ER Activity Research Articles

MP98-12 FOXA1 KNOCKOUT IS ASSOCIATED WITH INCREASED CARCINOGENIC SUSCEPTIBILITY AND ANDROGEN RECEPTOR EXPRESSION IN MURINE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology V1 Apr 2017MP98-12 FOXA1 KNOCKOUT IS ASSOCIATED WITH INCREASED CARCINOGENIC SUSCEPTIBILITY AND ANDROGEN RECEPTOR EXPRESSION IN MURINE BLADDER CANCER Lauren Shuman, Zongyu Zheng, Hironobu Yamashita, Joshua Warrick, Klaus Kaestner, and David DeGraff Lauren ShumanLauren Shuman More articles by this author , Zongyu ZhengZongyu Zheng More articles by this author , Hironobu YamashitaHironobu Yamashita More articles by this author , Joshua WarrickJoshua Warrick More articles by this author , Klaus KaestnerKlaus Kaestner More articles by this author , and David DeGraffDavid DeGraff More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3077AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In the US, men are 3 to 4 times more likely to be diagnosed with bladder cancer (BC), however, women frequently present with more advanced disease and have inferior clinical outcomes. Recent data indicates androgen (AR) and estrogen receptors (ERα and ERβ) play an important role in BC tumorigenesis and progression. These hormone receptors physically interact with transcription factor Forkhead box A1 (FOXA1), playing an important role in transcriptional activity of AR and ER. While the role for FOXA1/AR/ER complexes in BC is unknown, Foxa1 knockout (KO) results in sex-specific changes in murine urothelial differentiation. Interestingly, BC development in mice exposed to the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) also occurs in a sex-dependent manner. Therefore, we initiated a study to determine the impact of Foxa1 KO on sex-dependent development of BC in mice. METHODS To determine the effect of Foxa1 KO on BC development in males and females, Foxa1 ablation was achieved using a tamoxifen-inducible ubiquitin Cre (UBC-CreERT2) system. Control and KO mice were then exposed to BBN for 16 weeks and bladders were harvested for H&E, immunohistochemistry (IHC), and qPCR. RESULTS Following 16 weeks of BBN treatment, female control mice appeared relatively resistant to carcinogenesis compared to males, which exhibited pre-neoplastic changes including keratinizing squamous metaplasia. However, Foxa1 KO followed by BBN treatment resulted in development of keratinizing squamous metaplasia in females and progression to muscle invasive BC in males. IHC for Krt14 and Ki67 confirmed the presence of squamous differentiation and increased proliferative index in both male and female Foxa1 KO mice treated with BBN. Interestingly, our analysis also shows that nuclear AR expression is increased in male control bladder tissue compared to female control bladder tissue. However, Foxa1 KO increases AR expression independent of sex. CONCLUSIONS Overall, our data indicates that Foxa1 KO in adult male and female mice renders them more susceptible to carcinogen exposure. Interestingly, Foxa1 KO resulted in development of keratinizing squamous metaplasia in female mice. Additionally, AR was slightly increased in Foxa1 KO mice, independent of sex. These data indicate loss of FOXA1 in human BC may be associated with increased AR expression and activity, and subsequent disease progression. Future work includes determining ERα and ERβ expression following Foxa1 KO, as well as the mechanism by which Foxa1 KO alters AR expression and activity in BC. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1316 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Lauren Shuman More articles by this author Zongyu Zheng More articles by this author Hironobu Yamashita More articles by this author Joshua Warrick More articles by this author Klaus Kaestner More articles by this author David DeGraff More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Distribution of Ki-67 values within HER2 & ER/PgR expression variants of ductal breast cancers as a potential link between IHC features and breast cancer biology

BackgroundUnexpected differences in Ki-67 values among HER2 & ER/PgR defined subgroups were found. This study aims to detect possible subdivisions beyond the conventional breast cancer types.MethodsOne thousand one hundred eighty consecutive patients with invasive ductal breast carcinoma were included and distributed in 16 subgroups (four HER2 phenotypes (0+, 1+, 2+ and 3+) times four ER/PgR phenotypes). Complex distributions of Ki-67 values were tested by expectation maximization (EM) clustering.ResultsPooled Ki67 values of all patients showed the presence of three EM clusters (defined as LMA-low mitotic activity, IMA-intermediate mitotic activity and HMA-high mitotic activity) with expected mean Ki-67 values of 1.17%, 40.45% and 77.79%, respectively. Only ER-PgR- tumors significantly dispersed in three clusters (29.75% tumors in LMA, 46.95% in IMA and 23.30% in the HMA cluster), while almost no detected HMA tumors were of ER + PgR+ or ER + PgR- phenotypes.Among 799 ER + PgR+ patients distribution in clusters was HER2 dependent (p = 0.000243), due to increased number of IMA HER2 3+ tumors on the expense of LMA HER2 3+ tumors (52 IMA out of 162 HER2 3+ patients versus113 IMA out of 637 HER2 < 3+ patients). This was not found among ER + PgR- patients (p = 0.186968).Among ER-PgR- patients, HER2 overexpression also increased number of IMA tumor, but by reducing the number of HMA tumors (p < 0.000001). Here, difference between HER2 absent (0+) and HER2 3+ patients was evident (10 HMA out of 125 HER2 3+ patients versus 42 HMA out of 103 HER2 0+ patients).ConclusionsResults suggest that distributions of breast cancers in three clusters of mitotic activity depend on different mechanisms for ER + PgR+ and ER negative tumors. Although HER2 overexpression increases number of IMA tumors in both settings, in the former it is done by reducing number of LMA tumors, while in the latter it reduces the number of HMA tumors. Mitotic activity of ER + PgR- tumors seems unrelated to the HER2 status, possibly as an indicator that ER dysfunctionality in cancers that lack PgR expression. Among ER negative tumors, the absence of HER2 (0+) might be as important as the HER2 overexpression.

