Citations

Purpose Endoxifen is a tamoxifen metabolite with potent antiestrogenic activity. Patients and Methods We performed a phase I study of oral Z-endoxifen to determine its toxicities, maximum tolerated dose (MTD), pharmacokinetics, and clinical activity. Eligibility included endocrine-refractory, estrogen receptor–positive metastatic breast cancer. An accelerated titration schedule was applied until moderate or dose-limiting toxicity occurred, followed by a 3+3 design and expansion at 40, 80, and 100 mg per day. Tumor DNA from serum (circulating cell free [cf); all patients] and biopsies [160 mg/day and expansion]) was sequenced. Results Of 41 enrolled patients, 38 were evaluable for MTD determination. Prior endocrine regimens during which progression occurred included aromatase inhibitor (n = 36), fulvestrant (n = 21), and tamoxifen (n = 15). Patients received endoxifen once daily at seven dose levels (20 to 160 mg). Dose escalation ceased at 160 mg per day given lack of MTD and endoxifen concentrations > 1,900 ng/mL. Endoxifen clearance was unaffected by CYP2D6 genotype. One patient (60 mg) had cycle 1 dose-limiting toxicity (pulmonary embolus). Overall clinical benefit rate (stable > 6 months [n = 7] or partial response by RECIST criteria [n = 3]) was 26.3% (95% CI, 13.4% to 43.1%) including prior tamoxifen progression (n = 3). cfDNA mutations were observed in 13 patients ( PIK3CA [n = 8], ESR1 [n = 5], TP53 [n = 4], and AKT [n = 1]) with shorter progression-free survival ( v those without cfDNA mutations; median, 61 v 132 days; log-rank P = .046). Clinical benefit was observed in those with ESR1 amplification (tumor; 80 mg/day) and ESR1 mutation (cfDNA; 160 mg/day). Comparing tumor biopsies and cfDNA, some mutations ( PIK3CA, TP53, and AKT) were undetected by cfDNA, whereas cfDNA mutations ( ESR1, TP53, and AKT) were undetected by biopsy. Conclusion In endocrine-refractory metastatic breast cancer, Z-endoxifen provides substantial drug exposure unaffected by CYP2D6 metabolism, acceptable toxicity, and promising antitumor activity.

Abstract TGFβ–SMAD signaling exerts a contextual effect that suppresses malignant growth early in epithelial tumorigenesis but promotes metastasis at later stages. Longstanding challenges in resolving this functional dichotomy may uncover new strategies to treat advanced carcinomas. The Krüppel-like transcription factor, KLF10, is a pivotal effector of TGFβ/SMAD signaling that mediates antiproliferative effects of TGFβ. In this study, we show how KLF10 opposes the prometastatic effects of TGFβ by limiting its ability to induce epithelial-to-mesenchymal transition (EMT). KLF10 depletion accentuated induction of EMT as assessed by multiple metrics. KLF10 occupied GC-rich sequences in the promoter region of the EMT-promoting transcription factor SLUG/SNAI2, repressing its transcription by recruiting HDAC1 and licensing the removal of activating histone acetylation marks. In clinical specimens of lung adenocarcinoma, low KLF10 expression associated with decreased patient survival, consistent with a pivotal role for KLF10 in distinguishing the antiproliferative versus prometastatic functions of TGFβ. Our results establish that KLF10 functions to suppress TGFβ-induced EMT, establishing a molecular basis for the dichotomy of TGFβ function during tumor progression. Cancer Res; 77(9); 2387–400. ©2017 AACR.

