The scaffold protein MEK Partner 1 is required for the survival of estrogen receptor positive breast cancer cells

ABSTRACT: MEK Partner 1 (MP1 or MAPKSP1) is a scaffold protein that has been reported to function in multiple signaling pathways, including the ERK, PAK and mTORC pathways. Several of these pathways influence the biology of breast cancer, but MP1's functional significance in breast cancer cells has not been investigated. In this report, we demonstrate a requirement for MP1 expression in estrogen receptor (ER) positive breast cancer cells. MP1 is widely expressed in both ER-positive and negative breast cancer cell lines, and in non-tumorigenic mammary epithelial cell lines. However, inhibition of its expression using siRNA duplexes resulted in detachment and apoptosis of several ER-positive breast cancer cell lines, but not ER-negative breast cancer cells or non-tumorigenic mammary epithelial cells. Inhibition of MP1 expression in ER-positive MCF-7 cells did not affect ERK activity, but resulted in reduced Akt1 activity and reduced ER expression and activity. Inhibition of ER expression did not result in cell death, suggesting that decreased ER expression is not the cause of cell death. In contrast, pharmacological inhibition of PI3K signaling did induce cell death in MCF-7 cells, and expression of a constitutively active form of Akt1 partially rescued the cell death observed when the MP1 gene was silenced in these cells. Together, these results suggest that MP1 is required for pro-survival signaling from the PI3K/Akt pathway in ER-positive breast cancer cells.     

Downregulation of CXXC Finger Protein 4 Leads to a Tamoxifen-resistant Phenotype in Breast Cancer Cells Through Activation of the Wnt/β-catenin Pathway

Tamoxifen is a successful endocrine therapy drug for estrogen receptor–positive (ER+) breast cancer. However, resistance to tamoxifen compromises the efficacy of endocrine treatment. In the present study, we identified potential tamoxifen resistance–related gene markers and investigated their mechanistic details. First, we established two ER + breast cancer cell lines resistant to tamoxifen, named MCF-7/TMR and BT474/TMR. Gene expression profiling showed that CXXC finger protein 4 (CXXC4) expression is lower in MCF-7/TMR cells than in MCF-7 cells. Furthermore, CXXC4 mRNA and protein expression are lower in the resistant cell lines than in the corresponding parental cell lines. We also investigated the correlation between CXXC4 and endocrine resistance in ER + breast cancer cells. CXXC4 knockdown accelerates cell proliferation in vitro and in vivo and renders breast cancer cells insensitive to tamoxifen, whereas CXXC4 overexpression inhibits cancer cell growth and increases tamoxifen sensitivity of resistant cells. In addition, we demonstrated that CXXC4 inhibits Wnt/β-catenin signaling in cancer cells by modulating the phosphorylation of GSK-3β, influencing the integrity of the β-catenin degradation complex. Silencing the CXXC4 gene upregulates expression of cyclinD1 and c-myc (the downstream targets of Wnt signaling) and promotes cell cycle progression. Conversely, ectopic expression of CXXC4 downregulates the expression of these proteins and arrests the cell cycle in the G0/G1 phase. Finally, the small-molecule inhibitor XAV939 suppresses Wnt signaling and sensitizes resistant cells to tamoxifen. These results indicate that components of Wnt pathway that are early in response to tamoxifen could be involved as an intrinsic factor of the transition to endocrine resistance, and inhibition of Wnt signaling may be an effective therapeutic strategy to overcome tamoxifen resistance.     

G0S2 represses PI3K/mTOR signaling and increases sensitivity to PI3K/mTOR pathway inhibitors in breast cancer

G0/G1 switch gene 2 (G0S2) is a direct retinoic acid target implicated in cancer biology and therapy based on frequent methylation-mediated silencing in diverse solid tumors. We recently reported that low G0S2 expression in breast cancer, particularly estrogen receptor-positive (ER+) breast cancer, correlates with increased rates of recurrence, indicating that G0S2 plays a role in breast cancer progression. However, the function(s) and mechanism(s) of G0S2 tumor suppression remain unclear. In order to determine potential mechanisms of G0S2 anti-oncogenic activity, we performed genome-wide expression analysis that revealed an enrichment of gene signatures related to PI3K/mTOR pathway activation in G0S2 null cells as compared to G0S2 wild-type cells. G0S2 null cells also exhibited a dramatic decreased sensitivity to PI3K/mTOR pathway inhibitors. Conversely, restoring G0S2 expression in human ER+ breast cancer cells decreased basal mTOR signaling and sensitized the cells to pharmacologic mTOR pathway inhibitors. Notably, we provide evidence here that the increase in recurrence seen with low G0S2 expression is especially prominent in patients who have undergone antiestrogen therapy. Further, ER+ breast cancer cells with restored G0S2 expression had a relative increased sensitivity to tamoxifen. These findings reveal that in breast cancer G0S2 functions as a tumor suppressor in part by repressing PI3K/mTOR activity, and that G0S2 enhances therapeutic responses to PI3K/mTOR inhibitors. Recent studies implicate hyperactivation of PI3K/mTOR signaling as promoting resistance to antiestrogen therapies in ER+ breast cancer. Our data establishes G0S2 as opposing this form of antiestrogen resistance. This promotes further investigation of the role of G0S2 as an antineoplastic breast cancer target and a biomarker for recurrence and therapy response.     

