Hsuan-Heng Yeh

Autocrine IL-6-induced Stat3 activation contributes to the pathogenesis of lung adenocarcinoma and malignant pleural effusion

Malignant pleural effusion (MPE) is a poor prognostic sign for patients with non-small-cell lung cancer (NSCLC). The generation of MPE is largely regulated by vascular endothelial growth factor (VEGF), and upregulation of VEGF by Stat3 has been observed in several types of tumor cells. In this study, we demonstrate constitutively activated Stat3 in several human lung cancer cell lines and in tumor cells infiltrated in the pleurae of patients with adenocarcinoma cell lung cancer (ADCLC) and MPE. The observations suggest that activated Stat3 plays a role in the pathogenesis of ADCLC. In PC14PE6/AS2 cells, a Stat3-positive human ADCLC cell line, autocrine IL-6 activated Stat3 via JAKs, not via Src kinase. PC14PE6/AS2 cells express higher VEGF mRNA and protein than do Stat3-negative PC14PE6/AS2/dnStat3 cells. In an animal model, PC14P6/AS2/dnStat3 cells produced no MPE and less lung metastasis than did PC14P6/AS2 cells. PC14PE6/AS2 cells also expressed higher VEGF protein, microvessel density, and vascular permeability in tumors than did PC14P6/AS2/dnStat3 cells. Therefore, we hypothesize that autocrine IL-6 activation of Stat3 in ADCLC may be involved in the formation of malignant pleural effusion by upregulating VEGF. Higher levels of IL-6 and VEGF were also found in the pleural fluids of patients with ADCLC than in patients with congestive heart failure. The autocrine IL-6/Stat3/VEGF signaling pathway may also be activated in patients with ADCLC and MPE. These findings provide novel targets for the management of MPE.

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.

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.

Abstract 4103: IL-6 dynamics regulate neuroendocrine transformation in gefitinib acquired resistance EGFR mutant lung cancer cells

Transformation to small-cell lung cancer (SCLC, one of aggressive neuroendocrine [NE] tumor) is reported when activating epidermal growth factor receptor (EGFR) mutant non-small-cell lung cancer acquired resistance to tyrosine kinase inhibitors (TKI, such as gefitinib). IL-6 activation confers to acquire TKI resistance and associates with p53 and RB inactivation those are SCLC hallmark changes. Whether NE transformation could phenocopy in isogenic acquired resistance cell line and the role of IL-6 in this process remain unknown. We established 827GRs (including 827GR, 827GR + and 827GR.M6) acquired resistance to gefitinib from HCC827 cells by long term stepwise treated with gefitinib and they still had EGFR exon 19 deletion without acquired T790M. 827GR was parental resistance line with unstable gefitinib resistance in drug-free medium by passage. We maintained 827GR in medium with or without 1μM gefitinib over 6 months to generate stable clones: 827GR + and 827GR.M6. 827GRs had SCLC hallmark changes, i.e., inactivation of p53, RB and Notch by western blot and gene set enrichment analysis. Compared to HCC827, 827GRs were more sensitive to cisplatin and etoposide but not paclitaxel. IL-6 level was positive correlated with gefitinib resistance among 827GRs by cytokine array and ELISA. Interestingly, among 827GRs, 827GR.M6 harbored low IL-6 secretion had obviously NOTCH-ASCL1-DLL3 alteration, high NE marker expression and significant inter-rater agreement with selected Byers’ SCLC gene signature than high IL-6 secretion 827GR + , suggesting IL-6 dynamics might regulate NE marker expression. IL-6 genetic manipulation in HCC827 and 827GR + also demonstrated this phenomenon. Moreover, IL-6 dynamics correlate with NE expression also showed in patient derived lung cancer cell line in published microarray dataset (GSE64322). In conclusion, our work demonstrated activating EGFR mutant lung cancer acquired resistance to TKI with NE transformation could phenocopy in isogenic cell line model and IL-6 dynamics might regulate this process. Citation Format: Shang-Yin Wu, Hsuan-Heng Yeh, Chun-Hua Hung, Chien-Chung Lin, Wen-Pin Su, Wu-Chou Su. IL-6 dynamics regulate neuroendocrine transformation in gefitinib acquired resistance EGFR mutant lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4103. doi:10.1158/1538-7445.AM2017-4103

