in CL1-5 but not in CL1-0 cells, whereas in CL1-5 cells, the overexpression of UCP-2 with complementary DNA (cDNA) blocked Stat3 activation. In lung cancer patients, low UCP-2 expression in cancer cells was a predictor of a poor response to chemotherapy. Therefore, UCP-2 modulates the ROS/Stat3 signaling pathway and response to chemotherapy treatment in lung cancer cells. Targeting UCP-2, ROS and Stat3 pathways may improve anticancer therapies.

Transcriptional activation of the Axl and PDGFR-α by c-Met through a ras- and Src-independent mechanism in human bladder cancer.

BACKGROUND: A cross-talk between different receptor tyrosine kinases (RTKs) plays an important role in the pathogenesis of human cancers. METHODS: Both NIH-Met5 and T24-Met3 cell lines harboring an inducible human c-Met gene were established. C-Met-related RTKs were screened by RTK microarray analysis. The cross-talk of RTKs was demonstrated by Western blotting and confirmed by small interfering RNA (siRNA) silencing, followed by elucidation of the underlying mechanism. The impact of this cross-talk on biological function was demonstrated by Trans-well migration assay. Finally, the potential clinical importance was examined in a cohort of 65 cases of locally advanced and metastatic bladder cancer patients. RESULTS: A positive association of Axl or platelet-derived growth factor receptor-alpha (PDGFR-α) with c-Met expression was demonstrated at translational level, and confirmed by specific siRNA knock-down. The transactivation of c-Met on Axl or PDGFR-α in vitro was through a ras- and Src-independent activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. In human bladder cancer, co-expression of these RTKs was associated with poor patient survival (p < 0.05), and overexpression of c-Met/Axl/PDGFR-α or c-Met alone showed the most significant correlation with poor survival (p < 0.01). CONCLUSIONS: In addition to c-Met, the cross-talk with Axl and/or PDGFR-α also contributes to the progression of human bladder cancer. Evaluation of Axl and PDGFR-α expression status may identify a subset of c-Met-positive bladder cancer patients who may require co-targeting therapy.

Signal transducer and activator of transcription 3 activation up-regulates interleukin-6 autocrine production: a biochemical and genetic study of established cancer cell lines and clinical isolated human cancer cells.

BACKGROUND: Spontaneous interleukin-6 (IL-6) production has been observed in various tumors and implicated in the pathogenesis, progression and drug resistance in cancer. However, the regulation of IL-6 autocrine production in cancer cells is not fully understood. IL-6 is auto-regulated in many types of cell. Two of the three major downstream pathways of IL-6, MEK/extracellular signal-related kinase (Erk) pathway and phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, have been shown to regulate IL-6 expression through the activation of AP-1 and NF-κB. However, it is not clear what the role of Janus kinase (Jak) 2/signal transducer and activator of transcription (Stat) 3 pathway. This study was designed to determine the role of Jak2/Stat3 pathway in the regulation of IL-6 autocrine production in cancer cells. RESULTS: Inhibitors of Jak2/Stat3, MEK/Erk and PI3-K/Akt pathways down-regulated IL-6 secretion in the lung adenocarcinoma PC14PE6/AS2 (AS2) cells, which spontaneously secreted IL-6 and possessed constitutively activated Stat3. Transfection with dominant-negative Stat3, Stat3 siRNA, or Stat3 shRNA decreased IL-6 expression in AS2 cells. Conversely, transfection with constitutively-activated Stat3 increased the production of IL-6. In AS2 derived cells, resistance to paclitaxel was positively correlated with Stat3 activation status and the expression of IL-6, which is commonly secreted in drug resistant cancer cells. The pharmacological inhibition of NF-κB, PI3-K/Akt and MEK/Erk and the pharmacological inhibition and genetic inhibition (Stat3 siRNA) of Jak2/Stat3 pathway decreased IL-6 autocrine production in various drug resistant cancer cell lines and similarly decreased IL-6 autocrine production in clinically isolated lung cancer cells. CONCLUSIONS: This study is the first to directly address the role Stat3 plays on the autocrine production of IL-6, which occurs through a positive-feedback loop. Our biochemical and genetic studies clearly demonstrated that Jak2/Stat3, in combination with other IL-6 downstream pathways, contributed frequently and substantially to IL-6 autocrine production in a broad spectrum of cancer cell lines as well as in clinical cancer samples. Our findings suggest that Stat3 could potentially be regulated to suppress IL-6 autocrine production in cancer cells to inhibit the progression of cancer and reduce drug resistance.

SUMO-1 overexpression increases RbAp46 protein stability and suppresses cell growth.