Abstract Background: The primary mechanism by which tamoxifen (Tam) and its metabolites exert their biologic effects is through estrogen receptor (ER) binding and inhibition of ER signaling. We and others demonstrated that endoxifen (Endx) has greater antitumor activity in vitro and in vivo compared to Tam and the first-in-human Endx phase I study demonstrated its antitumor activity in patients with prior progression on Tam (Goetz SABC 2015). PKCs are a family of serine/threonine-specific protein kinases that regulate signaling pathways involved in cell proliferation and tumorigenic transformation. Our prior protein docking studies suggested endoxifen may be a substrate for PKCs. Here we report the effects of Tam and Endx on PKCβ1 binding, kinase activity, as well as interactions between PKCβ1 and ERα. Methods: Surface Plasmon Resonance (SPR, Biacore T200, GE Healthcare) was used to evaluate binding of Tam, N-desmethyl Tam (NDMT), 4-HT, and Endx to PKCβ1 and PKCβ2. The effects of Tam and Endx on PKCβ1 kinase activity were determined. Proliferation and colony formation in MCF7 parental and PKCβ1 overexpressing cells were evaluated. siRNA silencing was used to knockdown PKCβ1 expression in the following cells: MCF7 aromatase expressing cells that were either sensitive (MCF7/AC1) or resistant to letrozole (MCF7/AC1 L-resistant); T47D; and MDA-MB-361. Coimmunoprecipitation assay and DUOlink in situ proximity ligation were used to investigate the interaction between PKCβ1 and ERα. Results: Endx more potently inhibited PKCβ1 kinase activity compared to Tam ( IC50 350 nM vs 47.8 μM ) with KDs for PKCβ1 binding as follows: Endx (100 nM ), Tam ( 2 μM ), 4-HT ( 2 μM ) and NDMT (> 7 μM ). None of the SERMs exhibited PKCβ2 binding. In the MCF7/AC1 and MCF7/AC1 L-resistant cells, PKCβ1 knockdown resulted in ERα degradation and potently inhibited cell proliferation. These results were confirmed in T47D and MDA-MB-361 cells. Notably, PKCβ1 knockdown in MCF7/AC1 cells resulted in significantly greater E2 induced proliferation comparing siRNA knockdown vs. control. To further explore these effects, we evaluated the effects of PKCβ1 overexpression in MCF7 cells and demonstrated that PKCβ1 overexpression reduced cell proliferation and colony formation compared to parental MCF-7 cells without affecting ERα protein stability. Coimmunoprecipitation assays in transient transfected MCF-7 cells with exogenous PKCβ1 as well as PKCβ1 expressing MDA-MB-231 cells transiently or stably transfected with ERα demonstrated PKCβ1 and ERα interaction, with confirmation by Duolink assay that this interaction occurs in the cytoplasm. Conclusions: Our findings demonstrated that endoxifen binds and inhibits PKCβ1 at relevant concentrations achieved in the endoxifen clinical trial studies. PKCβ1 interacts with cytoplasmic ERα and PKCβ1 knockdown inhibits cell proliferation and enhances ERα turnover. However, in PKCβ1 overexpressing cells, PKCβ1 may exhibit tumor suppressive effects. These data suggest a complex interaction between PKCβ1 and ERα and that endoxifen's effects on PKCβ1 may alter drug response of endocrine therapy. Further studies are ongoing to characterize the role of PKCβ1 and its role in ER biology and response to endoxifen. Citation Format: Guo C, Kuffel MJ, Kudgus RA, Huang Z, Bode AM, Cheng J, Suman VJ, Reid JM, Bruinsma ES, Subramaniam M, Ames MM, Hawse JR, Goetz MP. Identification and characterization of a novel endoxifen substrate, PKCβ1, and its interaction with the estrogen receptor [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 P1-08-03.

The prevalence of asthma is higher in pre‐pubescent and aging males, and in post‐pubertal females, strongly indicating that sex steroids (especially estrogen) may be an important modulator in lung disease. We recently demonstrated that airway smooth muscle (ASM) expresses both alpha and beta forms of the estrogen receptor (ERα and ERβ) in males and females, and that these receptors regulate intracellular [Ca 2+ ] and ASM contractility. Although both ERα and ERβ have multiple splice variants, it is unclear if and how the expression of these variants is modulated under conditions such as chronic inflammation/asthma. In order to test the hypothesis that the differential expression of ERα and ERβ variants contributes to the pathogenesis of asthma, we profiled the expression of various ERα and ERβ genes in asthmatic and inflamed (TNFα‐ or IL‐13‐treated) ASM. Gene expression was assessed at both the mRNA and protein levels in asthmatic ASM cells or non‐asthmatic cells treated with TNFα (20 ng/ml) or IL‐13 (50 ng/ml). We observed marked variation in the expression of ER isoforms in response to inflammatory stimuli, and in non‐asthmatic versus asthmatic ASM. Changes in protein levels of ERα and ERβ corresponded with the observed differential mRNA patterns. Pharmacological studies implicate cytosolic (p42/44 MAPK and PI3 K) and nuclear (NFκB, STAT6, and AP‐1) signaling pathways as putative mechanisms that mediate and/or regulate effects of inflammation on ER expression. We conclude that variations in ASM ER expression profiles occur with inflammation and that ER variants could contribute to estrogen signaling in airway diseases such as asthma. J. Cell. Physiol. 232: 1754–1760, 2017. © 2016 Wiley Periodicals, Inc.

