Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in cancer, promoting its emergence as a promising target for cancer treatment. Inhibiting constitutive STAT3 signaling represents a potential therapeutic approach. We used structure-based design to develop a nonpeptide, cell-permeable, small molecule, termed as LLL12, which targets STAT3. LLL12 was found to inhibit STAT3 phosphorylation (tyrosine 705) and induce apoptosis as indicated by the increases of cleaved caspase-3 and poly (ADP-ribose) polymerase in various breast, pancreatic, and glioblastoma cancer cell lines expressing elevated levels of STAT3 phosphorylation. LLL12 could also inhibit STAT3 phosphorylation induced by interleukin-6 in MDA-MB-453 breast cancer cells. The inhibition of STAT3 by LLL12 was confirmed by the inhibition of STAT3 DNA binding activity and STAT3-dependent transcriptional luciferase activity. Downstream targets of STAT3, cyclin D1, Bcl-2, and survivin were also downregulated by LLL12 at both protein and messenger RNA levels. LLL12 is a potent inhibitor of cell viability, with half-maximal inhibitory concentrations values ranging between 0.16 and 3.09 microM, which are lower than the reported JAK2 inhibitor WP1066 and STAT3 inhibitor S3I-201 in six cancer cell lines expressing elevated levels of STAT3 phosphorylation. In addition, LLL12 inhibits colony formation and cell migration and works synergistically with doxorubicin and gemcitabine. Furthermore, LLL12 demonstrated a potent inhibitory activity on breast and glioblastoma tumor growth in a mouse xenograft model. Our results indicate that LLL12 may be a potential therapeutic agent for human cancer cells expressing constitutive STAT3 signaling.

T he cellular mechanisms contributing to breast and prostate cancer involve a multistep process that includes the inactivation of tumor suppressor genes and the dysregulation of several oncogenic pathways. Among the signaling pathways implicated are the AKT, HER2/neu, and signal transducers and activators of transcription (STAT) 3 pathways.(1-5) Activated or phosphorylated AKT appears to play an important role in proliferation, chemoresistance, and resistance to hormone therapy of breast cancer cells. (6)(7)(8) The AKT pathway has emerged as one of the key oncogenic pathways in breast cancer, carrying with it the potential for therapeutic intervention.(9) Although AKT can be activated through a variety of mechanisms, its dysregulation in many breast cancers stems from overexpression of the HER2/neu protein. (10) HER2/neu is a 185-kDa surface membrane protein that is overexpressed in approximately 25-30% of breast cancers due to amplification of the HER2 gene.(5) Patients with breast cancers that overexpress HER2/neu generally have a poor prognosis, experiencing shorter relapse times and low survival rates. (11) Evidence suggests that cancer cells that overexpress HER2/neu may also be less sensitive to chemotherapy. (12,13) STAT3 is a latent transcription factor and is one of the downstream signaling proteins for cytokine and growth factor receptors. (14,15) Activation of these receptors induces the phosphorylation and subsequent dimerization of two STAT3 monomers through conserved SH2 domains. (16,17) Persistently active STAT3 has been found with high frequency in a wide range of human cancer cell lines and tissues, (18) where it has been implicated in stimulating cell proliferation, promoting angiogenesis, mediating immune evasion, and conferring increased resistance to apoptosis.(19-23) Mora et al. reported constitutive activation of STAT3 in 37 out of 45 (82%) prostate tumor samples.(3) Barton et al. also reported a similar observation except that all the prostate tumor samples they examined had constitutive activation of STAT3.(4) The constitutive activation of STAT3 is also frequently detected in breast cancer specimens with advanced diseases. (20,24,25) STAT3-associated activities are apparently required for the continued survival of certain cancer cells, as interference of the STAT3 pathway has been found to result in growth inhibition and induction of apoptosis, (26)(27)(28) furthering its interest as a potential chemotherapeutic target. (29)(30)(31)(32) Because the dysregulation of multiple oncogenic pathways is common among many cancers, an optimal therapeutic agent would need to be able to inhibit multiple pathways simultaneously while causing minimal deleterious side effects. One compound that may function in this capacity is curcumin, the bioactive component of the perennial herb Curcuma longa. Extensive research has revealed that the complex chemistry of curcumin allows it to influence multiple cell signaling pathways, giving it anti-inflammatory, antioxidant, chemopreventive, and chemotherapeutic ...

Persistent activation of the signal transducer and activator of transcription 3 (STAT3) signalling has been linked to oncogenesis and the development of chemotherapy resistance in glioblastoma and other cancers. Inhibition of the STAT3 pathway thus represents an attractive therapeutic approach for cancer. In this study, we investigated the inhibitory effects of a small molecule compound known as LLL-3, which is a structural analogue of the earlier reported STAT3 inhibitor, STA-21, on the cell viability of human glioblastoma cells, U87, U373, and U251 expressing constitutively activated STAT3. We also investigated the inhibitory effects of LLL-3 on U87 glioblastoma cell growth in a mouse tumour model as well as the impact it had on the survival time of the treated mice. We observed that LLL-3 inhibited STAT3-dependent transcriptional and DNA binding activities. LLL-3 also inhibited viability of U87, U373, and U251 glioblastoma cells as well as induced apoptosis of these glioblastoma cell lines as evidenced by increased poly (ADP-ribose) polymerase (PARP) and caspase-3 cleavages. Furthermore, the U87 glioblastoma tumour-bearing mice treated with LLL-3 exhibited prolonged survival relative to vehicle-treated mice (28.5 vs 16 days) and had smaller intracranial tumours and no evidence of contralateral invasion. These results suggest that LLL-3 may be a potential therapeutic agent in the treatment of glioblastoma with constitutive STAT3 activation.

Brain tumors remain the leading cause of cancer-related deaths in children and often are associated with long-term sequelae among survivors of current therapies. Hence, there is an urgent need to identify actionable targets and to develop more effective therapies. Telomerase and telomeres play important roles in cancer, representing attractive therapeutic targets to treat children with poor-prognosis brain tumors such as diffuse intrinsic pontine glioma (DIPG), high-grade glioma (HGG), and high-risk medulloblastoma. We have previously shown that DIPG, HGG, and medulloblastoma frequently express telomerase activity. Here, we show that the telomerase-dependent incorporation of 6-thio-2'deoxyguanosine (6-thio-dG), a telomerase substrate precursor analogue, into telomeres leads to telomere dysfunction-induced foci (TIF) along with extensive genomic DNA damage, cell growth inhibition, and cell death of primary stem-like cells derived from patients with DIPG, HGG, and medulloblastoma. Importantly, the effect of 6-thio-dG is persistent even after drug withdrawal. Treatment with 6-thio-dG elicits a sequential activation of ATR and ATM pathways and induces G-M arrest. treatment of mice bearing medulloblastoma xenografts with 6-thio-dG delays tumor growth and increases in-tumor TIFs and apoptosis. Furthermore, 6-thio-dG crosses the blood-brain barrier and specifically targets tumor cells in an orthotopic mouse model of DIPG. Together, our findings suggest that 6-thio-dG is a promising novel approach to treat therapy-resistant telomerase-positive pediatric brain tumors..