Abstract 4157: IL-6 glycoforms differentially modulate the polarization of tumor-associated macrophages in lung cancer

Interleukin-6 (IL-6) is critical in modulating both tumor growth and anti-tumor immunity. Here, we describe the glycosylation pattern of lung cancer cell-secreted IL-6 and examine the function of the IL-6 glycoforms in polarization of tumor-associated macrophages (TAMs). IL-6 with different molecular weights were detected in various lung cancer cells. Because in NetNGlyc 1.0 Server, one possible N-glycosylation site has been predicted at N73 on IL-6, we used tunicamycin treatment and site-directed mutagenesis on N73 to demonstrate that lung cancer cell-secreted IL-6 is modified by N-glycosylation. To explore more detailed attached glycans, we measured the expression of glycosyltransferases by qPCR and found that the expression of fucosyltransferase 8 (FUT8), responsible for core fucosylation on N-glycosylated proteins, was higher in lung cancer cells than normal bronchial cells. Core fucosylation on IL-6 was reduced by silencing FUT8. We generated AS2-IL6, AS2-IL6-shFUT8, and AS2-IL6-N73Q cells for producing full-glycosylated IL-6 (G-IL6), core fucose-depleted IL-6 (DeCF-IL6), and Asn73-N-glycan-depleted IL-6 (N73Q-IL6). To examine the effect of the IL-6 glycoforms on differentiation of TAMs, THP-1 monocytic leukemic cells were incubated with phorbol-12-myristate 13-acetate for transforming to macrophages, followed by co-culture with the AS2 cell derivatives to mimic the education of TAMs. The co-cultured TAMs of each AS2 derivative showed distinct morphology. Intriguingly, the G-IL6-producing cells promote the expression of M2-TAM markers CD204, CCR1, arginase-1, and M2-specific cytokines that benefit anti-tumor activity, while DeCF-IL6 and N73Q-IL6 favored the formation of M1 macrophages. The phagocytosis capacity of cocultured macrophages is also altered by different IL-6 glycoform-secreting cells. On the other hand, the Stat3 activation status induced by IL-6 glycoforms is concordant to our previous results, in which the G-IL6 can induce a persistant Stat3 activation that is not observed in the DeCF-IL6- and N73Q-IL6-induced macrophages. We subsequently examined the distribution and polarization of TAMs in in vivo xenograft tumor model by both subcutaneous and intravenous injection of the IL-6 glycoform-overexpressing cells. Higher proportion of M2 TAM was attracted by the N73Q-IL6 tumor than other glycoform tumors. Together, we report the presence of specific IL-6 glycoforms secreted from lung cancer cells. Moreover, the glycosylation on IL-6 changes its activity on the polarization of TAMs, suggesting the pivotal role of soluble factor(s) in orchestrating the tumor microenvironment of lung cancer. Citation Format: Chun-Hua Hung, Hao-Chen Wang, Hsuan-Heng Yeh, Chien-Chung Lin, Wu-Chou Su. IL-6 glycoforms differentially modulate the polarization of tumor-associated macrophages in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4157.