Abstract Background: Intrinsic resistance has been linked to ER down-regulation and poor clinical outcome in breast tumors. Aurora-A kinase (AURKA) promotes distant metastases through activation of epithelial to mesenchymal transition (EMT) programming and expansion of breast tumor initiating cells (BTICs) harboring a CD44high/CD24low phenotype. Moreover, AURKA induces down-regulation of ERα expression and resistance to endocrine therapy that is mediated by SMAD5 transcription factor (D’Assoro et al., Oncogene 2014: 33:599-610). The aim of this study was to evaluate the role of AURKA in endocrine resistance induced by breast cancer stemness and ER down-regulation. Materials and Methods: We employed parental MCF-7 and variant MCF-7 cells ex-vivo (vMCF- 7/LCC9, vMCF-7/FR) that have high endogenous levels of AURKA, are ERlow, and exhibit in vivo resistance to fulvestrant. Real-Time Cell Proliferation and Apoptosis assays were performed to assess response to endocrine and/or AURKA-targeted therapy using the innovative IncuCyte ZOOM System. Stemness activity was assessed by culturing breast cancer cells under non-adherent conditions to generate mammospheres. Expression of ER, CD44 and CD24 markers was characterized by FACS analysis and immunofluorescence. Results: Pharmacologic inhibition of AURKA activity with the small molecule, alisertib, significantly restored sensitivity to fulvestrant in vMCF-7/LCC9 and vMCF-7/FR cells. To establish whether fulvestrant resistance was linked to enrichment of BTICs, breast cancer cells were cultured under non-adherent conditions to generate mammospheres. vMCF-7/FR cells generated mammospheres at higher efficiency compared to parental cells suggesting that vMCF-7/FR cells exhibited high self-renewal activity. vMCF-7/FR-derived mammospheres displayed a stem cell-like CD44high/CD24low phenotype that was further characterized by low levels of ER expression and resistance to fulvestrant. When vMCF-7 FR-derived mammospheres were treated with alisertib alone or combined with fulvestrant, the combination was more effective in mediating restoration of endocrine sensitivity. The phenotype of the alisertib-treated mammospheres was functionally linked to loss of CD44 expression, restoration of CD24 and ER expression, and sensitivity to fulvestrant. Conclusions: These findings provide further evidence that AURKA activity plays a central role in breast cancer progression through the genesis and clonal expansion of BTICs that are characterized by ER down-regulation and resistance to endocrine therapy. These results are important for the treatment of endocrine resistant breast tumors because they provide the rationale for the development of an innovative therapeutic approach to combine endocrine therapy with a targeted inhibitor of AURKA activity. Citation Format: Antonino B. D’Assoro, Tufia Haddad, Matthew Goetz, Mary Kuffel, John Hawse, Vera Suman, Jeffrey Salisbury, James Ingle, Evanthia Galanis. Pharmacologic targeting of Aurora-A kinase restores ERα expression and endocrine sensitivity through impairment of breast cancer stemness. [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 3313.

