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Tamoxifen is a common adjuvant treatment for estrogen receptor (ER)α-positive patients with breast cancer; however, acquired resistance abrogates the efficacy of this therapeutic approach. We recently demonstrated that G protein–coupled estrogen receptor 1 (GPER1) mediates tamoxifen action in breast cancer cells by inducing insulin-like growth factor–binding protein-1 (IGFBP-1) to inhibit IGF-1–dependent signaling. To determine whether dysregulation of IGFBP-1 induction is associated with tamoxifen resistance, IGFBP-1 transcription was measured in tamoxifen-resistant MCF-7 cells (TamR) after tamoxifen (Tam) treatment. IGFBP-1 transcription was not stimulated in tamoxifen-treated TamR cells whereas decreased expression of FoxO1, a known modulator of IGFBP-1, was observed. Exogenous expression of FoxO1 rescued the ability of tamoxifen to induce IGFBP-1 transcription in TamR cells. As decreased IGF-1R expression is observed in tamoxifen-resistant cells, the requirement for IGF-1R expression on tamoxifen-stimulated IGFBP-1 transcription was investigated. In TamR and SK-BR-3 cells, both characterized by low IGF-1R levels, exogenous IGF-1R expression increased FoxO1 levels and IGFBP-1 expression, whereas IGF-1R knockdown in MCF-7 cells decreased tamoxifen-stimulated IGFBP-1 transcription. Interestingly, both 17β-estradiol (E2)-stimulated ERα phosphorylation and progesterone receptor (PR) expression were altered in TamR. PR is a transcription factor known to modulate FoxO1 transcription. In addition, IGF-1R knockdown decreased FoxO1 protein levels in MCF-7 cells. Furthermore, IGF-1R or FoxO1 knockdown inhibited the ability of tamoxifen to induce IGFBP-1 transcription and tamoxifen sensitivity in MCF-7 cells. These data provide a molecular mechanistic connection between IGF-1R expression and the FoxO1-mediated mechanism of tamoxifen action in breast cancer cells.Implications: Loss of IGF-1R expression is associated with decreased tamoxifen efficacy in patients with breast cancer and the development of tamoxifen resistance. This contribution identifies potential molecular mechanisms of altered tamoxifen sensitivity in breast cancer cells resulting from decreased IGF-1R expression. Mol Cancer Res; 15(4); 489–97. ©2017 AACR.

qPCR analysis of IGFBP-1 transcription after 24h treatment with vehicle or 10nM E2 in MCF-7 cells in serum-free condition. * = p < 0.05 and results are average or representative of three independent experiments. Error bars are SEM.

GPER1 and CREB expression and accumulation of p-CREB (S133) in MCF-7 and TamR cells. B, Accumulation of p-CREB (S133) after 2h Tam treatment in MCF-7 and TamR cells. * = p < 0.05 and results are average or representative of three independent experiments. Error bars are SEM.

FoxO1 protein levels after 48 h of treatment with 5μM PPP in MCF-7 cells grown in DMEM containing 10% FBS. Results are average or representative of three independent experiments. Error bars are SEM.

qPCR analysis of IGFBP-1 transcription after 24h treatment with vehicle or 1 µM Tam in absence and presence of actinomycin D (2 μg/ml) pretreatment in MCF-7 cells in serum-free condition. * = p < 0.05 and results are representative of two independent experiments. Error bars are SEM.

qPCR analysis of IGFBP-1 transcription after 24h treatment with IGF-1 (50 ng/ml) in MCF-7 cells in serum-free condition. * = p < 0.05 and results are average or representative of three independent experiments. Error bars are SEM.

Supplementary Figures 1-2, Table 1 from AMP-Activated Protein Kinase Signaling Results in Cytoplasmic Sequestration of p27

Tuberin, the Tsc2 gene product, integrates the phosphatidylinositol 3-kinase/mitogen-activated protein kinase (mitogenic) and LKB1/AMP-activated protein kinase (AMPK; energy) signaling pathways, and previous independent studies have shown that loss of tuberin is associated with elevated AMPK signaling and altered p27 function. In Tsc2-null tumors and tumor-derived cells from Eker rats, we observed elevated AMPK signaling and concordant cytoplasmic mislocalization of p27. Cytoplasmic localization of p27 in Tsc2-null cells was reversible pharmacologically using inhibitors of the LKB1/AMPK pathway, and localization of p27 to the cytoplasm could be induced directly by activating AMPK physiologically (glucose deprivation) or genetically (constitutively active AMPK) in Tsc2-proficient cells. Furthermore, AMPK phosphorylated p27 in vitro on at least three sites including T170 near the nuclear localization signal, and T170 was shown to determine p27 localization in response to AMPK signaling. p27 functions in the nucleus to suppress cyclin-dependent kinase-2 (Cdk2) activity and has been reported to mediate an antiapoptotic function when localized to the cytoplasm. We found that cells with elevated AMPK signaling and cytoplasmic p27 localization exhibited elevated Cdk2 activity, which could be suppressed by inhibiting AMPK signaling. In addition, cells with elevated AMPK signaling and cytoplasmic p27 localization were resistant to apoptosis, which could be overcome by inhibition of AMPK signaling and relocalization of p27 to the nucleus. These data show that AMPK signaling determines the subcellular localization of p27, and identifies loss of integration of pathways controlling energy balance, the cell cycle, and apoptosis due to aberrant AMPK and p27 function as a feature of cells that have lost the Tsc2 tumor suppressor gene. [Cancer Res 2008;68(16):6496–506]

