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 ...

Curcumin, a yellow pigment and the active component of turmeric, has been shown to protect against carcinogenesis and prevent tumor development in several types of cancer. However, its low bioavailability and potency prevent it from being effective in most chemotherapeutic applications. One potential means of circumventing this problem has been the creation of synthetic curcumin analogues. We tested the efficacy of two such analogues, known as FLLL11 and FLLL12, in human pancreatic cancer cell lines. We compared the impact of curcumin with FLLL11 and FLLL12 on cell viability in five different pancreatic cancer cell lines. Although all three compounds were capable of lowering viability in all cell lines tested, FLLL11 and FLLL12 (IC 50 values between 0.28-3.2 and 0.91-3.43λμmol/l, respectively) were substantially more potent than curcumin (IC 50 values between 8.67 and 20.35λμmol/l). In addition, FLLL11 and FLLL12 inhibited phosphorylation of signal transducer and activator of transcription 3 and AKT, two cell signaling pathways frequently found persistently active in many forms of cancer. Furthermore, FLLL11 and FLLL12 were found to be more effective than curcumin in inducing apoptosis as evidenced by increased cleavage of PARP and caspase-3 in pancreatic cancer cell lines. These results indicate that the curcumin analogues, FLLL11 and FLLL12, are more effective than curcumin in inhibiting cell viability and inducing apoptosis, and may have translational potential as chemopreventive or therapeutic agents for pancreatic cancer.

Abstract. Curcumin has numerous anti-carcinogenic properties, but low bioavailability prevents its use in chemotherapeutic applications. One strategy for circumventing this problem has been the creation of synthetic analogues. We tested the efficacy of an analogue known as GO-Y030 in human breast and pancreatic cancer cells. We compared the impact of curcumin and GO-Y030 on the breast cancer cell line MDA-MB-231 and pancreatic cancer cell lines, PANC-1, HPAC and BXPC-3. Both compounds reduced cell viability and induced apoptosis, but GO-Y030 was substantially more potent. We also demonstrated that GO-Y030 was capable of interfering with STAT3, a persistently activated transcription factor in many cancer types. GO-Y030 inhibited STAT3 phosphorylation and transcriptional activity whereas comparable dosages of curcumin had little or no effect. These results indicate that GO-Y030 is a potent inhibitor of cell viability and STAT3 activation, and may thus have potential as a therapeutic agent for cancers expressing high levels of activated STAT3.

The small GTP-binding proteins Rac1 and Cdc42 stimulate activity of the serine/threonine kinase p21-activated kinase-1 (PAK-1) to drive growth factor signaling networks and Ras-driven tumorigenesis. Genomic amplification and over-expression of PAK1 are prevalent in luminal breast cancer and correlate with poor clinical outcome. Here we use a novel and selective small molecule inhibitor, G-5555, of the group I PAKs (PAK1, 2, and 3) to evaluate the importance of PAK1 in promoting growth of PAK1 amplified breast cancer cells. Cell lines with amplification of PAK1 were found to be more sensitive to PAK1 inhibition than non-amplified cell lines. Additionally, reverse phase protein array (RPPA) was used to assess the effects of PAK1 inhibition on a wide range of signaling pathways in both amplified and non-amplified cell lines. Reduced levels of phosphorylation of MEK S298 was observed in all cell lines exposed to G-5555 irrespective of amplification status, consistent with PAK1 inhibition in these cell lines. However, modulation of this downstream PAK1 substrate did not correlate with inhibition of cell proliferation or induction of cell death. Cell lines that showed inhibition of proliferation in response to G-5555 also showed enhanced levels of cell death along with apoptosis. Moreover, G-5555 reduced tumor growth in the PAK1 amplified MDA-MB-175 xenograft tumor model. Finally, we compared the in vitro activity of G-5555 with palbociclib, a recently approved inhibitor of the cyclin-dependent kinases CDK4 and CDK6, in PAK1 amplified luminal breast cancer cell lines. Our data supports PAK1 as an attractive target in PAK1 amplified cells and tumors and suggests that inhibiting PAK1 rather than CDK4/6 in this context may be a more attractive therapeutic strategy. Citation Format: Ong CC, Blackwood E, Jakubiak D, Daemen A, Ramaswamy S, Heise C, Schmidt M, Sanders L, Wilson TR, Huw L, Ndubaku C, Rudolph J, Hoeflich KP, Friedman L, O'Brien T. PAK-1 amplified breast cancer cell lines are preferentially sensitive to PAK inhibition with G-5555. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-04.