Neuromuscular choristoma (NMC) is a very rare, developmental malformation characterized by the endoneurial intercalation of mature muscle fibers among peripheral nerve fibers. NMC typically arises in the major proximal peripheral nerves, most commonly the sciatic nerve, and may involve the lumbosacral and brachial plexus. Patients present clinically with progressive neuropathy or plexopathy. NMC is strongly associated with development of a fibromatosis, histologically identical to conventional desmoid-type fibromatosis (NMC-fibromatosis). The development of NMC-fibromatosis is often precipitated by iatrogenic trauma (ie, biopsy). Desmoid-type fibromatosis is characterized by CTNNB1 exon 3 mutations, which result in aberrant nuclear β-catenin localization and dysregulated canonical Wnt signaling. In contrast, the pathogenesis of NMC and NMC-fibromatosis is unknown. Desmoid-type fibromatosis expresses estrogen receptors (ER), specifically the ER-beta isoform (ERβ), and endocrine therapies may be used in surgically unresectable cases. In contrast, the ER expression profile of NMC-fibromatosis is unknown. We evaluated a series of NMC and NMC-fibromatosis for CTNNB1 mutations, β-catenin expression, and ER isoform expression. Five NMCs occurred in 2 female and 3 male patients (median age: 14 y, range <1 to 42 y), as masses involving the sciatic nerve (N=4) or brachial plexus (N=1). Four (of 5) NMCs had CTNNB1 mutations: 3 c.134 C>T (p.S45F) and 1 c.121 A>G (p.T41A). Four patients subsequently developed NMC-fibromatosis, and all 4 cases contained CTNNB1 mutations, including 1 p.T41A and 3 p.S45F mutations. In 3 patients, the NMC and NMC-fibromatosis had identical CTNNB1 mutations. Only 1 NMC had no detectable CTNNB1 mutation; however, the patient’s subsequent NMC-fibromatosis had a CTNNB1 p.T41A mutation. All NMC and NMC-fibromatosis showed aberrant nuclear localization of β-catenin, nuclear ERβ expression, and no ERα expression. The presence of CTNNB1 mutations both in NMC and NMC-fibromatosis may be a shared molecular genetic abnormality underlying their pathogenesis.

Abstract Background: AI's are more effective than TAM in ER+ breast cancer. In AI refractory MBC, the response rate to TAM is 0% (Osborne 2011). Z-Endx is an active metabolite of TAM and among TAM treated women in the adjuvant and metastatic settings, reduced CYP2D6 metabolism and low Endx concentrations (Css &lt;20 nM) have been associated with increased likelihood of disease recurrence. Preclinical studies have demonstrated greater Z-Endx exposure and anti-tumor activity with oral Z-Endx compared to equivalent doses of oral TAM (Reid 2014) Methods: We conducted a phase I trial to determine the maximum-tolerated dose (MTD) and evaluate the toxicities, clinical activity, and pharmacokinetics (PK) of Z-Endx in patients (pts) with ER+, AI refractory MBC. Unlimited prior endocrine regimens were allowed. An accelerated titration schedule was applied (2 pts/dose level) until moderate toxicity or DLT, followed by a 3+3 design and then to expansion cohorts (40, 80, and 100 mg/day). Z-Endx was administered orally once daily (28 day cycle). Eye exams were performed at baseline, and end of cycles 2 and 6. PK was performed during cycle 1 and prior to subsequent cycles. For pts in the expansion cohorts, tumor biopsies were obtained at baseline for DNA sequencing (Foundation Medicine). Plasma cholesterol levels were obtained at baseline and after 1 cycle. Results: From March 2011 to Dec 2014, 41 pts (38 evaluable), median age 60, received Z-Endx once daily encompassing 7 dose levels (20-160 mg/daily). The median number of prior hormonal regimens was 2 and 3 for the dose escalation and expansion cohorts, respectively. Dose escalation was stopped at 160 mg/day given MTD not reached and attainment of mean Endx Css of 3.6 uM. Cycle 1 DLT (PE) was observed in one patient (60 mg). No eye toxicity was observed. PK demonstrated mean Endx Css of &gt; 1 uM at all dose levels ≥ 40 mg/day. Antitumor activity was observed at multiple dose levels including 3 confirmed partial responses and an additional 7 with stable disease for ≥6 cycles. Of these 10 pts, 9 had prior progression on both AI and fulvestrant and 3 additionally on TAM. After 1 cycle, total and LDL cholesterol decreased &gt; 20 points in 54% and 40% of pts, respectively. Tumor sequencing in the expansion cohorts (n=14) did not identify ESR1 mutations; however, ESR1 amplification was identified in 1 pt with