The active metabolite of tamoxifen, 4‐hydroxytamoxifen, functions as an anti‐estrogen in breast cancer cells and thus inhibits proliferation. While tamoxifen continues to be successfully used to treat estrogen‐dependent breast cancer, most patients receiving treatment will develop chemoresistance over time. Two commonly reported biomarkers of tamoxifen resistance are decreased expression of insulin‐like growth factor 1 receptor (IGF‐1R) and increased expression of epidermal growth factor receptor (EGFR). In prior work we have shown that these receptors facilitate chemoresistance and have unique regulatory functions measurable in resistant cell lines compared with nonresistant. Thus, we hypothesized that these receptors and a newly identified biomarker, integrin β1, may be used to search for the presence of resistant breast cancer cells within a population of cells that are sensitive to tamoxifen therapy. We tested this by designing a straightforward cell‐labeling approach to measure differences in the receptor expression of resistant vs. sensitive cells cytometrically. Our results show that separation is possible when observing the expression of IGF‐1R as well as integrin β1. Interestingly, we found no detectable difference in EGFR expression between tamoxifen resistant and ‐sensitive cells when measured with cytometry despite the fact that EGFR is upregulated in resistant cells. Our long‐term goal is to utilize sorting to isolate tamoxifen resistant subpopulations of cells by receptor expression level. Isolating rare resistant cells that reside within a population of drug‐sensitive cells will offer new insights into why chemoresistance occurs.

G protein-coupled estrogen receptor 1 (GPER1) is a seven-transmembrane receptor that mediates rapid cell signaling events stimulated by estrogens. While the role that GPER1 has in the modulation of E2-responsive tissues and cancers is well documented, the molecular mechanisms that regulate GPER1 expression are currently not well defined. The recently identified GPER1-dependent mechanism of tamoxifen action in breast cancer cells underscores the importance of identifying mechanisms that regulate GPER1 expression in this cell type. We hypothesized that GPER1 expression in breast cancer cells is sensitive to [D-glucose] and provide data showing increased GPER1 expression when cells were cultured in low [D-glucose]. To determine if the observed accumulation of GPER1 was AMP-activated protein kinase (AMPK)-dependent, small molecule stimulation or inhibition of AMPK was performed. AMPK inhibition decreased GPER1 accumulation in cells grown in low [D-glucose] while the AMPK-activating compound AICAR increased GPER1 accumulation in cells grown in high [D-glucose] media. Additionally, transfection of cells with a plasmid expressing constitutively active AMPK resulted in increased GPER1 accumulation. To determine if [D-glucose]-dependent GPER1 accumulation altered breast cancer cell response to tamoxifen, cells grown in the presence of decreasing [D-glucose] were co-treated with tamoxifen and IGFBP-1 transcription was measured. The results from these experiments reveal that D-glucose deprivation increased GPER1-mediated and tamoxifen-induced IGFBP-1 transcription suggesting that [D-glucose] may increase breast cancer cell sensitivity to tamoxifen. Taken together, these results identify a previously unknown mechanism that regulates GPER1 expression that modifies one aspect tamoxifen action in breast cancer cells.

Insulin-like growth factors (IGFs) are signaling proteins which bind to insulin-like growth factor 1 receptor (IGF-1R) and induce a signaling cascade to promote proliferation and survival in breast cancer cells. Insulin-Like Growth Factor Binding Protein 6 (IGFBP-6) induces apoptosis by inhibiting IGF-1R upon binding to both IGF-1 and IGF-2, however, the role of IGFBP-6 in breast cancer has not been adequately studied. While IGFBP-6 can bind to both IGF-1 and IGF-2, the binding affinity for IGF2 is 20-fold higher compared to IGF1. To determine if IGFBP-6 modulates breast cancer cells, MCF-7 and T-47D cells were stably transfected with an IGFBP-6 expression plasmid. IGFBP6 over-expression resulted in decreased proliferation rates in both cell lines when compared to empty vector controls. To identify a mechanism of decreased proliferation rates, the activation of the IGF-1R-dependent AKT and MAPK mediators was measured. Reduced phosphorylation of MAPK and AKT was observed in both cell lines over-expressing IGFBP-6 compared to empty vector controls. MAPK and AKT phosphorylate ERα at serine 118 and serine 167 respectively, therefore, ERα phosphorylation at these residues was determined in the presence of IGFBP6 expression. Reduction in phosphorylation was greater in MCF-7 cells than T-47D cells when IGFBP6 was constitutively expressed. To determine if reduced ERα phosphorylation altered expression of known targets of ERα-modulated expression, such as IGF-1R and Progesterone Receptor (PR) was measured. Unexpectedly, IGF-1R and PR protein and transcript levels were greater in IGFBP6 over-expressing cells. Studies have shown that differences in ERα phosphorylation change the recruitment of transcriptional modulators associated with ERα-mediated transcription. These results suggest that despite reducing IGF-1R-dependent cell signaling, compensatory mechanisms may be induced to enhance ERα-mediated expression. Citation Format: Francisco Javier Lariz, Kevin D. Houston. IGFBP6 modulates estrogen receptor alpha phosphorylation and transcriptional activity in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 102.

Insulin-Like Growth Factor Binding Protein 4 (IGFBP-4) is one of six binding proteins involved in sequestering insulin-like growth factors 1 and 2 (IGFs). IGFBP-4 expression is induced by estrogen receptor α (ERα), and its classical function is to prevent activation of IGF-1 receptor (IGF-1R) by sequestration of IGF-1. However, the aggressive triple-negative breast cancer cell line MDA-MB-231 shows high expression of IGFBP-4 along with its protease, pregnancy-associated plasma protein A (PAPP-A). Further, inhibition of IGFBP-4 in MDA-MB-231 cells is sufficient to reduce migration. To better understand the function of IGFBP-4 as a pro-cancer or anti-cancer protein, its anti-proliferative effects in ERα positive cells must be reconciled with its pro-migratory effects in triple negative cells. The role of IGFBP-4 in proliferation across four breast cancer cell lines representing ERα positive and triple negative subtypes was investigated. Stable transfection with IGFBP-4 expression construct in the MCF-7 and T-47D cell lines, which have silenced the PAPP-A gene, shows a reduction in proliferation. Further, MCF-7 parental cells cultured in conditioned media containing IGFBP-4 also show reduced proliferation, suggesting that the anti-proliferative effect of IGFBP-4 is associated with the absence of PAPP-A. Stable IGFBP-4 transfection of triple-negative MDA-MB-468 cell line, which has low extracellular IGFBP-4 when compared to MDA-MB-231 but does express PAPP-A, was sufficient to increase proliferation. Taken together, these results support the conclusion that proliferative effects of IGFBP-4 are dependent upon the expression status of PAPP-A. Citation Format: Keenan L. Flynn, Kevin D. Houston. Insulin-like growth factor binding protein 4 exerts differential effects on breast cancer cell proliferation based on the expression status of pregnancy-associated plasma protein A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 108.