Abstract 3452: N-glycosylation on lung cancer cell-secreted IL-6 prolongs its activation on JAK/STAT pathway

Interleukin-6 (IL-6) is overexpressed in various cancer cells, intriguingly contributing to both tumor growth and modulating anti-tumor immunity. Cell-type-specific glycosylations on IL-6 have been deduced in early studies; however, the glycosylation pattern and biological function of cancer cell-secreted IL-6 remain unclear. Here, we describe the glycosylation pattern of lung cancer cell-secreted IL-6 and its impact on the activation of JAK/STAT pathway. IL-6 molecules with different molecular weights were detected in the conditional media of IL-6-overexpressed lung cancer cell lines by immunoblot. Because in NetNGlyc 1.0 Server, one possible N-glycosylation site has been predicted at N73 on IL-6, we used treatment of N-glycosylation-specific inhibitors and site-directed mutagenesis on N73 to demonstrate that lung cancer cell-secreted IL-6 is modified by N-glycosylation. To examine which glycotransferases may participate in the modification, we had screened the expression of glycosyltransferases using qPCR and found that the expression of fucosyltransferase 8 (FUT8), responsible for core fucosylation on N-glycosylated proteins, was higher in lung cancer cells compared to normal bronchial cell. We then reduced core fucosylation on lung cancer cell-secreted IL-6 by silencing FUT8 with shRNA transduction. Subsequently, cells were treated with conditional media containing fully-glycosylated or core fucose-depleted IL-6 to uncover the potential influences on cellular signaling from glycosylation on IL-6. The fully-gycosylated IL-6 induced prolonged STAT3Y705 phosphorylation and distinct gene population compared to core fucose-depleted IL-6. The nuclear retention of STAT3 was concordant with the prolonged STAT3 activation in cells treated with fully-gycosylated IL-6. In paired normal (N) and tumor (T) tissues from lung cancer patients, higher FUT8 mRNA was detected in tumor part than normal part. Besides, we found similar glycosylation pattern in the secreted IL-6 of short-term cultured lung cancer cells derived from malignant pleural effusions. Together, we report the presence of specific IL-6 glycoforms secreted from lung cancer cell lines and lung cancer cells from clinical samples. Moreover, the glycosylation on IL-6 changes its activity on the regulation of JAK/STAT pathway. Citation Format: Chun-Hua Hung, Hsuan-Heng Yeh, Hao-Chen Wang, Chien-Chung Lin, Tsung-Lin Tsai, Wei-Lun Huang, Chuan-Fa Chang, Wu-Chou Su. N-glycosylation on lung cancer cell-secreted IL-6 prolongs its activation on JAK/STAT pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3452. doi:10.1158/1538-7445.AM2014-3452

Abstract 2968: FTY720 inhibits mutant Kras-induced lung cancer via disrupting Stat3-S1PR1 vicious cycle and downregulating tumor PD-L1 expression

Aberrant activation of signal transducer and activator of transcription 3 (Stat3) occurs in many cancers and plays a critical role in tumor progression. The system that Stat3-induced Sphingosine-1-phosphate receptor 1 (S1PR1) expression and the S1P-S1PR1 pathway reciprocally regulate Stat3 activity was considered a major positive feedback loop for persistent Stat3 activation in cancer cells and the cells of tumor microenvironment. Tumor cells expressing the ligand for the receptor programmed death-1 (PD-1), PD-L1, have been shown to increase apoptosis of antigen-specific human T-cell to evade the immune system. In this study, we determined the influence of disrupting Stat3-S1PR1 vicious cycle on tumor formation and PD-L1 expression in lung cancer. We have established mutant Kras-induced lung cancer mice model and found that Stat3 phosphorylation was elevated in tumor tissues of lung cancer. Treatment of lung cancer cells with AG490 (JAK2 inhibitor) inhibited Stat3 activation and AG490 administration to the mice decreased the numbers of lung cancer nodules, indicating that blockage of Stat3 activation could inhibit mutant Kras-induced lung cancer in mice. Similar effects were also detected by using Stat3 inhibitor, S3I-201. We found that lung cancer cells expressed more pStat3 showed higher PD-L1 expression levels than that of lower pStat3 expressing cells. Inhibition of Stat3 activation by AG490 decreased PD-L1 expression in vitro and in vivo, indicating that PD-L1 expression could be regulated by Stat3. Moreover, FTY720 (S1P antagonist) could inhibit S1PR1 expression, down-regulate Stat3 activity, pJAK1 expression and inhibit IL-6 secretion in lung cancer cell lines and causes inhibition of the mutant Kras-induced lung cancer in mice. Furthermore, FTY720 administration dose-dependently suppressed tumor PD-L1 expression in mice lung cancer tissues. Taken together, our study indicated that FTY720 could suppress mutant Kras induced-lung cancer formation via inhibiting persistent Stat3 activation and down-regulating tumor PD-L1 expression. Citation Format: Hsuan-Heng Yeh, Tsung-I Hsu, Jan-Jong Hung, Wen-Pin Su, Wu-Chou Su. FTY720 inhibits mutant Kras-induced lung cancer via disrupting Stat3-S1PR1 vicious cycle and downregulating tumor PD-L1 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2968. doi:10.1158/1538-7445.AM2014-2968

