Jiayuh Lin

Inhibition of constitutively active Stat3 suppresses growth of human ovarian and breast cancer cells

Signal transducers and activators of transcription (STATs) are transcription factors activated in response to cytokines and growth factors. Constitutively active Stat3 has been shown to mediate oncogenic transformation in cultured cells and induce tumor formation in mice. An increasing number of tumor-derived cell lines as well as samples from human cancer have been reported to express constitutively active Stat3 protein. We previously demonstrated that ovarian cancer cell lines express high levels of constitutively active Stat3. In this study, we show that inhibition of the Stat3 signaling pathway using the Janus Kinase-selective inhibitor, AG490, and a dominant negative Stat3 (Stat3β) significantly suppresses the growth of ovarian and breast cancer cell lines harboring constitutively active Stat3. In the ovarian cancer cell lines, AG490 also diminished the phosphorylation of Stat3, Stat3 DNA binding activity, and the expression of Bcl-xL. Further, AG490 induced significant apoptosis in ovarian and breast cancer cell lines expressing high levels of constitutively active Stat3 but had a less profound effect on normal cells lacking constitutively active Stat3. AG490 also enhanced apoptosis induced by cisplatin in ovarian cancer cells. These results suggest that inhibition of Stat3 signaling may provide a potential therapeutic approach for treating ovarian and breast cancers.

Curcumin is a potent DNA hypomethylation agent

Molecular docking of the interaction of curcumin and DNMT1 suggested that curcumin covalently blocks the catalytic thiolate of C1226 of DNMT1 to exert its inhibitory effect. This was validated by showing that curcumin inhibits the activity of M. SssI with an IC(50) of 30 nM, but no inhibitory activity of hexahydrocurcumin up to 100 microM. In addition, curcumin can induce global DNA hypomethylation in a leukemia cell line.

Stat3 activation in human endometrial and cervical cancers

The activation of signal transducer and activator of transcription 3 (Stat3) has been implicated in the oncogenesis of cancer and is regarded as a novel target for cancer therapy. Stat3 is classified as a proto-oncogene, because an activated form of Stat3 can mediate oncogenic transformation in cultured cells and tumour formation in nude mice. The constitutive activation of Stat3 has been frequently detected in various types of human cancers. However, the constitutive activation of Stat3 in endometrial and cervical cancers has not been studied. We examined tyrosine phosphorylation of Stat3 (activated form of Stat3) in multiple endometrial and cervical cancer tissues using tissue microarray slides as well as cancer cell lines to explore the possible activation of Stat3. Our results indicated that elevated phosphorylation of Stat3 was detected in cervical and endometrial cancer cell lines. Our results also showed that elevated levels of phosphorylation of Stat3 protein were detected in the endometrial and cervical cancer specimens. This is the first study to demonstrate that Stat3 is activated in human endometrial and cervical cancer tissues. Immunohistochemical staining showed that activated Stat3 is associated with increased expression of downstream antiapoptotic genes, Bcl-xL, survivin, and Mcl-1 in these tissues. Expression of a dominant-negative Stat3 mutant using adenovirus-mediated gene transfer inhibited cell growth and induced apoptosis in HeLa and SiHa cervical cancer cell lines expressing elevated levels of Stat3 phosphorylation. Further, a JAK/Stat3 small molecular inhibitor, JSI-124, induced apoptosis more selectively in HeLa and SiHa cancer cell lines than Ishikawa cell line without elevated levels of Stat3 phosphorylation. These results indicate that Stat3 is activated in human endometrial and cervical cancers and the inhibition of constitutive Stat3 signaling may be an effective target for cancer intervention in these two cancers.

p53 regulates Stat3 phosphorylation and DNA binding activity in human prostate cancer cells expressing constitutively active Stat3

Constitutive activation of the signal transducer and activator of transcription 3 (Stat3) and mutation of the p53 are both commonly detected in human prostate cancer cells. We sought to investigate whether there is functional regulation of Stat3 by wild-type (wt) p53. Our results demonstrate that expression of wt p53 but not mutant p53 significantly reduced tyrosine phosphorylation of Stat3 and inhibited Stat3 DNA binding activity in both DU145 and Tsu prostate cancer cell lines that express constitutively active Stat3. Expression of the p53 downstream target, p21WAF-1, did not have any inhibitory effect on Stat3 phosphorylation. Wt p53 but not p21WAF-1 induced dramatic apoptosis in these prostate cancer cells. Expression of wt p53 did not cause a reduction of phosphorylation-independent Stat3 protein and reduction of phosphorylation of three unrelated protein kinases, ERK1, ERK2 (ERK1/2), and AKT. Interestingly, p53-dependent apoptosis occurred in the presence of high levels of phosphorylated AKT and ERK1/2 in both DU145 and Tsu prostate cancer cells. Further, we evaluated a series of established human prostate, breast, and ovarian cancer cell lines and found that all cancer cell lines expressing constitutively active Stat3, only harbor mutated or deleted p53. One implication of these results is that the anti-proliferative activities of p53 may not be compatible with the constitutive Stat3 signal in cancer cells.