Insulin-like growth factor (IGF) system plays a significant role in many cellular processes, including proliferation, and survival. In estrogen receptor positive breast cancer, the level of circulating IGF-1 is positively associated with the incidence and at least 50% of cases have elevated IGF-1R signaling. Tamoxifen, a selective estrogen receptor modulator and antagonist for estrogen receptor alpha (ERα) in breast tissue, is a commonly prescribed adjuvant treatment for patients presenting with ERα-positive breast cancer. Unfortunately, tamoxifen resistance is a frequent occurrence in patients receiving treatment and the molecular mechanisms that underlie tamoxifen resistance not adequately defined. It has recently been reported that the inhibition of IGF-1R activation and the proliferation of breast cancer cells upon tamoxifen treatment is mediated by the accumulation of extracellular insulin-like growth factor binding protein 1 (IGFBP-1). Elevated IGFBP-1 expression was observed in tamoxifen-resistant (TamR) MCF-7 and T-47D cells lines suggesting that the tamoxifen-resistant state is associated with IGFBP-1 accumulation. MCF-7 and T-47D breast cancer cells stably transfected with and IGFBP-1 expression vector were generated (MCF7-BP1 and T47D-BP1) to determine the impact of breast cancer cell culture in the presence of increased IGFBP-1 expression. In these cells, the expression of IGF-1R was significantly reduced compared to controls and was similar to our observations in tamoxifen-resistant MCF-7 and T-47D cells. Also similar to TamR breast cancer cells, MCF7-BP1 and T47D-BP1 were resistant to tamoxifen treatment, had elevated epidermal growth factor receptor (EGFR) expression, increased phospho-EGFR (pEGFR), and phospho-Erk (pErk). Furthermore, tamoxifen sensitivity was restored in the MCF7-BP1 and T47D-BP1 upon inhibition of Erk phosphorylation. Lastly, the transient knockdown of IGFBP-1 in MCF7-BP1 and T47D-BP1 inhibited pErk accumulation and increased tamoxifen sensitivity. Taken together, these data support the conclusion that IGFBP-1 is a key component of the development of tamoxifen resistance in breast cancer cells.

A multi-color spectral flow cytometry panel targeting receptors on MCF-7 breast cancer cells is used to potentially identify a tamoxifen resistant subpopulation. Results using separate resistant and normal cells indicate detection and sorting is plausible.

Autofluorescence from the intracellular metabolite, NAD(P)H, is a biomarker that is widely used and known to reliably screen and report metabolic activity as well as metabolic fluctuations within cells. As a ubiquitous endogenous fluorophore, NAD(P)H has a unique rate of fluorescence decay that is altered when bound to coenzymes. In this work we measure the shift in the fluorescence decay, or average fluorescence lifetime (1–3 ns), of NAD(P)H and correlate this shift to changes in metabolism that cells undergo during apoptosis. Our measurements are made with a flow cytometer designed specifically for fluorescence lifetime acquisition within the ultraviolet to violet spectrum. Our methods involved culture, treatment, and preparation of cells for cytometry and microscopy measurements. The evaluation we performed included observations and quantification of the changes in endogenous emission owing to the induction of apoptosis as well as changes in the decay kinetics of the emission measured by flow cytometry. Shifts in NAD(P)H fluorescence lifetime were observed as early as 15 min post‐treatment with an apoptosis inducing agent. Results also include a phasor analysis to evaluate free to bound ratios of NAD(P)H at different time points. We defined the free to bound ratios as the ratio of ‘short‐to‐long’ (S/L) fluorescence lifetime, where S/L was found to consistently decrease with an increase in apoptosis. With a quantitative framework such as phasor analysis, the short and long lifetime components of NAD(P)H can be used to map the cycling of free and bound NAD(P)H during the early‐to‐late stages of apoptosis. The combination of lifetime screening and phasor analyses provides the first step in high throughput metabolic profiling of single cells and can be leveraged for screening and sorting for a range of applications in biomedicine. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Tamoxifen is a common adjuvant treatment for estrogen receptor (ER)α-positive patients with breast cancer; however, acquired resistance abrogates the efficacy of this therapeutic approach. We recently demonstrated that G protein–coupled estrogen receptor 1 (GPER1) mediates tamoxifen action in breast cancer cells by inducing insulin-like growth factor–binding protein-1 (IGFBP-1) to inhibit IGF-1–dependent signaling. To determine whether dysregulation of IGFBP-1 induction is associated with tamoxifen resistance, IGFBP-1 transcription was measured in tamoxifen-resistant MCF-7 cells (TamR) after tamoxifen (Tam) treatment. IGFBP-1 transcription was not stimulated in tamoxifen-treated TamR cells whereas decreased expression of FoxO1, a known modulator of IGFBP-1, was observed. Exogenous expression of FoxO1 rescued the ability of tamoxifen to induce IGFBP-1 transcription in TamR cells. As decreased IGF-1R expression is observed in tamoxifen-resistant cells, the requirement for IGF-1R expression on tamoxifen-stimulated IGFBP-1 transcription was investigated. In TamR and SK-BR-3 cells, both characterized by low IGF-1R levels, exogenous IGF-1R expression increased FoxO1 levels and IGFBP-1 expression, whereas IGF-1R knockdown in MCF-7 cells decreased tamoxifen-stimulated IGFBP-1 transcription. Interestingly, both 17β-estradiol (E2)-stimulated ERα phosphorylation and progesterone receptor (PR) expression were altered in TamR. PR is a transcription factor known to modulate FoxO1 transcription. In addition, IGF-1R knockdown decreased FoxO1 protein levels in MCF-7 cells. Furthermore, IGF-1R or FoxO1 knockdown inhibited the ability of tamoxifen to induce IGFBP-1 transcription and tamoxifen sensitivity in MCF-7 cells. These data provide a molecular mechanistic connection between IGF-1R expression and the FoxO1-mediated mechanism of tamoxifen action in breast cancer cells. Implications: Loss of IGF-1R expression is associated with decreased tamoxifen efficacy in patients with breast cancer and the development of tamoxifen resistance. This contribution identifies potential molecular mechanisms of altered tamoxifen sensitivity in breast cancer cells resulting from decreased IGF-1R expression. Mol Cancer Res; 15(4); 489–97. ©2017 AACR.