Phosphorylation by p38 mitogen-activated protein kinase promotes estrogen receptor α turnover and functional activity via the SCF(Skp2) proteasomal complex

The nuclear hormone receptor estrogen receptor α (ERα) mediates the actions of estrogens in target cells and is a master regulator of the gene expression and proliferative programs of breast cancer cells. The presence of ERα in breast cancer cells is crucial for the effectiveness of endocrine therapies, and its loss is a hallmark of endocrine-insensitive breast tumors. However, the molecular mechanisms underlying the regulation of the cellular levels of ERα are not fully understood. Our findings reveal a unique cellular pathway involving the p38 mitogen-activated protein kinase (p38MAPK)-mediated phosphorylation of ERα at Ser-294 that specifies its turnover by the SCF(Skp2) proteasome complex. Consistently, we observed an inverse relationship between ERα and Skp2 or active p38MAPK in breast cancer cell lines and human tumors. ERα regulation by Skp2 was cell cycle stage dependent and critical for promoting the mitogenic effects of estradiol via ERα. Interestingly, by the knockdown of Skp2 or the inhibition of p38MAPK, we restored functional ERα protein levels and the control of gene expression and proliferation by estrogen and antiestrogen in ERα-negative breast cancer cells. Our findings highlight a novel pathway with therapeutic potential for restoring ERα and the responsiveness to endocrine therapy in some endocrine-insensitive ERα-negative breast cancers.     

Suppression of epidermal growth factor receptor‐mediated β‐catenin nuclear accumulation enhances the anti‐tumor activity of phosphoinositide 3‐kinase inhibitor in breast cancer

Phosphoinositide 3‐kinase (PI3K) signaling is frequently deregulated in breast cancer and plays a critical role in tumor progression. However, resistance to PI3K inhibitors in breast cancer has emerged, which is due to the enhanced β‐catenin nuclear accumulation. Until now, the mechanisms underlying PI3K inhibition‐induced β‐catenin nuclear accumulation remains largely unknown. In the present study, we found inhibition of PI3K with LY294002 promoted β‐catenin nuclear accumulation in MCF‐7 and MDA‐MB‐231 breast cancer cells. Combining PI3K inhibitor LY294002 with XAV‐939, an inhibitor against β‐catenin nuclear accumulation, produced an additive anti‐proliferation effect against breast cancer cells. Subsequent experiments suggested β‐catenin nuclear accumulation induced by PI3K inhibition depended on the feedback activation of epidermal growth factor receptor (EGFR) signaling pathway in breast cancer cells. Inhibition of EGFR phosphorylation with Gefitinib enhanced anti‐proliferation effect of PI3K inhibitor LY294002 in MCF‐7 and MDA‐MB‐231 cells. Taken together, our findings may elucidate a possible mechanism explaining the poor outcome of PI3K inhibitors in breast cancer treatment.     

Cryptotanshinone inhibition of mammalian target of rapamycin pathway is dependent on oestrogen receptor alpha in breast cancer

Cryptotanshinone (CPT) has been demonstrated to inhibit proliferation and mammalian target of rapamycin (mTOR) pathway in MCF‐7 breast cancer cells. However, the same results are unable to be repeated in MDA‐MB‐231 cells. Given the main difference of oestrogen receptor α (ERα) between two types of breast cancer cells, It is possibly suggested that CPT inhibits mTOR pathway dependent on ERα in breast cancer. CPT could significantly inhibit cell proliferation of ERα‐positive cancer cells, whereas ERα‐negative cancer cells are insensitive to CPT. The molecular docking results indicated that CPT has a high affinity with ERα, and the oestrogen receptor element luciferase reporter verified CPT distinct anti‐oestrogen effect. Furthermore, CPT inhibits mTOR signalling in MCF‐7 cells, but not in MDA‐MB‐231 cells, which is independent on binding to the FKBP12 and disrupting the mTOR complex. Meanwhile, increased expression of phosphorylation AKT and insulin receptor substrate (IRS1) induced by insulin‐like growth factor 1 (IGF‐1) was antagonized by CPT, but other molecules of IGF‐1/AKT/mTOR signalling pathway such as phosphatase and tensin homolog (PTEN) and phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K) were negatively affected. Finally, the MCF‐7 cells transfected with shERα for silencing ERα show resistant to CPT, and p‐AKT, phosphorylation of p70 S6 kinase 1 (p‐S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E‐BP1) were partially recovered, suggesting ERα is required for CPT inhibition of mTOR signalling. Overall, CPT inhibition of mTOR is dependent on ERα in breast cancer and should be a potential anti‐oestrogen agent and a natural adjuvant for application in endocrine resistance therapy.     