The retinoblastoma suppressor (Rb)-associated protein 46 (RbAp46) is a nuclear protein of 46 kDa and contains four repeats that end with Trp-Asp (WD) residues. In this study, we reveal that the RbAp46 protein level upon SUMO-1 expression was increased. The increasing level of RbAp46 protein by SUMO-1 was not regulated at the transcriptional level. SUMO-1 does not affect the degradation of RbAp46. Co-localization of RbAp46 and SUMO-1 in the nuclei of stable NIH/3T3 cells harboring the inducible Ha-ras(Val12) oncogene (pSVlacOras) designated as 7-4, and protein-protein interaction between RbAp46 and SUMO-1 was also detected by co-immunoprecipitation in these cells. However, SUMO-l-related sumoylation was not involved in the modification of RbAp46. It is possibly that SUMO-1 acts through formation of complex with RbAp46 to stabilize RbAp46 protein. Overexpression of RbAp46 protein suppressed the NIH/3T3 cell growth induced by Ha-Ras(V12). SUMO-1 further enhances the suppression of cell growth through stabilization of RbAp46 protein. This is the first report to demonstrate that SUMO-1 can suppress Ras-related cell proliferation through stabilization of RbAp46 protein.

Upregulation of tissue factor by activated Stat3 contributes to malignant pleural effusion generation via enhancing tumor metastasis and vascular permeability in lung adenocarcinoma.

Malignant pleural effusion (MPE) is a poor prognostic sign for patients with lung cancer. Tissue factor (TF) is a coagulation factor that participates in angiogenesis and vascular permeability and is abundant in MPE. We previously demonstrated that autocrine IL-6-activated Stat3 contributes to tumor metastasis and upregulation of VEGF, resulting in the generation of MPE in lung adenocarcinoma. In this study, we found IL-6-triggered Stat3 activation also induces TF expression. By using pharmacologic inhibitors, it was shown that JAK2 kinase, but not Src kinase, contributed to autocrine IL-6-induced TF expression. Inhibition of Stat3 activation by dominant negative Stat3 (S3D) in lung adenocarcinoma suppressed TF-induced coagulation, anchorage-independent growth in vitro, and tumor growth in vivo. Consistently, knockdown of TF expression by siRNA resulted in a reduction of anchorage-independent growth of lung adenocarcinoma cells. Inhibition of TF expression also decreased the adhesion ability of cancer cells in normal lung tissues. In the nude mouse model, both lung metastasis and MPE generation were decreased when PC14PE6/AS2-siTF cells (TF expression was silenced) were intravenously injected. PC14PE6/AS2-siTF cells also produced less malignant ascites through inhibition of vascular permeability. In summary, we showed that TF expression plays a pivotal role in the pathogenesis of MPE generation via regulating of tumor metastasis and vascular permeability in lung adenocarcinoma bearing activated Stat3.

Activation of the signal transducer and activator of transcription 3 pathway up-regulates estrogen receptor-beta expression in lung adenocarcinoma cells.

Estrogens contribute to the pathogenesis of female lung cancer and function mainly through estrogen receptor-ß (ERß). However, the way in which ERß expression is regulated in lung cancer cells remains to be explored. We have found that signal transducer and activator of transcription 3 (Stat3) activation up-regulates ERß expression in PC14PE6/AS2 lung cancer cells in a preliminary Affymetrix oligonucleotide array study, and we sought to confirm the findings. In this study, we show that IL-6 induced ERß mRNA and protein expression in lung cancer cells. The induction of ERß in response to IL-6 was abolished by Janus kinase 2 inhibitor-AG490, dominant-negative mutant of Stat3, and Stat3-targeting short interfering RNA. The luciferase reporter assay and chromatin immunoprecipitation assay confirmed that IL-6-activated Stat3 binds to the ERß promoter. Besides the Janus kinase 2/Stat3 pathway, the MEK/Erk pathway contributes to ERß up-regulation induced by IL-6; however, the phosphoinositide 3'-kinase/Akt pathway does not. We also found that epidermal growth factor (EGF) stimulation or L858R mutation in EGF receptor (EGFR) induced Stat3 activation as well as ERß expression in lung cancer cells. Inhibiting Stat3 activity by pharmacological or genetic approaches reduced EGF- and L858R mutant EGFR-induced ERß expression, indicating that Stat3 activation is required for EGFR signaling-mediated ERß up-regulation. Silencing ERß decreased cell proliferation in lung cancer cells that overexpress L858R mutant EGFR. In conclusion, we have identified that Stat3 activation is essential for ERß induction by IL-6, EGF, and the presence of EGFR mutation. The findings shed light on new therapeutic targets for female lung cancer, especially for those with EGFR mutations.

Ha-ras oncogene-induced Stat3 phosphorylation enhances oncogenicity of the cell.

The ras oncogene needs a second factor to induce transformation and tumorigenicity of the cell. In this study, we show that mouse fibroblast 7-4-Stat3C cells overexpressing both Ha-ras(val12) oncogene and active-form Stat3 (Stat3C) showed higher colony formation in soft agar and xenograft tumor growth in BALB/c mice. Further studies show that both serine-727 and tyrosine-705 of Stat3 were phosphorylated while Ha-ras was overexpressed. Interleukin-6 (IL-6)-induced phosphorylation of tyrosine-705 and serine-727, as well as DNA-binding and transcriptional activity of Stat3 were further enhanced by Ha-ras overexpression. In addition, overexpression of Stat3C in 7-4-Stat3C cells prevented the cells from morphological change and apoptosis triggered by the Ha-ras oncogene under serum-depleted conditions. We demonstrate that Ha-ras and Stat3 acting together synergistically induce Stat3 phosphorylation at serine-727 phosphorylation and cyclin D1 expression and further enhance transformation and tumorigenicity of the cell. Ha-ras-induced Stat3 phosphorylation at serine-727 plays a pivotal role in transcriptional activation of cyclin D1 and suppression of cell apoptosis. The effect of Ha-ras on Stat3 phosphorylation at serine-727 was also detected in human bladder (T24) and lung (H460) cancer cells. Stat3 phosphorylation at serine-727 is important in Ras-related tumorigenesis.