Transcriptional regulation of mixed lineage kinase 3 by estrogen and its implication in ER-positive breast cancer pathogenesis.

Mixed Lineage Kinase 3 (MLK3), also called as MAP3K11 is a tightly regulated MAP3K member but its cellular function is still not fully understood. Earlier we reported post-translational regulation of MLK3 by estrogen (E2) that inhibited the kinase activity and favored survival of ER+ breast cancer cells. Here we report that MLK3 is also transcriptionally downregulated by E2 in ER+ breast cancer cells. Publicly available data and in situ hybridization of human breast tumors showed significant down regulation of MLK3 transcripts in ER+ tumors. The basal level of MLK3 transcripts and protein in ER+ breast cancer cell lines were significantly lower, and the protein expression was further down regulated by E2 in a time-dependent manner. Analysis of the promoter of MLK3 revealed two ERE sites which were regulated by E2 in ER+ but not in ER− breast cancer cell lines. Both ERα and ERβ were able to bind to MLK3 promoter and recruit nuclear receptor co-repressors (NCoR, SMRT and LCoR), leading to down-regulation of MLK3 transcripts. Collectively these results suggest that recruitment of nuclear receptor co-repressor is a key feature of ligand-dependent transcriptional repression of MLK3 by ERs. Therefore coordinated transcriptional and post-translational repression of pro-apoptotic MLK3 probably is one of the mechanisms by which ER+ breast cancer cells proliferate and survive.

Abstract P6-02-08: Modulation of indoleamine 2, 3-dioxygenase (IDO1) expression in breast cancer cells by activated CD8+ T cells is controlled by DNA promoter methylation

Abstract Tumor infiltrating lymphocytes (TILs) play a critical role in regulating the immunomodulatory properties of triple negative breast cancer (TNBC). However, the specific adaptations that TNBC tumors undergo when challenged by lymphocyte infiltration remain unclear. In order to address this gap in knowledge, we conducted an immuno-phenotype comparison using mRNA sequencing between the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were co-cultured with activated human T-cells. Although the cytokine-induced immune signature of the two cell lines were similar, MDA-MD-231 cells were able to transcribe the tryptophan catabolizing enzyme IDO1 at a significantly higher level than MCF7 cells. Stimulation with IFNg was able to differentially induce IDO protein expression and enzymatic activity in ER- cell lines compared to ER+ cell lines, though no differences were observed in upstream JAK/STAT1 signaling or IDO1 mRNA stability between the two cell lines. Further experiments showed that treatment with the demethylating agent 5-aza-deoxycytidine was able to reverse suppression of IDO1 expression in MCF7 cells, suggesting that DNA methylation serves as a potential determinant in IDO1 induction. Analysis of TCGA and other previously published breast cancer datasets revealed subtype-specific mRNA and promoter methylation differences in IDO1, with TNBC/basal-like subtypes exhibiting lower promoter methylation and higher mRNA expression than ER+/luminal subtypes. Bisulfite pyrosequencing validated the subtype-specific association of decreased promoter methylation with increased IDO1 expression in breast cancer cell lines and an independent cohort of primary breast tumors. In addition, decreased IDO1 promoter methylation and elevated IDO1 expression in basal-like breast tumors was found to be associated with increased levels of kynurenine, the metabolic product of IDO1, as well as higher numbers of CD8+ TILs. Furthermore, high kynurenine levels in breast tumors were associated with worse patient survival. Taken together, these findings suggest that subtype-specific IDO1 promoter methylation regulates the ability of breast tumors to escape from antitumor immune responses driven by CD8+ TILs and could be used as a predictive biomarker for IDO inhibitor-based immunotherapy. Citation Format: Gu F, Noonepalle SK, Lee E-J, Choi J-H, Shull AY, Pei L, Sreekumar A, Ambs S, Shi H. Modulation of indoleamine 2, 3-dioxygenase (IDO1) expression in breast cancer cells by activated CD8+ T cells is controlled by DNA promoter methylation [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-02-08.