The DEK Oncogene Is a Target of Steroid Hormone Receptor Signaling in Breast Cancer

Expression of estrogen and progesterone hormone receptors indicates a favorable prognosis due to the successful use of hormonal therapies such as tamoxifen and aromatase inhibitors. Unfortunately, 15–20% of patients will experience breast cancer recurrence despite continued use of tamoxifen. Drug resistance to hormonal therapies is of great clinical concern so it is imperative to identify novel molecular factors that contribute to tumorigenesis in hormone receptor positive cancers and/or mediate drug sensitivity. The hope is that targeted therapies, in combination with hormonal therapies, will improve survival and prevent recurrence. We have previously shown that the DEK oncogene, which is a chromatin remodeling protein, supports breast cancer cell proliferation, invasion and the maintenance of the breast cancer stem cell population. In this report, we demonstrate that DEK expression is associated with positive hormone receptor status in primary breast cancers and is up-regulated in vitro following exposure to the hormones estrogen, progesterone, and androgen. Chromatin immunoprecipitation experiments identify DEK as a novel estrogen receptor α (ERα) target gene whose expression promotes estrogen-induced proliferation. Finally, we report for the first time that DEK depletion enhances tamoxifen-induced cell death in ER+ breast cancer cell lines. Together, our data suggest that DEK promotes the pathogenesis of ER+ breast cancer and that the targeted inhibition of DEK may enhance the efficacy of conventional hormone therapies.     

BC-N102 suppress breast cancer tumorigenesis by interfering with cell cycle regulatory proteins and hormonal signaling,and induction of time-course arrest of cell cycle at G1/G0 phase

Mechanisms of breast cancer progression and invasion, often involve alteration of hormonal signaling, and upregulation and/or activation of signal transduction pathways that input to cell cycle regulation. Herein, we describe a rationally designed first-in-class novel small molecule inhibitor for targeting oncogenic and hormonal signaling in ER-positive breast cancer. BC-N102 treatment exhibits dose-dependent cytotoxic effects against ER+ breast cancer cell lines. BC-N102 exhibited time course- and dose-dependent cell cycle arrest via downregulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), phosphatidylinositol 3-kinase (PI3K), phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-Akt, CDK2, and CDK4 while increasing p38 mitogen-activated protein kinase (MAPK), and mineralocorticoid receptor (MR) signaling in breast cancer cell line. In addition, we found that BC-N102 suppressed breast cancer tumorigenesis in vivo and prolonged the survival of animals. Our results suggest that the proper application of BC-N102 may be a beneficial chemotherapeutic strategy for ER+ breast cancer patients.     

An epigenomic approach to therapy for tamoxifen-resistant breast cancer

Tamoxifen has been a frontline treatment for estrogen receptor alpha (ERα)-positive breast tumors in premenopausal women. However, resistance to tamoxifen occurs in many patients. ER still plays a critical role in the growth of breast cancer cells with acquired tamoxifen resistance, suggesting that ERα remains a valid target for treatment of tamoxifen-resistant (Tam-R) breast cancer. In an effort to identify novel regulators of ERα signaling, through a small-scale siRNA screen against histone methyl modifiers, we found WHSC1, a histone H3K36 methyltransferase, as a positive regulator of ERα signaling in breast cancer cells. We demonstrated that WHSC1 is recruited to the ERα gene by the BET protein BRD3/4, and facilitates ERα gene expression. The small-molecule BET protein inhibitor JQ1 potently suppressed the classic ERα signaling pathway and the growth of Tam-R breast cancer cells in culture. Using a Tam-R breast cancer xenograft mouse model, we demonstrated in vivo anti-breast cancer activity by JQ1 and a strong long-lasting effect of combination therapy with JQ1 and the ER degrader fulvestrant. Taken together, we provide evidence that the epigenomic proteins BRD3/4 and WHSC1 are essential regulators of estrogen receptor signaling and are novel therapeutic targets for treatment of Tam-R breast cancer.     