Oncogenic Ras-induced morphologic change is through MEK/ERK signaling pathway to downregulate Stat3 at a posttranslational level in NIH3T3 cells.

Ras is a key regulator of the MAP kinase-signaling cascade and may cause morphologic change of Ras-transformed cells. Signal transducer and activator of transcription 3 (Stat3) can be activated by cytokine stimulation. In this study, we unravel that Ha-ras(V12) overexpression can downregulate the expression of Stat3 protein at a posttranslational level in NIH3T3 cells. Furthermore, we demonstrate that Stat3 expression downregulated by Ha-ras(V12) overexpression is through proteosome degradation and not through a mTOR/p70S6K-related signaling pathway. The suppression of Stat3 accompanied by the morphologic change induced by Ha-ras(V12) was through mitogen extracellular kinase (MEK)/extracellular-regulated kinase (ERK) signaling pathway. Microtubule disruption is involved in Ha-ras(V12)-induced morphologic change, which could be reversed by overexpression of Stat3. Taken together, we are the first to demonstrate that Stat3 protein plays a critical role in Ha-ras(V12)-induced morphologic change. Oncogenic Ras-triggered morphologic change is through the activation of MEK/ERK to posttranslationally downregulate Stat3 expression. Our finding may shed light on developing novel therapeutic strategies against Ras-related tumorigenesis.

HER-2/neu raises SHP-2, stops IFN-gamma anti-proliferation in bladder cancer.

Gene amplification or HER-2/neu protein overexpression signals a poor outcome for bladder cancer patients. We investigated the anti-proliferative effect of IFN-gamma in HER-2/neu-transfected human bladder cancer cells (TCC-N5 and TCC-N10). The cells continued growing after IFN-gamma stimulation but did not activate the Janus kinase (Jak)/Stat pathway. We found Jak/Stat protein phosphatase in TCC-N5 and TCC-N10 cells with upregulated Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2). After the cells had been treated with AG825, a HER-2/neu-specific inhibitor, SHP-2 expression declined, and Jak2/Stat1 reactivated. Similar results were reported in a mouse bladder cancer cell line, MBT2, with constitutive HER-2/neu overexpression. Further, AG825 pretreatment restored the anti-proliferation activity of IFN-gamma in TCC-N5 and TCC-N10 cells. Therefore, the suppression of IFN-gamma signaling in HER-2/neu-overexpressing bladder cancer cells might be due to SHP-2 upregulation. The regulation of SHP-2 by HER-2/neu provides a new target for blocking the HER-2/neu oncogenic pathway.

Cell confluence-induced activation of signal transducer and activator of transcription-3 (Stat3) triggers epithelial dome formation via augmentation of sodium hydrogen exchanger-3 (NHE3) expression.

Cell confluence induces the activation of signal transducer and activator of transcription-3 (Stat3) in various cancer and epithelial cells, yet the biological implications and the associated regulatory mechanisms remain unclear. Because confluent polarized epithelia demonstrate dome formation and sodium influx that mimic the onset of differentiation, we sought to elucidate the role of Stat3 in association with the regulation of selective epithelial transporters in this biological phenomenon. This study established the correlation between Stat3 activation and cell confluence-induced dome formation in Madin-Darby canine kidney cells (MDCK) by following Stat3 activation events in dome-forming cells. Epifluorescent and confocal microscopy provided evidence showing specific localization of phosphorylated Stat3 Tyr(705) in the nuclei of dome-forming cells at initial stages. The relationship was further elucidated by the establishment of tetracycline-inducible expression of constitutive Stat3 mutant (Stat3-C) in MDCK cells or expression of dominant negative Stat3 (Stat3-D) stable cell lines (MDCK and NMuMG). Dome formation was promoted by the expression of Stat3-C but inhibited by Stat3-D. Two trans-epithelial transporters, NHE3 and ENaC alpha-subunit, were found to be increased during cell confluence. Interestingly, NHE3 expression could be specifically up-regulated by Stat3-C but inhibited by Stat3-D through promoter regulation, whereas NHE1 and ENaC alpha-subunit were not affected by Stat3 expression. Application of NHE3 shRNA, NHE3 inhibitors (EIPA and S3226) suppressed confluence-induced dome formation in MDCK or NMuMG cells. These results demonstrate a cell confluence-induced Stat3 signaling pathway in epithelial cells in triggering dome formation through NHE3 augmentation.