Stress responses are critical for estrogen (E2)-induced apoptosis in E2-deprived breast cancer cells. Nuclear factor-kappa B (NF-κB) is an important therapeutic target to prevent stress responses in chronic inflammatory diseases including cancer. However, whether E2 activates NF-κB to participate in stress-associated apoptosis in E2-deprived breast cancer cells is unknown. Here, we demonstrated that E2 differentially modulates NF-κB activity according to treatment time. E2 initially has significant potential to suppress NF-κB activation; it completely blocks tumor necrosis factor alpha (TNFα)-induced activation of NF-κB. We found that E2 preferentially and constantly enhances the expression of the adipogenic transcription factor CCAAT/enhancer binding protein beta (C/EBPβ), which is responsible for the suppression of NF-κB activation by E2 in MCF-7:5C cells. Interestingly, NF-κB p65 DNA-binding activity is increased when E2 is administered for 48 h, leading to the induction of TNFα and associated apoptosis. Blocking the nuclear translocation of NF-κB can completely prevent the induction of TNFα and apoptosis induced by E2. Further examination revealed that protein kinase RNA-like endoplasmic reticulum kinase (PERK), a stress sensor of unfolded protein response (UPR), plays an essential role in the late activation of NF-κB by E2. This modulation between PERK and NF-κB is mainly mediated by a stress responsive transcription factor, transducer and activator of transcription 3 (STAT3), independently of the classic canonical IκBα signaling pathway. Thus, inhibition of PERK kinase activity completely blocks the DNA binding of both STAT3 and NF-κB, thereby preventing induction of NF-κB-dependent genes and E2-induced apoptosis. All of these findings suggest that PERK is a key regulator to convey stress signals from the endoplasmic reticulum to the nucleus and illustrate a crucial role for the novel PERK/STAT3/NF-κB/TNFα axis in E2-induced apoptosis in E2-deprived breast cancer cells.

Stress responses are critical for estrogen (E2) to induce apoptosis in E2-deprived breast cancer cells. Nuclear factor-kappa B (NF-κB) is well known as a therapeutic target to prevent stress responses in chronic inflammatory diseases including cancer. However, whether E2 activates NF-κB to participate in stress-associated apoptosis in E2-deprived breast cancer cells is unclear. We demonstrated that E2 differentially modulates NF-κB activity in E2-deprived breast cancer cells according to the treatment time. Because E2 initially has significant potential to down modulate the NF-κB activation, it completely suppresses the tumor necrosis factor alpha (TNFα)-induced NF-κB activation. We found that E2 preferentially and constantly enhances the expression of transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) which is responsible for suppression of NF-κB activation by E2 in MCF-7:5C cells. The mTOR signaling pathway promotes repression of NF-κB by C/EBPβ which is confirmed by the evidence that inhibition of mTOR is synergistic with E2 to upregulate NF-κB-dependent genes, such as TNFα. Interestingly, NF-κB p65 activity is upregulated when E2-treatment is administered for 48 hours, leading to induction of TNFα. Blocking the nuclear translocation of NF-κB completely prevents E2 from induction of TNFα and apoptosis. Importantly, protein kinase RNA-like endoplasmic reticulum kinase (PERK), a stress sensor of unfolded protein response, is activated by E2 and plays an essential role in increasing NF-κB p65 DNA binding through the activation of STAT3, independently of canonical IκBα signal pathway. Thus, inhibition of PERK kinase activity completely blocks nuclear activation of NF-κB and NF-κB-dependent induction of TNFα, thereby preventing E2-induced apoptosis. All of these findings illustrate a crucial role for the novel PERK/NF-κB/TNFα axis in E2-induced apoptosis which is integrally modulated by the stress responsive transcription factor C/EBPβ and endoplasmic reticulum stress. This study provides an important rationale for exercising caution in clinical trials when considering targeting PERK or NF-κB following the development of acquired resistance to aromatase inhibitors whereas mTOR may be a target to enhance the therapeutic effects of E2 in antihormone resistant breast cancer. Citation Format: Ping Fan, Amit K. Tyagi, Fadeke A. Agboke, Niranjana Pokharel, V. Craig Jordan. Integral modulation of nuclear factor-kappa B activation by C/EBPβ and the endoplasmic reticulum stress sensor PERK to mediate estrogen-induced apoptosis in estrogen-deprived breast 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 2332. doi:10.1158/1538-7445.AM2017-2332

Factor quinolinone inhibitors (FQIs), a first-in-class set of small molecule inhibitors targeted to the transcription factor LSF (TFCP2), exhibit promising cancer chemotherapeutic properties. FQI1, the initial lead compound identified, unexpectedly induced a concentration-dependent delay in mitotic progression. Here, we show that FQI1 can rapidly and reversibly lead to mitotic arrest, even when added directly to mitotic cells, implying that FQI1-mediated mitotic defects are not transcriptionally based. Furthermore, treatment with FQIs resulted in a striking, concentration-dependent diminishment of spindle microtubules, accompanied by a concentration-dependent increase in multi-aster formation. Aberrant γ-tubulin localization was also observed. These phenotypes suggest that perturbation of spindle microtubules is the primary event leading to the mitotic delays upon FQI1 treatment. Previously, FQIs were shown to specifically inhibit not only LSF DNA-binding activity, which requires LSF oligomerization to tetramers, but also other specific LSF-protein interactions. Other transcription factors participate in mitosis through non-transcriptional means, and we recently reported that LSF directly binds α-tubulin and is present in purified cellular tubulin preparations. Consistent with a microtubule role for LSF, here we show that LSF enhanced the rate of tubulin polymerization in vitro, and FQI1 inhibited such polymerization. To probe whether the FQI1-mediated spindle abnormalities could result from inhibition of mitotic LSF-protein interactions, mass spectrometry was performed using as bait an inducible, tagged form of LSF that is biotinylated by endogenous enzymes. The global proteomics analysis yielded expected associations for a transcription factor, notably with RNA processing machinery, but also to nontranscriptional components. In particular, and consistent with spindle disruption due to FQI treatment, mitotic, FQI1-sensitive interactions were identified between the biotinylated LSF and microtubule-associated proteins that regulate spindle assembly, positioning, and dynamics, as well as centrosome-associated proteins. Probing the mitotic LSF interactome using small molecule inhibitors therefore supported a non-transcriptional role for LSF in mediating progression through mitosis.

Factor quinolinone inhibitors are promising anti-cancer compounds, initially characterized as specific inhibitors of the oncogenic transcription factor LSF (TFCP2). These compounds exert anti-proliferative activity at least in part by disrupting mitotic spindles. Herein, we report additional interphase consequences of the initial lead compound, FQI1, in two telomerase immortalized cell lines. Within minutes of FQI1 addition, the microtubule network is disrupted, resulting in a substantial, although not complete, depletion of microtubules as evidenced both by microtubule sedimentation assays and microscopy. Surprisingly, this microtubule breakdown is quickly followed by an increase in tubulin acetylation in the remaining microtubules. The sudden breakdown and partial depolymerization of the microtubule network precedes FQI1-induced morphological changes. These involve rapid reduction of cell spreading of interphase fetal hepatocytes and increase in circularity of retinal pigment epithelial cells. Microtubule depolymerization gives rise to FH-B cell compaction, as pretreatment with taxol prevents this morphological change. Finally, FQI1 decreases the rate and range of locomotion of interphase cells, supporting an impact of FQI1-induced microtubule breakdown on cell motility. Taken together, our results show that FQI1 interferes with microtubule-associated functions in interphase, specifically cell morphology and motility.