Summary of aromatase inhibitor trials: the past and future

Antagonizing estrogen by inhibition of aromatase has become a mainstay of adjuvant endocrine therapy in women with hormone receptor positive (ER+) breast cancer. Recent trials have shown an incremental gain for the AIs over tamoxifen when given as an up-front alternative to tamoxifen, but additionally added benefit is achieved by giving them in sequence with tamoxifen after either an early switch (2–3 years) or as a late switch (5 years). The true clinical implications of accelerated bone resorption from AIs is becoming better understood and its management defined. AI minimally effect quality of life. The chronic relapsing nature of ER+ breast cancer implies long term therapy will be of benefit in selected patients. Outstanding issues under investigation include optimal duration of endocrine therapy, optimal sequence, optimal agents and whether combining anti-estrogens will yield advantage. The role of AIs is also under investigation in premenopausal women in combination with ovarian function suppression. Identifying prognostic and predictive factors of endocrine therapy is important as is the identification and overcoming of resistance mechanisms. Both tumor and host signatures are being pursued to this end. Optimizing, expanding and extending endocrine therapy is likely to add further to patient outcome.     

Relationships between Signaling Pathway Usage and Sensitivity to a Pathway Inhibitor: Examination of Trametinib Responses in Cultured Breast Cancer Lines

Cellular signaling pathways involving mTOR, PI3K and ERK have dominated recent studies of breast cancer biology, and inhibitors of these pathways have formed a focus of numerous clinical trials. We have chosen trametinib, a drug targeting MEK in the ERK pathway, to address two questions. Firstly, does inhibition of a signaling pathway, as measured by protein phosphorylation, predict the antiproliferative activity of trametinib? Secondly, do inhibitors of the mTOR and PI3K pathways synergize with trametinib in their effects on cell proliferation? A panel of 30 human breast cancer cell lines was chosen to include lines that could be classified according to whether they were ER and PR positive, HER2 over-expressing, and “triple negative”. Everolimus (targeting mTOR), NVP-BEZ235 and GSK2126458 (both targeting PI3K/mTOR) were chosen for combination experiments. Inhibition of cell proliferation was measured by IC₅₀ values and pathway utilization was measured by phosphorylation of signaling kinases. Overall, no correlation was found between trametinib IC₅₀ values and inhibition of ERK signaling. Inhibition of ERK phosphorylation was observed at trametinib concentrations not affecting proliferation, and sensitivity of cell proliferation to trametinib was found in cell lines with low ERK phosphorylation. Evidence was found for synergy between trametinib and either everolimus, NVP-BEZ235 or GSK2126458, but this was cell line specific. The results have implications for the clinical application of PI3K/mTOR and MEK inhibitors.     

AND-34 activates phosphatidylinositol 3-kinase and induces anti-estrogen resistance in a SH2 and GDP exchange factor-like domain-dependent manner

AND-34, a 95-kDa protein with modest homology to Ras GDP exchange factors, associates with the focal adhesion protein p130Cas. Overexpression of AND-34 confers anti-estrogen resistance in breast cancer cell lines, a property linked to its ability to activate Rac. Here, we show that both the GDP exchange factor-like domain and the SH2 domain of AND-34 are required for Rac activation and for resistance to the estrogen receptor (ER) antagonist ICI 182,780. As phosphatidylinositol 3-kinase (PI3K) signaling can regulate Rac activation, we examined the effects of AND-34 on PI3K. Overexpression of AND-34 in MCF-7 cells increased PI3K activity and augmented Akt Ser(473) phosphorylation and kinase activity. Inhibition of PI3K with LY294002 or a dominant-negative p85 construct blocked AND-34-mediated Rac and Akt activation. Although R-Ras can activate PI3K, transfection with constitutively active R-Ras failed to induce Rac activation and AND-34 overexpression failed to induce R-Ras activation. Treatment of either vector-only or AND-34-transfected ZR-75-1 cells with ICI 182,780 markedly diminished ERalpha levels, suggesting that AND-34-induced anti-estrogen resistance is likely to occur by an ERalpha-independent mechanism. Treatment of a ZR-75-1 breast cancer cell line stably transfected with AND-34 plus 2 micromol/L LY294002 or 10 micromol/L NSC23766, a Rac-specific inhibitor, abrogated AND-34-induced resistance to ICI 182,780. Our studies suggest that AND-34-mediated PI3K activation induces Rac activation and anti-estrogen resistance in human breast cancer cell lines.