Abstract 3222: Metformin enhances cisplatin cytotoxicity of lung cancer through Stat3 pathway

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The antitumorigenic effects of metformin were mostly interpreted as attributing to the activation of LKB1-AMPK pathway and the studies investigating the AMPK-independent mechanism of metformin remained limited. In lung cancer, cisplatin-based chemotherapy remained the first line regimen for advanced lung cancer patients especially those without EGFR mutation. Previous study demonstrated the linkage between IL6-Stat3 autocrine and chemoresistance. In this study, we first showed that lung cancer line S3C (PC14PE6/AS2 cells with expressing plasmids containing constitutively-active Stat3) was more resistant to cisplatin than PC14PE6/AS2 cell. Treating lung cancer cells with Stat3 inhibitor (JSI-124) or metformin combined with cisplatin, the proliferation of cancer cells were inhibited additionally. And metformin inhibited Stat3 phosphorylation in PC14PE6/AS2 and A549 cells. To investigate the upstream mechanism associated Stat3 phosphorylation, we examined the secretion of IL-6 which was inhibited by metformin. And VEGF secretion, representing Stat3 downstream pathway, was inhibited by metformin in vitro and in vivo. Conversely, the secretion of VEGF was not inhibited in S3C cells after metformin treatment. However, using another AMPK activator-AICAR, the enhancement of cisplatin cytotoxicity is not obvious. In contrast to metformin, AICAR induced AMPK phosphorylation but Stat3 phosphorylation was not inhibited. To verify the LKB-AMPK independent effect of metformin on Stat3 phosphorylation, PC14PE6/AS2 cells were transfected with siRNA targeting LKB1 or AMPK. We demonstrated the inhibition of Stat3 phosphorylation of metformin even under the knockdown of LKB1-AMPK pathway. We also found that MTOR downstream signal including P70S6K and 4EBP1 were inhibited by metformin. Though previous study demonstrated MTOR pathway modulated Stat3 pathway, we found P70S6K and 4EBP1 phosphorylation were inhibited after treatment with metformin at 8hr and Stat3 phosphorylation change was detected after 24hr. Using MTOR inhibitor (rapamycin), the Stat3 tyrosine phosphorylation was not inhibited. Finally, we used subcutaneous tumor xenografts to evaluate the effect of combination of metformin with cisplatin in vivo. Body weight, blood sugar, and tumor volume were recorded in four groups including control, metformin, cisplatin, and metformin combined with cisplatin. Body weights and blood sugar were not different between combination treatment group and control group. In combination group, tumor stopped growing at 18th day and the tumor sizes were obvious less than the other three groups. Immunohistochemical staining of ki67 verified the lower proliferation in combination group. Our study demonstrated that metformin, independent of LKB1-AMPK pathway, inhibits Stat3 phosphorylation which contributes the enhancement of cisplatin cytotoxicity in lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3222. doi:1538-7445.AM2012-3222