Tamoxifen, a selective estrogen receptor modulator, is a commonly prescribed adjuvant therapy for estrogen receptor-α (ERα)-positive breast cancer patients. To determine if extracellular factors contribute to the modulation of IGF-1 signaling after tamoxifen treatment, MCF-7 cells were treated with IGF-1 in conditioned medium (CM) obtained from 4-OHT-treated MCF-7 cells and the accumulation of phospho-Akt (S473) was measured. CM inhibited IGF-1-dependent cell signaling and suggesting the involvement of extracellular factors (ie. IGFBPs). A significant increase in IGFBP-1 mRNA and extracellular IGFBP-1 protein was observed in 4-OHT-treated MCF-7 cells. Knockdown experiments demonstrated that both GPER1 and CREB mediate IGFBP-1 induction. Furthermore, experiments showed that 4-OHT-dependent IGFBP-1 transcription is downstream of GPER1-activation in breast cancer cells. Additionally, neutralization and knockdown experiments demonstrated a role for IGFBP-1 in the observed inhibition of IGF-1 signaling. These results suggested that 4-OHT inhibits IGF-1 signaling via GPER1 and CREB mediated extracellular IGFBP-1 accumulation in breast cancer cells.

Flow cytometers are powerful high-throughput devices that capture spectroscopic information from individual particles or cells. These instruments provide a means of multi-parametric analyses for various cellular biomarkers or labeled organelles and cellular proteins. However, the spectral overlap of fluorophores limits the number of fluorophores that can be used simultaneously during experimentation. Time-resolved parameters enable the quantification of fluorescence decay kinetics, thus circumventing common issues associated with intensity-based measurements. This contribution introduces fluorescence lifetime excitation cytometry by kinetic dithering (FLECKD) as a method to capture multiple fluorescence lifetimes using a hybrid time-domain approach. The FLECKD approach excites fluorophores by delivering short pulses of light to cells or particles by rapid dithering and facilitates measurement of complex fluorescence decay kinetics by flow cytometry. Our simulations demonstrated a resolvable fluorescence lifetime value as low as 1.8 ns (±0.3 ns) with less than 20% absolute error. Using the FLECKD instrument, we measured the shortest average fluorescence lifetime value of 2.4 ns and found the system measurement error to be ±0.3 ns (SEM), from hundreds of monodisperse and chemically stable fluorescent microspheres. Additionally, we demonstrate the ability to detect two distinct excited state lifetimes from fluorophores in single cells using FLECKD. This approach presents a new ability to resolve multiple fluorescence lifetimes while retaining the fluidic throughput of a cytometry system. The ability to discriminate more than one average fluorescence lifetime expands the current capabilities of high-throughput and intensity-based cytometry assays as the need to tag one single cell with multiple fluorophores is now widespread.

Macrophage Cells Secrete Specific Cytokines and Accumulate Activated Interferon Regulatory Factor 3 after Multi-Walled Carbon Nanotube Exposure The health consequences of human exposure to carbon-based nanomaterials are not fully understood and the effects that such materials have on the immune system have not been adequately characterized. To determine if the innate immune system is modulated by exposure to carbon-based nanomaterials, the extracellular accumulation of an array of cytokines was measured in cell culture media obtained from mouse macrophage cells (RAW264.7) following exposure to carbon nanotubes (CNTs) or fullerene (C60). Accumulation of a specific subset of cytokines was observed after exposure to multi-walled CNTs (MWCNT), but was not observed when cells were exposed to single-walled CNTs (SWCNTs) or C60. Additionally, the accumulation of the activated (phosphorylated) form of the interferon regulatory factor 3 (IRF3) transcription factor and associated interferon beta (IFNβ) was observed after MWCNT exposure. These data suggest that IRF3 is a mediator of MWCNTactivated signal transduction pathways in macrophage cells. Furthermore, our data show that MWCNTs induce an innate immune response at subtoxic doses and suggests that MWCNT exposure may result in chronic inflammation and compromised immunity.

The tuberous sclerosis complex 2 (Tsc2) gene product, tuberin, acts as a negative regulator of mTOR signaling, and loss of tuberin function leads to tumors of the brain, skin, kidney, heart, and lungs. Previous studies have shown that loss of tuberin function affects the stability and subcellular localization of the cyclin-dependent kinase inhibitor (CKI) p27, although the mechanism(s) by which tuberin modulates p27 stability has/have not been elucidated. Previous studies have also shown that AMP-activated protein kinase (AMPK), which functions in an energy-sensing pathway in the cell, becomes activated in the absence of tuberin. Here we show that in Tsc2-null tumors and cell lines, AMPK activation correlates with an increase in p27 levels, and inhibition of AMPK signaling decreases p27 levels in these cells. In addition, activation of AMPK led to phosphorylation of p27 at the conserved terminal threonine residue of murine p27 (T197) in both in vitro kinase assays and in cells. Phosphorylation of p27 at T197 led to increased interaction between p27 and 14-3-3 proteins and increased the protein stability of p27. Furthermore, activation of AMPK signaling promoted the interaction between p27 and 14-3-3 proteins and increased the stability of the p27 protein in a manner that was dependent on T197. These data identify a conserved mechanism for the regulation of p27 stability via phosphorylation at the terminal threonine (mT197/hT198) and binding of 14-3-3 proteins, which when AMPK is activated results in stabilization of the p27 protein.