Characterization of STAT3 activation and expression in canine and human osteosarcoma

BackgroundDysregulation of signal transducer and activator of transcription 3 (STAT3) has been implicated as a key participant in tumor cell survival, proliferation, and metastasis and is often correlated with a more malignant tumor phenotype. STAT3 phosphorylation has been demonstrated in a subset of human osteosarcoma (OSA) tissues and cell lines. OSA in the canine population is known to exhibit a similar clinical behavior and molecular biology when compared to its human counterpart, and is often used as a model for preclinical testing of novel therapeutics. The purpose of this study was to investigate the potential role of STAT3 in canine and human OSA, and to evaluate the biologic activity of a novel small molecule STAT3 inhibitor.MethodsTo examine STAT3 and Src expression in OSA, we performed Western blotting and RT-PCR. OSA cells were treated with either STAT3 siRNA or small molecule Src (SU6656) or STAT3 (LLL3) inhibitors and cell proliferation (CyQUANT), caspase 3/7 activity (ELISA), apoptosis (Western blotting for PARP cleavage) and/or viability (Wst-1) were determined. Additionally, STAT3 DNA binding after treatment was determined using EMSA. Expression of STAT3 targets after treatment was demonstrated with Western blotting, RT-PCR, or gel zymography.ResultsOur data demonstrate that constitutive activation of STAT3 is present in a subset of canine OSA tumors and human and canine cell lines, but not normal canine osteoblasts. In both canine and human OSA cell lines, downregulation of STAT3 activity through inhibition of upstream Src family kinases using SU6656, inhibition of STAT3 DNA binding and transcriptional activities using LLL3, or modulation of STAT3 expression using siRNA, all resulted in decreased cell proliferation and viability, ultimately inducing caspase-3/7 mediated apoptosis in treated cells. Furthermore, inhibition of either Src or STAT3 activity downregulated the expression of survivin, VEGF, and MMP2, all known transcriptional targets of STAT3.ConclusionThese data suggest that STAT3 activation contributes to the survival and proliferation of human and canine OSA cells, thereby providing a potentially promising target for therapeutic intervention. Future investigational trials of LLL3 in dogs with spontaneous OSA will help to more accurately define the role of STAT3 in the clinical setting.

Signal transducer and activator of transcription 3 is involved in cell growth and survival of human rhabdomyosarcoma and osteosarcoma cells

BackgroundStat3 has been classified as a proto-oncogene and constitutive Stat3 signaling appears to be involved in oncogenesis of human cancers. However, whether constitutive Stat3 signaling plays a role in the survival and growth of osteosarcomas, rhabdomyosarcomas, and soft-tissue sarcomas is still unclear.MethodsTo examine whether Stat3 is activated in osteosarcomas, rhabdomyosarcomas and other soft-tissue sarcomas we analyzed sarcoma tissue microarray slides and sarcoma cell lines using immunohistochemistry and Western blot analysis, respectively, with a phospho-specific Stat3 antibody. To examine whether the activated Stat3 pathway is important for sarcoma cell growth and survival, adenovirus-mediated expression of a dominant-negative Stat3 (Y705F) and a small molecule inhibitor (termed STA-21) were used to inhibit constitutive Stat3 signaling in human sarcoma cell lines expressing elevated levels of Stat3 phosphorylation. Cell viability was determined by MTT assays and induction of apoptosis was analyzed by western blotting using antibodies that specifically recognize cleaved caspases-3, 8, and 9.ResultsStat3 phosphorylation is elevated in 19% (21/113) of osteosarcoma, 27% (17/64) of rhabdomyosarcoma, and 15% (22/151) of other soft-tissue sarcoma tissues as well as in sarcoma cell lines. Expression of the dominant-negative Stat3 and treatment of STA-21 inhibited cell viability and growth and induced apoptosis through caspases 3, 8 and 9 pathways in human sarcoma cell lines expressing elevated levels of phosphorylated Stat3.ConclusionThis study demonstrates that Stat3 phosphorylation is elevated in human rhabdomyosarcoma, osteosarcomas and soft-tissue sarcomas. Furthermore, the activated Stat3 pathway is important for cell growth and survival of human sarcoma cells.