Abstract P3-04-05: Identification of SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that has strong antitumor activity in wild-type and mutant ER+ breast cancer models

Abstract Estrogen receptor positive (ER+) breast cancer accounts for 70% of all breast cancers and is primarily treated with endocrine therapy. Approximately 40% of patients on endocrine therapy will become resistant via a number of mechanisms. There is evidence that in many cases ER continues to play a central role, including mutations in ER leading to a constitutively active receptor. Estrogen receptor degraders like fulvestrant are effective in shutting down ER signaling; however, poor pharmaceutical properties limit fulvestrant clinical activity and prevent it from achieving maximum receptor blockade. We describe the discovery of SAR439859, a novel, orally bioavailable SERD that is a potent antagonist and degrader of ER both in vitro and in vivo. SAR439859 has robust activity in multiple ER+ breast cancer cell lines including cells that are resistant to tamoxifen as well as cell lines harboring ER mutants. Across a large panel of ER+ cells, SAR439859 demonstrated broad and superior ER degradation activity than most SERDs undergoing clinical testing. This leads to a profound inhibition of ER signaling, better inhibition of cell growth and results in improved in vivo efficacy. SAR439859 demonstrated tumor regression in all ER+ BC models including MCF7-ESR1 mutant-Y537S model, as well as patient-derived xenograft model that is resistant to endocrine therapies. Furthermore, SAR439859 displays limited cross-resistance with other class of SERDs. Taken together, these results suggest that SAR439859 would be of therapeutic benefit in metastatic BC setting for patients harboring wild type or mutant ER. SAR439859 is being advanced toward the clinic. Citation Format: Shomali M, Cheng J, Koundinya M, Weinstein M, Malkova N, Sun F, Hebert A, Cindachao M, Hoffman D, McManus J, Levit M, Pollard J, Vincent S, Besret L, Adrian F, Winter C, El-Ahmad Y, Halley F, Hsu K, Lager J, Garcia-Echeverria C, Bouaboula M. Identification of SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that has strong antitumor activity in wild-type and mutant ER+ breast cancer models [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-05.

Abstract PD2-02: SFX-01 targets Wnt signalling to inhibit stem-like cells in breast cancer patient-derived xenograft tumours

Abstract Background: SFX-01 is a novel therapeutic comprising synthetic sulforaphane (SFN) stabilised within a-cyclodextrin. Breast cancer stem-like cells (CSCs) have been identified in all molecular subtypes and are likely drivers of breast cancer metastasis and treatment resistance. We recently established that CSC activity in ER+ BC, represent a source of therapeutic resistance (Simões et al, Cell Reports, 2015). Material and methods: We investigated SFX-01 effects on breast CSC activity using mammosphere formation efficiency (MFE) and aldehyde dehydrogenase (ALDH) activity using the ALDEFLUOR assay in patient samples and patient-derived xenograft (PDX) tumours. Cells from primary (n=12) and metastatic (n=15) samples were treated with SFX-01 (5 μM) or vehicle control.Using a 2 or 8 week in vivo treatment, early (HBCx34) and metastatic (BB3RC31) ER+ PDX tumours were treated with SFX-01 (300mg/Kg/day) alone or in combination with tamoxifen (TAM, 10 mg/kg/day) or fulvestrant (FULV, 200 mg/kg/week). Tumours were dissociated and MFE and ALDH activity assessed. Results: SFX-01 in vitro reduced MFE of both primary (0.19%±0.02 vs control 0.52%±0.06: p&amp;lt;0.001) and metastatic patient samples (0.43±0.04 vs control 0.93%±0.07: p&amp;lt;0.001). SFX-01 treatment in vivo for 2 weeks reduced MFE of HBCx34 (0.35%±0.03 vs control 0.64%±0.09; p&amp;lt;0.01) and BB3RC31 (0.78%±0.04 vs control 0.89%±0.06: p&amp;lt;0.05) and also ALDH activity of HBCx34 (3%±0.6 vs control 6.3%±0.4: p&amp;lt;0.01) and BB3RC31 (1%±0.2 vs control 3%±0.6: p&amp;lt;0.05). TAM and FULV increased MFE and ALDH activity after 2 weeks of treatment in vivo, which was abrogated by combination with SFX-01; for example HBCx34 MFE with TAM alone: 0.81%±0.07 vs TAM+SFX-01: 0.34%±0.02 (p&amp;lt;0.01) and ALDH+ with TAM alone 10%±0.4 vs TAM+SFX 4.2%±0.4 (p&amp;lt;0.01). TAM+SFX-01 suppressed tumour growth at 8 weeks vs TAM alone in HBCx34 but not BB3RC31. FULV treatment maintained tumour growth suppression at 8 weeks and no additive effect was seen with SFX-01, although MFE and ALDH activity were suppressed. Mechanistically, SFX-01 potently inhibited the canonical Wnt pathway in MCF-7 cells and their endocrine-resistant derivatives and we are currently exploring SFX-01 activity on other CSC regulatory pathways. SFX-01 has been shown to be well tolerated in SAD and MAD studies in normal volunteers and clinical studies designed to test tolerability and efficacy in combination with the three major classes of endocrine therapy (AI, TAM and FULV) in advanced BC will begin in Q4 2016. Conclusions: Our data demonstrate the potential of SFX-01 for clinically meaningful improvements to endocrine therapy in ER+ breast cancer by reversing CSC mediated resistance. Citation Format: Howell SJ, Simoes BM, Alferez D, Eyre R, Spence K, Santiago-Gomez A, Sarmiento-Castro A, Tanaka I, Howat D, Clarke RB. SFX-01 targets Wnt signalling to inhibit stem-like cells in breast cancer patient-derived xenograft tumours [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD2-02.