Tuberin, the Tsc2 gene product, integrates the phosphatidylinositol 3-kinase/mitogen-activated protein kinase (mitogenic) and LKB1/AMP-activated protein kinase (AMPK; energy) signaling pathways, and previous independent studies have shown that loss of tuberin is associated with elevated AMPK signaling and altered p27 function. In Tsc2-null tumors and tumor-derived cells from Eker rats, we observed elevated AMPK signaling and concordant cytoplasmic mislocalization of p27. Cytoplasmic localization of p27 in Tsc2-null cells was reversible pharmacologically using inhibitors of the LKB1/AMPK pathway, and localization of p27 to the cytoplasm could be induced directly by activating AMPK physiologically (glucose deprivation) or genetically (constitutively active AMPK) in Tsc2-proficient cells. Furthermore, AMPK phosphorylated p27 in vitro on at least three sites including T170 near the nuclear localization signal, and T170 was shown to determine p27 localization in response to AMPK signaling. p27 functions in the nucleus to suppress cyclin-dependent kinase-2 (Cdk2) activity and has been reported to mediate an antiapoptotic function when localized to the cytoplasm. We found that cells with elevated AMPK signaling and cytoplasmic p27 localization exhibited elevated Cdk2 activity, which could be suppressed by inhibiting AMPK signaling. In addition, cells with elevated AMPK signaling and cytoplasmic p27 localization were resistant to apoptosis, which could be overcome by inhibition of AMPK signaling and relocalization of p27 to the nucleus. These data show that AMPK signaling determines the subcellular localization of p27, and identifies loss of integration of pathways controlling energy balance, the cell cycle, and apoptosis due to aberrant AMPK and p27 function as a feature of cells that have lost the Tsc2 tumor suppressor gene. [Cancer Res 2008;68(16):6496–506]

Dictyostelium discoideum cells secrete CfaD, a protein that is similar to cathepsin proteases. Cells that lack cfaD proliferate faster and reach a higher stationary-phase density than wild-type cells, whereas cells that overexpress CfaD proliferate slowly and reach the stationary phase when at a low density. On a per-nucleus basis, CfaD affects proliferation but not growth. The drawback of not having CfaD is a reduced spore viability. Recombinant CfaD has no detectable protease activity but, when added to cells, inhibits the proliferation of wild-type and cfaD– cells. The secreted protein AprA also inhibits proliferation. AprA is necessary for the effect of CfaD on proliferation. Molecular-sieve chromatography indicates that in conditioned growth medium, the 60 kDa CfaD is part of a ∼150 kDa complex, and both chromatography and pull-down assays suggest that CfaD interacts with AprA. These results suggest that two interacting proteins may function together as a chalone signal in a negative feedback loop that slows Dictyostelium cell proliferation.

We used an animal model to study uterine leiomyoma in the context of pregnancy-associated changes in gene expression and to determine how they might modulate tumor growth. Spontaneous tumors and normal myometrium were collected from Eker rats and compared with myometrial samples from pregnant animals. A leiomyoma-derived cell line was also used to assess pregnancy-related changes in gene expression and to determine the impact of signaling by the oxytocin receptor. Eker rat leiomyomas expressed several pregnancy-related genes, including connexin 43, oxytocin receptor (OTR), and cyclooxygenase (COX)-1; however, the tumors did not express COX-2, which is expressed in the parturient myometrium. The leiomyoma-derived cell lines also expressed OTR, which responds to estrogen, binds to oxytocin, and exhibits a calcium flux when stimulated with oxytocin. The OTR signaling mediated by oxytocin inhibited estrogen-stimulated growth of leiomyoma cells. Leiomyoma cells expressed many genes of the parturient myometrium, including OTRs, but were deficient in COX-2 expression. Signaling via the OTR appears to inhibit estrogen-induced cell proliferation, suggesting that signaling by this receptor might help mediate the protective effect of pregnancy on this disease.

Uterine leiomyomas develop in reproductive-age women with high frequency and are dependent on the production of ovarian hormones. While it is generally accepted that these tumors are estrogen (E(2))-responsive, the role of progesterone (P(4)) in modulating tumor growth is less clear. In the present study, an in vivo/in vitro rat model was used to characterize progesterone receptor (PR) isoform expression in uterine leiomyoma and investigate PR signaling using progestins and antiprogestins in the leiomyoma-derived cell line ELT-3. PR-A was the predominant isoform expressed in normal myometrium, leiomyomas and ELT3 cells. In the normal myometrium, PR-A and PR-B levels varied during the estrous cycle with low ratios of PR-A relative to PR-B (PR-A/PR-B) coinciding with times of cell proliferation. Although PR ligands had no effect on basal levels of uterine leiomyoma cell proliferation in vitro, both progestins and antiprogestins inhibited E(2)-stimulated cell proliferation. In addition, E(2)-stimulated transactivation of an estrogen-response-element reporter gene as well as E(2)-induced upregulation of the PR were also inhibited by PR ligands. These data indicate that PR ligands can transdominantly suppress estrogen receptor signaling and stimulation of uterine leiomyoma cell growth.