Inhibition of AKT survival pathway by a small molecule inhibitor in human endometrial cancer cells

The PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumour suppressor is mutated in 40–50% of human endometrial cancers. PTEN exerts its effects in part via inhibition of the antiapoptotic protein AKT. We demonstrate that two endometrial cancer cell lines that harbour PTEN mutations, Ishikawa and RL95-2, have high levels of phosphorylated AKT and high AKT kinase activity. Two additional endometrial cancer cell lines that express wild-type PTEN, Hec1A and KLE, have little phosphorylated AKT and minimal demonstrable AKT kinase activity. We tested a potential inhibitor of the AKT pathway, API-59CJ-OMe, in these four cell lines. We found that API-59CJ-OMe inhibits AKT kinase activity and induces apoptosis in the Ishikawa and RL95-2 cell lines with high AKT activity, but has little effect on Hec1A and KLE cells without AKT activity. API-59CJ-OMe may therefore have therapeutic potential for those endometrial cancers that harbour PTEN mutations and AKT activation.

Stat3 upregulates MEK5 expression in human breast cancer cells.

The constitutive activation of signal transducer and activator of transcription 3 (Stat3) is frequently detected in breast cancer cell lines but not in normal breast epithelial cells. Stat3 has been classified as an oncogene, because constitutively active Stat3 can mediate oncogenic transformation in cultured cells and tumor formation in nude mice. Since Stat3 appears to play an important role in breast cancer, it is of interest to investigate Stat3-regulated genes and elucidate Stat3-mediated oncogenesis. In this study, we investigated the Stat3-regulated genes in human breast epithelial cells. Upon overexpression of Stat3-C, a constitutively active Stat3 form, in nonmalignant telomerase immortalized breast (TERT) cells, the total mRNA was extracted and subjected to Affymetrix microarray analysis. Our results showed that mitogen-activated protein kinase kinase 5 (MEK5) was markedly induced (more than 22-fold increase, P<0.001) by Stat3-C expression. RT–PCR result also demonstrated that MEK5 mRNA was significantly induced by Stat3-C in TERT cells. The upregulation of MEK5 by Stat3-C was further confirmed by Western blot in MCF10A breast epithelial cells. Furthermore, in MDA-MB-435s breast carcinoma cells, which express high levels of activated Stat3 and MEK5, MEK5 protein was significantly reduced by using Stat3 short interfering RNA. The reduction of MEK5 was consistent with Stat3 knockdown in this breast carcinoma cell line. We also investigated MEK5 expression in different breast carcinoma cell lines and breast cancer tissues using tissue array analysis. Compared with nonmalignant breast epithelial cells or normal tissues without constitutively active Stat3 signaling, MEK5 protein levels are remarkably higher in breast carcinoma cell lines and cancer tissues with constitutively activated Stat3. Taken together, our findings suggest that constitutively active Stat3 upregulates MEK5 in the breast epithelial cells. MEK5 may be one of the Stat3-regulated genes and plays its essential roles in oncogenesis mediated by aberrantly activated Stat3 signaling in breast carcinomatosis and malignancies.

STAT3 as a Therapeutic Target for Glioblastoma

Glioblastoma (GBM) is the most common type of primary malignant brain tumor. Despite advances in surgical resection, radiotherapy and chemotherapy, prognosis remains very poor. Accordingly, recent studies have been focused on the aberrant signal transduction pathways in glioblastoma. Many patient derived primary glioblastomas and cell lines express constitutively activated signal transducers and activators of transcription 3 (STAT3). Here we focused on the recent progresses regarding to the roles of STAT3 in glioblastoma and glioblastoma stem cells (GBM-SCs), the dysregulation of STAT3 in glioblastoma, and targeting STAT3 for glioblastoma therapy.

Reactivation of RASSF1A in Breast Cancer Cells by Curcumin

Reactivation of tumor suppressor genes (TSGs) involved in carcinogenesis by nontoxic bioactive food component represents a promising strategy for cancer chemoprevention. Recently, curcumin has been demonstrated to inhibit a bacterial DNA methyltransferase (M. Sss I) activity, induce global DNA hypomethylation in leukemia cells, and reactivate several hypermethylation silenced genes in lung and prostate cancer cells. Herein, we demonstrated that curcumin can enhance the mRNA and protein levels of ras-association domain family protein 1A (RASSF1A), 1 hypermethylation-silenced TSG, and decrease its promoter methylation in breast cancer cells. Mechanistic study demonstrated that curcumin can decrease DNA methylation activity of nuclear extract and downregulate the mRNA and protein levels of DNMT1 in MCF-7 cells, which may be associated with curcumin-induced disruption of NF-κB/Sp1 complex bound to the promoter region of DNMT1. Altogether, this study reveals a novel molecular mechanism of curcumin as a chemo-preventive agent for breast cancer through hypomethylation reactivation of RASSF1A.