Abstract P1-08-01: Regulation of estrogen receptor-α by NF1

Abstract Background. Although great strides have been made in targeting the ER pathway for treating ER+ breast cancer, relapse and death is common and is closely linked to resistance to ER-targeting agents. As a result, the majority of deaths from breast cancer still come from ER+ tumors. To discover drivers for endocrine resistance, we have sequenced tumor DNAs from a cohort of &amp;gt;600 patients treated with 5-year tamoxifen (Tam) monotherapy with a median 10.4 years follow up. Our preliminary data show that the worst outcome mutations (Hazard Ratio of ∼3 for relapse) were mostly those of the Neurofibromatosis type 1 (NF1) gene (encoding Neurofibromin), with nonsense/frame shift mutations creating early stop codons. Germline NF1mutations cause neurofibromatosis type 1, a common inherited disorder that predisposes individuals to both benign and malignant tumors of the nervous system, as well as an increased risk for breast cancer. Analysis of DNA sequencing data has also shown that the NF1 gene is mutated in a wide range of common cancers (e.g., melanoma, lymphoma, and cancers of the lung, breast, and colon). Thus, NF1-deficiency underlies the formation and/or progression of a large number of cancers, so that the development of therapies targeted to NF1-deficient malignancies would have broad impact. These observations support the hypothesis that NF1 gene inactivation is associated with aggressive tumor behaviors, such as endocrine therapy resistance in breast cancer. The key focus of this study is to define how the NF1 protein neurofibromin, regulates endocrine therapy resistance. Although neurofibromin is best known as a negative regulator for Ras, our data show that it may have other functions. Method. Our data suggest that many of the identified nonsense/frame shift create a NF1 null state; thus, we have used gene-silencing to recapitulate the effects of such NF1 mutations on the activities of ER+ breast cancer cells. NF1+ and NF1– ER+ breast cancer cells were grown in defined media to measure how estradiol (E2) and Tam impact their growth, transforming activities, and gene expression. The binding between neurofibromin and components of the ER transcriptional pathway was measured biochemically and using the mammalian two-hybrid system. Results. Our data showed that NF1-silenced cells use Tam as an agonist and can grow with very little E2, and these activities are driven by enhanced recruitment of ER to the ERE, leading to efficient expression of many classic ER-responsive genes. Expressing the NF1-GAP domain does not restore normal responses to Tam and E2 in NF1-silenced cells, suggesting that neurofibromincan regulate ER activity in a Ras-independent manner. To investigate the possibility that neurofibromin can directly regulate ER, we found that it can bind ER; furthermore, neurofibromin was more strongly recruited to the ERE by Tam than by E2. Conclusion. Our data support a model whereby neurofibromin acts like a co-repressor for ER. As such,NF1 loss may result in more aggressive tumor behaviors by activating, not only the Ras pathways, but also the ER transcriptional pathways. Simultaneous activation of two powerful oncogenic pathways by the loss of a single tumor suppressor may explain why neurofibromin is such a potent tumor suppressor lost in a wide range of cancers. Citation Format: Zheng Z-Y, Cakar B, Lavere P, Cao J, Yao J, Singh P, Lei JT, Toonen JA, Haricharan S, Anurag M, Shah K, Kavuri M, Chan DW, Chen X, Gutmann DH, Foulds CE, Ellis MJ, Chang EC. Regulation of estrogen receptor-α by NF1 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-01.

Abstract P3-04-11: ER is required for mTORC1 inhibitor-induced feedback activation of PI3K/AKT in ER+ breast cancer cells and patients' tumors