G protein-coupled estrogen receptor 1 (GPER1, aka GPR30), is a 7-transmembrane receptor that mediates rapid cell signaling events stimulated by 17β-estradiol (E2) in cancer and tumor cells. In breast cancer cells, GPER1 mediates E2-induced CTGF-dependent cell migration and cyclin E expression resulting in cell cycle progression. GPER1 has also been shown to mediate antiproliferative cell signaling such as p53-dependent inhibition of cell cycle progression in breast cancer cells treated with the GPER1-specific agonist G1 and the inhibition of IGF-1R signaling in tamoxifen-treated breast cancer cells. In addition to E2, both natural and synthetic compounds have been shown to activate GPER1 in breast cancer cells. GPER1 has also been shown to regulate cell signaling in cells that lack ERα. For example, GPER1 is antiproliferative in triple-negative breast cancer cells. While the importance of GPER1 in the modulation of E2-responsive tumor and cancer cells as been demonstrated, there is very little information regarding the regulation of GPER1 expression in these cell types. The work presented here is aimed at determining the molecular mechanisms that regulate GPER1 expression. To determine if GPER1 expression is sensitive to D-glucose concentration in breast cancer cells, MCF-7 and T47D human breast cancer cells were cultured in media containing increasing concentrations of D-glucose and GPER1 expression was measured using real-time PCR and immunoblot. Data from these experiments showed that GPER1 expression was significantly increased in breast cancer cells cultured in media containing low D-glucose concentrations (0 and 2.5mM) and significantly reduced in media containing high D-glucose (25 mM). Since low D-glucose concentration is known to activate the energy-sensing AMP kinase (AMPK), the observed GPER1 induction in low D-glucose conditions was determined after pretreatment with the AMPK inhibitor compound C. GPER1 expression was inhibited in both MCF-7 and T47D breast cancer cells cultured in low D-glucose media when pretreated with compound C. Additionally, the AMPK activator metformin induced GPER1 expression in MCF-7 and T47D breast cancer cells cultured in high D-glucose (25mM) conditions. These data suggest that AMPK mediates GPER1 expression in cells cultured in low D-glucose. The D-glucose sensitivity of GPER1 expression was also determined in the Eker rat-derived uterine leiomyoma cells (ELT-3 and ELT-6) which are characterized by hyperactive AMPK. In both ELT-3 and ELT-6 cells, GPER1 expression was not sensitive to D-glucose concentration nor was the activation state of AMPK sensitive to D-glucose concentrations. However, the inhibition of AMPK by compound C in both ELT-3 and ELT-6 cells resulted in decreased GPER1 expression. These findings reveal a previously unknown mechanism that regulates GPER1 expression in E2-responsive tumor and cancer cells. Citation Format: Yan Zheng, Kevin D. Houston. GPER1 expression is modulation by D-glucose concentration in estrogen-responsive cancer and tumor 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 2432. doi:10.1158/1538-7445.AM2017-2432

Breast cancer is one of the leading causes of cancer-related deaths in the world. The majority of breast cancers express estrogen receptor alpha (ERα) and tamoxifen, a selective estrogen receptor modulator, is commonly prescribed as adjuvant therapy for ERα-positive breast cancer patients. The development of chemoresistance is a common occurrence in treated patients, however, the molecular mechanisms associated with acquired tamoxifen resistance are not well defined. Previous work from our lab identified a novel mechanism of tamoxifen action that involved the accumulation of extracellular Insulin-like growth factor binding protein-1 (IGFBP-1) and modulates IGF-1-dependent signaling transduction. Tamoxifen-induced IGFBP-1 induction in this model was mediated by G protein-coupled estrogen receptor (GPER)-dependent CREB activation as shown by phosphorylation on serine 133. To determine IGFBP-1 induction is altered during the development of chemoresistance, this mechanism of action was studied in tamoxifen resistant MCF-7 (TamR) cells. Whereas CREB was constitutively phosphorylated in TamR cells, IGFBP-1 levels were not elevated when compared to parental cells and IGFBP-1 was not induced by tamoxifen treatment in TamR cells. These data suggest that another transcription factor(s) are also necessary for the induction of IGFBP-1 in breast cancer cells. FoxO1 is a known regulator of IGFBP-1 transcription and we tested whether FoxO1 contributes to tamoxifen-induced IGFBP-1 transcription. Our results indicate that FoxO1 expression levels were significantly reduced in TamR cells compared with parental cells. Furthermore, FoxO1 knockdown experiments demonstrated this transcription factor mediates tamoxifen-induced IGFBP-1 induction in MCF-7 cells. Consistent with previously published observations, IGF-1R expression was reduced in TamR cells compared to parental MCF-7 cells. To determine if loss of IGF-1R expression in involved in the loss of FoxO1 expression in TamR cells, IGF-1R was exogenously expressed and FoxO1 expression was measured. IGF-1R expression increases FoxO1 levels and partially restores tamoxifen-induced IGFBP-1 induction in TamR cells. These results suggest that FoxO1-mediated IGFBP-1 accumulation is altered during the development of tamoxifen resistance and is associated with decreased IGF-1R expression. Citation Format: Ali Vaziri-Gohar, Kevin D. Houston. Loss of FoxO1 expression is associated with decreased IGF-1R levels and alters IGFBP-1 induction in tamoxifen-resistant breast cancer cells. [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 1797.

The tuberous sclerosis complex 2 (Tsc2) gene product, tuberin, acts as a negative regulator of mTOR signaling, and loss of tuberin function leads to tumors of the brain, skin, kidney, heart, and lungs. Previous studies have shown that loss of tuberin function affects the stability and subcellular localization of the cyclin-dependent kinase inhibitor (CKI) p27, although the mechanism(s) by which tuberin modulates p27 stability has/have not been elucidated. Previous studies have also shown that AMP-activated protein kinase (AMPK), which functions in an energy-sensing pathway in the cell, becomes activated in the absence of tuberin. Here we show that in Tsc2-null tumors and cell lines, AMPK activation correlates with an increase in p27 levels, and inhibition of AMPK signaling decreases p27 levels in these cells. In addition, activation of AMPK led to phosphorylation of p27 at the conserved terminal threonine residue of murine p27 (T197) in both in vitro kinase assays and in cells. Phosphorylation of p27 at T197 led to increased interaction between p27 and 14-3-3 proteins and increased the protein stability of p27. Furthermore, activation of AMPK signaling promoted the interaction between p27 and 14-3-3 proteins and increased the stability of the p27 protein in a manner that was dependent on T197. These data identify a conserved mechanism for the regulation of p27 stability via phosphorylation at the terminal threonine (mT197/hT198) and binding of 14-3-3 proteins, which when AMPK is activated results in stabilization of the p27 protein.