prolonged stable disease (&gt;200 days). Of 6 pts with rapid progression (≤2 cycles), 4/6 had either CCND1 amplification (n=1) or at least one of the following activating mutations: ERBB2 L755S (n=1), AKT1 E17K (n=1), MTOR E1799K (n=1). Conclusions: The direct administration of Z-END provides substantial drug exposure, acceptable toxicity, and "proof of principle" antitumor activity in endocrine resistant MBC. While the MTD was not determined, the goal of achieving Endx Css concentrations of &gt; 1 uM was achieved. Tumor sequencing identified pts with predicted and confirmed endocrine resistance. A randomized phase II comparing endoxifen (80 mg/day) with TAM in AI refractory MBC was recently activated (NCT02311933). Supported in part by CA 133049, CA186686, CA15083, CA116201, and CA15083. Citation Format: Goetz MP, Suman VJ, Reid JM, Northfelt DW, Mahr MA, Dockter T, Kuffel M, Buhrow SA, Safgren SL, McGovern RM, Collins JM, Streicher H, Hawse JR, Haddad TC, Erlichman C, Ames MM, Ingle JN. Final results of a first-in-human phase I study of the tamoxifen (TAM) metabolite, Z-Endoxifen hydrochloride (Z-Endx) in women with aromatase inhibitor (AI) refractory metastatic breast cancer (MBC) (NCT01327781). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD2-03.

Abstract Background: Triple negative breast cancer (TNBC) accounts for approximately 20% of all breast cancer diagnoses. Clinical management of TNBC is limited to surgery, chemotherapy and radiation due to lack of estrogen receptor alpha and HER2 expression. Recently, we have shown that approximately 40% of TNBCs express estrogen receptor beta (ERβ) and have begun to explore the possibility that this receptor could be utilized as a novel therapeutic target for this disease. Methods: To examine the biological functions of ERβ in TNBC, novel ERβ expressing TN cell lines (MDA-MB-231 and Hs578T) were developed. In vitro experiments were employed to determine alterations in the global gene expression profiles, biological pathways, proliferation rates, and cell cycle progression following estrogen or ERβ-specific agonist treatment. Cell line xenografts were also established in athymic ovariectomized nude mice to examine tumoral responses to ERβ targeting agents and to investigate gene and protein expression patterns as well as potential serum biomarkers indicative of therapeutic response. Additionally, using the resources of the Mayo Clinic Breast Cancer Genome Guided Therapy Study (BEAUTY), we have identified, and begun to analyze, ERβ+ and ERβ- patient derived xenografts (PDX) established from women with TNBC. Results: Our studies have revealed that both estrogen and multiple ERβ-specific agonists elicit significant anti-proliferative effects in ERβ+ TNBC cells primarily through a G1/S phase cell cycle arrest. These anti-proliferative effects appear to be mediated by cystatins, a family of small secreted cysteine protease inhibitors which are highly induced following estrogen and ERβ-specific agonist treatment. Conditioned media isolated from estrogen or ERβ-specific agonist treated cells decreased the proliferation rates of multiple non-ERβ expressing cell lines; effects that were completely reversed when cystatins were depleted from the media. In addition, we have shown that activation of ERβ, and the subsequent induction of cystatin gene expression, leads to suppression of canonical TGFβ signaling through multiple mechanisms including suppression of TGFβR2 expression, induction of Smad7 expression and blockade of TGFβ ligand-mediated activation of this pathway both in vitro and in vivo. Finally, ERβ+ TNBC PDXs exhibit significantly decreased tumor growth rates in estrogen-treated mice compared to ERβ- TN breast tumors. Conclusions: Our in vitro and in vivo data show that estrogen and ERβ-specific agonists elicit anti-cancer effects in ERβ+ TNBC. These effects appear to be mediated, in part, by cystatins through their inhibitory effects on canonical TGFB signaling, a pathway known to drive TNBC progression. Importantly, these data lay the foundation for studies aimed at examining the ability to therapeutically target ERβ in TNBC patients. Citation Format: Reese J, Bruinsma E, Subramaniam M, Suman V, Pitel K, Kalari K, Yu J, Wang L, Goetz M, Ingle J, Hawse J. ERβ elicits tumor suppressive effects in triple negative breast cancer through the induction of cystatins and suppression of TGFβ signaling. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-01.