Abstract The mTORC1 inhibitor everolimus (afinitor) is approved for the treatment of patients with advanced/metastatic ER+/HER2- breast cancer in combination with the steroidal aromatase inhibitor exemestane following progression on a non-steroidal aromatase inhibitor. The BOLERO-2 and TAMRAD studies demonstrated that combined everolimus/anti-estrogen therapy provided longer PFS compared to anti-estrogen alone. However, it has not been clarified whether continued treatment with an anti-estrogen backbone is beneficial in the setting of mTORC1 inhibition. Upon activation by mTORC1, p70S6K phosphorylates the insulin-like growth factor-1 receptor (IGF-1R)/insulin receptor (InsR) effector IRS-1 to promote IRS-1 degradation, which in turn decreases activation of phosphatidylinositol 3-kinase (PI3K), AKT, and mTORC1. IGF1R, IRS1, and IRS2 are ER-inducible genes, and crosstalk between the ER and IGF-1R pathways has been described. We hypothesized that mTORC1 inhibition with everolimus will upregulate IGF-1R/InsR/IRS-1/2 signaling to activate PI3K/AKT and promote cancer cell survival, while combined inhibition of ER and mTORC1 will block PI3K/AKT activation by decreasing IGF-1R and IRS-1/2, providing rationale for combined targeting of ER and mTORC1. In 3 ER+ breast cancer cell lines, everolimus treatment increased phospho-AKT levels. ER inhibition with fulvestrant suppressed the induction of P-AKT by everolimus. IGF-1R/InsR inhibition with OSI-906, and RNAi-mediated knockdown of IGF-1R, InsR, or IRS-1/2, decreased everolimus-induced P-AKT. Everolimus sensitized IGF-1R/InsR to IGF-1 that was suppressed by fulvestrant but enhanced by 17b-estradiol. Although fulvestrant decreased IGF-1R and InsR protein levels, phospho-receptor tyrosine kinase profiling showed that fulvestrant increased P-IGF-1R and P-InsR. Acting downstream of IGF-1R/InsR, fulvestrant prevented everolimus-induced PI3K/AKT activation by blocking binding between the p85 regulatory subunit of PI3K and IRS-1, possibly by decreasing IRS-1/2 levels. In summary, everolimus-induced activation of PI3K/AKT requires IGF-1R/InsR/IRS-1/2 signaling facilitated by ER. Combined treatment with fulvestrant and everolimus synergistically inhibited growth in 4 ER+ cell lines. To determine whether ER promotes PI3K/AKT activation induced by mTORC1 inhibition in patients' tumors without exposing patients to everolimus, we analyzed live tumor tissues from post-menopausal patients with ER+/HER2- breast cancer treated +/- letrozole for 10-21 d before surgical tumor resection. Tumor cores (1 mm diameter) were used for ex vivo culture in DMEM +/- everolimus +/- OSI-906 for 1 h, and lysates were analyzed by immunoblot. Everolimus significantly increased P-AKT in tumors from untreated patients (n=10). OSI-906 did not affect P-AKT, but OSI-906 suppressed everolimus-induced P-AKT. In tumors from letrozole-treated patients (n=7), neither everolimus nor OSI-906 affected P-AKT. These data collectively suggest that ER activation is required for activation of PI3K/AKT induced by mTORC1 inhibition, and provide rationale for therapeutic combinations of anti-estrogens and mTORC1 inhibitors. Citation Format: Yang W, Chen VS, Schwartz GN, Marotti JD, Rosenkranz KM, Gui J, Miller TW. ER is required for mTORC1 inhibitor-induced feedback activation of PI3K/AKT in ER+ breast cancer cells and patients' tumors [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-11.

Abstract PD2-01: Clinically dormant ER+ breast tumors exhibit AMPK activation

Abstract While adjuvant anti-estrogen therapy has shown immense clinical benefit for patients with estrogen receptor-positive (ER+) breast cancer, &amp;gt;30% of patients experience cancer recurrence within 15 years of initial diagnosis. Anti-estrogen therapies inhibit ER activity either directly (tamoxifen, fulvestrant) or by reducing systemic estrogen levels (aromatase inhibitors, AIs). Extending adjuvant anti-estrogen therapy from 5 to 10 years further prevents recurrence; however, with both 5- and 10-year treatment regimens, a large proportion of patients who relapse do so after cessation of therapy (“late recurrence”). Additionally, disseminated tumor cells in bone marrow have been found after 4 years of adjuvant anti-estrogen therapy in “disease-free” patients. These data collectively indicate that anti-estrogens elicit clinical benefit as adjuvant therapies, in part, by maintaining a population of residual micrometastatic cancer cells in a “clinically dormant” state (i.e., undetectable by routine clinical methods). Understanding how such dormant cancer cells survive will enable the development of more effective adjuvant therapies. We developed several luciferase-labeled xenograft models of ER+ breast cancer that recapitulate clinical dormancy in vivo. Low systemic levels of estrogen in mice can be further suppressed by ovariectomy, mimicking the effects of AI-induced estrogen deprivation seen in patients. In ovariectomized mice, palpable tumors form upon 17b-estradiol supplementation, but quickly regress upon estrogen withdrawal. While regressed tumors become non-palpable within 2 wk, a small proportion of cancer cells survive these estrogen-deprived conditions for &amp;gt;4 months in a clinically dormant, growth-suppressed state. This estrogen deprivation-induced clinically dormant cell population retains tumorigenic potential, as 17b-estradiol retreatment induces tumor recurrence. RNA expression profiling revealed AMPK alpha 2 as one of the most highly expressed genes in clinically dormant residual tumor cells compared to acutely estrogen-withdrawn tumors. Increased AMPK kinase activity was confirmed through immunohistochemical analysis of phospho-ACC, and AMPK substrate. AMPK activation using glucose deprivation or the anti-diabetes drug metformin promoted estrogen-independent survival and growth of ER+ breast cancer cells in vitro. Metformin treatment may also slow estrogen withdrawal-induced tumor regression and promote tumor cell survival in ER+ breast cancer xenografts. As a cellular energy sensor, AMPK has been shown to promote autophagy, a process linked with anti-estrogen resistance. Immunofluorescent staining of estrogen deprivation-induced clinically dormant residual tumor cells revealed decreased levels of the autophagy marker p62 compared to 17b-estradiol-driven tumors. Early data suggest that inhibition of autophagy with hydroxychloroquine abrogates cell survival conferred by metformin in estrogen-depleted conditions. Thus, AMPK may be promoting the survival of ER+ breast cancer cells in estrogen-deprived conditions by increasing autophagic flux. These data have implications for the interpretation of data from ongoing clinical studies testing metformin for the treatment of cancer. Citation Format: Hampsch RA, Dillon LM, Miller TW. Clinically dormant ER+ breast tumors exhibit AMPK activation [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD2-01.