Carbon nanotubes (CNTs) have unique properties that make them ideal for applications like constructing medical devices and drug delivery. Human health consequences resulting from exposure to CNTs in a medical setting remain unclear. Thus far, studies aimed at determining the biocompatibility of CNTs have relied on traditional toxicological endpoints such as cell death. Such studies have not led to a definitive answer regarding specific biological effects or health consequences of exposure to sub-toxic doses of CNTs. One potential health consequence of exposure to CNTs is the activation of the innate immune system that could result in chronic inflammation and compromised immunity. In experiments performed to identify biomarkers for MWCNT exposure and to determine if such exposure activates an innate immune response, the levels cytokines/chemokines were determined after MWCNT treatment of mouse macrophage cells (RAW264.7). Increased extracellular levels of a subset of cytokines/chemokines were observed upon MWCNT treatment. This subset of induced cytokines/chemokines represent potential biomarkers for CNT exposure. Furthermore, MWCNT treatment did not lead to the induction of all cytokines/chemokines tested suggesting that exposure to MWCNTs results in the activation of specific pathways of the innate immune system and not general inflammation.

Tamoxifen is a commonly prescribed anti-estrogen treatment for breast cancer patients; this selective estrogen receptor modulator functions as an ERα-antagonist in breast tissue and inhibits cell proliferation and survival of breast cancer cells. More recently, the active metabolite of tamoxifen, 4-OHT (Tam), has been shown to activate the G protein-coupled estrogen receptor (GPER). Previous studies from our laboratory identified a novel, GPER-dependent mechanism of Tam action in breast cancer cells that requires the extracellular accumulation of the insulin-like growth factor binding protein-1 (IGFBP-1). The molecular mechanisms of Tam-induced IGFBP-1 production mediated by GPER have not been determined. GPER activation has been shown to stimulate cAMP production and PKA activation in several cell types. We hypothesized the cAMP response element-binding protein (CREB) activation is required for Tam-induced IGFBP-1 production in breast caner cells. To determine if CREB activation may be involved in IGFBP-1 transcription, CREB phosphorylation (S133) status and nuclear accumulation were determined in MCF-7 cells after Tam treatment. The phosphorylation and nuclear accumulation of CREB were observed in Tam-treated MCF-7 cells suggesting that CREB activation may be required for Tam-induced IGFBP-1 transcription. Increased CREB phosphorylation and IGFBP-1 transcription were inhibited by pretreatment with the PKA inhibitor H-89 supporting a role for CREB in Tam-induced IGFBP-1 expression. H-89 pretreatment also significantly decreased the previously reported inhibition of IGF-1-stimulated signaling by Tam treatment in breast cancer cells. We have previously reported that Tam-induced IGFBP-1 expression is dysregulated in Tam-resistant MCF-7 cells (MCF-7TamR). To determine if aberrant CREB signaling results in the observed dysregulation, similar experiments were performed in MCF-7TamR cells. Our results show that CREB phosphorylation and nuclear accumulation are decreased in Tam-resistant cells compared to Tam-sensitive cells. Taken together, these results suggest that activation of GPER results in CREB-dependent IGFBP-1 production and subsequent inhibition of IGF-1-stimulated signaling in breast cancer cells treated with Tam. Furthermore, dysregulation of GPER-mediated CREB activation may be involved in the development of Tam resistance in breast cancer. Citation Format: Ali Vaziri-Gohar, Kevin D. Houston. GPER-mediated activation of the cAMP response element-binding protein (CREB) and subsequent IGFBP-1 expression is altered during the development of tamoxifen resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4421. doi:10.1158/1538-7445.AM2015-4421

Peroxisome proliferator-activated receptor (PPAR) gamma is an important signaling molecule in cells of mesenchymal origin, inducing differentiation and regulating cell proliferation in several cell types such as vascular smooth muscle cells. Leiomyomas arise from smooth muscle cells of the uterine myometrium with an incidence rate as high as 70% in women of reproductive age. PPAR signaling has not been characterized in these tumors, although prostaglandins, natural PPAR ligands, are known effectors of key biological functions in the normal myometrium. Leiomyomas and tumor-derived cells isolated from a rat model for this disease were characterized by Western analysis and found to express all three PPAR isoforms, suggesting that signaling pathways mediated by these receptors were intact in this tumor type. In vitro experiments with a leiomyoma-derived cell line demonstrated that the pan-PPAR ligand cis-4,7,10,13,16,19-docosahexaenoic acid and PPARgamma-specific ligands 15-deoxy-delta(12,14)-prostaglandin J(2), troglitazone, and ciglitazone inhibited 17beta-estradiol-stimulated cell proliferation. This inhibitory effect was not observed with PPARalpha- or PPARbeta-specific ligands. Although both PPAR and estrogen receptor (ER) signaling pathways were intact in leiomyoma cells, in addition to growth inhibition, stimulation of PPARgamma signaling also inhibited ER-mediated gene expression. Human leiomyomas were also found to express all three PPAR isoforms, and primary cultures of these cells were sensitive to the inhibitory effects of PPARgamma ligands. These results suggest that in uterine leiomyomas PPARgamma activation is growth inhibitory and that this inhibition is mediated at least in part by negative cross-talk between ER and PPAR signaling pathways.