Synthesis of diamond in double carbon-rare earth element systems

For the first time, a possibility of diamond synthesis under high pressures was studied in binary systems “rare earth element (REE)-carbon” with refractory carbides. It was found that at pressure 8GPa in the Eu-C system graphite transforms into diamond at about melting temperature of EuC2 carbide (1900–2200°C), while in the Er-C and Tm-C systems diamond formation takes place well below melting temperature of carbides ErC2 or TmC2 (1600–1800°C). Simultaneous crystallization of diamond and carbides ErC2 or TmC2 from vanishing carbon-saturated liquid phase was proposed as a mechanism for the diamond formation in the latter systems. As compared with Eu, more strong catalytic activity of Er and Tm for diamond synthesis was explained by matching their crystal structures to the diamond one.

Ethanol-extracted Cameroonian propolis exerts estrogenic effects and alleviates hot flushes in ovariectomized Wistar rats

BackgroundSince the biological properties of propolis depend to the plants that can be found in a specific region, propolis from unexplored regions attracts the attention of scientists. Ethanolic extract of Cameroonian propolis (EEP) is used to treat various ailments including gynecological problems and amenorrhea. Since there were no scientific data to support the above claims, the present study was therefore undertaken to assess estrogenic properties of Cameroonian propolis.MethodsTo achieve our goal, the ability of EEP to induce MCF-7 cells proliferation in E-screen assay as well as to activate estrogen receptors α (ERα) and β (ERβ) in cell-based reporter gene assays using human embryonic kidney cells (HEK293T) transfected with ERs was tested. Further, a 3-day uterotrophic assay was performed and the ability of EEP to alleviate hot flushes in ovariectomized adult rats was evaluated.ResultsIn vitro, EEP showed an antiestrogenic activity in both HEK293T ER-α and ER-β cells. In vivo, EEP induced a significant increase in a bell shape dose response manner of the uterine wet weight, the total protein levels in the uterus, the uterine and vaginal epithelium height and acini border cells of mammary gland with the presence of abundant eosinophil secretions. Moreover, EEP induced a significant decrease in the total number, average duration as well as frequency of hot flushes after 3 days of treatment in rat (equivalent to a month in woman). The dose of 150 mg/kg exhibited the most potent estrogenic effects among all the tested doses. The UPLC-HRMS analysis showed the presence of caffeic acid derivatives and trirtepernoids in EEP, which are well known endowed with estrogenic properties.ConclusionThese results suggest that Ethanolic extract of Cameroonian propolis has estrogen-like effects in vivo and may alleviate some menopausal problems such as vaginal dryness and hot flushes.Graphical abstractEthanol-extracted Cameroobian propolis exhibited in vitro and in vivo estrogen-like effects. This extract may contain promising phytoestrogens.

Effects of thermal annealing on the structural and electronic properties of rare earth-implanted MoO3 nanoplates

The compositional, structural and morphological recovery of α-MoO3 nanoplates implanted with Eu and Er ions has been assessed as a function of post-implantation thermal treatments. Rapid thermal annealing induces the formation of phases different from α-MoO3 and seems to be unable to recover the original oxygen content of the nanostructures. In contrast, conventional annealing in air at 450 °C for 4 hours fully recovers the original molybdenum trioxide structure and leads to an effective optical activation of both Er and Eu ions. Synchrotron-based X-ray photoelectron spectroscopy and X-ray absorption studies confirm the incorporation of the rare earth ions into their trivalent charge state and reveal the modification of the original valence band structure of the nanoplates.

The role of soybean extracts and isoflavones in hormone-dependent breast cancer: aromatase activity and biological effects.

Estrogen receptor-positive (ER+) breast cancer is the most common cause of cancer death in women worldwide. Nowadays, the relationship between soya diet and breast cancer is controversial due to the unknown role of its isoflavones, genistein (G) and daidzein (D). In this work, we investigated not only the anti-tumor properties of a soybean extract (NBSE) but also whether the biotransformation of extract (BSE) by the fungus Aspergillus awamori increased its effectiveness. The BSE showed a stronger anti-aromatase activity and anti-proliferative efficacy in ER+ aromatase-overexpressing breast cancer cells. D and G were weak aromatase inhibitors, but inhibited cancer cell growth, being G the isoflavone that contributed to the BSE-induced effects. This work demonstrated that the biotransformation increased the anti-aromatase activity and the anti-tumoral efficacy of soybean extract in breast cancer cells. Moreover, it elucidated the potential use of soya in the prevention and/or treatment of ER+ breast cancer.

Ricolinostat (ACY-1215), a Selective HDAC6 Inhibitor, Alone and in Combination with Bendamustine Is Effective in Preclinical Studies in Lymphoma Cell Lines

Background. Histone deacetylase inhibitors (HDACi) have emerged as a new class of anticancer agents, targeting the biological processes including cell cycle and apoptosis. The discovery of isoform selective compounds may offer a therapeutic advantage by minimizing toxicity. Ricolinostat (ACY-1215) inhibits HDAC6, resulting in tubulin hyperacetylation. Previous studies have investigated the synergistic effects of the combination of HDACis and alkylating agents offering new therapeutic strategies. The aim of this in vitro study was to investigate the activity of ricolinostat alone and in combination with bendamustine in a panel of non-Hodgkin's lymphoma (NHL) cell lines.Methods. Ricolinostat was kindly provided by Acetylon Pharmaceuticals (Boston). NHL cell lines (WSU-NHL, RL, Granta-519, Jeko-1, Hut-78 and Karpas-299) were treated with ricolinostat and bendamustine alone and in combination. The interaction between the drugs was evaluated by the Chou-Talalay method and cell viability and clonogenicity were also evaluated. Apoptosis, ROS generation, Bcl-2 protein expression, cell cycle progression and tubulin expression were determined by flow cytometry. The effects of ricolinostat alone and in combination on caspases, PI3K/Akt, ER stress and UPR pathways were assessed by immunoblotting.Results. Ricolinostat was able to affect the cell viability in a dose dependent manner, with IC50 values ranging from 1.51 to 8.65 μM. Ricolinostat with bendamustine synergistically induced anti-proliferative and pro-apoptotic effects in lymphoma cells, even in the presence of the bone marrow microenvironment. Combination treatment decreased the percentage of viable peripheral blood mononuclear cells (PBMCs) from patients with lymphoma but had minimal or no cytotoxic effect on PBMCs from healthy donors. Clonogenic assay revealed that the drug combination significantly inhibited the colony formation compared with the drugs alone. Drug combination reduced the proportion of cells in the G0/G1 and S phases and caused an increase of "sub-G0/G1" peak with a decrease of cyclin D1 and cyclin E and an increase of p21 and p27 proteins. The synergistic effect was accompanied by increased reactive oxygen species (ROS) generation, which is linked to a decrease of thioredoxin-1 (Trx1) expression, activation of caspase -8, -9, and -3, the cleavage of PARP and modulation of the Bcl-2 protein family. Apoptosis was associated with increased hallmarks of ER stress and activation of UPR sensors and was mediated by dephosphorylation of the AKT. In addition, the exposure of ricolinostat induced the acetylation level of α-tubulin, the extent of which was not further modified by bendamustine. The direct cytotoxicity of ricolinostat/bendamustine may be mediated by an effect on microtubule stabilization. Finally, ricolinostat alone induced a significant down-regulation of IL-10 that was especially evident in WSU-NHL with a fold decrease of 6.6 compared to control. The drug combination affected IL-10 production in all three cell lines with a fold decrease of 5.77 in WSU-NL; 11.5 in Hut-78; and 10.9 in Jeko-1 cells compared with ricolinostat alone.Conclusion. These preclinical studies suggest that bendamustine in combination with epigenetic therapy, such as ricolinostat, may be a promising treatment regime for managing lymphomas. DisclosuresQuayle:Acetylon Pharmaceuticals: Employment, Equity Ownership.

Abstract A08: Mechanistic basis of Palbociclib combinatorial activity in ER+ breast cancer and non-breast indications

Abstract Phosphorylation of the retinoblastoma protein (Rb) by cyclin-dependent kinases 4 and 6 (CDK4/6) is a critical checkpoint for G1/S cell cycle progression and commitment to cellular proliferation. Human malignancies often subvert these control mechanisms through a range of genetic and biochemical adaptations. Accordingly, tumors that depend on CDK4/6 activity for proliferation and survival are particularly sensitive to inhibition of this pathway by palbociclib (Ibrance™), a highly selective inhibitor of CDK4/6 kinase activities. Treatment regimen of palbociclib with letrozole significantly improved progression-free survival in a randomized phase 2 study of women with advanced estrogen receptor-positive (ER+), HER2-negative breast cancer. Likewise, in ER+ breast cancer models palbociclib and estrogen antagonists combine for greater anti-proliferative activity, increased hallmarks of cellular senescence and prolonged durability of response following drug removal. Dual inhibition of CDK4/6 and ER signaling demonstrated robust anti-tumor activity in xenograft studies. The addition of Palbociclib to other targeted therapeutics elicits improved activity in pre-clinical models of several non-breast indications and these effects also manifest through modulation of cellular proliferation, senescence and growth arrest. Data will be presented on the molecular basis of combination benefit with Palbociclib in ER+ breast and other oncology indications. Citation Format: Stephen Dann, John Chionis, Liu Choating, Enhong Chen, Ping Wei, Koleen Eisele, David J. Shields, Paul A. Rejto, Todd VanArsdale. Mechanistic basis of Palbociclib combinatorial activity in ER+ breast cancer and non-breast indications. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A08.