Uterine leiomyomas, also called fi broids, are the most common reproductive tract neoplasm and the leading indication for hysterectomy in premenopausal women. The discovery and development of medicinal therapies for uterine leiomyoma have been hampered by a lack of understanding regarding the etiology and molecular mechanisms underlying the development of these lesions. Although the estrogen responsiveness of uterine leiomyoma is well established, the impact of environmental estrogens and their contribution to the development of these tumors is currently unknown. The Eker rat model of uterine leiomyoma has proven useful for addressing these issues and understanding the pathophysiology of this disease. The Eker rat is the only animal model that develops spontaneous uterine leiomyomas, and these tumors share many characteristics with those found in humans. The availability of tumor-derived cell lines from these rats has made this a valuable in vitro/in vivo model system for experimental studies to investigate molecular mechanisms of disease and to design interventional and preventative strategies for this clinically relevant tumor.

Uterine leiomyomas are ovarian hormone-responsive tumors originating in the smooth muscle layer of the uterus called the myometrium. These benign tumors require surgery or other clinical intervention in ∼25% of all premenopausal women in the United States. Despite the high frequency and clinical impact of these tumors, the pathogenesis of uterine leiomyomas is not understood. The estrogen-responsive growth of uterine leiomyomas has been well established, however the role of G protein-coupled estrogen receptor (GPER) in leiomyoma cell regulation has not been studied. The membrane bound estrogen receptor GPER, formerly known as GPR30, is a 7-transmembrane G protein-coupled receptor that is activated by 17β-estradiol (E2). A role for GPER has been previously established in cancers that originate in E2-dependent tissues such as the breast and uterus. Other reports show that GPER is expressed in myometrial cells and activation of GPER modulates myometrial contraction suggesting an important regulatory role for this receptor in the myometrium. Based on these reports, we hypothesized that GPER regulates uterine leiomyoma cells and may represent a putative therapeutic target for this tumor type. The Eker rat model-derived uterine leiomyoma (ELT-3) cell line was used as a model system to study GPER in uterine leiomyoma cells. First, the expression of GPER in uterine leiomyoma cells was determined using RNA and protein samples from ELT-3 cells and real-time PCR and immunoblot analysis. Our findings indicate that GPER transcript and protein were expressed in ELT-3 cells. To determine if activation of GPER regulated uterine leiomyoma cell proliferation, ELT-3 cells were treated with G1, a GPER-specific ligand, or vehicle control and counted 5 days after treatment. Data from these experiments indicated that G1 treatment, and potentially GPER activation, reduced the number of cells over time compared to vehicle control. GPER activation is known to result in the accumulation of pERK and intracellular free Ca2+ and such accumulation is used as a biomarker for GPER activation. Upon G1 treatment, pERK and intracellular free Ca2+ were elevated compared to vehicle treatment in ELT-3 cells suggesting that GPER is functional in uterine leiomyoma cells and is activated by G1. In an effort to determine the mechanism of inhibition of uterine leiomyoma cell proliferation by G1 treatment, we hypothesized that treatment of estrogen-stimulated ELT-3 cells with G1 would inhibit the characteristic E2-induced proliferation of this cell type. Growth curve experiments were performed and data obtained in these experiments suggested that G1 treatment resulted in the inhibition of E2-induced ELT-3 cell proliferation. Taken together, these data suggest that GPER is an important regulator of uterine leiomyoma cell proliferation and is a putative target for novel molecular therapeutics for this tumor type. Citation Format: Maryann Castillo, Angelique M. Wimbley, Jacob J. Mayfield, Jenifer C. Lascano, Kevin D. Houston. Activation of G-protein coupled estrogen receptor (GPER) inhibits ELT-3 uterine leiomyoma cell proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1305. doi:10.1158/1538-7445.AM2013-1305

The ability to label biomolecules with fluorescent tags has become essential in basic cancer research and offers great promise for improvements in clinical diagnostics. The structure and physicochemical properties of the fluorescent tag have the potential of altering binding and activity characteristics of the conjugates, therefore it is desirable to use small, neutral, membrane permeable and biocompatible fluorescent tags with favorable photophysical properties, and versatile coupling chemistries. . Estradiol (E2) is a well-characterized ovarian steroid hormone with known measureable biological endpoints at the cellular, transcriptional, and signal transduction levels. We synthesized a new class of fluorescent estrogen derivatives from 17-alpha-ethynylestradiol conjugated to triazaborolopyridinium (HPY) dyes. The estrogenic activity of the covalently linked E2-HPY fluorophores were characterized using the MCF-7 breast cancer cell line, an E2-dependent cell line that is often used as a model system to study E2-induced cell responses. The MCF-7 cells were treated with a series of E2-HPY derivatives (10nM), E2 (10nM) or vehicle control. The time points for treatment was as short as 15 minutes for effect on signal transduction to 5 days for cell proliferation endpoints. After treatment for specified time points, E2-induced proliferation was determined by growth curve analysis (1-5 days), E2-mediated progesterone receptor (PR) transcription was determined by real-time PCR (24 hours), and E2-dependent accumulation of pERK was measured by immunoblot analysis (15min). Our results indicate that the bioactivity of E2-HPY was not significantly different from unconjugated E2 for the three endpoints tested. The accumulation of E2-HPY in cells was demonstrated by flow cytometry and fluorescence microscopy. These data indicate that E2-HPY is taken up by cells and is easily detectable. Additionally, transcriptome analysis was performed to identify potential differences in general transcriptional activation between E2 and E2-HPY. These data suggest the exciting possibility that HPY fluorophores can be covalently linked to cancer-related biomolecules to enhance basic research and for potential use as a cancer diagnostic tool in the clinical. Citation Format: Jason E. Stailey, Sudath Hapuarachchige, Chinnasamy Ramesh, Sanjay Shrestha, Angelique M. Wimbley, Charles B. Shuster, Jeffrey B. Arterburn, Kevin D. Houston. Synthesis and characterization of novel, membrane permeable estradiol fluorophores that maintain estrogenic activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2237. doi:10.1158/1538-7445.AM2013-2237