Cooperative Dynamics of AR and ER Activity in Breast Cancer.

This study suggests that AR plays a previously unrecognized role in supporting E2-mediated ER activity in ER+/AR+ breast cancer cells, and that enzalutamide may be an effective therapeutic in ER+/AR+ breast cancers. Mol Cancer Res; 14(11); 1054-67. ©2016 AACR.

Tumor suppressor ING4 inhibits estrogen receptor activity in breast cancer cells.

Resistance to antiestrogen therapy remains a significant problem in breast cancer. Low expression of inhibitor of growth 4 (ING4) in primary tumors has been correlated with increased rates of recurrence in estrogen receptor-positive (ER+) breast cancer patients, suggesting a role for ING4 in ER signaling. This study provides evidence that ING4 inhibits ER activity. ING4 overexpression increased the sensitivity of T47D and MCF7 ER+ breast cancer cells to hormone deprivation. ING4 attenuated maximal estrogen-dependent cell growth without affecting the dose–response of estrogen. These results indicated that ING4 functions as a noncompetitive inhibitor of estrogen signaling and may inhibit estrogen-independent ER activity. Supportive of this, treatment with fulvestrant but not tamoxifen rendered T47D cells sensitive to hormone deprivation as did ING4 overexpression. ING4 did not affect nuclear ERα protein expression, but repressed selective ER-target gene transcription. Taken together, these results demonstrated that ING4 inhibited estrogen-independent ER activity, suggesting that ING4-low breast tumors recur faster due to estrogen-independent ER activity that renders tamoxifen less effective. This study puts forth fulvestrant as a proposed therapy choice for patients with ING4-low ER+ breast tumors.

Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations.

TM7SF3, a novel p53-regulated homeostatic factor, attenuates cellular stress and the subsequent induction of the unfolded protein response.

Earlier reported small interfering RNA (siRNA) high-throughput screens, identified seven-transmembrane superfamily member 3 (TM7SF3) as a novel inhibitor of pancreatic β-cell death. Here we show that TM7SF3 maintains protein homeostasis and promotes cell survival through attenuation of ER stress. Overexpression of TM7SF3 inhibits caspase 3/7 activation. In contrast, siRNA-mediated silencing of TM7SF3 accelerates ER stress and activation of the unfolded protein response (UPR). This involves inhibitory phosphorylation of eukaryotic translation initiation factor 2α activity and increased expression of activating transcription factor-3 (ATF3), ATF4 and C/EBP homologous protein, followed by induction of apoptosis. This process is observed both in human pancreatic islets and in a number of cell lines. Some of the effects of TM7SF3 silencing are evident both under basal conditions, in otherwise untreated cells, as well as under different stress conditions induced by thapsigargin, tunicamycin or a mixture of pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin-1 beta and interferon gamma). Notably, TM7SF3 is a downstream target of p53: activation of p53 by Nutlin increases TM7SF3 expression in a time-dependent manner, although silencing of p53 abrogates this effect. Furthermore, p53 is found in physical association with the TM7SF3 promoter. Interestingly, silencing of TM7SF3 promotes p53 activity, suggesting the existence of a negative-feedback loop, whereby p53 promotes expression of TM7SF3 that acts to restrict p53 activity. Our findings implicate TM7SF3 as a novel p53-regulated pro-survival homeostatic factor that attenuates the development of cellular stress and the subsequent induction of the UPR.

Pushing estrogen receptor around in breast cancer.

The estrogen receptor-α (herein called ER) is a nuclear sex steroid receptor (SSR) that is expressed in approximately 75% of breast cancers. Therapies that modulate ER action have substantially improved the survival of patients with ER-positive breast cancer, but resistance to treatment still remains a major clinical problem. Treating resistant breast cancer requires co-targeting of ER and alternate signalling pathways that contribute to resistance to improve the efficacy and benefit of currently available treatments. Emerging data have shown that other SSRs may regulate the sites at which ER binds to DNA in ways that can powerfully suppress the oncogenic activity of ER in breast cancer. This includes the progesterone receptor (PR) that was recently shown to reprogram the ER DNA binding landscape towards genes associated with a favourable outcome. Another attractive candidate is the androgen receptor (AR), which is expressed in the majority of breast cancers and inhibits growth of the normal breast and ER-positive tumours when activated by ligand. These findings have led to the initiation of breast cancer clinical trials evaluating therapies that selectively harness the ability of SSRs to 'push' ER towards anti-tumorigenic activity. Our review will focus on the established and emerging clinical evidence for activating PR or AR in ER-positive breast cancer to inhibit the tumour growth-promoting functions of ER.